WO2026030369A1

WO2026030369A1 - Methods and compositions for in situ analyte detection

Info

Publication number: WO2026030369A1
Application number: PCT/US2025/039731
Authority: WO
Inventors: Jason C. BELL; Jeremy Lackey; Michael Schnall-Levin
Original assignee: 10X Genomics Inc
Current assignee: 10X Genomics Inc
Priority date: 2024-07-31
Filing date: 2025-07-29
Publication date: 2026-02-05
Anticipated expiration: 2027-01-31

Abstract

The present disclosure relates in some aspects to methods and compositions for analyzing target nucleic acids in a biological sample. In some aspects, the presence, amount, and/or identity of a plurality of target nucleic acids are analyzed in situ in a sample. Also provided are oligonucleotides for hybridization, sets of oligonucleotides, sequencing reagents, compositions, and systems for use in accordance with the methods.

Description

202412023840 METHODS AND COMPOSITIONS FOR IN SITU ANALYTE DETECTION CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application No. 63/677,827 filed July 31, 2024, entitled “METHODS AND COMPOSITIONS FOR IN SITU ANALYTE DETECTION”, and U.S. Provisional Patent Application No. 63/692,458 filed September 9, 2024, entitled “METHODS AND COMPOSITIONS FOR IN SITU ANALYTE DETECTION”, each of which is herein incorporated by reference in its entirety for all purposes. FIELD [0002] The present disclosure relates in some aspects to methods and compositions for nucleic acid analysis in situ in biological samples, such as multiplex target nucleic acid detection and readout in situ in a cell or tissue sample. BACKGROUND [0003] Methods are available for detecting nucleic acids present in a biological sample. For instance, advances in single molecule fluorescent in situ hybridization (smFISH) have enabled nanoscale-resolution imaging of RNA in cells and tissues. However, analysis of various target analyte types, including large panels of transcripts and short variant sequences (e.g., single nucleotide differences such as single nucleotide variation (SNVs) or point mutations) in situ has remained challenging. Improved methods for identifying these various analytes and analyzing their spatial distribution in cell or tissue samples are needed. Provided herein are methods, compositions, and kits that address such and other needs. SUMMARY [0004] Provided herein is a method for analyzing a biological sample comprising (a) binding a plurality of circularizable probes to a plurality of target nucleic acids in the biological sample, wherein: the plurality of circularizable probes comprises a first probe that binds to a first target nucleic acid and a second probe that binds to a second target nucleic acid, wherein the first probe comprises a 5’ end and a 3’ end; (b) ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template; (c) extending the second probe using a sequence of the second target nucleic acid as template to generate an extended probe comprising a gap filled sequence; (d) ligating the extended probe to generate a second circularized template; (e) 1 ^{MOFO-357975700} 202412023840 performing rolling circle amplification (RCA) of the first circularized template and the second circularized template in the biological sample to generate a first RCA product and a second RCA product, respectively; (f) contacting the biological sample with an oligonucleotide probe that directly or indirectly binds to a sequence in the first RCA product and detecting a first signal associated with the oligonucleotide probe, thereby detecting the first target nucleic acid, and (g) sequencing the second RCA product to determine the gap filled sequence or a complement thereof, thereby detecting the second target nucleic acid. In some instances, (b) and (d) are performed simultaneously. In some instances, (b), (c), and (d) are performed in an order selected from the group consisting of: (b) - (c) - (d); (c) - (d) - (b); and (c) - (b) - (d). [0005] In some embodiments, the first probe comprises a barcode sequence corresponding to the first target nucleic acid or a sequence thereof. In some aspects, the barcode sequence is not complementary to the target nucleic acid or sequence thereof. In some embodiments, the oligonucleotide probe directly or indirectly binds to the barcode sequence or a complement thereof. [0006] In some aspects, the first probe comprises (i) a first hybridization region complementary to a first sequence of the first target nucleic acid and (ii) a second hybridization region complementary to a second sequence of the first target nucleic acid. In some embodiments, the oligonucleotide probe directly or indirectly binds to a sequence comprising: (i) the first sequence of the first target nucleic acid or a portion thereof, and/or (ii) the second sequence of the first target nucleic acid or a portion thereof. In some embodiments, the second probe does not comprise a barcode sequence corresponding to the second target nucleic acid or a sequence thereof. In some aspects, the second probe comprises (i) a first hybridization region complementary to a first sequence of the second target nucleic acid and (ii) a second hybridization region complementary to a second sequence of the second target nucleic acid, and the first hybridization region and the second hybridization region of the second probe are common among a plurality of second probes each targeting a molecule comprising a different variant sequence of the second target nucleic acid. In some embodiments, the plurality of second probes are capable of hybridizing to both a wildtype molecule and a mutant molecule of the second target nucleic acid. In some embodiments, the first sequence and the second sequence of ^{MOFO-357975700} 202412023840 the second target nucleic acid are separated by a gap sequence of at least 2 nucleotides in the second target nucleic acid. [0007] In some instances, the gap sequence is between about 2 and about 40 nucleotides in length. In some aspects, the second target nucleic acid comprises a variant sequence at the 3’ or 5’ end of the gap sequence. In some embodiments, the second target nucleic acid comprises a variant sequence at or near the central nucleotide(s) of the gap sequence. In some embodiments, the second target nucleic acid comprises a variant sequence at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or more phosphodiester bonds from the 3’ or 5’ end of the gap sequence. In some embodiments, the first hybridization region and the second hybridization region in the first probe are equal in length. In some embodiments, the first hybridization region is shorter or longer than the second hybridization region in the first probe. In some embodiments, the first hybridization region and the second hybridization region in the second probe are equal in length. In some embodiments, the first hybridization region is shorter or longer than the second hybridization region in the second probe. [0008] In some embodiments, the first hybridization region and/or the second hybridization region in the first probe is between about 5 and about 50 nucleotides in length. In some embodiments, the first hybridization region and/or the second hybridization region in the second probe is between about 5 and about 50 nucleotides in length. [0009] In some aspects, the first probe is provided in two or more parts. In some instances, the first probe comprises a first part and a second part. In some instances, the first probe is provided in three parts. In some instances, the first probe is provided in two parts. In some embodiments, the second probe is provided in two or more parts. In some instances, the second probe comprises a first part and a second part. In some instances, the second probe is provided in three parts. In some instances, the second probe is provided in two parts. In some embodiments, the method comprises ligating the 5’ end of the first probe to the 3’ end of the first probe using the first target nucleic acid as template. [0010] In some aspects, the biological sample comprises different molecules of the second target nucleic acid each comprising a mutant sequence of a plurality of different mutant sequences. In some embodiments, extending the second probe is performed using a library of splint oligonucleotides comprising a plurality of different mutant splint oligonucleotides each ^{MOFO-357975700} 202412023840 comprising a hybridization region complementary to sequence of a plurality of different second target nucleic acids, and a splint oligonucleotide of the library of splint oligonucleotides that is complementary to the second target nucleic acid is ligated to the second probe, thereby circularizing the second probe to generate the second circularized template. In some embodiments, each splint oligonucleotide of the library of splint oligonucleotides comprises a 3’ hydroxyl group and a 5’ phosphate group, optionally wherein the splint oligonucleotide comprises a 5’ flap and/or one or more RNA residues at and/or near its 3’ end. In some cases, the splint oligonucleotide is between about 2 and about 40 nucleotides in length. In some instances, the splint oligonucleotide is ligated to the second probe by a ligase using the second target nucleic acid as a template. In some cases, the second target nucleic acid is an RNA and the ligase has an RNA-templated ligase activity. In some cases, the second target nucleic acid is a DNA, and the ligase has a DNA-templated ligase activity. [0011] In some embodiments, the library of splint oligonucleotides comprises at least or about 2, at least or about 5, at least or about 10, at least or about 15, at least or about 20, at least or about 25, at least or about 30, at least or about 35, at least or about 40, at least or about 45, at least or about 50, or more splint oligonucleotides of different sequences. In some aspects, the biological sample is washed after contacting with the library of splint oligonucleotides, optionally wherein the washing is performed under less than stringent conditions. [0012] In some embodiments, extending the second probe comprises extending an end of the second probe by a polymerase using the second target nucleic acid as a template. In some aspects, the second target nucleic acid is an RNA and the polymerase has an RNA- templated polymerase activity. In some aspects, the second target nucleic acid is a DNA and the polymerase has a DNA-templated polymerase activity. In some instances, the polymerase is a reverse transcriptase. In some cases, the polymerase incorporates two or more residues into the 3’ end of the second probe to generate the extended probe. In some embodiments, the polymerase has no or little strand displacement activity. [0013] In some embodiments, the extended probe is ligated by a ligase using the second target nucleic acid as a template, wherein the target nucleic acid is an RNA or a DNA, and the ligase has an RNA-templated ligase activity and/or a DNA-templated ligase activity. ^{MOFO-357975700} 202412023840 [0014] In some embodiments, the second target nucleic acid comprises a nucleotide variation, a nucleotide polymorphism, a mutation, a substitution, an insertion, a deletion, a translocation, a rearrangement, a duplication, an inversion, and/or a repetitive sequence. In some embodiments, the second target nucleic acid comprises a variant sequence and the variant sequence comprises is a single nucleotide, optionally wherein the variant sequence is a single nucleotide variation (SNV), a single nucleotide polymorphism (SNP), a point mutation, a single nucleotide substitution, a single nucleotide insertion, or a single nucleotide deletion. [0015] In some embodiments, the second target nucleic acid comprises a sequence of an immune molecule. In some instances, the sequence of the immune molecule is an antigen receptor transcript. In some instances, the antigen receptor transcript is a T cell receptor (TCR) transcript, optionally wherein the TCR transcript comprises a TCRα VJ join, a TCRβ VDJ join, a TCRγ VJ join, or a TCRδ VDJ join. In some instances, the second RCA product comprises multiple copies of a unit sequence comprising a sequence of a VDJ join or a complement thereof. In some instances, the unit sequence comprises the D segment of the VDJ join. In some instances, the unit sequence comprises the V segment or a portion thereof and/or the J segment or a portion thereof of the VDJ join. In some instances, the antigen receptor transcript is an immunoglobulin (Ig) transcript, optionally wherein the Ig transcript comprises an Igκ VJ join, an Igλ VJ join, or an IgH VDJ join. In some instances, multiple different antigen receptor transcripts present at a plurality of locations in the biological sample are identified. In some instances, a plurality of VDJ joins of the multiple different antigen receptor transcripts comprises at least about 100, at least about 500, at least about 1,000, at least about 5,000, at least about 10,000, or more VDJ joins of different sequences. [0016] In some aspects, the second target nucleic acid comprises one or more exon- exon boundaries of a nucleic acid. In some aspects, the second target nucleic acid comprises a sequence of a perturbation agent introduced to the biological sample. In some aspects, the second target nucleic acid comprises a CRISPR molecule, a nucleic acid molecule edited using the CRISPR molecule, and/or a precursor or derivative thereof. In some instances, the second target nucleic acid is a transcript comprising a unique barcode specific to a guide RNA. In some instances, the second target nucleic acid is an RNA transcript less than 60 nucleotides in length. In some instances, the second target nucleic acid is an RNA transcript less than 80 nucleotides in ^{MOFO-357975700} 202412023840 length. In some instances, the second target nucleic acid is an RNA transcript less than 100 nucleotides in length. In some instances, the second target nucleic acid is an RNA transcript less than 200 nucleotides in length. In some instances, the first target nucleic acid and/or the second target nucleic acid is RNA. [0017] In some instances, the first target nucleic acid and the second target nucleic acid are RNA transcripts of different genes. In some instances, the biological sample is contacted with a plurality of different first probes comprising the first probe, wherein each different first probe binds to an RNA transcript of one of a plurality of different genes, and the biological sample is contacted with the second probe, wherein the second probe is capable of binding to at least two different RNA transcripts of a first gene of the plurality of different genes. In some instances, the biological sample is contacted with a plurality of different second probes comprising the second probe and an additional second probe, wherein the additional second probe is capable of binding to at least two different RNA transcripts of a second gene of the plurality of different genes. In some instances, molecules of second probe are configured to bind to at least two different RNA transcripts of the same second gene (e.g., first hybridization region and second hybridization region are common for binding the first sequence and the second sequence among the different RNA transcripts). In some embodiments, the second target nucleic acid comprises a barcode sequence. In some cases, the barcode sequence is for lineage tracing. [0018] In some instances, an RNase H cleaves the first target nucleic and the second target nucleic acid to generate a first cleaved target RNA and a second cleaved target RNA. In some cases, RCA of the first circularized template and the second circularized template is performed using the first cleaved target RNA and the second cleaved target RNA, respectively, as a primer. In some cases, the first RCA product and the second RCA product is generated in situ in the biological sample or a matrix embedding the biological sample. [0019] In some embodiments, the method comprises imaging the biological sample to detect the first RCA product and the second RCA product in situ in the biological sample or a matrix embedding the biological sample. In some cases, the first target nucleic acid and/or the second target nucleic acid is attached directly or indirectly to the biological sample or to a matrix embedding the biological sample. In some instances, the first target nucleic acid and/or the second target nucleic acid is crosslinked in the biological sample or in a matrix embedding the ^{MOFO-357975700} 202412023840 biological sample. In some instances, the first RCA product and the second RCA product is covalently linked to the first target nucleic acid and the second target nucleic acid, respectively. [0020] In some instances, the barcode sequence comprises a first subunit and a second subunit, wherein a portion of the first subunit overlaps with some but not all of the second subunit. In some instances, the barcode sequence is assigned a signal code sequence, wherein the first subunit or a complement thereof corresponds to a first signal code of the signal code sequence, and the second subunit or a complement thereof corresponds to a second signal code of the signal code sequence. In some instances, the oligonucleotide probe comprises a detectable label associated with the first signal code. In some instances, the detectable label associated with the first signal code that identifies the first target nucleic acid is detected. In some instances, decoding is performed using the second signal code detected from a sequential cycle of hybridization, detection, and removal of a second oligonucleotide probe labeled with a second detectable label. [0021] In some instances, the oligonucleotide probe hybridizes to a barcode sequence or complement thereof of the first probe. In some instances, a universal pool of detectably labeled probes is used for detection. In some instances, the number of different detectably labeled probes in the universal pool is four. In some instances, the oligonucleotide probe comprises i) a hybridization regions complementary to the barcode sequences of the first probe or complement thereof and ii) a reporter region complementary to a detectably labeled probe of the universal pool of detectably labeled probes. In some instances, the detectably labeled probe comprises a sequence complementary to the reporter region and a detectable label. [0022] In some instances, a complex formed between the barcode sequence or complement thereof, the oligonucleotide probe, and the detectably labeled probe is detected. In some instances, a series of signal codes that identifies the first probe or the first target nucleic acid is detected. In some instances, decoding a series of signal codes detected from a plurality of sequential cycles of hybridization, detection, and removal of sequential pools of oligonucleotide probes and the universal pool of detectably labeled probes is performed. In some instances, the series of signal codes are fluorophore sequences assigned to the corresponding first target nucleic acid. In some instances, the detectably labeled probes are fluorescently labeled. ^{MOFO-357975700} 202412023840 [0023] In some instances, the plurality of sequential cycles comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cycles. In some instances, a detectable label of the detectably labeled probes corresponds to a sequence of the oligonucleotide probe. In some instances, a plurality of first target nucleic acids are present in the biological sample and each first target nucleic acid of the plurality of first target nucleic acids are assigned a signal code sequence, and detecting the first target nucleic acids comprises (i) contacting the biological sample with a first oligonucleotide probe and a first detectably labeled probe to generate a first complex comprising the first oligonucleotide probe hybridized to the first RCA product and the first detectably labeled probe hybridized to the first oligonucleotide probe, (ii) imaging the biological sample to detect a first signal from the first detectably labeled probe, wherein the first signal corresponds to a first signal code in the signal code sequence assigned to the first target nucleic acid; (iii) contacting the biological sample with a second oligonucleotide probe and a second detectably labeled probe to generate a second complex comprising the second oligonucleotide probe hybridized to the second RCA product and the second detectably labeled probe hybridized to the second oligonucleotide probe; and (iv) imaging the biological sample to detect a second signal from the second detectably labeled probe, wherein the second signal corresponds to a second signal code in the signal code sequence assigned to the first target nucleic acid, wherein the signal code sequence comprising at least the first signal code and the second signal code is determined based on signals detected at a location in the biological sample, thereby identifying the one or more sequences of the first target nucleic acid at the location in the biological sample. [0024] In some instances, the gap filled sequence or a complement thereof in the second RCA product is detected using sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof. In some instances, the gap filled sequence or a complement thereof in the second RCA product is sequenced using in situ sequencing by synthesis (SBS) in the biological sample. In some instances, the gap filled sequence or a complement thereof in the second RCA product is detected using single nucleotide sequencing by synthesis. In some instances, the gap filled sequence or a complement thereof in the second RCA product is sequenced using in situ sequencing by ligation (SBL) in the biological sample. In some instances, the gap filled sequence or a complement thereof in the second RCA product is sequenced using in situ sequencing by binding (SBB) in the biological sample. In some ^{MOFO-357975700} 202412023840 instances, a priming strand bound to the second RCA product comprising the gap filled sequence or a complement thereof is contacted with (i) a polymerase and (ii) a first plurality of nucleotide molecules to form a complex comprising a 3’ terminus of the priming strand, the second RCA product, the polymerase, and a nucleotide molecule of the first plurality of nucleotide molecules and detecting presence of the nucleotide molecules in the complex to identify a complementary nucleotide in the second RCA product. In some embodiments, the priming strand is bound to a sequencing primer binding sequence in the second RCA product, wherein at least a portion of the sequencing primer binding sequence in the second RCA product is the same as a sequence in the second target nucleic acid complementary to the second probe. In some embodiments, the priming strand is bound to the second RCA product at a sequence that is not the same as a sequence of the second target nucleic acid. In some embodiments, the priming strand is extended by two or more nucleotides that are not fluorescently labeled in consecutive sequencing cycles, using a sequence of the second RCA product as a template. In some instances, the priming strand comprises a 3’ terminal nucleotide that is reversibly blocked. In some instances, the method further comprises removing the complex, unblocking the reversibly blocked 3’ terminal nucleotide molecule and contacting the priming strand bound to the second RCA product with a polymerase and a second plurality of nucleotide molecules. In some instances, contacting the priming strand with an additional plurality of nucleotide molecules to identify additional complementary nucleotides of the gap filled sequence in the second RCA product is repeated for at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or at least 150 additional cycles. [0025] In some instances, the first target nucleic acid detected and the second target nucleic acid detected in are registered to the same cell. [0026] In some instances, a labeling agent comprising a reporter oligonucleotide, wherein the labeling agent is bound directly or indirectly to a non-nucleic acid analyte in the biological sample, is detected. In some instances, a sequence of the reporter oligonucleotide or a complement thereof is detected by sequencing. In some instances, the sequence of the reporter oligonucleotide or a complement thereof is detected by using single nucleotide sequencing by synthesis. ^{MOFO-357975700} 202412023840 [0027] In some embodiments, the biological sample is contacted with a library of first probes to detect a panel of at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, at least 2,000, at least 3,000, at least 4,000, at least 5,000 or more first target nucleic acids. In some embodiments, the biological sample is a cell or tissue sample, e.g., a sample comprising cells or cellular components. In some cases, the biological sample is a tissue section. In some instances, the biological sample is a formalin-fixed, paraffin- embedded (FFPE) sample, a frozen tissue sample, or a fresh tissue sample. In some aspects, the biological sample is fixed and/or permeabilized. In some embodiments, the biological sample is crosslinked and/or embedded in a matrix, optionally wherein the matrix comprises a hydrogel. In some instances, the biological sample is cleared. [0028] Provided herein is a system comprising a plurality of circularizable probes, wherein the plurality of circularizable probes comprises a first probe that binds to a first target nucleic acid and a second probe that binds to a second target nucleic acid, wherein the first probe comprises a 5’ end and a 3’ end; one or more reagents for gap filling the second probe to generate an extended probe comprising a gap filled sequence; one or more reagents for circularizing the plurality of circularizable probes and extended probe to generate a plurality of circularized templates; one or more reagents for generating a plurality of rolling circle amplification products (RCPs) of the plurality of circularized template; a plurality of oligonucleotide probes for detecting a first subset of the generated RCPs; and one or more reagents for sequencing a second subset of the generated RCPs. In some instances, each oligonucleotide probe comprises i) a hybridization region complementary to a sequence in a first RCA product of the first subset of the generated RCPs; and ii) a detectable region. In some instances, the system comprises a plurality detectably labeled probes each comprising a detectable label and a sequence for hybridizing to a sequence in the first subset of the generated RCPs. In some cases, the system comprises a plurality of a sequencing primer, a plurality of detectably labeled nucleotides, and a polymerase. In some embodiments, the one or more reagents for gap filling the second probe comprises an engineered family B polymerase and dNTPs. In some instances, the one or more reagents for circularizing the plurality of circularizable probes comprises a ligase. In some embodiments, the engineered family B ^{MOFO-357975700} 202412023840 polymerase is an engineered Tgo polymerase comprising one or more mutations that confer reverse transcriptase activity. [0029] In some embodiments, the system comprises an optical detection system configured to detect the generated RCA products. In some embodiments, the system comprises a solid support having a biological sample attached thereto, wherein the biological sample comprises the first target nucleic acid and the second target nucleic acid. In some instances, the biological sample is a cell or tissue sample, e.g., a sample comprising cells or cellular components. In some instances, the biological sample is a tissue section. In some instances, the biological sample is crosslinked and/or embedded in a matrix. In some instances, the matrix comprises a hydrogel. In some instances, the biological sample comprises different molecules of the second target nucleic acid each comprising a mutant sequence of a plurality of different mutant sequences. In some instances, the second target nucleic acid comprises a sequence of a perturbation agent introduced to the biological sample. [0030] In some embodiments, the one or more reagents for sequencing is for performing sequencing by synthesis (SBS). In some instances, the one or more reagents for sequencing is for performing single nucleotide sequencing by synthesis. In some instances, the one or more reagents for sequencing is for performing sequencing by ligation (SBL). In some instances, the one or more reagents for sequencing is for performing in situ sequencing by binding (SBB). BRIEF DESCRIPTION OF THE DRAWINGS [0031] The drawings illustrate certain features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. [0032] FIG. 1A shows an example of a method for multiplex detection of different first and second target nucleic acids using a circularizable probe (e.g., first probe) with ends that are ligated and detected using a hybridization based approach (top) and a circularizable probe (e.g., second probe) that is gap filled and detected using a sequencing based approach (bottom). [0033] FIG. 1B shows a schematic of determining the gap filled sequence by performing a sequencing reaction. ^{MOFO-357975700} 202412023840 [0034] FIG. 2 is an example of a workflow for analysis of a biological sample (e.g., a cell or tissue sample) using an opto-fluidic instrument, according to various embodiments. DETAILED DESCRIPTION [0035] All publications, comprising patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference. [0036] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. OVERVIEW [0037] Certain methods for detecting analytes aim to provide specificity or sensitivity but may have certain trade offs. For example, assays for detecting a large panel of analytes may have a reduced ability to detect shorter and/or variable sequences (e.g., a particular SNV or unknown sequences). In some cases, assays aimed at detecting specific sequences may be limited in plexy (e.g., number of analytes to be detected). In some aspects, probes and readout methods designed for particular targets vary in structure and require different optimization. In some instances, hybridization based methods used for detection may not be optimal for detecting variable or unknown analyte sequences that require high sensitivity. Improved methods for analyzing various analytes (e.g., nucleic acids) present in a biological sample are needed. [0038] In some aspects, sequencing based detection (e.g., base-by-base sequencing reaction; e.g., as described in Section III.B) allows for detection of sequences at specified locations but with unspecified sequences (e.g., unknown or highly variable gap sequences flanked by known sequences) while hybridization based detection methods (e.g., as described in Section III.A) allow for simplicity and/or efficiency, particularly when a large number of analytes are being detected (e.g., for higher plex measurements). In some examples, an assay with combined detection using hybridization and sequencing allows for hybridization based ^{MOFO-357975700} 202412023840 detection for gene expression counting and sequencing allows for additional sequence detection where hybridization based readouts may be lacking. [0039] In some embodiments, provided herein are methods comprising binding a plurality of circularizable probes to a plurality of target nucleic acids in the biological sample, wherein the plurality of circularizable probes comprises a first probe that binds to a first target nucleic acid and a second probe that binds a second target nucleic acid, wherein the first probe comprises a 5’ end and a 3’ end; ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template; extending the second probe using a sequence of the second target nucleic acid as template to generate an extended probe comprising a gap filled sequence; ligating the extended probe to generate a second circularized template; performing rolling circle amplification (RCA) of the first circularized template and the second circularized template in the biological sample to generate a first RCA product and a second RCA product; contacting the biological sample with an oligonucleotide probe that directly or indirectly hybridize to a sequence in the first RCA product and detecting a first signal associated with the oligonucleotide probe, thereby detecting the first target nucleic acid, and sequencing the second RCA product to determine the gap filled sequence or a complement thereof, thereby detecting the second target nucleic acid. [0040] In some instances, ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template and ligating the extended probe to generate a second circularized template are performed simultaneously. In some instances, ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template, extending the second probe using a sequence of the second target nucleic acid as template to generate an extended probe comprising a gap filled sequence, and ligating the extended probe to generate a second circularized template are performed in the same step. In some instances, ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template, extending the second probe using a sequence of the second target nucleic acid as template to generate an extended probe comprising a gap filled sequence, and ligating the extended probe to generate a second circularized template are performed in the same step. In some instances, ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template is performed prior to extending the second probe using a sequence of the second target nucleic acid as template to ^{MOFO-357975700} 202412023840 generate an extended probe comprising a gap filled sequence and ligating the extended probe to generate a second circularized template. In some instances, extending the second probe using a sequence of the second target nucleic acid as template to generate an extended probe comprising a gap filled sequence is performed prior to ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template and ligating the extended probe to generate a second circularized template. In some aspects, extending the second probe is performed prior to ligating the extended probe to generate a second circularized template. [0041] In some instances, the plurality of circularizable probes comprising first probes and second probes are contacted with the biological sample at the same time. In some instances, the plurality of circularizable probes comprising first probes and second probes are contacted with the biological sample separately. In some instances, extending the second probe using a sequence of the second target nucleic acid as template to generate an extended probe comprising a gap filled sequence is performed prior to ligating both the extended probe to generate the second circularized template and the 5’ end of the first probe to the 3’ end to generate the first circularized template. In some embodiments, ligating the extended probe to generate the second circularized template and the 5’ end of the first probe to the 3’ end to generate the first circularized template is performed at the same time (e.g., in the same step). [0042] Provided herein is a method for analyzing a biological sample comprising: binding a plurality of circularizable probes to a plurality of target nucleic acids in the biological sample, wherein: the plurality of circularizable probes comprises a first probe that binds to a first target nucleic acid and a second probe that binds to a second target nucleic acid, wherein the first probe comprises a 5’ end and a 3’ end; extending the second probe using a sequence of the second target nucleic acid as template to generate an extended probe comprising a gap filled sequence; ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template and ligating the extended probe to generate a second circularized template; performing rolling circle amplification (RCA) of the first circularized template and the second circularized template in the biological sample to generate a first RCA product and a second RCA product, respectively; contacting the biological sample with an oligonucleotide probe that directly or indirectly binds to a sequence in the first RCA product and detecting a first signal associated with the oligonucleotide probe; and sequencing the second RCA product to determine the gap ^{MOFO-357975700} 202412023840 filled sequence or a complement thereof. In some embodiments, extending the second probe using a sequence of the second target nucleic acid as template to generate an extended probe comprising a gap filled sequence is prior to performing the ligation to generate a first circularized template and/or the second circularized template. In some instances, a plurality of first probes targeting a panel of target nucleic acids in the biological sample are contacted with the biological sample. [0043] Provided herein is a method for analyzing a biological sample comprising: binding a plurality of circularizable probes to a plurality of target nucleic acids in the biological sample, wherein: the plurality of circularizable probes comprises a first probe that binds to a first target nucleic acid and a second probe that binds to a second target nucleic acid, wherein the first probe comprises a 5’ end and a 3’ end; extending the second probe using a sequence of the second target nucleic acid as template to generate an extended probe comprising a gap filled sequence; ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template and ligating the extended probe to generate a second circularized template; performing rolling circle amplification (RCA) of the first circularized template and the second circularized template in the biological sample to generate a first RCA product and a second RCA product, respectively; contacting the biological sample with an oligonucleotide probe that directly or indirectly binds to a sequence in the first RCA product and detecting a first signal associated with the oligonucleotide probe; and performing in situ sequencing by synthesis (SBS) in the biological sample to determine the gap filled sequence or a complement thereof. [0044] Provided herein is a method for analyzing a biological sample comprising: binding a plurality of circularizable probes to a plurality of target nucleic acids in the biological sample, wherein: the plurality of circularizable probes comprises a first probe that binds to a first target nucleic acid and a second probe that binds to a second target nucleic acid, wherein the first probe comprises a 5’ end and a 3’ end; extending the second probe using a sequence of the second target nucleic acid as template to generate an extended probe comprising a gap filled sequence; ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template and ligating the extended probe to generate a second circularized template; performing rolling circle amplification (RCA) of the first circularized template and the second circularized template in the biological sample to generate a first RCA product and a second RCA product, ^{MOFO-357975700} 202412023840 respectively; contacting the biological sample with an oligonucleotide probe that directly or indirectly binds to a sequence in the first RCA product and detecting a first signal associated with the oligonucleotide probe; and performing in situ sequencing by binding (SBB) in the biological sample to determine the gap filled sequence or a complement thereof. [0045] Provided herein is a method for analyzing a biological sample comprising: binding a plurality of circularizable probes to a plurality of target nucleic acids in the biological sample, wherein: the plurality of circularizable probes comprises a first probe that binds to a first target nucleic acid and a second probe that binds to a second target nucleic acid, wherein the first probe comprises a 5’ end and a 3’ end; extending the second probe using a sequence of the second target nucleic acid as template to generate an extended probe comprising a gap filled sequence; ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template and ligating the extended probe to generate a second circularized template; performing rolling circle amplification (RCA) of the first circularized template and the second circularized template in the biological sample to generate a first RCA product and a second RCA product, respectively; contacting the biological sample with an oligonucleotide probe that directly or indirectly binds to a sequence in the first RCA product and detecting a first signal associated with the oligonucleotide probe; and performing in situ avidity sequencing in the biological sample to determine the gap filled sequence or a complement thereof. [0046] Provided herein is a method for analyzing a biological sample comprising: binding a plurality of circularizable probes to a plurality of target nucleic acids in the biological sample, wherein: the plurality of circularizable probes comprises a first probe that binds to a first target nucleic acid and a second probe that binds to a second target nucleic acid, wherein the first probe comprises a 5’ end and a 3’ end; extending the second probe using a sequence of the second target nucleic acid as template to generate an extended probe comprising a gap filled sequence, and wherein the second target nucleic acid comprises a variant sequence (e.g., a single nucleotide variation (SNV) or a single nucleotide polymorphism (SNP)); ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template and ligating the extended probe to generate a second circularized template; performing rolling circle amplification (RCA) of the first circularized template and the second circularized template in the biological sample to generate a first RCA product and a second RCA product, respectively; contacting the biological 16 ^{MOFO-357975700} 202412023840 sample with an oligonucleotide probe that directly or indirectly binds to a sequence in the first RCA product and detecting a first signal associated with the oligonucleotide probe; and sequencing the second RCA product to determine the variant sequence. In some instances, a plurality of first probes targeting a panel of target nucleic acids in the biological sample and a plurality of second probes each targeting a molecule comprising a different variant sequence of the second target nucleic acid are contacted with the biological sample. [0047] Provided herein is a method for analyzing a biological sample comprising: binding a plurality of circularizable probes to a plurality of target nucleic acids in the biological sample, wherein: the plurality of circularizable probes comprises a first probe that binds to a first target nucleic acid and a second probe that binds to a second target nucleic acid, wherein the first probe comprises a 5’ end and a 3’ end; extending the second probe using a sequence of the second target nucleic acid as template to generate an extended probe comprising a gap filled sequence, and wherein the second target nucleic acid comprises a sequence of an immune molecule (e.g., an antigen receptor transcript); ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template and ligating the extended probe to generate a second circularized template; performing rolling circle amplification (RCA) of the first circularized template and the second circularized template in the biological sample to generate a first RCA product and a second RCA product, respectively; contacting the biological sample with an oligonucleotide probe that directly or indirectly binds to a sequence in the first RCA product and detecting a first signal associated with the oligonucleotide probe; and sequencing the second RCA product to determine the sequence of the immune molecule. [0048] Provided herein is a method for analyzing a biological sample comprising: binding a plurality of circularizable probes to a plurality of target nucleic acids in the biological sample, wherein: the plurality of circularizable probes comprises a first probe that binds to a first target nucleic acid and a second probe that binds to a second target nucleic acid, wherein the first probe comprises a 5’ end and a 3’ end; extending the second probe using a sequence of the second target nucleic acid as template to generate an extended probe comprising a gap filled sequence, and wherein the second target nucleic acid comprises a sequence of a perturbation agent introduced to the biological sample (e.g., a guide RNA); ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template and ligating the extended probe to 17 ^{MOFO-357975700} 202412023840 generate a second circularized template; performing rolling circle amplification (RCA) of the first circularized template and the second circularized template in the biological sample to generate a first RCA product and a second RCA product, respectively; contacting the biological sample with an oligonucleotide probe that directly or indirectly binds to a sequence in the first RCA product and detecting a first signal associated with the oligonucleotide probe; and sequencing the second RCA product to determine the sequence of the perturbation agent. II. PROBES AND ANALYTES [0049] In some embodiments, provided herein are probe molecules (e.g., first probe and second probe) that are capable of being amplified. In some embodiments, provided herein are probe molecules (e.g., first probe and second probe) that are capable of being circularized, e.g., to generate a circularized template for rolling circle amplification. In some cases, a probe (e.g., a first probe) comprises a 5’ end and a 3’ end and the 5’ end and 3’ end are ligated to generate a circularized template. In some embodiments, a circularizable probe (e.g., a second probe) is hybridized to a target nucleic acid molecule comprising a gap sequence. In some instances, the gap sequence comprises a variant sequence of interest. In some embodiments, the circularizable probe is circularized to generate a circularized probe comprising a gap filled sequence complementary to the gap sequence in the target nucleic acid molecule. In some embodiments, a second probe contacted with the biological sample is circularized to generate a circularized probe comprising a gap filled sequence complementary to the gap sequence in the target nucleic acid molecule. In some embodiments, the circularized template comprising at least portions of the complement of the gap sequence is amplified (e.g., through RCA) and the RCA product is detected in order to detect the variant sequence in the target nucleic acid molecule. [0050] In some embodiments, the first probe is configured to be ligated without gap fill to generate a first circularized template and a second probe is configured to be extended (e.g., gap filled) and ligated to generated a second circularized template. In some embodiments, the first probe and the second probe are configured to bind to different first and second target nucleic acids. In some embodiments, the first probe comprises a barcode sequence corresponding to the ^{MOFO-357975700} 202412023840 first target nucleic acid or a sequence thereof and the second probe does not comprise a barcode sequence corresponding to the second target nucleic acid or a sequence thereof. [0051] In some embodiments, the first probe is provided in one or more parts (e.g., one or more separate nucleic acid molecules). In some embodiments, the second probe is provided in one or more parts (e.g., one or more separate nucleic acid molecules). In some embodiments, the first probe is provided as at least two parts (e.g., at least two nucleic acid molecules). In some embodiments, the second probe is provided as at least two parts (e.g., at least two nucleic acid molecules). For example, a first part of the first probe comprises a first hybridization region and a second part of the first probe comprises a second hybridization region for binding to the target nucleic acid. In some instances, a first part of the second probe comprises a first hybridization region and a second part of the second probe comprises a second hybridization region for binding to the target nucleic acid, wherein the first and second hybridization regions are separated by a gap upon hybridization to the second target nucleic acid. [0052] In some aspects, a first probe is capable of being amplified enzymatically or non-enzymatically. In some aspects, a first probe is selected from the group consisting of a circular probe, a circularizable probe, and a linear probe. In some embodiments, a circular probe can be one that is pre-circularized prior to hybridization to a target nucleic acid and/or one or more other probes. In some embodiments, a circularizable probe can be one that can be circularized upon hybridization to a target nucleic acid and/or one or more other probes such as a splint. In some embodiments, a linear probe can be one that comprises a target recognition sequence and a sequence that does not hybridize to a target nucleic acid, such as a 5’ overhang, a 3’ overhang, and/or a linker or spacer (which may comprise a nucleic acid sequence or a non- nucleic acid moiety). In some embodiments, the sequence (e.g., the 5’ overhang, 3’ overhang, and/or linker or spacer) is non-hybridizing to the target nucleic acid but may hybridize to one another and/or one or more other probes, such as detectably labeled probes (e.g., as described in Section III). [0053] In some aspects, the method comprises contacting the biological sample with a circularizable probe (e.g., a first probe or second probe) comprising a first hybridization region and a second hybridization region (e.g., 5’ and 3’ arms of a padlock probe) that hybridize to a first sequence and a second sequence, respectively, in a target nucleic acid (e.g., an RNA or ^{MOFO-357975700} 202412023840 cDNA) in the biological sample. In some embodiments, the first hybridization region and/or the second hybridization region in the circularizable probe comprises one or more RNA residues at and/or near its 3’ end. In some cases, a probe comprises a barcode sequence corresponding to the target nucleic acid or a sequence thereof. In some instances, a probe (e.g., a first probe) comprises from a 5’ end to a 3’ end: a first hybridization region – a barcode region comprising one or more barcode sequences – a second hybridization region. In some embodiments, the barcode sequence is associated with, corresponds to, and/or identifies a target nucleic acid or a sequence therein. In some aspects, upon hybridization to the target nucleic acid, the first hybridization region and the second hybridization region are positioned adjacent to each other for direct ligation. In some aspects, the 5’ end of the first probe is ligated to the 3’ end of the first probe are ligated using the first target nucleic acid. In some aspects, upon hybridization to the target nucleic acid, the 5’ end of the first probe is ligated to the 3’ end of the first probe using the first target nucleic acid as template. [0054] In some embodiments, a circularizable probe (e.g., first probe) disclosed herein comprises one or more barcode sequences. In some embodiments, the first probe comprises one or more barcode sequences. In some embodiments, a circularizable probe comprises two or more barcode sequences. The barcode sequences, if present, may be of any length. If more than one barcode sequence is used, the barcode sequences may independently have the same or different lengths, such as at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50 nucleotides in length. In some embodiments, the barcode sequence may be no more than 120, no more than 112, no more than 104, no more than 96, no more than 88, no more than 80, no more than 72, no more than 64, no more than 56, no more than 48, no more than 40, no more than 32, no more than 24, no more than 16, or no more than 8 nucleotides in length. Combinations of any of these are also possible, e.g., the barcode sequence may be between 5 and 10 nucleotides, between 8 and 15 nucleotides, etc. [0055] The barcode sequence may be arbitrary or random. In certain cases, the barcode sequences are chosen so as to reduce or minimize homology with other components in a sample, e.g., such that the barcode sequences do not themselves bind to or hybridize with other nucleic acids suspected of being within the cell or other sample. In some embodiments, between a particular barcode sequence and another sequence (e.g., a cellular nucleic acid sequence in a ^{MOFO-357975700} 202412023840 sample or other barcode sequences in probes added to the sample), the homology may be less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the homology may be less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or less than 2 bases, and in some embodiments, the bases are consecutive bases. [0056] In some instances, each probe may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. or more barcode sequences. In some embodiments, a population of nucleic acid probes may each contain the same number of barcode sequences, although in other cases, there may be different numbers of barcode sequences present on the various probes. In some embodiments, the barcode sequences or any subset thereof in the population of nucleic acid probes can be independently and/or combinatorially detected and/or decoded. In some embodiments, the first probe does not contain a barcode sequence corresponding to the first target nucleic acid or a sequence thereof. In some embodiments, the first probe does not comprise any nucleic acid barcode sequence. In some embodiments, the first probe is detected by hybridizing, directly or indirectly, an oligonucleotide probe to a sequence of the first and/or second hybridization regions or a complement or portion thereof. [0057] In some embodiments, the second probe does not contain a barcode sequence corresponding to the second target nucleic acid or a sequence thereof. [0058] In some embodiments, the biological sample is contacted with a library of first probes to detect a panel of at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, at least 2,000, at least 3,000, at least 4,000, at least 5,000 or more first target nucleic acids. In some embodiments, the biological sample is contacted with a library of first probes to detect a panel of at least 500 or more first target nucleic acids (e.g., mRNAs). [0059] In some aspects, the method comprises contacting the biological sample with a circularizable probe (e.g., a second probe) comprising a first hybridization region and a second hybridization region (e.g., 5’ and 3’ arms of a padlock probe) that hybridize to a first sequence and a second sequence, respectively, in a target nucleic acid (e.g., an RNA or cDNA) in the ^{MOFO-357975700} 202412023840 biological sample, wherein the first sequence and the second sequence of the second target nucleic acid are separated by a gap sequence. In some cases, the gap sequence serves as a template for gap filling the probe. In some embodiments, the first and second hybridization regions are common among a plurality of circularizable probes that target a plurality of target nucleic acids that comprise different gap (e.g., variant) sequences. In some embodiments, each of the plurality of target nucleic acids comprises a common first target sequence (among the plurality of target nucleic acids) and a common second target sequence (among the plurality of target nucleic acids) that are complementary to the common first and second probe regions, respectively, among the plurality of circularizable probes. In some embodiments, a plurality of circularizable probes for gap filling comprise molecules of the same nucleic acid sequence. In some embodiments, the plurality of circularizable probes comprise molecules of different nucleic acid sequences. In some embodiments, any two or more different nucleic acid sequences of the circularizable probes comprise common first and second hybridization regions. In some cases, a plurality of probes (e.g., second probes) are capable of hybridizing to both a wildtype molecule and a mutant molecule of the target nucleic acid. For example, the target nucleic acid (e.g., second target nucleic acid) comprises from a 5’ end to a 3’ end: a first sequence for binding to a first hybridization region of a probe – a gap sequence – a second sequence for binding to a second hybridization region of the probe. In some instances, a second probe comprises from a 3’ end to a 5’ end: a first hybridization region – a non-hybridizing region (which does not hybridize to the second target nucleic acid) – a second hybridization region, and upon hybridization of the second probe to the second target nucleic acid, the 3’ and 5’ ends of the second probe are configured to be connected by a gap filled sequence. In some instances, a second probe comprises a sequencing primer binding sequence or a complement thereof, wherein the sequencing primer binding sequence is used as a primer binding site, and the complement of the sequencing primer binding sequence is part of a sequencing primer. In some embodiments, a second probe comprises a sequence configured to be used as part of a sequencing primer. In some embodiments, a sequence of the hybridization region (in the second probe) that hybridizes to the second target nucleic acid is configured to be used as the sequencing primer sequence or a part thereof. In some embodiments, a sequence of the non-hybridizing region (in the second probe) that does not hybridize to the second target nucleic acid is configured to be used as the ^{MOFO-357975700} 202412023840 sequencing primer sequence or a part thereof. In some instances, a second probe comprises from a 3’ end to a 5’ end: a first hybridization region (which in some embodiments, comprise a sequence of a sequencing primer) – a non-hybridizing region – a second hybridization region. In some instances, a second probe comprises from a 3’ end to a 5’ end: a first hybridization region – a non-hybridizing region (which does not hybridize to the second target nucleic acid) comprising a sequence of a sequencing primer – a second hybridization region. In some instances, a second probe comprises from a 5’ end to a 3’ end: a first hybridization region – a non-hybridizing region (which does not hybridize to the second target nucleic acid) comprising a sequence of a sequencing primer – a second hybridization region. [0060] In some cases, the first hybridization region and the second hybridization region in the first probe are equal in length. In some cases, the first hybridization region and the second hybridization region in the second probe are equal in length. In some cases, the first hybridization region is shorter or longer than the second hybridization region in the first probe and/or second probe. In some cases, the first hybridization region is shorter than the second hybridization region in the first probe. In some cases, the first hybridization region is longer than the second hybridization region in the first probe. In some cases, the first hybridization region is shorter than the second hybridization region in the second probe. In some cases, the first hybridization region is longer than the second hybridization region in the second probe. In some instances, the first hybridization region and/or the second hybridization region of the circularizable probe (e.g., first probe and/or second probe) is individually between about 5 and about 50 nucleotides in length. In some aspects, the first hybridization region and/or the second hybridization region of the circularizable probe (e.g., first probe and/or second probe) is individually between about 15 and about 25 nucleotides in length. In some aspects, the first hybridization region and/or the second hybridization region of the circularizable probe is individually between about 6 and about 18 nucleotides in length. [0061] In some embodiments, a gap sequence of a target nucleic acid (e.g., second nucleic acid) comprises a nucleotide variation, a nucleotide polymorphism, a mutation, a substitution, an insertion, a deletion, a translocation, a duplication, an inversion, a rearrangement and/or a repetitive sequence, for identifying a variant sequence among a plurality of different sequences in situ in a biological sample. In some embodiments, the gap sequence comprises ^{MOFO-357975700} 202412023840 variant sequence of a single nucleotide, for instance, a single nucleotide variation (SNV), a single nucleotide polymorphism (SNP), a point mutation, a single nucleotide substitution, a single nucleotide insertion, or a single nucleotide deletion. In some embodiments, a gap sequence of a target nucleic acid (e.g., second nucleic acid) comprises one or more exon-exon boundaries. In some embodiments, a gap sequence of a target nucleic acid (e.g., second nucleic acid) comprises a sequence of an exon-exon boundary. [0062] In some embodiments, circularizable probes (e.g., second probe) that target common regions adjacent to hotspots for mutation are used. In some embodiments, the common regions flank a gap sequence in the target nucleic acid (e.g., second target nucleic acid). In some embodiments, the gap sequence comprises one or more hotspots for mutation. In some embodiments, the gap sequence comprises a variant sequence among a plurality of different variant sequences. In some embodiments, gaps in the circularizable probes upon hybridization to their nucleic acid targets are filled by polymerization (e.g., as described in Section II.A). In some embodiments, the gaps are filled by splint ligation, using a library of splint oligonucleotides that are diverse in sequences and comprise a plurality of possible variant sequences (e.g., possible mutations for the hotspots). In some embodiments, the library of splint oligonucleotides are incubated with the sample for hybridization to target nucleic acid molecules, allowing the best matching splint oligonucleotide to outcompete other splint oligonucleotides in the library (e.g., as described in Section II.B). In some embodiments, after washing the sample, the best matching splint oligonucleotides are ligated into the circularizable probes and the circularized probes are amplified. [0063] In some embodiments, the first and second sequences in the target nucleic acid (e.g., second target nucleic acid) bound by the first hybridization region and second hybridization region of the circularizable probe are separated by a gap sequence in the target nucleic acid. In some embodiments, the gap sequence is about or at least 2, about or at least 4, about or at least 6, about or at least 8, about or at least 10, about or at least 12, about or at least 14, about or at least 16, about or at least 18, about or at least 20, or more nucleotides in length. In some instances, the gap sequence is between about 2 and about 40 nucleotides in length. In some cases, the target nucleic acid comprises a variant sequence at the 3’ or 5’ end of the gap sequence. In some cases, the target nucleic acid comprises a variant sequence at or near the ^{MOFO-357975700} 202412023840 central nucleotide(s) of the gap sequence. In some instances, the target nucleic acid comprises a variant sequence at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or more phosphodiester bonds from the 3’ or 5’ end of the gap sequence. [0064] In some embodiments, the gap sequence comprises a variant sequence among a plurality of different variant sequences. In some embodiments, the plurality of circularizable probes that target a plurality of target nucleic acids that comprise different variant sequences do not hybridize to the gap sequences (which comprise the different variant sequences), and instead hybridize to common first and second sequences that flank the gap sequence. [0065] In some embodiments, the gap sequence comprises a barcode sequence. In some embodiments, the second target nucleic acid comprises a barcode sequence. For example, the barcode sequence comprises one barcode sequence among a plurality of different barcode sequences of an exogenous library of nucleic acid constructs introduced to the biological sample. In some aspects, the barcode sequence is used for lineage tracing. In some aspects, the biological sample is contacted with a library of constructs, wherein the constructs comprise barcoded nucleic acid molecules, before the plurality of circularizable probes (comprising the first probes and second probes) are bound to the target nucleic acids. In some embodiments, the second target nucleic acid comprises a unique barcode sequence or a unique collection of barcode sequences specific to the second target nucleic acid. In some instances, the second target nucleic acid is or is derived from a construct (e.g., a vector) comprising a barcode sequence and the gap sequence of the second target nucleic acid comprises the barcode sequence of the construct or a complement thereof. [0066] In some embodiments, the gap sequence comprises a sequence of an immune molecule. In TCR and BCR RNA transcripts, the V(D)J sequences are 5’ to the constant region exon(s) and the 3’ poly(A) tail of the transcript. The number of V(D)J transcripts of a particular V(D)J join sequence in a sample comprising T cells or B cells of various antigen specificities can be low, and sequence information in particular V(D)J joins can be lost and become unavailable for subsequent in situ detection. In some embodiments, the present disclosure provides methods for high-throughput profiling of V(D)J transcripts in a large number of clonal T cell populations comprising TCRs with varying antigenic specificities. The methods and compositions disclosed herein may be used in research, diagnostics, and drug target discovery. Analyzing the spatial ^{MOFO-357975700} 202412023840 distribution of V(D)J transcripts in situ in various tissues could be used for development of therapeutic and/or prophylactic agents, e.g., TCR therapeutic treatment modalities and/or anti- disease vaccination. [0067] Provided herein is a method for analysis of immune molecule sequences by contacting the biological sample with a circularizable probe (e.g., second probe), extending the second probe using a sequence of an immune molecule as template to generate an extended probe comprising a gap filled sequence, ligating the extended probe, performing rolling circle amplification, and sequencing the amplification product to determine the gap filled sequence or a complement thereof to detect the sequence of the immune molecule. In some aspects, the provided methods for immune molecule analysis allows for sensitive detection even if the number of particular transcripts are low in the biological sample. In some embodiments, the second target nucleic acid comprises a sequence of an immune molecule. In some aspects, the second target nucleic acids in a biological sample are highly variable. For example, the sequence of the immune molecule is an antigen receptor transcript. In some cases, the antigen receptor transcript is a T cell receptor (TCR) transcript, optionally wherein the TCR transcript comprises a TCRα VJ join, a TCRβ VDJ join, a TCRγ VJ join, or a TCRδ VDJ join. In some cases, the antigen receptor transcript is an immunoglobulin (Ig) transcript, optionally wherein the Ig transcript comprises an Igκ VJ join, an Igλ VJ join, or an IgH VDJ join. In some embodiments, the methods are used for identifying multiple different antigen receptor transcripts present at a plurality of locations in the biological sample. [0068] In some embodiments, the second probes comprise gap fill circularizable probes that target conserved regions in the V and J sequences and the probes are gap filled to fill in the D sequence in order to identify the D region sequences. In some embodiments, gaps can be filled by polymerization, e.g., primer extension by a DNA polymerase using the 3’ end of a circularizable probe as a primer and a cDNA comprising a VDJ join as a template. In some embodiments, gaps can be filled by splint ligation, using a diversity library of gap fill oligonucleotides that comprises numerous possible D sequence variants. In some embodiments, a library of oligonucleotides are incubated with the sample for hybridization to cDNA molecules comprising VDJ joins, allowing the best matching oligonucleotide to outcompete other oligonucleotides in the library and hybridize to the corresponding VDJ sequence. After washing ^{MOFO-357975700} 202412023840 the sample, the best matching oligonucleotides can be ligated into the gap fill circularizable probes and the circularized probes can be amplified. In some embodiments, amplicons (e.g., RCA products) comprising V(D)J sequences or complements thereof are detected in situ using sequencing. [0069] In some aspects, a nucleic molecule (e.g., second target nucleic acid) comprising a V(D)J join sequence disclosed herein, e.g., a cDNA, is a product of a TCR transcript. There are two subsets of T cells based on the exact pair of receptor chains expressed. These are either the alpha (α) and beta (β) chain pair, or the gamma (γ) and delta (δ) chain pair, identifying the αβ or γδ T cells, respectively. The expression of the β and δ chain is limited to one chain in each of their respective subsets and this is referred to as allelic exclusion. These two chains are also characterized by the use of an additional DNA segment - the diversity (D) region - during the rearrangement process. The D region is flanked by N nucleotides which constitutes the NDN region of the CDR3 in these two chains. In some aspects, the CDR3 of each of the two receptor chains defines the T cell clonotype of cells expressing TCRs comprising the CDR3. For αβ T cells the CDR3 is in most contact with the peptide bound to the MHC; as such, CDR3 sequences are generally a focus for analyzing immunological sequences. In some embodiments, the TCR transcript disclosed herein comprises a TCRα VJ join. In some embodiments, the TCR transcript disclosed herein comprises a TCRβ VDJ join. In some embodiments, the TCR transcript disclosed herein comprises a TCRγ VJ join. In some embodiments, the TCR transcript disclosed herein comprises a TCRδ VDJ join. [0070] In some aspects, a nucleic molecule (e.g., second target nucleic acid) comprising a V(D)J join sequence disclosed herein, e.g., a cDNA, is a product of a BCR or immunoglobulin transcript. B cells are highly diverse, each expressing a practically unique BCR or immunoglobulin. There are approximately 10¹⁰-10¹¹ B cells in a human adult. Each B cell in an organism (e.g., human) expresses a different BCR that allows it to recognize a particular set of molecular patterns. Individual B cells gain this specificity during their development in the bone marrow, where they undergo a somatic rearrangement process that combines multiple germline-encoded gene segments to procures the BCR. Human BCR and antibody molecules are composed of heavy and light chains (each of which contains both constant (C) and variable (V) regions), which are encoded by genes on three loci: the immunoglobulin heavy locus IgH, 27 ^{MOFO-357975700} 202412023840 containing the gene segments for the immunoglobulin heavy chain; the immunoglobulin kappa (κ) locus (Igκ), containing the gene segments for the κ light chain; and the immunoglobulin lambda (λ) locus (Ig λ), containing the gene segments for the λ light chain. Each heavy chain and light chain gene contains multiple copies of three different types of gene segments for the variable regions of the antibody proteins. For example, the human immunoglobulin heavy chain region contains Constant (e.g., Cμ and Cδ) gene segments and 44 Variable (V) gene segments plus 27 Diversity (D) gene segments and 6 Joining (J) gene segments. The light chains also possess Constant (e.g., Cμ and Cδ) gene segments and numerous V and J gene segments, but do not have D gene segments. DNA rearrangement causes one copy of each type of gene segment to go in any given lymphocyte, generating an enormous antibody repertoire, although some are removed due to self-reactivity. [0071] Because of the rearrangement undergone of the V(D)J segment in T cells and B cells, only parts of the V(D)J segments (the V, D, and J segments) can be traced back to segments encoded in highly repetitive regions of the germline that are not typically sequenced directly from the germ line DNA. Furthermore, the V, D, and J segments can be significantly modified during the V(D)J rearrangement process and through, in the case of B cells, somatic hypermutation (SHM). As such, there are typically no pre-existing full-length templates to align to sequence reads of the V(D)J segments of T cell receptors and B cell immunoglobulins. In some embodiments, clonal grouping or clonotyping can involve clustering the set of V(D)J sequences into clones, which are defined as a group of cells that are descended from a common ancestor. Unlike the case of T cells, members of a B cell clone may differ in their V(D)J sequences due to SHM. [0072] In addition to V(D)J recombination and SHM, gene rearrangements editing the immunoglobulin (Ig) genes include class switch recombination (CSR). Like V(D)J recombination, CSR requires the formation of DNA double strand breaks (DSBs) as the key initiating step. Under physiological conditions, DSBs are introduced at the Ig genes by the activity of B lymphocyte cell specific enzymes such as recombinase activating gene 1/2 (RAG1/2, for V(D)J recombination) and activation-induced cytidine deaminase (AID, for CSR). During CSR, AID generates DSBs in the Ig locus by targeting repetitive sequences in the switch (S) regions that precede each Ig heavy (IgH) coding sequence. Paired DSBs in the switch ^{MOFO-357975700} 202412023840 regions are then joined by the classical and alternative non-homologous end joining (NHEJ) pathways to generate a switch of the IgH. This long range joining is thought to be part of a general mechanism of DNA repair where two DSBs are joined in cis over long chromosome distances. [0073] In the immature B cell's antibody immunoglobulin (Ig) heavy chain (IgH) locus, the order of arrangement of the nucleic acid sequence encoding the heavy chain segments (order of the heavy chain exons) are as follows: for human, they are μ (for IgM), δ (for IgD), γ3 (for IgG3), γ1 (for IgG1), α1 (for IgA1), γ2 (for IgG2), γ4 (for IgG4), ε (for IgE), and α2 (for IgA2); and for mouse, they are μ (for IgM), δ (for IgD), γ3 (for IgG3), γ1 (for IgG1), γ2b (for IgG2b), γ2a (for IgG2a), ε (for IgE), and α (for IgA). Class switching occurs after the activation of a mature B cell via its membrane-bound antibody molecule (BCRs) to generate the different classes of antibodies. Ligand or antigen binding to the cell surface BCR triggers an intracellular cell signaling process that brings about CSR and produces the various classes of antibodies. The various classes of antibodies all have the same variable domains as the original antibody generated in the immature B cell during the process of V(D)J recombination, but possessing distinct constant domains in their heavy chains. [0074] In some embodiments, disclosed herein is a method involving detecting a sequence of a V(D)J transcript in a biological sample. In some embodiments, V(D)J sequences include those in V(D)J transcripts comprising V(D)J joins. In some embodiments, V(D)J sequences include those in cDNA generated from V(D)J transcripts comprising V(D)J joins. In some embodiments, V(D)J sequences are incorporated into circularized probes generated during gap fill polymerization or gap fill oligonucleotide ligation. In some embodiments, V(D)J sequences are present in amplification products of V(D)J transcripts, amplification products of cDNA of V(D)J transcripts, or amplification products of the probes that hybridize to V(D)J transcripts or cDNA of V(D)J transcripts. [0075] In some aspects, methods provided herein further comprise generating rolling circle amplification (RCA) products of the circularized probes extended with a sequence of an immune molecule and corresponding products thereof (e.g., RCA products) are detected for analyzing the spatial organization of V(D)J sequences in samples (e.g., tissues such as tumors ^{MOFO-357975700} 202412023840 comprising infiltrating immune cells). Such insights can be crucial to understanding disease development and establishing new treatment strategies. [0076] In some embodiments, a probe disclosed herein (e.g., a second probe) includes a sequence complementary to a region of a target nucleic acid sequence encoding a constant region of an antibody or a fragment thereof. In some embodiments, a probe disclosed herein (e.g., a second probe) comprises a sequence complementary to a region of a target nucleic acid sequence encoding a constant region of an immune cell receptor. In some embodiments, a probe disclosed herein (e.g., a second probe) includes a sequence complementary to a region of a target nucleic acid sequence encoding a constant region of a B cell receptor. In some embodiments, a probe disclosed herein (e.g., a second probe) includes a sequence complementary to a region of a target nucleic acid sequence encoding a constant region of a T cell receptor. [0077] Provided herein is a method for analysis of one or more perturbation agents introduced to a cell by contacting the biological sample with a circularizable probe (e.g., second probe), extending the second probe using a sequence of the perturbation agent or a corresponding molecule as template to generate an extended probe comprising a gap filled sequence, ligating the extended probe, performing amplification, and sequencing the amplification product to determine the gap filled sequence or a complement thereof to detect the sequence of the perturbation agent. In some aspects, the biological sample is contacted with a library of perturbation agents. In some aspects, the assays described herein are used for detecting CRISPR guides, e.g., guide RNAs (gRNAs). In some embodiments, the assays described herein are used for detecting perturbations introduced by CRISPR libraries and/or cellular RNA transcripts. In some cases, a sequence of a CRISPR guide RNA is incorporated by gap fill into a circularizable probe (e.g., second probe) by incorporating a sequence complementarity to the CRISPR guide RNA. [0078] In some embodiments, the second target nucleic acid comprises a sequence of a perturbation agent or a corresponding molecule (e.g., a precursor or derivative thereof) as template for the gap fill of the circularizable probe. For example, the second target nucleic acid comprises a sequence of or associated with a perturbation agent introduced to the biological sample before circularizable probes are introduced. In some embodiments, a CRISPR molecule (e.g., a CRISPR RNA), a nucleic acid molecule edited using the CRISPR molecule, and/or a ^{MOFO-357975700} 202412023840 precursor or derivative thereof is detected. In some aspects, the second target nucleic acid comprises a sequence of a CRISPR molecule (e.g., a CRISPR RNA), a nucleic acid molecule edited using the CRISPR molecule, and/or a precursor or derivative thereof. In some instances, the second target nucleic acid is an RNA molecule derived from an exogenously introduced nucleic acid molecule. In some embodiments, the exogenously introduced nucleic acid molecule is an RNA derived from a plasmid, an integrated DNA sequence (e.g. using viral transduction in a cell), a gRNA from a CRISPR genetic element, etc. In some embodiments, the perturbation agent comprises a spacer sequence that is an element (e.g., about 20 nucleotides) that can be found as a component of gRNA. In some aspects, the spacer sequence found on the gRNA corresponds to a protospacer sequence that is found in the target region. The target region (the protospacer sequence) is the region of interest, e.g., a region of the cellular DNA designed to be targeted by the guide RNA. In some examples, the protospacer is found in the cellular DNA that is complementary to the protospacer that is found in the guide RNA. CRISPR enzymes can target a nucleic acid molecule using a guide RNA containing a spacer sequence that hybridizes to a target sequence of the nucleic acid molecule site. A CRISPR enzyme can be a Cas fusion protein. The system for introducing perturbations may further comprise a CRISPR enzyme. In some aspects, a CRISPR RNA comprises a spacer sequence to anneal to a target nucleic acid molecule and a scaffold sequence to bind to the Cas fusion protein. In some embodiments, a CRISPR RNA comprises a barcode sequence or a sequence for binding to a barcoded nucleic acid molecule. [0079] In some embodiments, a circularizable probe (e.g., second probe) binds to or hybridizes to a protospacer sequence or hybridization regions flanking a protospacer sequence, or a complement thereof. In some embodiments, a circularizable probe (e.g., second probe) binds to a conserved region of the guide RNA (e.g., a common sequence shared by a plurality of different guide RNAs). In some aspects, CRISPR libraries are generated in cells of a biological sample. In some aspects, a CRISPR library may comprise hundreds, thousands, or tens of thousands of different spacer sequences. In some embodiments, a circularizable probe (e.g., second probe) hybridizes to the complement or reverse complement of a guide RNA spacer sequence. In some examples, a nucleic acid molecule to be analyzed is introduced and/or ^{MOFO-357975700} 202412023840 delivered into a cell or a cell constituent (e.g., a nucleus of a cell) using any of a variety of techniques. [0080] In some embodiments, the second target nucleic acid is a transcript comprising a unique barcode specific to the perturbation agent. In some embodiments, the second target nucleic acid is a transcript comprising a unique barcode specific to a guide RNA. In some embodiments, the second target nucleic acid is a transcript comprising a guide RNA sequence. In some instances, a guide RNA and guide RNA barcode is expressed from the same vector and the barcode or a complement thereof is used as template to generate an extended probe comprising a gap filled sequence. For example, perturbation agents are described in U.S. Patent Application Publication No 2021/0171938. [0081] In some embodiments, upon hybridization to the target nucleic acid, the circularizable probe is circularized to generate a circularized probe comprising a gap filled region complementary to the gap sequence. In some cases, the gap filled region is generated using gap filling by polymerization, or gap fill splint ligation, or a combination thereof. In some embodiments, a rolling circle amplification product (RCP) of the circularized probe is generated in the biological sample, and the RCP comprises multiple copies of the gap sequence or complement thereof. [0082] In some embodiments, the circularizable probe (e.g., second probe) comprises a 5’ region and a 3’ region that hybridize to sequences adjacent to a gap sequence in the target nucleic acid. In some embodiments, upon hybridization of a circularizable probe to the target nucleic acid molecule, the 3’ terminal nucleotide and the 5’ terminal nucleotide of the circularizable probe are not juxtaposed directly next to each other; as such, a ligase alone cannot catalyze the formation of a phosphodiester bond directly between the 5’ phosphate group of the 5’ terminal nucleotide and the 3’ hydroxyl group of the 3’ terminal nucleotide. In some embodiments, upon hybridization of a circularizable probe to the target nucleic acid molecule, the 3’ terminal nucleotide and the 5’ terminal nucleotide of the circularizable probe are separated from each other by a gap of between about 1 and about 150 nucleotides in length. In some embodiments, upon hybridization of a circularizable probe to the target nucleic acid molecule, the 3’ terminal nucleotide and the 5’ terminal nucleotide of the circularizable probe are separated from each other by a gap of between about 1 and about 40 nucleotides in length. In some ^{MOFO-357975700} 202412023840 embodiments, the gap is about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, or of any integer (or range of integers) of nucleotides in between the indicated values. In some embodiments, the gap is no more than about 40 nucleotides in length. In some embodiments, the gap is no more than about 30 nucleotides in length. In some embodiments, the gap is about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20, about 22, about 24, or of any integer (or range of integers) of nucleotides in between the indicated values. In some embodiments, the gap is between about 10 to about 150 nucleotides in length. In some embodiments, the gap is between about 100 to about 150 nucleotides in length. In some embodiments, the gap is between about 10 to about 30 nucleotides in length. In some embodiments, the gap is between about 15 to about 25 nucleotides in length. In some embodiments, the gap is no more than about 10 nucleotides in length. In some embodiments, the gap is about 5 nucleotides in length. [0083] In some embodiments, a circularizable probe (e.g., second probe) disclosed herein does not comprise any nucleic acid barcode sequence. In some embodiments, circularizable probes for hybridizing to multiple different target nucleic acids comprise a common sequence that is not complementary to the target nucleic acids. For instance, the backbone sequences of a plurality of circularizable probes for detecting different variant sequences of a target nucleic acid is a common backbone sequence. In some embodiments, the backbone sequences of the plurality of gap fill padlock probes do not contain any nucleic acid barcode sequence that uniquely corresponds to a particular target nucleic acid or a particular sequence variant thereof. [0084] In some aspects, the binding (e.g., coupling) between two or more nucleic acids, e.g., oligonucleotides and/or polynucleotides is detected. In some embodiments, the binding is indirect binding. In some embodiments, the binding is direct (e.g., binding comprising direct hybridization of nucleic acid sequences). The nature of the binding may vary. In some instances, a first nucleic acid sequence directly binds to a second nucleic acid sequence via hybridization of complementary sequences. In some instances, a first nucleic acid sequence indirectly binds to a second nucleic acid sequence via one or more intermediate nucleic acids. For example, an intermediate nucleic acid comprises a first region that binds to the first nucleic acid sequence and has a second region for binding to the second nucleic acid sequence, thereby ^{MOFO-357975700} 202412023840 forming a complex comprising the first and second nucleic acid sequences. In some embodiments, hybridization of substantially complementary or complementary nucleic acid sequences within two different molecules is analyzed. For example, hybridization of an endogenous analyte with a probe is analyzed. Pairing can be achieved by any process in which a nucleic acid sequence joins with a substantially or fully complementary sequence through base pairing to form a hybridization complex. For purposes of hybridization, two nucleic acid sequences are “substantially complementary” if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of their individual bases are complementary to one another. [0085] Various probes can be hybridized to an endogenous analyte and/or a labeling agent and each probe may comprise one or more barcode sequences. For example, barcoded probes or probe sets may be based on a padlock probe, a gapped padlock probe, a SNAIL (Splint Nucleotide Assisted Intramolecular Ligation) probe set, a PLAYR (Proximity Ligation Assay for RNA) probe set, a PLISH (Proximity Ligation in situ Hybridization) probe set, and RNA- templated ligation probes. The specific probe design can vary. [0086] A biological sample may comprise one or a plurality of analytes of interest. Methods for performing multiplexed assays to analyze two or more different analytes in a single biological sample are provided. The methods and compositions disclosed herein can be used to detect and analyze a wide variety of different analytes. In some aspects, an analyte can include any biological substance, structure, moiety, or component to be analyzed. In some aspects, a target disclosed herein may similarly include any analyte of interest. In some examples, a target or analyte can be directly or indirectly detected. [0087] Analytes can be derived from a specific type of cell and/or a specific sub- cellular region. For example, analytes can be derived from cytosol, from cell nuclei, from mitochondria, from microsomes, and more generally, from any other compartment, organelle, or portion of a cell. Permeabilizing agents that specifically target certain cell compartments and organelles can be used to selectively release analytes from cells for analysis, and/or allow access of one or more reagents (e.g., probes for analyte detection) to the analytes in the cell or cell compartment or organelle. [0088] The analyte may include any biomolecule or chemical compound, including a macromolecule such as a protein or peptide, a lipid or a nucleic acid molecule, or a small ^{MOFO-357975700} 202412023840 molecule, including organic or inorganic molecules. The analyte may be a cell or a microorganism, including a virus, or a fragment or product thereof. An analyte can be any substance or entity for which a specific binding partner (e.g. an affinity binding partner) can be developed. Such a specific binding partner may be a nucleic acid probe (for a nucleic acid analyte) and may lead directly to the generation of a RCA template (e.g. a padlock or other circularizable probe). Alternatively, the specific binding partner may be coupled to a nucleic acid, which may be detected using an RCA strategy, e.g. in an assay which uses or generates a circular nucleic acid molecule which can be the RCA template. [0089] Analytes of particular interest may include nucleic acid molecules, such as DNA (e.g. genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, etc.) and RNA (e.g. mRNA, microRNA, rRNA, snRNA, viral RNA, etc.), and synthetic and/or modified nucleic acid molecules, (e.g. including nucleic acid domains comprising or consisting of synthetic or modified nucleotides such as LNA, PNA, morpholino, etc.), proteinaceous molecules such as peptides, polypeptides, proteins or prions or any molecule which includes a protein or polypeptide component, etc., or fragments thereof, or a lipid or carbohydrate molecule, or any molecule which comprise a lipid or carbohydrate component. The analyte may be a single molecule or a complex that contains two or more molecular subunits, e.g. including but not limited to protein-DNA complexes, which may or may not be covalently bound to one another, and which may be the same or different. Thus in addition to cells or microorganisms, such a complex analyte may also be a protein complex or protein interaction. Such a complex or interaction may thus be a homo- or hetero-multimer. Aggregates of molecules, e.g. proteins may also be target analytes, for example aggregates of the same protein or different proteins. The analyte may also be a complex between proteins or peptides and nucleic acid molecules such as DNA or RNA, e.g. interactions between proteins and nucleic acids, e.g. regulatory factors, such as transcription factors, and DNA or RNA. [0090] In some embodiments, an analyte herein is endogenous to a biological sample and can include nucleic acid analytes and non-nucleic acid analytes. Methods and compositions disclosed herein can be used to analyze nucleic acid analytes (e.g., using a nucleic acid probe that directly or indirectly hybridizes to a nucleic acid analyte) and/or non-nucleic acid analytes ^{MOFO-357975700} 202412023840 (e.g., using a labeling agent that comprises a reporter oligonucleotide and binds directly or indirectly to a non-nucleic acid analyte) in any suitable combination. [0091] Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral coat proteins, extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some embodiments, the analyte is inside a cell or on a cell surface, such as a transmembrane analyte or one that is attached to the cell membrane. In some embodiments, the analyte is an organelle (e.g., nuclei or mitochondria). In some embodiments, the analyte is an extracellular analyte, such as a secreted analyte. Examples of analytes include, but are not limited to, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, an extracellular matrix protein, a posttranslational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation or lipidation) state of a cell surface protein, a gap junction, and an adherens junction. [0092] Examples of nucleic acid analytes include DNA analytes such as single- stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA/DNA hybrids. The DNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA such as mRNA) present in a tissue sample. [0093] Examples of nucleic acid analytes also include RNA analytes such as various types of coding and non-coding RNA. Examples of the different types of RNA analytes include messenger RNA (mRNA), including a nascent RNA, a pre-mRNA, a primary-transcript RNA, and a processed RNA, such as a capped mRNA (e.g., with a 5’ 7-methyl guanosine cap), a polyadenylated mRNA (poly-A tail at the 3’ end), and a spliced mRNA in which one or more introns have been removed. Also included in the analytes disclosed herein are non-capped ^{MOFO-357975700} 202412023840 mRNA, a non-polyadenylated mRNA, and a non-spliced mRNA. The RNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA such as viral RNA) present in a tissue sample. Examples of a non-coding RNAs (ncRNA) that is not translated into a protein include transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), as well as small non-coding RNAs such as microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), extracellular RNA (exRNA), small Cajal body-specific RNAs (scaRNAs), and the long ncRNAs such as Xist and HOTAIR. The RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). Examples of small RNAs include 5.8S ribosomal RNA (rRNA), 5S rRNA, tRNA, miRNA, siRNA, snoRNAs, piRNA, tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNA or single-stranded RNA. In some embodiments, the RNA comprises circular RNA. In some embodiments, the RNA is a bacterial rRNA (e.g., 16s rRNA or 23s rRNA). [0094] In some embodiments, the second target nucleic acid is an RNA transcript less than 500 nucleotides in length, less than 400 nucleotides in length, less than 300 nucleotides in length, or less than 200 nucleotides in length. In some embodiments, the second target nucleic acid is an RNA transcript less than 200 nucleotides in length. In some embodiments, the second target nucleic acid is an RNA transcript less than 100 nucleotides in length. In some embodiments, the second target nucleic acid is an RNA transcript less than 80 nucleotides in length. In some embodiments, the second target nucleic acid is an RNA transcript less than 60 nucleotides in length. [0095] In some embodiments described herein, an analyte may be a denatured nucleic acid, wherein the resulting denatured nucleic acid is single-stranded. The nucleic acid may be denatured, for example, optionally using formamide, heat, or both formamide and heat. In some embodiments, the nucleic acid is not denatured for use in a method disclosed herein. [0096] Methods and compositions disclosed herein can be used to analyze any number of analytes. For example, the number of analytes that are analyzed can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, ^{MOFO-357975700} 202412023840 at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000 or more different analytes. [0097] In some embodiments, provided herein are methods and compositions for analyzing endogenous analytes (e.g., RNA, ssDNA, cell surface or intracellular proteins, and/or metabolites) in a sample using one or more labeling agents. In some embodiments, an analyte labeling agent may include an agent that interacts with an analyte (e.g., an endogenous analyte in a sample). In some embodiments, the labeling agents can comprise a reporter oligonucleotide that is indicative of the analyte or portion thereof interacting with the labeling agent. For example, the reporter oligonucleotide may comprise a barcode sequence that permits identification of the labeling agent. In some cases, the sample contacted by the labeling agent can be further contacted with a probe (e.g., a single-stranded probe sequence), that hybridizes to a reporter oligonucleotide of the labeling agent, in order to identify the analyte associated with the labeling agent. In some embodiments, the analyte labeling agent comprises an analyte binding moiety and a labeling agent barcode domain comprising one or more barcode sequences, e.g., a barcode sequence that corresponds to the analyte binding moiety and/or the analyte. An analyte binding moiety barcode includes to a barcode that is associated with or otherwise identifies the analyte binding moiety. In some embodiments, by identifying an analyte binding moiety by identifying its associated analyte binding moiety barcode, the analyte to which the analyte binding moiety binds can also be identified. An analyte binding moiety barcode can be a nucleic acid sequence of a given length and/or sequence that is associated with the analyte binding moiety. An analyte binding moiety barcode can generally include any of the variety of aspects of barcodes described herein. A. Gap fill Polymerization [0098] In some embodiments, a gap in a circularizable probe (e.g., second probe) hybridized to the target nucleic acid molecule may be filled by extending a 3' end of the circularizable probe to generate an extended probe comprising a gap filled sequence. In some embodiments, a polymerase is used to extend the 3’ end using the target nucleic acid molecule as a template, thereby filling the gap using the nucleotide sequence in the target nucleic acid molecule. In some embodiments, gap filling by the polymerase incorporates nucleotides residues into the circularizable probe, and the incorporated nucleotide sequence is ^{MOFO-357975700} 202412023840 complementary to the gap sequence or a portion thereof in the target nucleic acid molecule. In some embodiments, a polymerase is used to extend the 3’ end using the gap sequence in the target nucleic acid molecule as a template, thereby filling the gap using the nucleotide sequence in the target nucleic acid molecule. In some embodiments, a polymerase is configured to extend the 3’ end of the second probe using the gap sequence in the target nucleic acid molecule as a template, thereby filling the gap using the nucleotide sequence in the target nucleic acid molecule. [0099] In some instances, the gap filling is performed using a polymerase (e.g., DNA polymerase) in the presence of appropriate dNTPs and other cofactors, under isothermal conditions or non-isothermal conditions. Exemplary DNA polymerases include but are not limited to: E.coli DNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, VENT™ DNA polymerase, DEEPVENT™ DNA polymerase, LongAmp® Taq DNA polymerase, LongAmp® Hot Start Taq DNA polymerase, Crimson LongAmp® Taq DNA polymerase, Crimson Taq DNA polymerase, OneTaq® DNA polymerase, OneTaq® Quick- Load® DNA polymerase, Hemo KlenTaq® DNA polymerase, REDTaq® DNA polymerase, Phusion® DNA polymerase, Phusion® High-Fidelity DNA polymerase, Platinum Pfx DNA polymerase, AccuPrime Pfx DNA polymerase, Phi29 DNA polymerase, Klenow fragment, Pwo DNA polymerase, Pfu DNA polymerase, T4 DNA polymerase and T7 DNA polymerase enzymes. [0100] In some instances, the gap filling is performed using a DNA polymerase capable of incorporating at least about 25, at least about 50, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 300, at least about 400, at least about 500, at least about 600, or at least about 1,000 nucleotides in a single binding event before dissociating from the target nucleic acid molecule. [0101] Incorporation of the correct nucleotides to a growing strand of DNA, as determined by the template, is known as sequence fidelity. In some embodiments, a high fidelity DNA polymerase is used for gap filling and examples include but are not limited to: Taq DNA polymerase, Phusion® High-Fidelity DNA Polymerase, KAPA Taq, KAPA Taq HotStart DNA Polymerase, KAPA HiFi, and/or Q5® High-Fidelity DNA Polymerase. ^{MOFO-357975700} 202412023840 [0102] In some embodiments, an enzyme lacking strand displacement is used as the gap fill polymerase. Thus, when an extension reaction occurs from a first probe region to a second probe region to fill a gap, the second probe region is not displaced. Examples of non- strand displacing enzymes include Phusion® polymerase (Thermo Fisher, Waltham, MA) (which is generally described as non-strand displacing), 9°N, Vent® or Pfu DNA polymerases. Additional DNA polymerases without strand displacement activity include T7, Q5 or T4 DNA polymerase. In some aspects, absence of strand displacement enables efficient ligation. Suitable gap fill polymerases (e.g., with minimal strand displacement activity) are described in PCT publication WO2024238992, the content of which is herein incorporated by reference in its entirety. [0103] In some instances, the gap filling is performed using a polymerase having no or limited strand displacement activity, such that an extended 3’ region of the circularizable probe does not displace the 5’ region hybridized to the nucleic acid molecule. For example, T4 and T7 DNA Polymerases lack strand displacement activity and can be used for this purpose. In some embodiments, especially where the target nucleic acid is RNA, the polymerase can be a reverse transcriptase. Reverse transcriptases having reduced strand displacement activity can be used, see, e.g., Martín-Alonso et al., ACS Infect. Dis. 2020, 6, 5, 1140–1153, which is incorporated herein by reference in its entirety. [0104] In some embodiments, the enzyme has minimal strand displacement activity. An example of a gap fill polymerase that has minimal strand displacement activity is a Tgo RTX as described in PCT/US2024/030100. In some embodiments of any of the methods described herein, the gap fill polymerase: (a) substantially lacks strand displacement activity; or (b) displaces: (i) no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides; (ii) 1-2, 1-3, 1-4, 1-5, 1-6, 1- 7, 1-8, 1-9, or 1-10 nucleotides; or (iii) 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides; (iv) about 6 nucleotides; or (v) about 10 nucleotides. [0105] In some embodiments, a gap in a circularizable probe (e.g., second probe) hybridized to the target nucleic acid molecule is filled by extension using a polymerase. In some embodiments, the polymerase for extending the second probe is an engineered family B polymerase. In some instances, the engineered family B polymerase is selected from the group consisting of Pyrococcus furiosus (pfu) polymerase, Thermococcus gorgonarius polymerase ^{MOFO-357975700} 202412023840 (Tgo polymerase), a Thermococus kodakarensis (K0D1) polymerase, a Thermococcus litoralis (VENT®) polymerase, a Pyrococcus sp. (Deep Vent) polymerase, a Thermococcus sp. (9°N) polymerase, or a Thermococcus argininiproducens (Targ) polymerase. In some embodiments, the engineered family B polymerase comprises one or more mutations compared to a wild type family B polymerase. In some embodiments, the engineered family B polymerase comprises one or more mutations that confer reverse transcriptase activity. In some embodiments, a gap in a circularizable probe (e.g., second probe) hybridized to the target nucleic acid molecule is filled by extension using a Tgo polymerase. In some embodiment, the Tgo polymerase comprises one or more mutations that confer reverse transcriptase activity and a ligase. [0106] In certain embodiments the gap sequence is a gap of 1-1000, 1-750, 1-500, 1- 300, 1-200, 1-100, 1- 90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1- 11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 nucleotides; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides. [0107] In some embodiments, the 3’ region of the circularizable probe extended by the polymerase is juxtaposed to the 5’ region of the circularizable probe (e.g., second probe). In some embodiments, the 3’ region of the circularizable probe extended by the polymerase is juxtaposed to the 5’ region of the circularizable probe, forming a nick. In some embodiments, the ligation involves template dependent ligation, e.g., using the gap sequence in the target nucleic acid as template. In some embodiments, the ligation involves template independent ligation. The nick can be ligated using chemical ligation. In some embodiments, the chemical ligation involves click chemistry. [0108] In some embodiments, the ligation involves enzymatic ligation. In some embodiments, the enzymatic ligation involves use of a ligase. In some aspects, the ligase used herein comprises an enzyme that is commonly used to join polynucleotides together or to join the ends of a single polynucleotide. In some aspects, the ligase used herein is a DNA ligase. In some aspects, the ligase used herein is an ATP-dependent double-strand polynucleotide ligases, NAD-i-dependent double-strand DNA or RNA ligases and single-strand polynucleotide ligases, for example any of the ligases described in EC 6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2 ^{MOFO-357975700} 202412023840 (NAD+-dependent ligases), EC 6.5.1.3 (RNA ligases). Specific examples of ligases comprise bacterial ligases such as E. coli DNA ligase, Tth DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9°N™ DNA ligase, New England Biolabs), Taq DNA ligase, Ampligase™ (Epicentre Biotechnologies) and phage ligases such as T3 DNA ligase, T4 DNA ligase and T7 DNA ligase and mutants thereof. In some embodiments, the ligase is a T4 RNA ligase. In some embodiments, the ligase is a splintR ligase. In some embodiments, the ligase is a single stranded DNA ligase. In some embodiments, the ligase is a T4 DNA ligase. In some embodiments, the ligase is a ligase that has an DNA-splinted DNA ligase activity. In some embodiments, the ligase is a ligase that has an RNA-splinted DNA ligase activity. In some embodiments, the ligase is a ssDNA ligase. In some embodiments, the ssDNA ligase is a bacteriophage TS2126 RNA ligase or an archaebacterium RNA ligase or a variant or derivative thereof. In some embodiments, the ligase is Methanobacterium thermoautotrophicum RNA ligase 1, CircLigase™ I, CircLigase™ II, T4 RNA ligase 1, or T4 RNA ligase 2, or a variant or derivative thereof. B. Splint Oligonucleotide Ligation [0109] In some embodiments, a gap in a circularizable probe (e.g., second probe) hybridized to the target nucleic acid molecule is filled by a splint oligonucleotide. In some embodiments, the splint oligonucleotide is ligated to a circularizable probe to generate an extended probe comprising a gap filled sequence. [0110] In some embodiments, the splint oligonucleotide comprises a sequence complementary to a nucleotide variation, a nucleotide polymorphism, a mutation, a substitution, an insertion, a deletion, a translocation, a duplication, an inversion, and/or a repetitive sequence, for identifying a variant sequence among a plurality of different sequences in situ in a biological sample. In some embodiments, the splint oligonucleotide can comprise a sequence complementary to a single nucleotide, for instance, a single nucleotide variation (SNV), a single nucleotide polymorphism (SNP), a point mutation, a single nucleotide substitution, a single nucleotide insertion, or a single nucleotide deletion. In some embodiments, the splint oligonucleotide can comprise a sequence complementary to a sequence comprising multiple nucleotides, and each nucleotide can be independently at the position of an SNV, an SNP, a point mutation, a single nucleotide substitution, a single nucleotide insertion, or a single nucleotide deletion. In some embodiments, the splint oligonucleotide comprises a sequence complementary ^{MOFO-357975700} 202412023840 to a sequence of or associated with an immune molecule. In some embodiments, the splint oligonucleotide comprises a sequence complementary to a sequence of or associated with a perturbation agent. [0111] In some embodiments, provided herein is a library of splint oligonucleotides comprising i) a splint oligonucleotide comprising a sequence complementary to a nucleotide variation, a nucleotide polymorphism, a mutation, a substitution, an insertion, a deletion, a translocation, a duplication, an inversion, and/or a repetitive sequence, and ii) another splint oligonucleotide which does not comprise a sequence complementary to the nucleotide variation, nucleotide polymorphism, mutation, substitution, insertion, deletion, translocation, duplication, inversion, and/or repetitive sequence. In some embodiments, the library of splint oligonucleotides can comprise i) a splint oligonucleotide comprising a sequence complementary to a variant sequence or deletion or insertion, and ii) another splint oligonucleotide which does not comprise a sequence complementary to the variant sequence or deletion or insertion. For example, wildtype and variant splint oligonucleotides in the library, when contacted with the biological sample, can compete with one another for hybridization to a gap sequence comprising a variant sequence, and the complementary variant splint oligonucleotide can outcompete the wildtype splint oligonucleotide which is not complementary to the variant sequence (e.g., one or more nucleotides) in the gap sequence. The competition among splint oligonucleotides can allow the use of short (e.g., 2 nucleotides) splint oligonucleotides, while achieving specificity of splint oligonucleotide hybridization and/or ligation, for instance, when splint oligonucleotide hybridization and ligation are performed in the same reaction mix and/or the same reaction condition. In some embodiments, using a low hybridization temperature, less denaturation, and/or more co-factors such as Mg²⁺ or other factors that promote hybridization can enable the use of shorter splint oligonucleotides. [0112] In some embodiments, upon hybridization to the target nucleic acid molecule, the 5’ terminal nucleotide of the splint oligonucleotide is adjacent to the 3’ terminal nucleotide of the circularizable probe, and the 3’ terminal nucleotide of the splint oligonucleotide is adjacent to the 5’ terminal nucleotide of the circularizable probe. In some embodiments, the 5’ terminal nucleotide of the splint oligonucleotide and the 3’ terminal nucleotide of the circularizable probe are separated by a nick or a gap of one or more nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or ^{MOFO-357975700} 202412023840 more nucleotides. In some embodiments, the 3’ terminal nucleotide of the splint oligonucleotide and the 5’ terminal nucleotide of the circularizable probe are separated by a nick or a gap of one or more nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides. The nick can be ligated using any suitable ligase disclosed herein, and the gap can be filled using any suitable polymerase followed by ligation. [0113] In some embodiments, upon hybridization to the target nucleic acid, the circularizable probe is circularized to generate an extended probe comprising a gap filled region complementary to the gap sequence. [0114] In some embodiments, the circularizable probe is hybridized to the target nucleic acid, followed by contacting the biological sample with a library of splint oligonucleotides that compete for hybridization to the target nucleic acid (e.g., hybridization to the gap sequence in the target nucleic acid). In some embodiments, the hybridization of a splint oligonucleotide to the target nucleic acid and the ligation of the splint oligonucleotide to the circularizable probe are performed sequentially, e.g., the splint oligonucleotide hybridization is performed in a reaction condition or reaction mix, and the splint oligonucleotide ligation is performed in a different reaction condition or different reaction mix. In some embodiments, the hybridization of a splint oligonucleotide to the target nucleic acid and the ligation of the splint oligonucleotide to the circularizable probe are performed in the same reaction condition or the same reaction mix. In some embodiments, any one or more of the splint oligonucleotides in the library are 2 nucleotides or more in length. In some embodiments, the library of splint oligonucleotides is used to detect different variant sequences of a single target nucleic acid within the biological sample. In some embodiments, the different variant sequences of the single target nucleic acid are at different positions in the gap sequence. In some embodiments, a circularizable probe comprises a first hybridization region and a second hybridization region that hybridize to the common first sequence and second sequence shared by target nucleic acids comprising different variant sequences. [0115] In some embodiments, the circularizable probe and the library of splint oligonucleotides are contacted with the target nucleic acid at the same time, in the same reaction mix or separately. For example, the circularizable probe and the library of splint oligonucleotides are premixed before contacting the biological sample with the mixture. In ^{MOFO-357975700} 202412023840 another example, two separate compositions comprising the circularizable probe and the library of splint oligonucleotides, respectively, are contacted with the biological sample. In some embodiments, the hybridization of a splint oligonucleotide to the target nucleic acid and the ligation of the splint oligonucleotide to the circularizable probe are performed in the same reaction condition or the same reaction mix. In some embodiments, any one or more of the splint oligonucleotides in the library can be 2 nucleotides or more in length. In some embodiments, the library of splint oligonucleotides comprises at least or about 2, at least or about 5, at least or about 10, at least or about 15, at least or about 20, at least or about 25, at least or about 30, at least or about 35, at least or about 40, at least or about 45, at least or about 50, or more splint oligonucleotides of different sequences. In some embodiments, the molar concentration of the library of splint oligonucleotides is about equal to or about 2, about 4, about 8, about 10, or more times the molar concentration of the circularizable probe. [0116] In some aspects, a high fidelity ligase, such as a thermostable DNA ligase (e.g., a Taq DNA ligase), is used. Thermostable DNA ligases are active at elevated temperatures, allowing further discrimination by incubating the ligation at a temperature near the melting temperature (Tm) of the DNA strands. This selectively reduces the concentration of annealed mismatched substrates (expected to have a slightly lower Tm around the mismatch) over annealed fully base-paired substrates. Thus, high-fidelity ligation can be achieved through a combination of the intrinsic selectivity of the ligase active site and balanced conditions to reduce the incidence of annealed mismatched dsDNA. [0117] In some embodiments, the splint oligonucleotide comprises a sequence complementary to the gap sequence in the target nucleic acid molecule (e.g., second target nucleic acid). In some embodiments, the biological sample is contacted with a library of splint oligonucleotides. In some embodiments, the library comprises at least about 2, at least about 4, at least about 10, at least about 20, at least about 50, at least about 100, or more oligonucleotides of different sequences. In some embodiments, the sequence diversity of the splint oligonucleotides in the library is such that at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, or about 100% of the possible variant sequences in the gap sequence of the target nucleic acid in a sample have corresponding splint oligonucleotides in the library, e.g., the splint oligonucleotides comprise sequences that are complementary to the ^{MOFO-357975700} 202412023840 variant sequences in the target nucleic acid. [0118] In some embodiments, the gap filling is performed under conditions permissive for specific hybridization of a splint oligonucleotide to its complementary sequence in the gap sequence in the target nucleic acid molecule, and/or specific hybridization of a circularizable probe to the target nucleic acid molecule. In some embodiments, the circularizable probe comprises hybridization regions that hybridize to the target nucleic acid molecule at sequences outside the gap sequence (e.g., at constant region sequences flanking the gap sequence), whereas the variant sequences in the gap sequence are complementary to the splint oligonucleotides (e.g., wildtype or mutant) in the library. In some embodiments, the circularized probe is amplified by RCA (e.g., as described in Section II.C), and the RCA product comprises multiple copies of the gap sequence in the target nucleic acid, as shown in the bottom panel of FIG. 1A. In some embodiments, a sequence in the gap sequence in the RCA product is determined in situ, e.g., by sequencing the gap sequence as described in Section III. [0119] In some embodiments, the splint oligonucleotides is between about 6 and about 24 nucleotides in length. In some embodiments, any one or more of the splint oligonucleotides in the library is about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20, about 22, or about 24 nucleotides in length. Any two or more of the splint oligonucleotides in the library can have the same length or different lengths. In some embodiments, the splint oligonucleotides in the library can be of the same length. [0120] In some embodiments, the variant sequence is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or more phosphodiester bonds from the 3’ or 5’ end of the gap sequence. In some embodiments, the sequence complementary to the variant sequence is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or more phosphodiester bonds from the 5’ or 3’ end of the splint oligonucleotide. In some embodiments, the variant sequence is at or near the central nucleotide(s) of the gap sequence. In some embodiments, the sequence complementary to the variant sequence is at or near the central nucleotide(s) of the splint oligonucleotide. In some embodiments, the sequence complementary to the variant sequence is at or near the central 1, 2, 3, 4, or 5 nucleotide(s) of the splint oligonucleotide. In some embodiments, the sequence complementary to the variant sequence is no more than 1, no more than 2, no more than 3, no ^{MOFO-357975700} 202412023840 more than 4, no more than 5, or no more than 6 nucleotides from the central nucleotide(s) of the splint oligonucleotide. C. Amplification [0121] In some instances, the first amplification product generated using the first probe is a first RCA product comprising multiple copies of the barcode sequence (or complements thereof) of the first probe. In some instances, the second amplification product generated using the second probe is a second RCA product comprising multiple copies of the gap filled sequence (or complements thereof). In some embodiments, RCA is performed, and following formation of the circularized probes (e.g., first and second probes), in some instances, a primer oligonucleotide is added for amplification. In some instances, the primer oligonucleotide is added with the circularizable probe. In some instances, the primer oligonucleotide is added before or after the circularizable probe is contacted with the sample. In some instances, the primer oligonucleotide for amplification of the circularized template (e.g., circularized probe) comprises a sequence complementary to a target nucleic acid, as well as a sequence complementary to the circularizable probe that hybridizes to the target nucleic acid. In some embodiments, amplification of the circularized probe is primed by the target nucleic acid. In some embodiments, a washing step is performed to remove any unbound probes, primers, etc. In some embodiments, the wash is a stringency wash. Washing steps can be performed at any point during the process to remove non-specifically bound probes, probes that have ligated, etc. [0122] In some instances, a primer oligonucleotide for amplification of the circularized template comprises a single-stranded nucleic acid sequence having a 3’ end that can be used as a substrate for a nucleic acid polymerase in a nucleic acid extension reaction. The primer oligonucleotide can comprise both RNA nucleotides and DNA nucleotides (e.g., in a random or designed pattern). The primer oligonucleotide can also comprise other natural or synthetic nucleotides described herein that can have additional functionality. In some cases, the primer oligonucleotide is about 6 bases to about 100 bases, such as about 25 bases. [0123] In some embodiments, amplification of the first and/or second circularized template is primed by the target nucleic acid (e.g., target RNA). The target nucleic acid can optionally be immobilized in the biological sample. In some embodiments, the target RNA is cleaved by an enzyme (e.g., RNase H). In some embodiments, the target nucleic acid is cleaved 47 ^{MOFO-357975700} 202412023840 at a position downstream of the sequences bound to the circularized probe. In some aspects, the methods disclosed herein allow targeting of RNase H activity to a particular region in a target RNA that is adjacent to or overlapping with a target sequence for a probe. For example, a nucleic acid oligonucleotide is designed to hybridize to a complementary oligonucleotide hybridization region in the target RNA. In some embodiments, a nucleic acid oligonucleotide is used to provide a DNA-RNA duplex for RNase H cleavage of the target RNA in the DNA-RNA duplex. In some embodiments, the oligonucleotide binds to the target RNA at a position that overlaps with the target sequence of the probe by about 1 to about 20 nucleotides or by about 8 to about 10 nucleotides. The cleaved target RNA itself can then be used to prime RCA of the circular probe generated from a circularized probe (e.g., target-primed RCA). In some cases, a plurality of nucleic acid oligonucleotides can be used to perform target-primed RCA for a plurality of different target nucleic acids. [0124] In any of the embodiments herein, the biological sample is contacted with the RNase H (and optionally with the nucleic acid oligonucleotide) before or during formation of the circularized probe and/or circularized gap filled probe (e.g., as described in Section II). In some embodiments, the biological sample is contacted with the oligonucleotide and with the RNase H simultaneously or sequentially (in either order) before contacting the sample with the probe. In any of the embodiments herein, the biological sample is contacted with the RNase H (and optionally with the nucleic acid oligonucleotide) after formation of the circularized template from ligating the probe. In any of the embodiments herein, the RNase H comprises an RNase H1 and/or an RNAse H2. In some embodiments, RNase inactivating agents or inhibitors are added to the sample after cleaving the target RNA. [0125] In some instances, upon addition of a DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, the amplification primer is elongated by replication of multiple copies of the template. The amplification step can utilize isothermal amplification or non-isothermal amplification. In some embodiments, after the formation of the hybridization complex and any subsequent circularization (such as ligation of, e.g., a circularizable probe), the circularized probe is rolling-circle amplified to generate a RCA product (e.g., RCP) containing multiple copies of the sequence of the circularized template. ^{MOFO-357975700} 202412023840 [0126] In some embodiments, RCPs are generated using a polymerase selected from the group consisting of Phi29 DNA polymerase, Phi29-like DNA polymerase, M2 DNA polymerase, B103 DNA polymerase, GA-1 DNA polymerase, phi-PRD1 polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, KlenTaq DNA polymerase, DNA polymerase I, Klenow fragment of DNA polymerase I, DNA polymerase III, T3 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Bst polymerase, rBST DNA polymerase, N29 DNA polymerase, TopoTaq DNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, and a variant or derivative thereof. In some embodiments, the polymerase is Phi29 DNA polymerase. [0127] In some embodiments, the polymerase comprises a modified recombinant Phi29-type polymerase. In some embodiments, the polymerase comprises a modified recombinant Phi29, B103, GA-1, PZA, Phi15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp- 5, Cp-7, PR4, PR5, PR722, or L17 polymerase. In some embodiments, the polymerase comprises a modified recombinant DNA polymerase having at least one amino acid substitution or combination of substitutions as compared to a wildtype Phi29 polymerase. Suitable polymerases are described in U.S. Patent Nos. 8,257,954; 8,133,672; 8,343,746; 8,658,365; 8,921,086; and 9,279,155, all of which are herein incorporated by reference. In some embodiments, the polymerase is not directly or indirectly immobilized to a substrate, such as a bead or planar substrate (e.g., glass slide), prior to contacting a sample, although the sample may be immobilized on a substrate. [0128] In some embodiments, the amplification is performed at a temperature between or between about 20ºC and about 60ºC. In some embodiments, the amplification is performed at a temperature between or between about 30ºC and about 40ºC. In some aspects, the amplification step, such as the rolling circle amplification (RCA) is performed at a temperature between at or about 25°C and at or about 50°C, such as at or about 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, 37°C, 39°C, 41°C, 43°C, 45°C, 47°C, or 49°C. [0129] In some aspects, during the amplification step, modified nucleotides can be added to the reaction to incorporate the modified nucleotides in the amplification product (e.g., nanoball). Examples of the modified nucleotides comprise amine-modified nucleotides. In some aspects of the methods, for example, for anchoring or cross-linking of the generated ^{MOFO-357975700} 202412023840 amplification product (e.g., nanoball) to a scaffold, to cellular structures and/or to other amplification products (e.g., other nanoballs). In some aspects, the amplification products comprises a modified nucleotide, such as an amine-modified nucleotide. In some embodiments, the amine-modified nucleotide reacts with an acrylic acid N-hydroxysuccinimide moiety. Examples of other amine-modified nucleotides comprise, but are not limited to, a 5-Aminoallyl- dUTP moiety modification, a 5-Propargylamino-dCTP moiety modification, a N⁶-6- Aminohexyl-dATP moiety modification, or a 7-Deaza-7-Propargylamino-dATP moiety modification. In some embodiments, the modified nucleotides comprises base modifications, such as azide and/or alkyne base modifications, dibenzylcyclooctyl (DBCO) modifications, vinyl modifications, trans-Cyclooctene (TCO), and so on. [0130] In some embodiments, the extension reaction mixture comprises a deoxynucleoside triphosphate (dNTP) or derivative, variant, or analogue thereof. In some embodiments, the primer extension reaction mixture can comprise a catalytic cofactor of the polymerase. In any of the preceding embodiments, the primer extension reaction mixture can comprise a catalytic di-cation, such as Mg²⁺ and/or Mn²⁺. [0131] In some aspects, the RCA product is anchored to a polymer matrix. The amplification products may be immobilized within the matrix generally at the location of the nucleic acid being amplified, thereby creating a localized colony of amplicons. The amplification products may be immobilized within the matrix by steric factors. The amplification products may also be immobilized within the matrix by covalent or noncovalent bonding. In this manner, the amplification products may be considered to be attached to the matrix. By being immobilized to the matrix, such as by covalent bonding or cross-linking, the size and spatial relationship of the original amplicons is maintained. By being immobilized to the matrix, such as by covalent bonding or cross-linking, the amplification products are resistant to movement or unraveling under mechanical stress. [0132] In some aspects, the RCA products are copolymerized and/or covalently attached to the surrounding matrix thereby preserving their spatial relationship and any information inherent thereto. In some embodiments, the RCA products are generated from DNA or RNA within a cell embedded in the matrix. In some embodiments, the RCA products are functionalized to form covalent attachment to the matrix preserving their spatial information ^{MOFO-357975700} 202412023840 within the cell thereby providing a subcellular localization distribution pattern. In some embodiments, the provided methods involve embedding RCA products in the presence of hydrogel subunits to form one or more hydrogel-embedded amplification products. In some embodiments, the hydrogel-tissue chemistry described comprises covalently attaching nucleic acids to in situ synthesized hydrogel for tissue clearing, enzyme diffusion, and multiple-cycle sequencing or probe hybridization while an existing hydrogel-tissue chemistry method cannot. In some embodiments, to enable amplification product embedding in the tissue-hydrogel setting, amine-modified nucleotides are comprised in the RCA product, functionalized with an acrylamide moiety using acrylic acid N-hydroxysuccinimide esters, and copolymerized with acrylamide monomers to form a hydrogel. [0133] In some embodiments, the generated amplification products of the first probe and the second probe are subject to analysis and/or sequence determination comprising detecting a sequence in all or a portion of the nucleic acid concatemer or in situ hybridization to the generated RCA products. In some embodiments, a first RCA product generated is subject to analysis and/or sequence determination comprising in situ hybridization to the RCP. In some embodiments, a second RCA product generated is subject to analysis and/or sequence determination comprising in situ sequencing of the RCP to determine the gap filled sequence or a complement thereof. In some embodiments, the detection involves contacting the biological sample with an oligonucleotide probe that directly or indirectly hybridize to a sequence in the first RCA product and detecting a first signal associated with the oligonucleotide probe, thereby detecting the first target nucleic acid, and sequencing the second RCA product to determine the gap filled sequence or a complement thereof, thereby detecting the second target nucleic acid. In some instances, the gap filled sequence or a complement thereof is detected by sequencing by ligation, sequencing by synthesis or sequencing by binding. III. DETECTION AND ANALYSIS [0134] In some aspects, the provided methods involve analyzing, e.g., detecting or determining, one or more nucleic acid sequences such as sequences associated with the first target nucleic acid(s) and gap sequence(s) associated with the second target nucleic acid(s). In some aspects, the provided methods involve analyzing, e.g., detecting or determining, two or ^{MOFO-357975700} 202412023840 more different nucleic acid sequences associated with two or more different target analytes (e.g., two or more different RNAs). In some cases, the analysis is performed on one or more images captured, and may comprise processing the image(s) and/or quantifying signals observed. In some embodiments, the analysis comprises detecting a sequence (e.g., a gap sequence) present in the sample. In some embodiments, the analysis comprises detecting a barcode sequence present in the sample. In some embodiments, the analysis comprises quantification of puncta (e.g., if RCPs are detected). In some cases, the information may comprise signals from a cell, a region, and/or comprise readouts from multiple detectable labels. In some cases, the multiple detection rounds are performed to obtain signals from a cell, a region, and/or comprise different readouts. In some cases, the analysis further comprises displaying the information from the analysis or detection step. In some embodiments, software may be used to automate the processing, analysis, and/or display of data. [0135] In some embodiments, the detection comprises contacting the biological sample with an oligonucleotide probe that directly or indirectly hybridize to a sequence in the first RCA product and detecting a first signal associated with the oligonucleotide probe, thereby detecting the first target nucleic acid. In some embodiments, the detection comprises sequencing the second RCA product to determine the gap filled sequence or a complement thereof, thereby detecting the second target nucleic acid. In some instances, the contacting the biological sample with an oligonucleotide probe that directly or indirectly hybridize to a sequence in the first RCA product and detecting a signal associated with the oligonucleotide probe and sequencing the second RCA product to determine the gap filled sequence or a complement thereof are performed simultaneously. In some instances, sequencing the second RCA product to determine the gap filled sequence or a complement thereof is performed after one or more cycles of detection by hybridization (e.g., cycles of contacting the biological sample with an oligonucleotide probe that directly or indirectly hybridize to a sequence in the first RCA product and detecting a signal associated with the oligonucleotide probe). A. Detection by Hybridization [0136] In some aspects, the provided methods involve analyzing, e.g., detecting or determining, one or more sequences present in the first probes. In some instances, the method ^{MOFO-357975700} 202412023840 comprises contacting the biological sample with an oligonucleotide probe that directly or indirectly hybridizes to a sequence in the first RCA product and detecting a first signal associated with the oligonucleotide probe, thereby detecting the first target nucleic acid. In some embodiments, the detecting is performed at one or more locations in the biological sample. In some embodiments, the biological sample is a cell or tissue sample. In some embodiments, the locations are the locations of RNA transcripts in the biological sample. In some embodiments, the locations are the locations at which the probes (e.g., first probes) hybridize to the RNA transcripts in the biological sample, and are ligated and amplified by rolling circle amplification. In some instances, provided herein are probe panels for detecting a plurality of target nucleic acids in the biological sample. In some instances, a plurality of sequences present a plurality of first probes targeting a panel of target nucleic acids in the biological sample are detected. [0137] In some embodiments, detecting the one or more sequences present in the probes (e.g., first probes) in the biological sample is performed, and the detected sequences are compared to an expected set of detected sequences. In some embodiments, detecting one or more barcode sequences present in the probes (e.g., first probes) in the biological sample is performed. In some embodiments, the expected set of sequences is based on the barcode sequences of the panels of probes in the plurality of circularizable probes. In some embodiments, the one or more sequences are one or more barcode sequences or complements thereof. [0138] In some embodiments, detecting the one or more sequences present in the probes (e.g., first probes) in the biological sample is performed by detecting the first hybridization region and second hybridization region (e.g., 5’ and 3’ arms of a padlock probe) or a portion thereof hybridized to the first sequence and second sequence in the target nucleic acid (e.g., an RNA or cDNA). In some cases, the detected sequence is a sequence or a complement thereof of the first sequence and second sequence in the target nucleic acid (e.g., an RNA or cDNA). In some embodiments, detection of the sequences of the first probe or complements thereof in the first RCA product is performed by sequential hybridization of oligonucleotide probes to a sequence corresponding to the first hybridization region and second hybridization region (e.g., 5’ and 3’ arms of a padlock probe) or a portion thereof. In some embodiments, detection of the sequences of the first probe or complements thereof in the RCA product is performed by sequential hybridization of oligonucleotide probes to a sequence complementary to ^{MOFO-357975700} 202412023840 sequences of the first hybridization region and second hybridization region (e.g., 5’ and 3’ arms of a padlock probe) or a portion thereof. In some embodiments, detection of the sequences of the first probe or complements thereof in the RCA product is performed by sequential hybridization of oligonucleotide probes to the first sequence and second sequence in the generated RCA product comprising multiple copies of the endogenous sequences of the first target nucleic acid. In some embodiments, detection comprises binding an oligonucleotide probe directly or indirectly hybridizes to a sequence comprising: i) the first sequence of the first target nucleic acid or a portion thereof, and/or ii) the second sequence of the first target nucleic acid or a portion thereof. In some embodiments, detection comprises using an oligonucleotide probe comprising a sequence of the first hybridization region complementary to the first sequence of the first target nucleic acid or a portion thereof, and/or ii) the second hybridization region complementary to the second sequence of the first target nucleic acid or a portion thereof. [0139] In some embodiments, the detecting comprises a plurality of repeated cycles of hybridization and removal of oligonucleotide probes (e.g., detectably labeled probes, or oligonucleotide probes that bind to detectably labeled probes) to the probe hybridized to the target nucleic acid, or to a rolling circle amplification product generated from the probe hybridized to the target nucleic acid (e.g., using the generated circularized template). [0140] Methods for binding and identifying a target nucleic acid that uses various probes or oligonucleotides have been described in, e.g., US2003/0013091, US2007/0166708, US2010/0015607, US2010/0261026, US2010/0262374, US2010/0112710, US2010/0047924, and US2014/0371088, each of which is incorporated herein by reference in its entirety. In some embodiments, detectably-labeled probes are used for detecting multiple target nucleic acids and be detected in one or more hybridization cycles (e.g., sequential hybridization assays, or sequencing by hybridization). [0141] In some embodiments, the detecting comprises binding an oligonucleotide probe directly or indirectly to the circularized probe, binding a detectably labeled probe directly or indirectly to a detection region of the oligonucleotide probe, and detecting a signal associated with the detectably labeled probe. In some embodiments, the detecting comprises binding an oligonucleotide probe directly to the circularized probe, wherein the oligonucleotide probe comprises a detectable label, and detecting a signal associated with the detectably labeled probe. ^{MOFO-357975700} 202412023840 In some embodiments, the method comprises detecting a rolling circle amplification product (RCP) generated using the circularized probe as a template. In some embodiments, the method comprises detecting a rolling circle amplification product (RCP) generated using a circular or circularized probe as a template. In some embodiments, detecting the RCP comprises binding an oligonucleotide probe directly or indirectly to the RCP, binding a detectably labeled probe directly or indirectly to a detection region of the oligonucleotide probe, and detecting a signal associated with the detectably labeled probe. In some embodiments, the method comprises performing one or more wash steps to remove unbound and/or nonspecifically bound oligonucleotide probe molecules from the circularized probes or amplification products generated using the circularized template. [0142] In some embodiments, the detecting comprises detecting signals associated with detectably labeled probes that are hybridized to barcode regions or complements thereof in the circularized probe or a product thereof (e.g., an RCP); and/or detecting signals associated with detectably labeled probes that are hybridized to oligonucleotide probe which are in turn hybridized to the barcode regions or complements thereof. In some embodiments, the detectably labeled probes are fluorescently labeled. In some instances, at least two different oligonucleotide probes hybridize to the same barcode region or complement thereof. [0143] In some embodiments, the methods comprise detecting the sequence in all or a portion of a probe (e.g., first probe) or an RCP such as one or more barcode sequences present in the probe or generated RCP. In some embodiments, the sequence of the RCP, or barcode thereof, is indicative of a sequence of the target nucleic acid to which the probe is hybridized. In some embodiments, the detection step is by sequential fluorescent in situ hybridization (e.g., for combinatorial decoding of the barcode sequence or complement thereof). In some embodiments, a barcode sequence of the first probe is detected by performing cycles of sequential fluorescent in situ hybridization (e.g., combinatorial decoding of the barcode sequence or complement thereof). [0144] In some embodiments, the methods comprise detecting one or more barcode subunits of a barcode sequence or complement thereof present in the probe, the circularized template, or generated RCP. In some embodiments, the one or more barcode subunits are overlapping (e.g., a first barcode subunit overlaps in sequence with a second barcode subunit). ^{MOFO-357975700} 202412023840 In some embodiments, the barcode sequence comprises a first subunit and a second subunit. In some embodiments, the barcode sequence is assigned a signal code sequence (a sequential series of signals), wherein the signal code sequence identifies a corresponding target nucleic acid molecule. In some embodiments, each subunit of the barcode sequence is assigned to a signal code of the signal code sequence. In some embodiments, the first subunit corresponds to a first signal code of the signal code sequence, and the second subunit corresponds to a second signal code of the signal code sequence. In some embodiments, the signal code sequence is derived by interrogating (e.g., hybridizing) the one or more subunits on the barcode sequence or complement thereof with a plurality of oligonucleotide probes. In some embodiments, each oligonucleotide probe of the plurality of oligonucleotide probes comprises a detectable label. In some embodiments, the method comprises contacting the barcode sequence at the first subunit with an oligonucleotide probe and detecting the detectable label comprised on the oligonucleotide probe and associated with the first signal code to identify the first target nucleic acid. In some embodiments, the method further comprises contacting the barcode sequence at the second subunit with a second oligonucleotide probe and detecting the detectable label comprised on the second oligonucleotide probe and associated with the second signal code to identify the first target nucleic acid. [0145] In some embodiments, the detection or determination comprises hybridizing to an oligonucleotide probe directly or indirectly a detectably labeled probe labeled with a fluorophore, an isotope, a mass tag, or a combination thereof. In some embodiments, the detection or determination comprises imaging the oligonucleotide probe hybridized to the target nucleic acid, directly or indirectly (e.g., imaging one or more detectably labeled probes hybridized thereto). In some embodiments, the target nucleic acid is an mRNA in a tissue sample, and the detection or determination is performed when the target nucleic acid and/or the RCA product is in situ in the tissue sample. [0146] In some aspects, the provided methods comprise imaging a detectably labeled probe bound directly or indirectly to the first probe or product thereof and detecting the detectable label. In some embodiments, the detectably labeled probe comprises a detectable label that can be measured and quantitated. The label or detectable label can comprise a directly or indirectly detectable moiety, e.g., any fluorophores, radioactive isotopes, fluorescers, ^{MOFO-357975700} 202412023840 chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like. [0147] A fluorophore can comprise a substance or a portion thereof that is capable of exhibiting fluorescence in the detectable range. Particular examples of labels that may be used in accordance with the provided embodiments comprise, but are not limited to phycoerythrin, Alexa dyes, fluorescein, YPet, CyPet, Cascade blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas red, luminol, acradimum esters, biotin, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), firefly luciferase, Renilla luciferase, NADPH, beta-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenical acetyl transferase, and urease. [0148] Fluorescence detection in tissue samples can often be hindered by the presence of strong background fluorescence. Background fluorescence can include autofluorescence (that can arise from a variety of sources, including aldehyde fixation, extracellular matrix components, red blood cells, lipofuscin, and the like), as opposed to the desired immunofluorescence from the fluorescently labeled antibodies or probes. Tissue autofluorescence can lead to difficulties in distinguishing the signals due to fluorescent antibodies or probes from the general background. In some embodiments, a method disclosed herein utilizes one or more agents to reduce tissue autofluorescence, for example, Autofluorescence Eliminator (Sigma/EMD Millipore), TrueBlack Lipofuscin Autofluorescence Quencher (Biotium), MaxBlock Autofluorescence Reducing Reagent Kit (MaxVision Biosciences), and/or a very intense black dye (e.g., Sudan Black, or comparable dark chromophore). [0149] Examples of detectable labels comprise but are not limited to various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, protein-protein binding pairs and protein-antibody binding pairs. Examples of fluorescent proteins comprise, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), ^{MOFO-357975700} 202412023840 umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin. [0150] Examples of bioluminescent markers comprise, but are not limited to, luciferase (e.g., bacterial, firefly and click beetle), luciferin, aequorin and the like. Examples of enzyme systems having visually detectable signals comprise, but are not limited to, galactosidases, glucorimidases, phosphatases, peroxidases and cholinesterases. Identifiable markers also comprise radioactive compounds such as ¹²⁵I, ³⁵S, ¹⁴C, or ³H. Identifiable markers are commercially available from a variety of sources. [0151] In some embodiments, one or more fluorescent dyes are used as detectable labels. Commercially available fluorescent dyes include, but are not limited to 4,7- dichlorofluorescein dyes, spectrally resolvable rhodamine dyes, 4,7- dichlororhodamine dyes, cyanine dyes, ether-substituted fluorescein dyes, energy transfer dyes, and xanthine dyes. Labeling can also be carried out with quantum dots. In some embodiments, a fluorescent label comprises a signaling moiety that conveys information through the fluorescent absorption and/or emission properties of one or more molecules. Exemplary fluorescent properties comprise fluorescence intensity, fluorescence lifetime, emission spectrum characteristics and energy transfer. [0152] Examples of commercially available fluorescent nucleotide analogues readily incorporated into nucleotide and/or polynucleotide sequences comprise, but are not limited to, Cy3™-dCTP (cyanine 3-dCTP), Cy3™-dUTP (cyanine 3-dUTP), Cy5™-dCTP (cyanine 5- dCTP), Cy5™-dUTP (cyanine 5 dUTP) (Amersham Biosciences, Piscataway, N.J.), fluorescein- 12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED®-5-dUTP (red fluorescent dye-dUTP), CASCADE® BLUE-7-dUTP (blue fluorescent dye – dUTP), BODIPY™ FL-14-dUTP (green fluorescent dye-dUTP), BODIPY™ TMR-14-dUTP (orange fluorescent dye-dUTP), BODIPY™ TR-14-dUTP (red fluorescent dye-dUTP), RHODAMINE GREEN™-5-dUTP (green fluorescent dye-dUTP), OREGON GREEN™ 488-5-dUTP (green fluorescent dye-dUTP), TEXAS RED™- l2-dUTP (red fluorescent dye-dUTP), BODIPY™ 630/650-14-dUTP (far red fluorescent dye- dUTP), BODIPY™ 650/665-14-dUTP (far red fluorescent dye-dUTP), ALEXA FLUOR™ 488- 5-dUTP (green fluorescent dye-dUTP), ALEXA FLUOR™ 532-5-dUTP (yellow fluorescent dye-dUTP), ALEXA FLUOR™ 568-5-dUTP (red/orange fluorescent dye-dUTP), ALEXA ^{MOFO-357975700} 202412023840 FLUOR™ 594-5-dUTP (red fluorescent dye-dUTP), ALEXA FLUOR™ 546-14-dUTP (orange fluorescent dye-dUTP), fluorescein- 12-UTP, tetramethylrhodamine-6-UTP, TEXAS RED™-5- UTP (red fluorescent dye-UTP), mCherry, CASCADE® BLUE-7-UTP (blue fluorescent dye- UTP), BODIPY™ FL-14-UTP (green fluorescent protein-UTP), BODIPY™ TMR- 14-UTP (orange fluorescent dye-UTP), BODIPY™ TR-14-UTP (red fluorescent dye-UTP), RHODAMINE GREEN™-5-UTP (green fluorescent dye-UTP), ALEXA FLUOR™ 488-5-UTP (green fluorescent dye-UTP), and ALEXA FLUOR™ 546-14-UTP (orange fluorescent dye- UTP) (Molecular Probes, Inc. Eugene, Oreg.). Methods are known for custom synthesis of nucleotides having other fluorophores. [0153] Other fluorophores available for post-synthetic attachment comprise, but are not limited to, ALEXA FLUOR™ dyes (fluorescent dyes) such as ALEXA FLUOR™ 350 (blue fluorescent dye), ALEXA FLUOR™ 594 (red fluorescent dye), and ALEXA FLUOR™ 647 (far red fluorescent dye); BODIPY™ dyes (fluorescent dyes) such as BODIPY™ FL (green fluorescent dye), BODIPY™ TMR (orange fluorescent dye), and BODIPY™ 650/665 (far red fluorescent dye); Cascade® Blue (blue fluorescent dye), Cascade® Yellow (yellow fluorescent dye), Dansyl, lissamine rhodamine B, Marina Blue™ (blue fluorescent dye), Oregon Green™ 488, Oregon Green™ 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethyl rhodamine, Texas Red® (red fluorescent dye) (available from Molecular Probes, Inc., Eugene, Oreg.), Cy2™ (cyanine 2), Cy3.5™ (cyanine 3.5), Cy5.5™ (cyanine 5.5), and Cy7™ (cyanine 7) (Amersham Biosciences, Piscataway, N.J.). FRET tandem fluorophores may also be used, comprising, but not limited to, PerCP-Cy™5.5 (far red fluorescent tandem fluorophore), PE-Cy™5 (red fluorescent tandem fluorophore), PE-Cy™5.5 (red fluorescent tandem fluorophore), PE-Cy™7 (far red fluorescent tandem fluorophore), PE-Texas Red® (red fluorescent tandem fluorophore), APC-Cy™7 (far red fluorescent tandem fluorophore), PE- Alexa™ dyes (e.g., 610, 647, 680), and APC-Alexa™ dyes. [0154] In some cases, metallic silver or gold particles may be used to enhance signal from fluorescently labeled nucleotide and/or polynucleotide sequences (Lakowicz et al. (2003) Bio Techniques 34:62). [0155] Biotin, or a derivative thereof, may also be used as a label on a nucleotide and/or a polynucleotide sequence, and subsequently bound by a detectably labeled ^{MOFO-357975700} 202412023840 avidin/streptavidin derivative (e.g., phycoerythrin-conjugated streptavidin), or a detectably labeled anti-biotin antibody. Digoxigenin may be incorporated as a label and subsequently bound by a detectably labeled anti-digoxigenin antibody (e.g., fluoresceinated anti-digoxigenin). An aminoallyl-dUTP residue may be incorporated into a polynucleotide sequence and subsequently coupled to an N-hydroxy succinimide (NHS) derivatized fluorescent dye. In general, any member of a conjugate pair may be incorporated into a detection polynucleotide provided that a detectably labeled conjugate partner can be bound to permit detection. [0156] Other suitable labels for a polynucleotide sequence may comprise fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), and phosphor-amino acids (e.g., P-tyr, P-ser, P-thr). In some embodiments the following hapten/antibody pairs are used for detection, in which each of the antibodies is derivatized with a detectable label: biotin/a-biotin, digoxigenin/a- digoxigenin, dinitrophenol (DNP)/a-DNP, 5-Carboxyfluorescein (FAM)/a-FAM. [0157] In some embodiments, a polynucleotide sequence is indirectly labeled, such as with a hapten that is then bound by a capture agent. Many different hapten-capture agent pairs are available for use. Exemplary haptens comprise, but are not limited to, biotin, des-biotin and other derivatives, dinitrophenol, dansyl, fluorescein, cyanine dyes (e.g., Cy5™, and digoxigenin. For biotin, a capture agent may be avidin, streptavidin, or antibodies. Antibodies may be used as capture agents for the other haptens (many dye-antibody pairs being commercially available, e.g., Molecular Probes, Eugene, Oreg.). [0158] In some embodiments, detection of the barcode sequences of the first probe or complements thereof in the RCA product is performed by sequential hybridization of oligonucleotide probes to the barcode sequences or complements thereof and detecting complexes formed by the probes and barcode sequences or complements thereof. In some cases, each barcode sequence or complement thereof is assigned a sequence of signal codes that identifies the barcode sequence or complement thereof (e.g., a temporal signal signature or code that identifies the analyte), and detecting the barcode sequences or complements thereof can comprise decoding the barcode sequences of complements thereof by detecting the corresponding sequences of signal codes detected from sequential hybridization, detection, and removal of sequential pools of oligonucleotide probes and the universal pool of detectably ^{MOFO-357975700} 202412023840 labeled probes. In some cases, the sequences of signal codes are fluorophore sequences assigned to the corresponding barcode sequences or complements thereof. In some embodiments, the detectably labeled probes are fluorescently labeled. In some embodiments, the barcode sequence or complement thereof is performed by sequential probe hybridization as described in US 2021/0340618, the content of which is herein incorporated by reference in its entirety. [0159] In some embodiments, the oligonucleotide for detection of the barcode sequences of the first probe or complements thereof is between about 5 and about 30 nucleotides in length. In some embodiments, the oligonucleotide for detection of the barcode sequences of the first probe or complements thereof is at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 nucleotides in length. [0160] In some embodiments, the detecting comprises contacting the biological sample with one or more detectably labeled probes that directly or indirectly hybridize to the barcode sequences or complements thereof (e.g., in RCA products generated using the probes), and dehybridizing the one or more detectably labeled probes. In any of the embodiments herein, the contacting and dehybridizing steps are repeated with the one or more detectably labeled probes and/or one or more other detectably labeled probes that directly or indirectly hybridize to the barcode sequences or complements thereof. In some aspects, the method comprises sequential hybridization of detectably labeled probes to create a spatiotemporal signal signature or code that identifies the analyte. [0161] In some embodiments, the detecting comprises contacting the biological sample with one or more first detectably labeled probes that directly hybridize to the plurality of oligonucleotide probes. In some instances, the detecting step can comprise contacting the biological sample with one or more first detectably labeled probes that indirectly hybridize to the plurality of probes. In any of the embodiments herein, the detecting step can comprise contacting the biological sample with one or more first detectably labeled probes that directly or indirectly hybridize to the plurality of oligonucleotide probes. [0162] In some embodiments, the detecting step comprises contacting the biological sample with one or more oligonucleotide probes that directly or indirectly hybridize to the barcode sequences or complements thereof (e.g., of the plurality of probes or rolling circle amplification product generated using the plurality of probes), wherein the one or more ^{MOFO-357975700} 202412023840 oligonucleotide probes are detectable using one or more detectably labeled probes. In any of the embodiments herein, the detecting step can further comprise dehybridizing the one or more oligonucleotide probes and/or the one or more detectably labeled probes from the barcode sequences or complements thereof (e.g., of the plurality of probes or rolling circle amplification product generated using the plurality of probes). In any of the embodiments herein, the contacting and dehybridizing steps is repeated with the one or more oligonucleotide probes, the one or more detectably labeled probes, one or more other oligonucleotide probes, and/or one or more other detectably labeled probes. In some cases, the repeated contacting, detection and dehybridizing steps allows detection of barcode sequences or complements thereof and identification of the corresponding sequences of signal codes (e.g., fluorophore sequences assigned to the corresponding barcode sequences or complements thereof). [0163] In some embodiments, a detectably labeled probe hybridizes to a detectable region in an oligonucleotide probe. In some embodiments, the detectable region is in a 5’ overhang and/or a 3’ overhang of the oligonucleotide probe, upon hybridization of the oligonucleotide probe to the sequence or a portion thereof in an RCP. In some embodiments, the detectable region is a split region, e.g., a portion of the detectable region can be in the 5’ overhang and another portion of the detectable region can be in the 3’ overhang of an oligonucleotide probe. In some embodiments, the detectable region is in the 5’ overhang of the oligonucleotide probe. In some embodiments, the detectable region is in the 3’ overhang of the oligonucleotide probe. In some embodiments, a first portion of the detectable region is in the 3’ overhang and a second portion of the detectable region is in the 5’ overhang of the oligonucleotide probe. In some instances, readout signals are detected by hybridizing detectably labeled probes to oligonucleotide probes. In some embodiments, readout signals are detected by hybridizing detectably labeled probes directly to a sequence of the RCA product. [0164] In some embodiments, the method comprises contacting the sample with a first pool of oligonucleotide probes and a universal pool of detectably labeled probes, wherein the first pool of oligonucleotide probes comprises the first oligonucleotide probe and the universal pool of detectably labeled probes comprises the first detectably labeled probe and the second detectably labeled probe, wherein each oligonucleotide probe in the first pool of oligonucleotide probes comprises (i) a hybridization region complementary to a sequence in the ^{MOFO-357975700} 202412023840 RCA product (e.g., the first RCA product) and (ii) an overhang sequence complementary to a detectably labeled probe of the universal pool of detectably labeled probes; and the method comprises contacting the sample with a second pool of oligonucleotide probes and the universal pool of detectably labeled probes, wherein the second pool of oligonucleotide probes comprises the second oligonucleotide probe, and wherein each oligonucleotide probe in the second pool of oligonucleotide probes comprises (i) a hybridization region complementary to a sequence in the RCA product (e.g., the first RCA product) and (ii) an overhang sequence complementary to a detectably labeled probe of the universal pool of detectably labeled probes. In some embodiments, the first and second pool of oligonucleotide probes hybridize to a sequence in the RCA product. [0165] In some embodiments, the detection or determination comprises temporally detecting probes in a sequential manner for in situ analysis in a biological sample, e.g., in an intact tissue. In some aspects, provided herein is a method for detecting the detectably labeled probes, thereby generating a signal code. In some instances, each signal code corresponds to a target nucleic acid (e.g., in a panel of target nucleic acids). In some instances, the probes are optically detected (e.g., by detectably labeled probes) in a temporally-sequential manner. In some embodiments, the sample is contacted with a library of probes to detect the probes or products thereof (e.g., used or generated as described in Section II) associated with the first target nucleic acids. In some instances, a plurality of probes to detect a plurality of first target nucleic acids are applied to a sample simultaneously. In some instances, the probes are applied to a sample sequentially (e.g., in subsets of probes). In some aspects, the method comprises sequential hybridization of labelled probes to create a signal code sequence (e.g., a temporal pattern of signal codes corresponding to signals detected at a location) that identifies the first target nucleic acid or portion thereof. In some aspects, multiple cycles of probe hybridization and detection are performed to detect a plurality of target nucleic acids at location(s) in a biological sample, as shown in FIG. 1A, top panel. As depicted in FIG. 1A, the first probe is a circularizable probe and amplification of a barcode sequence of the first probe is performed by RCA, in some embodiments, the first probe is not used to generate a circularized template and is amplified by other amplification methods (e.g., as described in Section II). ^{MOFO-357975700} 202412023840 B. Detection by Sequencing [0166] In some aspects, the provided methods involve analyzing, e.g., detecting or determining, the gap filled sequence or a complement thereof in the second RCA product generated using the second probe. In some cases, the gap filled sequence is a variable sequence (e.g., can be different from cell to cell or sample to sample) and/or is unknown that is suitable for detecting by sequencing. In some embodiments, the gap filled sequence or a complement thereof in the second RCA product is detected using sequencing (e.g., single base-by-base sequencing), thereby detecting the second target nucleic acid. In some embodiments, the sequencing is performed at one or more locations in the biological sample. In some embodiments, the biological sample is a cell or tissue sample. In some embodiments, the locations are the locations of RNA transcripts in the biological sample. In some embodiments, the locations are the locations at which the probes (e.g., second probes) hybridize to the RNA transcripts in the biological sample, are gap filled, ligated and amplified by rolling circle amplification. In some aspects, multiple cycles of sequencing and detection are performed to detect a gap sequence or a portion thereof of one or more target nucleic acids at location(s) in a biological sample, as shown in FIG. 1A, bottom panel. In some instances, sequencing the second RCA product to determine the gap filled sequence or a complement thereof is performed after detecting the first target nucleic acid by hybridizing an oligonucleotide probe to the first RCA product (e.g., as described in Section III.A). [0167] In some aspects, a labeling agent comprising a reporter oligonucleotide bound directly or indirectly to a non-nucleic acid analyte (e.g., as described in Section II) is detected by sequencing at least a portion of the reporter oligonucleotide. In some instances, sequencing the second RCA product to determine the gap filled sequence or a complement thereof is performed at the same time as sequencing the reporter oligonucleotide to detect a labeling agent. In some embodiments, the labeling agent is bound directly or indirectly to a protein analyte in the biological sample. In some instances, a sequence of the reporter oligonucleotide or a complement thereof is detected by sequencing. In some instances, the sequence of the reporter oligonucleotide or a complement thereof is detected by using single nucleotide sequencing by synthesis. ^{MOFO-357975700} 202412023840 [0168] In some aspects, the provided methods involve analyzing, e.g., detecting or determining, one or more nucleic acid sequences such as gap sequences in target nucleic acids (e.g., second target nucleic acids). For example, the gap filled sequence or a complement thereof in the second RCA product is detected using sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof. In some embodiments, sequencing comprises capturing one or more images of the biological sample. In some cases, the analysis is performed on one or more images captured, and may comprise processing the image(s) and/or quantifying signals observed. In some embodiments, the analysis comprises detecting a sequence (e.g., a gap sequence) present in the biological sample. In some embodiments, the analysis comprises quantification of puncta (e.g., if RCA products are detected). In some embodiments, the obtained information may be compared to a positive and negative control, or to a threshold of a feature to determine if the sample exhibits a certain feature or phenotype. In some cases, the information may comprise signals from a cell, a region, and/or comprise readouts from multiple detectable labels. In some cases, the analysis further comprises displaying the information from the analysis or detection step. In some embodiments, software may be used to automate the processing, analysis, and/or display of data. [0169] In some embodiments, following amplification, the gap filled sequence or complement thereof of the RCA product (e.g., second RCA product generated using the second circularized template) or a portion thereof, is determined or otherwise analyzed. In some aspects, the analysis of the RCA product comprises sequencing-by-synthesis (SBS), sequencing- by-binding (SBB), avidity sequencing, or sequencing-by-ligation (SBL). In some embodiments, the gap filled sequence or a complement thereof in the second RCA product is detected using sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof. In some embodiments, the gap filled sequence or a complement thereof in the second RCA product is sequenced using in situ sequencing by synthesis (SBS) in the biological sample. In some embodiments, the gap filled sequence or a complement thereof in the second RCA product is detected using single nucleotide sequencing by synthesis. In some embodiments, the gap filled sequence or a complement thereof in the second RCA product is sequenced using in situ sequencing by ligation (SBL) in the biological sample. In some embodiments, the gap filled ^{MOFO-357975700} 202412023840 sequence or a complement thereof in the second RCA product is sequenced using in situ sequencing by binding (SBB) in the biological sample. [0170] In some examples, SBS or SBB is used to sequence the gap sequence or a complement thereof in the RCA product, and the biological sample is contacted with nucleotides in sequential cycles, where in each cycle a complex is formed, the complex comprising (i) the sequencing primer or an extension product thereof hybridized to the sequencing primer binding site 3’ to the gap sequence, (ii) a polymerase, and (iii) a cognate nucleotide that base pairs with a nucleotide in the gap sequence, and a signal (e.g., level of a signal) associated with the cognate nucleotide and/or the polymerase in the complex is detected at a particular location in the biological sample, wherein the signal corresponds to the base in the cognate nucleotide and the corresponding nucleotide in the gap sequence. In some embodiments, a signal code corresponding to the signal is detected at the particular location. In some embodiments, the signal code corresponds to a signal of a first color, a signal of a second color, a signal of a third color, or absence of signal, wherein the first, second, and third colors are different. In some embodiments, the signal code corresponds to a combination of signals of a first or second color, or absence of signal, wherein the first and second colors are different. In some embodiments, the signal code corresponds to a combination of signals detected in two or more imaging steps. [0171] In some embodiments, analyzing, e.g., detecting or determining the gap sequence or a complement thereof in the RCA product is performed using a base-by-base sequencing method, e.g., sequencing-by-synthesis (SBS), sequencing-by-avidity (SBA) or sequencing-by-binding (SBB). In some embodiments, the biological sample is contacted with a sequencing primer and base-by-base sequencing using a cyclic series of nucleotide incorporation or binding, respectively, thereby generating extension products of the sequencing primer is performed followed by removing, cleaving, or blocking the extension products of the sequencing primer. [0172] Generally in sequencing-by-synthesis methods, a first population of detectably labeled nucleotides (e.g., dNTPs) are introduced to contact a template nucleotide (e.g., a barcode sequence in the RCP) hybridized to a sequencing primer, and a first detectably labeled nucleotide (e.g., A, T, C, or G nucleotide) is incorporated by a polymerase to extend the sequencing primer in the 5’ to 3’ direction using a complementary nucleotide (a first nucleotide residue) in the ^{MOFO-357975700} 202412023840 template nucleotide as template. In some cases, a signal from the first detectably labeled nucleotide is detected. The first population of nucleotides may be continuously introduced, but in order for a second detectably labeled nucleotide to incorporate into the extended sequencing primer, nucleotides in the first population of nucleotides that have not incorporated into a sequencing primer are generally removed (e.g., by washing), and a second population of detectably labeled nucleotides are introduced into the reaction. Then, a second detectably labeled nucleotide (e.g., A, T, C, or G nucleotide) is incorporated by the same or a different polymerase to extend the already extended sequencing primer in the 5’ to 3’ direction using a complementary nucleotide (a second nucleotide residue) in the template nucleotide as template. Thus, in some embodiments, cycles of introducing and removing detectably labeled nucleotides are performed. [0173] In some instances, cycles of introducing and removing detectably labeled nucleotides in a temporally sequential manner for in situ analysis of an analyte in a biological sample, e.g., a target nucleic acid in a cell in an intact tissue is performed. In some aspects, provided herein is a method for detecting the detectably labeled nucleotides, thereby generating a signal signature associated with the labeled nucleotides. In some instances, the signal signature corresponds to an analyte of the plurality of analytes. In some instances, the methods described herein are based, in part, on the development of a multiplexed biological assay and readout, in which a sample is first contacted with a plurality of circularizable probes allowing the probes to directly or indirectly bind target analytes, which may then be optically detected (e.g., by sequencing) in a temporally-sequential manner. In some aspects, a circularized template comprising a gap filled sequence is formed from a circularizable probe, and optical detection (e.g. by sequencing) is performed on the gap filled sequence or a complement thereof in a temporally-sequential manner. In some aspects, provided herein is a method involving a multiplexed biological assay and sequencing readout including optically detecting labeled oligonucleotides in a temporally sequential manner. In some instances, as the positions of the analytes, probes, and/or products thereof can be maintained in a sample through the plurality of cycles of sequencing, the fluorescent spot corresponding to an analyte, probe, or product thereof remains in place during multiple rounds and can be aligned to read out a string of signals associated with each target analyte. ^{MOFO-357975700} 202412023840 [0174] In some embodiments, the base-by-base sequencing comprises using a polymerase that is fluorescently labeled. In some embodiments, the base-by-base sequencing comprises using a polymerase-nucleotide conjugate comprising a fluorescently labeled polymerase linked to a nucleotide moiety that is not fluorescently labeled. In some embodiments, the base-by-base sequencing comprises using a multivalent polymer-nucleotide conjugate comprising a polymer core, multiple nucleotide moieties, and one or more fluorescent labels. [0175] In some embodiments, the gap sequence or a complement thereof is sequenced by SBB using a polymerase that is fluorescently labeled and one or more nucleotides that are not fluorescently labeled. In some embodiments, during SBB, a cognate nucleotide is not incorporated by the polymerase into the sequencing primer or an extension product thereof. In some embodiments, incorporation of a cognate nucleotide by the polymerase into the sequencing primer or an extension product thereof is attenuated or inhibited. Various aspects of SBB are described in U.S. Pat. No. 10,655,176 B2, the content of which is herein incorporated by reference in its entirety. In some embodiments, SBB comprises performing repetitive cycles of detecting a stabilized complex that forms at each position along the template nucleic acid to be sequenced (e.g. a ternary complex that includes the primed template nucleic acid, a polymerase, and a cognate nucleotide for the position), under conditions that prevent covalent incorporation of the cognate nucleotide into the primer, and then extending the primer to allow detection of the next position along the template nucleic acid. In the sequencing-by-binding approach, detection of the nucleotide at each position of the template occurs prior to extension of the primer to the next position. Generally, the methodology is used to distinguish the four different nucleotide types that can be present at positions along a nucleic acid template by uniquely labeling each type of ternary complex (i.e. different types of ternary complexes differing in the type of nucleotide it contains) or by separately delivering the reagents needed to form each type of ternary complex. In some instances, the labeling comprises fluorescence labeling of, e.g., the cognate nucleotide or the polymerase that participate in the ternary complex. [0176] In some embodiments, the gap sequence or a complement thereof is sequenced by SBS, comprising contacting the biological sample with a nucleotide mix comprising a fluorescently labeled nucleotide and a nucleotide that is not fluorescently labeled. In some embodiments, during SBS, a cognate nucleotide is incorporated by the polymerase into ^{MOFO-357975700} 202412023840 the sequencing primer or an extension product thereof, and the cognate nucleotide may or may not be fluorescently labeled. In some embodiments, a priming strand (e.g., a primer) is provided and binds to the second RCA product comprising the gap filled sequence or a complement thereof. In some cases, the biological sample is contacted with (i) a polymerase and (ii) a first plurality of nucleotide in a nucleotide mix to form a complex comprising a 3’ terminus of the priming strand, the second RCA product, the polymerase, and a cognate nucleotide in the complex is detected to identify a complementary nucleotide in the second RCA product. Example SBS methods comprise those described for example, but not limited to, U.S. Pat. Pub. 2007/0166705, U.S. Pat. Pub. 2006/0188901, U.S. Patent No. 7,057,026, U.S. Pat. Pub. 2006/0240439, U.S. Pat. Pub. 2006/0281109, U.S. Pat. Pub. 2011/0059865, U.S. Pat. Pub. 2005/0100900, U.S. Patent No. 9,217,178, U.S. Pat. Pub. 2009/0118128, U.S. Pat. Pub. 2012/0270305, U.S. Pat. Pub. 2013/0260372, and U.S. Pat. Pub. 2013/0079232, each of which is herein incorporated by reference in its entirety. [0177] In some embodiments, the nucleotide mix comprises one or more nucleotides or analogs thereof, including a native nucleotide or a nucleotide analog or modified nucleotide (e.g., labeled with one or more detectable labels). In some embodiments, a nucleotide analog comprises a nitrogenous base, five-carbon sugar, and phosphate group, wherein any component of the nucleotide may be modified and/or replaced. In some embodiments, a method disclosed herein may comprise using one or more non-incorporable nucleotides. Non-incorporable nucleotides may be modified to become incorporable at any point during the sequencing method. [0178] Nucleotide analogs include, but are not limited to, alpha-phosphate modified nucleotides, alpha-beta nucleotide analogs, beta-phosphate modified nucleotides, beta-gamma nucleotide analogs, gamma-phosphate modified nucleotides, caged nucleotides, or ddNTPs. Examples of nucleotide analogs are described in U.S. Patent No. 8,071,755, which is incorporated by reference herein in its entirety. [0179] In some embodiments, the nucleotide mix comprises nucleotide analogs comprising reversible terminators, wherein the reversible terminators reversibly prevent nucleotide incorporation at the 3′-end of the primer. One type of reversible terminator is a 3′-O- blocked reversible terminator. Here the terminator moiety is linked to the oxygen atom of the 3′- OH end of the 5-carbon sugar of a nucleotide. For example, U.S. Patent Nos. 7,544,794 and ^{MOFO-357975700} 202412023840 8,034,923 (the disclosures of each of which is herein incorporated by reference in its entirety) describe reversible terminator dNTPs having the 3′-OH group replaced by a 3′-ONH2 group. Another type of reversible terminator is a 3′-unblocked reversible terminator, wherein the terminator moiety is linked to the nitrogenous base of a nucleotide. For example, U.S. Patent No. 8,808,989 (the disclosure of which is herein incorporated by reference in its entirety) discloses particular examples of base-modified reversible terminator nucleotides that may be used in connection with the methods described herein. Other reversible terminators that similarly can be used in connection with the methods described herein include those described in U.S. Patent Nos. 7,956,171, 8,071,755, and 9,399,798, each of which is herein incorporated by reference in its entirety. [0180] In some embodiments, a method disclosed herein may comprise using 1, 2, 3, 4 or more nucleotide analogs present in the SBS reaction. In some embodiments, a nucleotide analog is replaced, diluted, or sequestered during an incorporation step. In some embodiments, a nucleotide analog is replaced with a native nucleotide. In some embodiments, a nucleotide analog is modified during an incorporation step. The modified nucleotide analog can be similar to or the same as a native nucleotide. [0181] In some embodiments, a method disclosed herein may comprise using a nucleotide analog having a different binding affinity for a polymerase than a native nucleotide. In some embodiments, a nucleotide analog has a different interaction with a next base than a native nucleotide. Nucleotide analogs and/or non-incorporable nucleotides may base-pair with a complementary base of a template nucleic acid. [0182] In some embodiments, an extension product thereof of the sequencing primer comprises a reversible terminator nucleotide (e.g. a 3’ terminal nucleotide) that is reversibly blocked. In some aspects, the method further comprises unblocking the reversibly blocked 3’ terminal nucleotide molecule and contacting the extension product thereof of the sequencing primer bound to the second RCA product with a polymerase and a second plurality of nucleotide molecules. In some embodiments, the method comprises further repeating contacting the biological sample with an additional plurality of nucleotide molecules to identify additional complementary nucleotides of the gap filled sequence in the second RCA product for at least 10, at least 20, at least 30, at least 40, or at least 50 additional cycles. In some embodiments, no more ^{MOFO-357975700} 202412023840 than 50, no more than 100, no more than 200, or no more than 500 sequencing cycles are performed. In some embodiments, a gap sequence of no more than 20, no more than 50, no more than 100, no more than 200, or no more than 500 are determined by sequencing. After a plurality of cycles, the complementary nucleotides in the gap sequence of the second RCA product are identified. [0183] For readout using base-by-base sequencing (e.g., SBS) or SBL, sequencing primers can be designed to target the RCPs in the conserved regions shortly before the mutation hotspot, such as a few bases (e.g., 3-5 bases or more) 5’ to the gap sequence in the RCPs, such that a conserved region and/or the adjacent gap sequence can be determined as identifier sequences to identify which gene the RCP corresponds to, and as the sequencing reaction continues and reads into the gap sequence (e.g., containing the hotspot), the variant sequence(s) can be readout base-by-base. Using the bases before the variant sequence(s) as identifier sequences, this approach can be multiplexed, e.g., multiple sequence variants (e.g., in mutation or SNP hotspots) of one or more genes or transcripts thereof can be identified at the same time. In some embodiments, between about 10 and about 20 bases in a conserved region and/or the adjacent gap sequence can be determined to cover most or all of the sequence variants in a hotspot and provide enough sequence information to identify the gene as well as the sequence variants therein. [0184] In some embodiments, a sequencing reaction is performed to read at least a portion of the gap sequence in the RCA product or a complement thereof. In some examples, the gap sequence in the RCA product is a sequence that is complementary to the gap filled sequence in the extended second probe. In some embodiments, the second circularized template is used to generate a RCA product that comprises a sequencing primer binding site. In some instances, the RCA product generated using the second probe comprises a sequencing primer binding site 3’ of the gap sequence. In some instances, a sequencing strand (e.g., a sequencing primer) binds to the sequencing primer binding site of the generated RCA product for detection of the gap sequence. [0185] In some embodiments, the sequence adjacent to the gap sequence in the second RCA product is a primer binding site. In some embodiments, the primer binding site is a sequencing primer binding sequence. For example, a sequencing strand (e.g., primer or priming strand) hybridizes to the sequence adjacent to the gap sequence in the RCA product. In some ^{MOFO-357975700} 202412023840 embodiments, the sequence 3’ to the gap sequence in the RCA product is a primer binding site. In some embodiments, the sequence of the RCA product bound by the sequencing primer comprises at least a portion of the same sequence as the sequence in the target nucleic acid to which the second probe hybridizes. In some embodiments, the sequence of the RCA product bound by the sequencing strand (e.g., sequencing primer) is at least a portion of the same sequence as the sequence in the second target nucleic acid for hybridizing to a hybridization region in the second probe (the arm of the padlock probe). In some embodiments, the sequence of the RCA product bound by the sequencing strand (e.g., sequencing primer) is at least a portion of the same sequence as the sequence in the second target nucleic acid for hybridizing to the first hybridization region in the second probe (FIG. 1B left). In some embodiments, the sequence of the RCA product bound by the sequencing strand (e.g., sequencing primer) is at least a portion of the same sequence as the sequence in the second target nucleic acid for hybridizing to the second hybridization region in the second probe (FIG. 1B left). In some embodiments, the sequencing strand (e.g., sequencing primer) binds to the RCA product at a region of which the 5’ nucleic acid residue is 1 base, 2 bases, 3 bases, 4 bases, 5 bases, or more upstream or 3’ of the gap sequence in the RCA product. In some embodiments, the sequencing strand (e.g., primer or priming strand) is bound to the RCA product at a sequence that is not the same as a sequence of the second target nucleic acid. In some embodiments, the RCA product is a first RCA product. In some embodiments, the RCA product is a second RCA product. In some embodiments, the biological sample is contacted by a plurality of different second probes for binding to different target nucleic acids. In some instances, a plurality of different sequencing primers are used to sequence a plurality of generated RCA products that correspond to a plurality of different second target nucleic acids. For example, a first subset of the plurality of generated RCA products comprises a first sequencing primer binding site and a second subset of the plurality of generated RCA products comprises a second sequencing primer binding site, wherein the first and second sequencing primer bindings sites are different. In some embodiments, a plurality of sequencing primers are designed to bind the RCA product at regions 3’ to the gap sequence, such as a few bases (e.g., 1-5 bases or more) adjacent to the gap sequence in the RCA products, such that the adjacent gap sequence is detected in the sequencing reaction. In some embodiments, the plurality of sequencing primers are designed to bind the RCA product at a region of which the 5’ ^{MOFO-357975700} 202412023840 nucleic acid residue is 1 base, 2 bases, 3 bases, 4 bases, or 5 bases upstream or 3’ from the gap sequence in the RCA products. [0186] In some embodiments, the sequencing is performed by extending a sequencing strand (e.g., sequencing primer) complementary to a sequence of the generated second RCA product or an extension product thereof by performing one or more dark cycles (FIG. 1B right). In some embodiments, the sequence of the generated second RCA product or the extension product thereof to which the sequencing strand (e.g. sequencing primer) is complementary is comprised 3’ to the gap sequence. In some embodiments, the sequence of the generated second RCA product or the extension product thereof to which the sequencing strand (e.g., sequencing primer) is complementary is comprised 3’ to the second sequence in the target nucleic acid for hybridizing the first hybridization region or second hybridization region in second probe. In some instances, a dark cycle comprises extension by one or more nucleotides using a polymerase without detecting the nucleotide bound or incorporated in the dark cycle(s). In some cases, the dark cycle does not include performing detection (e.g., imaging). In some cases, performing one or more dark cycles reduces the amount of time required for the overall sequencing time and assay time. In some embodiments, the dark cycles are performed to advance through the sequence between the sequencing primer binding sequence and the gap sequence. In some embodiments, the dark cycles are performed to advance through the sequence between the sequencing primer binding sequence and the gap sequence. In some embodiments, the sequence between the sequencing primer binding sequence and the gap sequence is the second sequence in the target nucleic acid for hybridizing the second hybridization region in second probe. In some instances, the sequence of the RCA product bound by the sequencing primer is upstream of probe hybridization region of the target nucleic acid and one or more dark cycles is performed to advance through the sequence in the probe binding arm comprising the hybridization region (or a complement thereof in the RCA product). In some embodiments, the nucleotides in a nucleotide mix for performing a dark cycle do not include a detectable label. [0187] In some embodiments, a sequencing primer is extended in a plurality of consecutive dark cycles, wherein each dark cycle comprises incorporating one or more nucleotides using a polymerase, without detecting the nucleotides incorporated during the dark cycle. In some embodiments, the nucleotides incorporated during the dark cycles are not ^{MOFO-357975700} 202412023840 detectably labeled. In some embodiments, the nucleotides incorporated during the dark cycles are not fluorescently labeled, whereas at least some or all of the nucleotides incorporated during non-dark cycles are fluorescently labeled and detected. In some embodiments, one or more of the nucleotides incorporated during the dark cycles are detectably labeled (e.g., fluorescently labeled) but are not detected. In some embodiments, the plurality of dark cycles comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 dark cycles. In some instances, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more consecutive dark cycles are performed. In some embodiments, the priming strand is extended by two or more nucleotides that are not fluorescently labeled in consecutive sequencing cycles, using a sequence of the second RCA product as a template. In some instances, a nucleotide incorporated during a dark cycle comprises a reversible terminator moiety. In some embodiments, the dark cycles are not preceded by any sequencing cycles that comprises detecting a detectable label. In some embodiments, the dark cycles are not preceded by any sequencing cycles that identifies a complementary nucleotide in the second RCA product. In some embodiments, after the sequencing cycles to determine the gap filled sequence or a portion or complement thereof, additional dark cycles are not performed. [0188] In some embodiments, multiple different second target nucleic acids are detected using sequencing-based readout in the same biological sample. In some embodiments, one or more second target nucleic acids (target nucleic acids in “Block A”) are detected using sequencing strands (e.g., sequencing primers) that hybridize to the RCA products at regions complementary to the first or second hybridization regions in the second probes (which regions are 3’ to the gap sequences, e.g., as shown in FIG. 1B, left), whereas one or more other second target nucleic acids (target nucleic acids in “Block B” which are different from “Block A” target nucleic acids) are detected using sequencing strands (e.g., sequencing primers) that hybridize to the RCA products at regions 3’ to the regions complementary to the first or second hybridization regions in the second probes (e.g., as shown in FIG. 1B, right). In some embodiments, the RCA products of the target nucleic acids in “Block A” are sequenced and signals associated with nucleotide incorporation and/or binding are detected in sequencing cycles (e.g., as shown in FIG. 1B, left), while the RCA products of the target nucleic acids in “Block B” are subjected to consecutive dark cycles and signals associated with incorporation of the dark nucleotide are not ^{MOFO-357975700} 202412023840 detected, such that in some instances issues associated with optical crowding due to detecting signals associated with “Block A” and “Block B” RCA products simultaneously in the same sequencing cycles for the same biological sample can be addressed. In some embodiments, the plurality of dark cycles during sequencing of the “Block B” RCA products comprises N consecutive dark cycles, wherein N is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, and the lengths of the gap sequences in the “Block A” RCA products are no more than N, such that the gap sequences in the “Block A” RCA products are sequenced without interference of detectable signals associated with nucleotide incorporation and/or binding templated on “Block B” RCA products. [0189] In some embodiments, sequencing is performed by sequencing-by-avidity (SBA). Some aspects of SBA approaches are described in U.S. Pat. No. 10,768,173 B2, the content of which is herein incorporated by reference in its entirety. In some embodiments, SBA comprises detecting a multivalent binding complex formed between a fluorescently-labeled polymer-nucleotide conjugate, and a one or more primed target nucleic acid sequences (e.g., barcode sequences). Fluorescence imaging is used to detect the bound complex and thereby determine the identity of the N+1 nucleotide in the target nucleic acid sequence (where the primer extension strand is N nucleotides in length). Following the imaging step, the multivalent binding complex is disrupted and washed away, the correct blocked nucleotide is incorporated into the primer extension strand, and the sequencing cycle is repeated. [0190] In some examples where SBL is used to sequence the gap sequence in the RCA product, the biological sample is contacted with an anchor of known sequence and detectably labeled probes, one of which are complementary to a sequence in the gap sequence in the second RCA product. In some embodiments, the anchor is 3’ to the gap sequence to be sequenced. In some embodiments, the anchor is 5’ to the gap sequence to be sequenced. In some instances, after hybridization of the complementary detectably labeled probe to the gap sequence, it is ligated to the anchor or an extended product thereof, whereas detectably labeled probes that are not complementary to the gap sequence are not ligated and can be removed, e.g., by washing the sample. Signals associated with the complementary detectably labeled probe ligated to the anchor or extension product thereof can be detected, thereby detecting the corresponding sequence in the gap sequence. ^{MOFO-357975700} 202412023840 [0191] In some embodiments, provided herein is a method for analyzing a biological sample, comprising contacting the biological sample with a plurality of circularizable probes (e.g., second probes), wherein each circularizable probe comprises a first hybridization region and a second hybridization region that hybridize to a first sequence and a second sequence, respectively, in a target RNA in the biological sample, wherein the first and second sequences are separated by a gap sequence in the target RNA, extending the circularizable probe (e.g., second probe) using a sequence of the target nucleic acid as template to generate an extended probe comprising a gap filled sequence, ligating the extended probe to generate a circularized template thereby circularizing the circularizable probe to generate a circularized probe comprising a gap filled region complementary to the gap sequence in the target RNA. In some embodiments, a rolling circle amplification product (RCP) of the circularized probe is generated in the biological sample, wherein the RCP comprises multiple copies of the gap sequence that is detected by sequencing in the biological sample. C. Imaging and Readout [0192] In some aspects, provided herein are in situ assays using microscopy as a readout, e.g., hybridization and sequencing, or other detection or determination methods involving an optical readout. In some aspects, detection or determination of a sequence of one, two, three, four, five, or more nucleotides of a gap sequence in a target nucleic acid is performed in situ in a cell in an intact tissue. In some aspects, detection or determination of a sequence is performed such that the localization of a target nucleic acid (or product or a derivative thereof associated with the target nucleic acid) in the originating sample is detected. In some embodiments, the assay comprises detecting the presence or absence of a RCA product. In some embodiments, a provided method is quantitative and preserves the spatial information within a tissue sample without physically isolating cells or using homogenates^. In some embodiments, the present disclosure provides methods for high-throughput profiling of target nucleic acids in situ in a large number of cells, tissues, organs or organisms. [0193] In some aspects, the provided methods comprise imaging the RCA product via binding of an oligonucleotide probe and a detectably labeled probe (e.g., a detection oligonucleotide comprising a fluorescent label) or a labeled nucleotide, and detecting the detectable label. In some embodiments, a signal is detected, measured and quantitated. The terms ^{MOFO-357975700} 202412023840 "label" and "detectable label" comprise a directly or indirectly detectable moiety that is associated with (e.g., conjugated to) a molecule to be detected, comprising, but not limited to, fluorophores, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like. [0194] The term "fluorophore" comprises a substance or a portion thereof that is capable of exhibiting fluorescence in the detectable range. Particular examples of labels that may be used in accordance with the provided embodiments comprise, but are not limited to phycoerythrin, Alexa dyes, fluorescein, YPet, CyPet, Cascade blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas red, luminol, acradimum esters, biotin, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), firefly luciferase, Renilla luciferase, NADPH, beta-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenical acetyl transferase, and urease. [0195] Fluorescence detection in tissue samples can often be hindered by the presence of strong background fluorescence. “Autofluorescence” is the general term used to distinguish background fluorescence (that can arise from a variety of sources, including aldehyde fixation, extracellular matrix components, red blood cells, lipofuscin, and the like) from the desired immunofluorescence from the fluorescently labeled antibodies or probes. Tissue autofluorescence can lead to difficulties in distinguishing the signals due to fluorescent antibodies or probes from the general background. In some embodiments, a method disclosed herein utilizes one or more agents to reduce tissue autofluorescence, for example, Autofluorescence Eliminator (Sigma/EMD Millipore), TrueBlack Lipofuscin Autofluorescence Quencher (Biotium), MaxBlock Autofluorescence Reducing Reagent Kit (MaxVision Biosciences), and/or a very intense black dye (e.g., Sudan Black, or comparable dark chromophore). [0196] Examples of detectable labels comprise but are not limited to various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, protein-protein binding pairs and protein-antibody ^{MOFO-357975700} 202412023840 binding pairs. Examples of fluorescent proteins comprise, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin. [0197] Examples of bioluminescent markers comprise, but are not limited to, luciferase (e.g., bacterial, firefly and click beetle), luciferin, aequorin and the like. Examples of enzyme systems having visually detectable signals comprise, but are not limited to, galactosidases, glucorimidases, phosphatases, peroxidases and cholinesterases. Identifiable markers also comprise radioactive compounds such as ¹²⁵I, ³⁵S, ¹⁴C, or ³H. Identifiable markers are commercially available from a variety of sources. [0198] Examples of fluorescent labels and nucleotides and/or polynucleotides conjugated to such fluorescent labels comprise those described in, for example, Hoagland, Handbook of Fluorescent Probes and Research Chemicals, Ninth Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227- 259 (1991). In some embodiments, techniques and methods applicable to the provided embodiments comprise those described in, for example, US 4,757,141, US 5,151,507 and US 5,091,519. In some embodiments, one or more fluorescent dyes are used as labels for labeled target sequences, for example, as described in US 5,188,934 (4,7-dichlorofluorescein dyes); US 5,366,860 (spectrally resolvable rhodamine dyes); US 5,847,162 (4,7- dichlororhodamine dyes); US 4,318,846 (ether-substituted fluorescein dyes); US 5,800,996 (energy transfer dyes); US 5,066,580 (xanthine dyes); and US 5,688,648 (energy transfer dyes). Labelling can also be carried out with quantum dots, as described in US 6,322,901, US 6,576,291, US 6,423,551, US 6,251,303, US 6,319,426, US 6,426,513, US 6,444,143, US 5,990,479, US 6,207,392, US 2002/0045045 and US 2003/0017264. As used herein, the term "fluorescent label" comprises a signaling moiety that conveys information through the fluorescent absorption and/or emission properties of one or more molecules. Exemplary fluorescent properties comprise fluorescence intensity, fluorescence lifetime, emission spectrum characteristics and energy transfer. ^{MOFO-357975700} 202412023840 [0199] In some embodiments, one or more detectably labelled molecules are detected, e.g., fluorescent nucleotides or probes. [0200] Examples of commercially available fluorescent nucleotide analogues readily incorporated into nucleotide and/or polynucleotide sequences comprise, but are not limited to, Cy3™-dCTP (cyanine 3-dCTP), Cy3™-dUTP (cyanine 3-dUTP), Cy5™-dCTP (cyanine 5- dCTP), Cy5™-dUTP (cyanine 5 dUTP) (Amersham Biosciences, Piscataway, N.J.), fluorescein- 12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED®-5-dUTP (red fluorescent dye-dUTP), CASCADE® BLUE-7-dUTP (blue fluorescent dye – dUTP), BODIPY™ FL-14-dUTP (green fluorescent dye-dUTP), BODIPY™ TMR-14-dUTP (orange fluorescent dye-dUTP), BODIPY™ TR-14-dUTP (red fluorescent dye-dUTP), RHODAMINE GREEN™-5-dUTP (green fluorescent dye-dUTP), OREGON GREEN™ 488-5-dUTP (green fluorescent dye-dUTP), TEXAS RED™- l2-dUTP (red fluorescent dye-dUTP), BODIPY™ 630/650-14-dUTP (far red fluorescent dye- dUTP), BODIPY™ 650/665-14-dUTP (far red fluorescent dye-dUTP), ALEXA FLUOR™ 488- 5-dUTP (green fluorescent dye-dUTP), ALEXA FLUOR™ 532-5-dUTP (yellow fluorescent dye-dUTP), ALEXA FLUOR™ 568-5-dUTP (red/orange fluorescent dye-dUTP), ALEXA FLUOR™ 594-5-dUTP (red fluorescent dye-dUTP), ALEXA FLUOR™ 546-14-dUTP (orange fluorescent dye-dUTP), fluorescein- 12-UTP, tetramethylrhodamine-6-UTP, TEXAS RED™-5- UTP (red fluorescent dye-UTP), mCherry, CASCADE® BLUE-7-UTP (blue fluorescent dye- UTP), BODIPY™ FL-14-UTP (green fluorescent protein-UTP), BODIPY™ TMR- 14-UTP (orange fluorescent dye-UTP), BODIPY™ TR-14-UTP (red fluorescent dye-UTP), RHODAMINE GREEN™-5-UTP (green fluorescent dye-UTP), ALEXA FLUOR™ 488-5-UTP (green fluorescent dye-UTP), and ALEXA FLUOR™ 546-14-UTP (orange fluorescent dye- UTP) (Molecular Probes, Inc. Eugene, Oreg.). Methods are known for custom synthesis of nucleotides having other fluorophores. [0201] Other fluorophores available for post-synthetic attachment comprise, but are not limited to, ALEXA FLUOR™ dyes (fluorescent dyes) such as ALEXA FLUOR™ 350 (blue fluorescent dye), ALEXA FLUOR™ 594 (red fluorescent dye), and ALEXA FLUOR™ 647 (far red fluorescent dye); BODIPY™ dyes (fluorescent dyes) such as BODIPY™ FL (green fluorescent dye), BODIPY™ TMR (orange fluorescent dye), and BODIPY™ 650/665 (far red fluorescent dye); Cascade® Blue (blue fluorescent dye), Cascade® Yellow (yellow fluorescent ^{MOFO-357975700} 202412023840 dye), Dansyl, lissamine rhodamine B, Marina Blue™ (blue fluorescent dye), Oregon Green™ 488, Oregon Green™ 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethyl rhodamine, Texas Red® (red fluorescent dye) (available from Molecular Probes, Inc., Eugene, Oreg.), Cy2™ (cyanine 2), Cy3.5™ (cyanine 3.5), Cy5.5™ (cyanine 5.5), and Cy7™ (cyanine 7) (Amersham Biosciences, Piscataway, N.J.). FRET tandem fluorophores may also be used, comprising, but not limited to, PerCP-Cy™5.5 (far red fluorescent tandem fluorophore), PE-Cy™5 (red fluorescent tandem fluorophore), PE-Cy™5.5 (red fluorescent tandem fluorophore), PE-Cy™7 (far red fluorescent tandem fluorophore), PE-Texas Red® (red fluorescent tandem fluorophore), APC-Cy™7 (far red fluorescent tandem fluorophore), PE- Alexa™ dyes (e.g., 610, 647, 680), and APC-Alexa™ dyes. [0202] In some cases, metallic silver or gold particles may be used to enhance signal from fluorescently labeled nucleotide and/or polynucleotide sequences (Lakowicz et al. (2003) Bio Techniques 34:62). [0203] Biotin, or a derivative thereof, may also be used as a label on a nucleotide and/or a polynucleotide sequence, and subsequently bound by a detectably labeled avidin/streptavidin derivative (e.g., phycoerythrin-conjugated streptavidin), or a detectably labeled anti-biotin antibody. Digoxigenin may be incorporated as a label and subsequently bound by a detectably labeled anti-digoxigenin antibody (e.g., fluoresceinated anti-digoxigenin). An aminoallyl-dUTP residue may be incorporated into a polynucleotide sequence and subsequently coupled to an N-hydroxy succinimide (NHS) derivatized fluorescent dye. In general, any member of a conjugate pair may be incorporated into a detection polynucleotide provided that a detectably labeled conjugate partner can be bound to permit detection. In some embodiments, the term antibody refers to an antibody molecule of any class, or any sub-fragment thereof, such as a Fab. [0204] Other suitable labels for a polynucleotide sequence may comprise fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), and phosphor-amino acids (e.g., P-tyr, P-ser, P-thr). In some embodiments the following hapten/antibody pairs are used for detection, in which each of the antibodies is derivatized with a detectable label: biotin/a-biotin, digoxigenin/a-digoxigenin, dinitrophenol (DNP)/a-DNP, 5-Carboxyfluorescein (FAM)/a-FAM. ^{MOFO-357975700} 202412023840 [0205] In some embodiments, a nucleotide and/or an polynucleotide sequence can be indirectly labeled, especially with a hapten that is then bound by a capture agent, e.g., as disclosed in US 5,344,757, US 5,702,888, US 5,354,657, US 5,198,537 and US 4,849,336, and PCT publication WO 91/17160. Many different hapten-capture agent pairs are available for use. Exemplary haptens comprise, but are not limited to, biotin, des-biotin and other derivatives, dinitrophenol, dansyl, fluorescein, Cy5, and digoxigenin. For biotin, a capture agent may be avidin, streptavidin, or antibodies. Antibodies may be used as capture agents for the other haptens (many dye-antibody pairs being commercially available, e.g., Molecular Probes, Eugene, Oreg.). [0206] In some aspects, the detecting involves using detection methods such as flow cytometry; sequencing; probe binding and electrochemical detection; pH alteration; catalysis induced by enzymes bound to DNA tags; quantum entanglement; Raman spectroscopy; terahertz wave technology; and/or scanning electron microscopy. In some aspects, the flow cytometry is mass cytometry or fluorescence-activated flow cytometry. In some aspects, the detecting comprises performing microscopy, scanning mass spectrometry or other imaging techniques described herein. In such aspects, the detecting comprises determining a signal, e.g., a fluorescent signal. [0207] In some aspects, the detection (comprising imaging) is carried out using any of a number of different types of microscopy, e.g., confocal microscopy, two-photon microscopy, light-field microscopy, intact tissue expansion microscopy, and/or CLARITY™- optimized light sheet microscopy (COLM). [0208] In some embodiments, fluorescence microscopy is used for detection and imaging. In some aspects, a fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances. In fluorescence microscopy, a sample is illuminated with light of a wavelength which excites fluorescence in the sample. The fluoresced light, which is usually at a longer wavelength than the illumination, is then imaged through a microscope objective. Two filters may be used in this technique; an illumination (or excitation) filter which ensures the illumination is near monochromatic and at the correct wavelength, and a second emission (or barrier) filter which ensures none of the excitation light source reaches the ^{MOFO-357975700} 202412023840 detector. Alternatively, these functions may both be accomplished by a single dichroic filter. The "fluorescence microscope" comprises any microscope that uses fluorescence to generate an image, whether it is a more simple set up like an epifluorescence microscope, or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescent image. [0209] In some embodiments, confocal microscopy is used for detection and imaging. Confocal microscopy uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal. As only light produced by fluorescence very close to the focal plane can be detected, the image's optical resolution, particularly in the sample depth direction, is much better than that of wide-field microscopes. However, as much of the light from sample fluorescence is blocked at the pinhole, this increased resolution is at the cost of decreased signal intensity - so long exposures are often required. As only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster (i.e., a rectangular pattern of parallel scanning lines) in the specimen. The achievable thickness of the focal plane is defined mostly by the wavelength of the used light divided by the numerical aperture of the objective lens, but also by the optical properties of the specimen. The thin optical sectioning possible makes these types of microscopes particularly good at 3D imaging and surface profiling of samples. CLARITY™-optimized light sheet microscopy (COLM) provides an alternative microscopy for fast 3D imaging of large clarified samples. COLM interrogates large immunostained tissues, permits increased speed of acquisition and results in a higher quality of generated data. [0210] Other types of microscopy that can be employed comprise bright field microscopy, oblique illumination microscopy, dark field microscopy, phase contrast, differential interference contrast (DIC) microscopy, interference reflection microscopy (also known as reflected interference contrast, or RIC), single plane illumination microscopy (SPIM), super- resolution microscopy, laser microscopy, electron microscopy (EM), Transmission electron microscopy (TEM), Scanning electron microscopy (SEM), reflection electron microscopy (REM), Scanning transmission electron microscopy (STEM) and low- voltage electron microscopy (LVEM), scanning probe microscopy (SPM), atomic force microscopy (ATM), ballistic electron emission microscopy (BEEM), chemical force microscopy (CFM), conductive ^{MOFO-357975700} 202412023840 atomic force microscopy (C- AFM), electrochemical scanning tunneling microscope (ECSTM), electrostatic force microscopy (EFM), fluidic force microscope (FluidFM), force modulation microscopy (FMM), feature-oriented scanning probe microscopy (FOSPM), kelvin probe force microscopy (KPFM), magnetic force microscopy (MFM), magnetic resonance force microscopy (MRFM), near-field scanning optical microscopy (NSOM) (or SNOM, scanning near-field optical microscopy, SNOM, Piezoresponse Force Microscopy (PFM), PSTM, photon scanning tunneling microscopy (PSTM), PTMS, photothermal microspectroscopy/ microscopy (PTMS), SCM, scanning capacitance microscopy (SCM), SECM, scanning electrochemical microscopy (SECM), SGM, scanning gate microscopy (SGM), SHPM, scanning Hall probe microscopy (SHPM), SICM, scanning ion-conductance microscopy (SICM), SPSM spin polarized scanning tunneling microscopy (SPSM), SSRM, scanning spreading resistance microscopy (SSRM), SThM, scanning thermal microscopy (SThM), STM, scanning tunneling microscopy (STM), STP, scanning tunneling potentiometry (STP), SVM, scanning voltage microscopy (SVM), and synchrotron x-ray scanning tunneling microscopy (SXSTM), and intact tissue expansion microscopy (exM). [0211] In some aspects, the provided methods involve analyzing, e.g., detecting or determining, one or more sequences present in the probes or products thereof (e.g RCA product thereof). In some embodiments, the detecting is performed at one or more locations in the biological sample. In some embodiments, the locations are the locations of RNA transcripts in the biological sample. In some embodiments, the locations are the locations at which the probes hybridize to the RNA transcripts in the biological sample, and are optionally ligated and amplified by rolling circle amplification. IV. SAMPLES [0212] A sample disclosed herein can be or derived from any biological sample. Methods and compositions disclosed herein may be used for analyzing a biological sample, which may be obtained from a subject using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and/or other biological material from the subject. In addition to the subjects described above, a biological sample can be obtained from a prokaryote such as a bacterium, an archaea, a virus, or ^{MOFO-357975700} 202412023840 a viroid. A biological sample can also be obtained from non-mammalian organisms (e.g., a plant, an insect, an arachnid, a nematode, a fungus, or an amphibian). A biological sample can also be obtained from a eukaryote, such as a tissue sample, a patient derived organoid (PDO) or patient derived xenograft (PDX). A biological sample from an organism may comprise one or more other organisms or components therefrom. For example, a mammalian tissue section may comprise a prion, a viroid, a virus, a bacterium, a fungus, or components from other organisms, in addition to mammalian cells and non-cellular tissue components. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., a patient with a disease such as cancer) or a pre- disposition to a disease, and/or individuals in need of therapy or suspected of needing therapy. [0213] The biological sample can include any number of macromolecules, for example, cellular macromolecules and organelles (e.g., mitochondria and nuclei). In some instances, the biological sample comprises nucleic acids (such as DNA or RNA), proteins/polypeptides, carbohydrates, and/or lipids. In some embodiments, the biological sample is obtained as a tissue sample, such as a tissue section, biopsy, a core biopsy, needle aspirate, or fine needle aspirate. In some embodiments, the biological sample is or comprise a cell pellet or a section of a cell pellet. In some embodiments, the biological sample is or comprise a cell block or a section of a cell block. The sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histology sample, a histopathology sample, a plasma or serum sample, a tumor sample, living cells, cultured cells, a clinical sample such as, for example, whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions. In some embodiments, the biological sample comprises cells which are deposited on a surface. [0214] Biological samples can be derived from a homogeneous culture or population of the subjects or organisms mentioned herein or alternatively from a collection of several different organisms. Biological samples can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and/or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells can be derived from solid tumors, hematological ^{MOFO-357975700} 202412023840 malignancies, cell lines, or obtained as circulating tumor cells. Biological samples can also include fetal cells and immune cells. [0215] In some embodiments, a substrate herein can be any support that is insoluble in aqueous liquid and which allows for positioning of biological samples, analytes, features, and/or reagents (e.g., probes) on the support. In some embodiments, a biological sample is attached to a substrate. Attachment of the biological sample can be irreversible or reversible, depending upon the nature of the sample and subsequent steps in the analytical method. In certain embodiments, the sample is attached to the substrate reversibly by applying a suitable polymer coating to the substrate, and contacting the sample to the polymer coating. The sample can then be detached from the substrate, e.g., using an organic solvent that at least partially dissolves the polymer coating. Hydrogels are examples of polymers that are suitable for this purpose. In some embodiments, the substrate is coated or functionalized with one or more substances to facilitate attachment of the sample to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, poly-lysine, antibodies, and polysaccharides. [0216] A variety of steps can be performed to prepare or process a biological sample for and/or during an assay. Except where indicated otherwise, the preparative or processing steps described below can generally be combined in any manner and in any order to appropriately prepare or process a particular sample for and/or analysis. (i) Preparation [0217] A biological sample can be harvested from a subject (e.g., via surgical biopsy, whole subject sectioning) or grown in vitro on a growth substrate or culture dish as a population of cells, and prepared for analysis as a tissue slice or tissue section. Grown samples may be sufficiently thin for analysis without further processing steps. Alternatively, grown samples, and samples obtained via biopsy or sectioning, can be prepared as thin tissue sections using a mechanical cutting apparatus such as a vibrating blade microtome. As another alternative, in some embodiments, a thin tissue section can be prepared by applying a touch imprint of a biological sample to a suitable substrate material. [0218] The thickness of the tissue section can be a fraction of (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) the maximum cross-sectional dimension of a cell. However, ^{MOFO-357975700} 202412023840 tissue sections having a thickness that is larger than the maximum cross-section cell dimension can also be used. For example, cryostat sections can be used, which can be, e.g., 10-20 µm thick. More generally, the thickness of a tissue section typically depends on the method used to prepare the section and the physical characteristics of the tissue, and therefore sections having a wide variety of different thicknesses can be prepared and used. For example, the thickness of the tissue section can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40, or 50 µm. Thicker sections can also be used if desired or convenient, e.g., at least 70, 80, 90, or 100 µm or more. Typically, the thickness of a tissue section is between 1-100 µm, 1-50 µm, 1-30 µm, 1-25 µm, 1-20 µm, 1-15 µm, 1-10 µm, 2-8 µm, 3-7 µm, or 4-6 µm, but as mentioned above, sections with thicknesses larger or smaller than these ranges can also be analyzed. [0219] In some instances, multiple sections are obtained from a single biological sample. For example, multiple tissue sections can be obtained from a surgical biopsy sample by performing serial sectioning of the biopsy sample using a sectioning blade. Spatial information among the serial sections can be preserved in this manner, and the sections can be analyzed successively to obtain three-dimensional information about the biological sample. [0220] In some embodiments, the biological sample (e.g., a tissue section as described above) is prepared by deep freezing at a temperature suitable to maintain or preserve the integrity (e.g., the physical characteristics) of the tissue structure. The frozen tissue sample can be sectioned, e.g., thinly sliced, onto a substrate surface using any number of suitable methods. For example, a tissue sample can be prepared using a chilled microtome (e.g., a cryostat) set at a temperature suitable to maintain both the structural integrity of the tissue sample and the chemical properties of the nucleic acids in the sample. Such a temperature can be, e.g., less than -15°C, less than -20°C, or less than -25°C. [0221] In some embodiments, the biological sample is prepared using formalin- fixation and paraffin-embedding (FFPE), which are established methods. In some embodiments, cell suspensions and other non-tissue samples can be prepared using formalin-fixation and paraffin-embedding. Following fixation of the sample and embedding in a paraffin or resin block, the sample can be sectioned as described above. Prior to analysis, the paraffin-embedding material can be removed from the tissue section (e.g., deparaffinization) by incubating the tissue ^{MOFO-357975700} 202412023840 section in an appropriate solvent (e.g., xylene) followed by a rinse (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes). In some embodiments, the biological sample (e.g., FFPE sample) is permeable after deparaffinization. In some embodiments, processing of the biological sample, such as de-waxing, allows the biological sample to become permeabilized. [0222] As an alternative to formalin fixation described above, a biological sample can be fixed in any of a variety of other fixatives to preserve the biological structure of the sample prior to analysis. For example, a sample can be fixed via immersion in ethanol, methanol, acetone, paraformaldehyde (PFA)-Triton, and combinations thereof. [0223] In some embodiments, the methods provided herein comprises one or more post-fixing (also referred to as postfixation) steps. In some embodiments, one or more post- fixing step is performed after contacting a sample with a polynucleotide disclosed herein, e.g., one or more probes such as a circular or padlock probe. In some embodiments, one or more post-fixing step is performed after a hybridization complex comprising a probe and a target is formed in a sample. In some embodiments, one or more post-fixing step is performed prior to a ligation reaction disclosed herein. [0224] In some embodiments, a method disclosed herein comprises de-crosslinking the reversibly cross-linked biological sample. The de-crosslinking does not need to be complete. In some embodiments, only a portion of crosslinked molecules in the reversibly cross-linked biological sample are de-crosslinked and allowed to migrate. [0225] In some embodiments, a biological sample is permeabilized to facilitate transfer of species (such as probes) into the sample. If a sample is not permeabilized sufficiently, the transfer of species (such as probes) into the sample may be too low to enable adequate analysis. Conversely, if the tissue sample is too permeable, the relative spatial relationship of the analytes within the tissue sample can be lost. Hence, a balance between permeabilizing the tissue sample enough to obtain good signal intensity while still maintaining the spatial resolution of the analyte distribution in the sample is desirable. [0226] In general, a biological sample can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., ^{MOFO-357975700} 202412023840 paraformaldehyde), detergents (e.g., saponin, Triton X-100™ or Tween-20™), and enzymes (e.g., trypsin, proteases). In some embodiments, the biological sample is incubated with a cellular permeabilizing agent to facilitate permeabilization of the sample. Additional methods for sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol. 588:63- 66, 2010, the entire contents of which are incorporated herein by reference. Any suitable method for sample permeabilization can generally be used in connection with the samples described herein. [0227] In some embodiments, the biological sample can be permeabilized by any suitable methods. In some embodiments, the biological sample is a permeable biological sample. For example, one or more lysis reagents can be added to the sample. Examples of suitable lysis agents include, but are not limited to, bioactive reagents such as lysis enzymes that are used for lysis of different cell types, e.g., gram positive or negative bacteria, plants, yeast, mammalian, such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and a variety of other commercially available lysis enzymes. Other lysis agents can additionally or alternatively be added to the biological sample to facilitate permeabilization. For example, surfactant-based lysis solutions can be used to lyse sample cells. Lysis solutions can include ionic surfactants such as, for example, sarcosyl and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents can include, without limitation, organic solvents, chelating agents, detergents, surfactants, and chaotropic agents. [0228] Additional reagents can be added to a biological sample to perform various functions prior to analysis of the sample. In some embodiments, DNase and RNase inactivating agents or inhibitors such as proteinase K, and/or chelating agents such as EDTA, is added to the sample. For example, a method disclosed herein may comprise a step for increasing accessibility of a nucleic acid for binding, e.g., a denaturation step to open up DNA in a cell for hybridization by a probe. For example, proteinase K treatment may be used to free up DNA with proteins bound thereto. (ii) Embedding [0229] In some embodiments, the biological sample is embedded in a matrix (e.g., a hydrogel matrix). Embedding the sample in this manner typically involves contacting the biological sample with a hydrogel such that the biological sample becomes surrounded by the ^{MOFO-357975700} 202412023840 hydrogel. For example, the sample can be embedded by contacting the sample with a suitable polymer material, and activating the polymer material to form a hydrogel. In some embodiments, the hydrogel is formed such that the hydrogel is internalized within the biological sample. Biological samples can include analytes (e.g., protein, RNA, and/or DNA) embedded in a 3D matrix. In some embodiments, amplicons (e.g., rolling circle amplification products) derived from or associated with analytes (e.g., protein, RNA, and/or DNA) can be embedded in a 3D matrix. In some embodiments, a 3D matrix may comprise a network of natural molecules and/or synthetic molecules that are chemically and/or enzymatically linked, e.g., by crosslinking. In some embodiments, a 3D matrix may comprise a synthetic polymer. In some embodiments, a 3D matrix comprises a hydrogel. [0230] In some aspects, a biological sample can be embedded in any of a variety of other embedding materials to provide structural substrate to the sample prior to sectioning and other handling steps. In some cases, the embedding material is removed e.g., prior to analysis of tissue sections obtained from the sample. Suitable embedding materials include, but are not limited to, waxes, resins (e.g., methacrylate resins), epoxies, and agar. [0231] In some embodiments, the biological sample is embedded in a matrix (e.g., a hydrogel matrix). Embedding the sample in this manner typically involves contacting the biological sample with a hydrogel such that the biological sample becomes surrounded by the hydrogel. For example, the sample can be embedded by contacting the sample with a suitable polymer material, and activating the polymer material to form a hydrogel. In some embodiments, the hydrogel is formed such that the hydrogel is internalized within the biological sample. [0232] In some embodiments, the biological sample is immobilized in the hydrogel via cross-linking of the polymer material that forms the hydrogel. Cross-linking can be performed chemically and/or photochemically, or alternatively by any other suitable hydrogel- formation method. [0233] In some embodiments, the biological sample is reversibly cross-linked prior to or during an in situ assay. In some aspects, the analytes, polynucleotides and/or amplification product (e.g., amplicon) of an analyte or a probe bound thereto can be anchored to a polymer matrix. For example, the polymer matrix can be a hydrogel. In some embodiments, one or more of the polynucleotide probe(s) and/or amplification product (e.g., amplicon) thereof can be ^{MOFO-357975700} 202412023840 modified to contain functional groups that can be used as an anchoring site to attach the polynucleotide probes and/or amplification product to a polymer matrix. In some embodiments, a modified probe comprising oligo dT may be used to bind to mRNA molecules of interest, followed by reversible or irreversible crosslinking of the mRNA molecules. [0234] In some embodiments, the biological sample is immobilized in a hydrogel via cross-linking of the polymer material that forms the hydrogel. Cross-linking can be performed chemically and/or photochemically, or alternatively by any other suitable hydrogel-formation method. A hydrogel may include a macromolecular polymer gel including a network. Within the network, some polymer chains can optionally be cross-linked, although cross-linking does not always occur. [0235] In some embodiments, a hydrogel can include hydrogel subunits, such as, but not limited to, acrylamide, bis-acrylamide, polyacrylamide and derivatives thereof, poly(ethylene glycol) and derivatives thereof (e.g. PEG-acrylate (PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), and poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, and the like, and combinations thereof. [0236] In some embodiments, a hydrogel includes a hybrid material, e.g., the hydrogel material includes elements of both synthetic and natural polymers. Examples of suitable hydrogels are described, for example, in U.S. Patent Nos. 6,391,937, 9,512,422, and 9,889,422, and in U.S. Patent Application Publication Nos. 2017/0253918, 2018/0052081 and 2010/0055733, the entire contents of each of which are incorporated herein by reference. [0237] The composition and application of the hydrogel-matrix to a biological sample typically depends on the nature and preparation of the biological sample (e.g., sectioned, non-sectioned, type of fixation). As one example, where the biological sample is a tissue section, the hydrogel-matrix can include a monomer solution and an ammonium persulfate (APS) initiator/tetramethylethylenediamine (TEMED) accelerator solution. As another example, where the biological sample consists of cells (e.g., cultured cells or cells disassociated from a tissue ^{MOFO-357975700} 202412023840 sample), the cells can be incubated with the monomer solution and APS/TEMED solutions. For cells, hydrogel-matrix gels are formed in compartments, including but not limited to devices used to culture, maintain, or transport the cells. For example, hydrogel-matrices can be formed with monomer solution plus APS/TEMED added to the compartment to a depth ranging from about 0.1 μm to about 2 mm. [0238] Additional methods and aspects of hydrogel embedding of biological samples are described for example in Chen et al., Science 347(6221):543–548, 2015, the entire contents of which are incorporated herein by reference. [0239] In some embodiments, the hydrogel can form the substrate. In some embodiments, the substrate includes a hydrogel and one or more second materials. In some embodiments, the hydrogel is placed on top of one or more second materials. For example, the hydrogel can be pre-formed and then placed on top of, underneath, or in any other configuration with one or more second materials. In some embodiments, hydrogel formation occurs after contacting one or more second materials during formation of the substrate. Hydrogel formation can also occur within a structure (e.g., wells, ridges, projections, and/or markings) located on a substrate. [0240] In some embodiments, hydrogel formation on a substrate occurs before, contemporaneously with, or after probes are provided to the sample. For example, hydrogel formation can be performed on the substrate already containing the probes. [0241] In some embodiments, hydrogel formation occurs within a biological sample. In some embodiments, a biological sample (e.g., tissue section) is embedded in a hydrogel. In some embodiments, hydrogel subunits are infused into the biological sample, and polymerization of the hydrogel is initiated by an external or internal stimulus. [0242] In embodiments in which a hydrogel is formed within a biological sample, functionalization chemistry can be used. In some embodiments, functionalization chemistry includes hydrogel-tissue chemistry (HTC). Any hydrogel-tissue backbone (e.g., synthetic or native) suitable for HTC can be used for anchoring biological macromolecules and modulating functionalization. Non-limiting examples of methods using HTC backbone variants include CLARITY, PACT, ExM, SWITCH and ePACT. In some embodiments, hydrogel formation within a biological sample is permanent. For example, biological macromolecules can ^{MOFO-357975700} 202412023840 permanently adhere to the hydrogel allowing multiple rounds of interrogation. In some embodiments, hydrogel formation within a biological sample is reversible. In some embodiments, HTC reagents are added to the hydrogel before, contemporaneously with, and/or after polymerization. In some embodiments, a cell labeling agent is added to the hydrogel before, contemporaneously with, and/or after polymerization. In some embodiments, a cell-penetrating agent is added to the hydrogel before, contemporaneously with, and/or after polymerization. [0243] In some embodiments, additional reagents are added to the hydrogel subunits before, contemporaneously with, and/or after polymerization. For example, additional reagents can include but are not limited to oligonucleotides (e.g., probes), endonucleases to fragment DNA, fragmentation buffer for DNA, DNA polymerase enzymes, dNTPs used to amplify the nucleic acid and to attach the barcode to the amplified fragments. Other enzymes can be used, including without limitation, RNA polymerase, ligase, proteinase K, and DNAse. Additional reagents can also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers, and oligonucleotides. In some embodiments, optical labels are added to the hydrogel subunits before, contemporaneously with, and/or after polymerization. [0244] Hydrogels embedded within biological samples can be cleared using any suitable method. For example, electrophoretic tissue clearing methods can be used to remove biological macromolecules from the hydrogel-embedded sample. In some embodiments, a hydrogel-embedded sample is stored before or after clearing of hydrogel, in a medium (e.g., a mounting medium, methylcellulose, or other semi-solid mediums). [0245] In some embodiments, a biological sample embedded in a matrix (e.g., a hydrogel) is isometrically expanded. Isometric expansion methods that can be used include hydration, a preparative step in expansion microscopy, as described in, e.g., Chen et al., Science 347(6221):543–548, 2015 and U.S. Pat. 10,059,990, which are herein incorporated by reference in their entireties. Isometric expansion of the sample can increase the spatial resolution of the subsequent analysis of the sample. The increased resolution in spatial profiling can be determined by comparison of an isometrically expanded sample with a sample that has not been isometrically expanded. In some embodiments, a biological sample is isometrically expanded to a size at least 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9x its non- ^{MOFO-357975700} 202412023840 expanded size. In some embodiments, the sample is isometrically expanded to at least 2x and less than 20x of its non-expanded size. (iii) Staining and Immunohistochemistry (IHC) [0246] To facilitate visualization, biological samples can be stained using a wide variety of stains and staining techniques. In some embodiments, for example, a sample can be stained using any number of stains and/or immunohistochemical reagents. In some embodiments, one or more staining steps are performed to prepare or process a biological sample for an assay described herein or may be performed during and/or after an assay. In some embodiments, the sample is contacted with one or more nucleic acid stains, membrane stains (e.g., cellular or nuclear membrane), cytological stains, or combinations thereof. In some examples, the stain may be specific to proteins, phospholipids, DNA (e.g., dsDNA, ssDNA), RNA, an organelle or compartment of the cell. The sample may be contacted with one or more labeled antibodies (e.g., a primary antibody specific for the analyte of interest and a labeled secondary antibody specific for the primary antibody). In some embodiments, cells in the sample is segmented using one or more images taken of the stained sample. [0247] In some embodiments, the stain is performed using a lipophilic dye. In some examples, the staining is performed with a lipophilic carbocyanine or aminostyryl dye, or analogs thereof (e.g, DiI, DiO, DiR, DiD). Other cell membrane stains may include FM and RH dyes or immunohistochemical reagents specific for cell membrane proteins. In some examples, the stain may include but is not limited to, acridine orange, acid fuchsin, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, haematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, ruthenium red, propidium iodide, rhodamine (e.g., rhodamine B), or safranine, or derivatives thereof. In some embodiments, the sample may be stained with haematoxylin and eosin (H&E). [0248] The sample can be stained using hematoxylin and eosin (H&E) staining techniques, using Papanicolaou staining techniques, Masson’s trichrome staining techniques, silver staining techniques, Sudan staining techniques, and/or using Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation. In ^{MOFO-357975700} 202412023840 some embodiments, the sample can be stained using Romanowsky stain, including Wright’s stain, Jenner’s stain, Can-Grunwald stain, Leishman stain, and Giemsa stain. [0249] In some embodiments, biological samples is destained. Any suitable methods of destaining or discoloring a biological sample may be utilized and generally depend on the nature of the stain(s) applied to the sample. For example, in some embodiments, one or more immunofluorescent stains are applied to the sample via antibody coupling. Such stains can be removed using techniques such as cleavage of disulfide linkages via treatment with a reducing agent and detergent washing, chaotropic salt treatment, treatment with antigen retrieval solution, and treatment with an acidic glycine buffer. Methods for multiplexed staining and destaining are described, for example, in Bolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin et al., Nat Commun. 2015; 6:8390, Pirici et al., J. Histochem. Cytochem. 2009; 57:567–75, and Glass et al., J. Histochem. Cytochem. 2009; 57:899–905, the entire contents of each of which are incorporated herein by reference. V. COMPOSITIONS, SYSTEMS, AND KITS [0250] In some aspects, provided herein are compositions and kits comprising any of the probes (e.g., first and second probes) and reagents for performing a ligation reaction, reagents for performing a gap fill reaction, reagents for amplification (e.g., of circularized templates), and reagents for detection of generated RCA products described herein. Also provided herein are systems for analyzing an analyte in a biological sample according to any of the methods described herein. In some embodiments, provided herein is a system comprising a plurality of circularizable probes, wherein the plurality of circularizable probes comprises a first probe that binds to a first target nucleic acid and a second probe that binds a second target nucleic acid, wherein the first probe comprises a 5’ end and a 3’ end; one or more reagents for gap filling the second probe to generate an extended probe comprising a gap filled sequence; one or more reagents for circularizing the plurality of circularizable probes and extended probe to generate a plurality of circularized templates; one or more reagents for generating a plurality of rolling circle amplification products (RCPs) of the plurality of circularized template; a plurality of oligonucleotide probes for detecting a first portion of the generated RCPs; and one or more reagents for sequencing a second portion of the generated RCPs. In some embodiments, a system ^{MOFO-357975700} 202412023840 provided herein comprises a panel of first probes, wherein each first probe of the panel of first probes each binds to a first target nucleic acid, and wherein the panel of first probes comprises a plurality of different first probes that bind to a plurality of different first target nucleic acids. In some embodiment, the plurality of different first probes that bind to a plurality of different first target nucleic acids each comprise a different barcode that corresponds to each first target nucleic acid. The various components of the system may be present in separate containers or certain compatible components may be pre-combined into a single container. In some embodiments, the system or kit further contains instructions for using the components to practice the provided methods. [0251] In some embodiments, the system comprises reagents and/or consumables required for performing one or more steps of the provided methods. In some embodiments, the system comprises reagents for fixing, embedding, and/or permeabilizing the biological sample. In some embodiments, the system comprises reagents, such as enzymes and buffers for ligation and/or amplification, such as ligases and/or polymerases. In some aspects, the system comprises any of the reagents described herein, e.g., wash buffer and ligation buffer. In some embodiments, the system comprises reagents for detection by hybridization of probes and sequencing, such as barcode detection probes and labeled nucleotides. In some embodiments, the system comprises optionally other components, for example nucleic acid primers for sequencing. [0252] In some embodiments, the system comprises a solid support having a biological sample attached thereto, wherein the biological sample comprises the first target nucleic acid and the second target nucleic acid. In some instances, the biological sample is a cell or tissue sample comprising cells or cellular components. In some instances, the biological sample is a tissue section. In some instances, the biological sample is crosslinked and/or embedded in a matrix. In some instances, the matrix comprises a hydrogel. In some instances, the biological sample comprises different molecules of the second target nucleic acid each comprising a mutant sequence of a plurality of different mutant sequences. In some instances, the second target nucleic acid comprises a sequence of a perturbation agent introduced to the biological sample. [0253] Provided herein are systems and kits, for example comprising one or more oligonucleotides, e.g., any described in Sections I-IV, and instructions for performing the ^{MOFO-357975700} 202412023840 methods provided herein. In some embodiments, the system or kit comprises one or more reagents for performing the methods provided herein. In some embodiments, the system or kit comprises one or more reagents required for one or more steps comprising hybridization, ligation, extension, amplification, detection, and/or sample preparation, e.g., as described in Sections II-IV. In some embodiments, the system or kit comprises any one or more of the oligonucleotide probes and detectably labeled oligonucleotides disclosed herein, e.g., as described in Section III. In some embodiments, any or all of the oligonucleotides are DNA molecules. In some embodiments, the system comprises an enzyme such as a ligase and/or a polymerase described herein. In some embodiments, the system comprises a polymerase, for instance configured to perform extension in a sequencing reaction. In some embodiments, the system comprises reagents for forming a functionalized matrix (e.g., a hydrogel), such as any suitable functional moieties. In some examples, also provided are buffers and reagents for tethering RCA products to a matrix (e.g., a hydrogel). The various components of the kit may be present in separate containers or certain compatible components may be pre-combined into a single container. In some embodiments, the system comprises instructions for using the components to practice the provided methods. [0254] In some embodiments, provided herein is a system for analyzing a biological sample, comprising: (a) a circularizable probe comprising a first hybridization region and a second hybridization region that hybridize to a first sequence and a second sequence, respectively, in a target nucleic acid (e.g., RNA) in the biological sample, wherein the first and second sequences are separated by a gap sequence in the target nucleic acid (e.g., RNA), and the gap sequence comprises a variant sequence among a plurality of different variant sequences; (b) one or more reagents for circularizing the extended probe to generate a circularized template comprising a gap filled region complementary to the gap sequence; and/or (c) one or more reagents for generating a rolling circle amplification product (RCP) of the circularized template, wherein the RCP comprises multiple copies of the gap sequence. In some instances, the one or more reagents for circularizing the plurality of circularizable probes comprises a ligase. [0255] In some embodiments, the system comprises an engineered family B polymerase and dNTPs. In some instances, the engineered family B polymerase is an engineered Tgo polymerase comprising one or more mutations that confer reverse transcriptase activity. ^{MOFO-357975700} 202412023840 [0256] In some embodiments, a system disclosed herein comprises a plurality of oligonucleotide probes. In some embodiments, a system disclosed herein comprises a pool of detectably labeled probes each comprising a detectable label. [0257] In some embodiments, a system disclosed herein comprises a library of splint oligonucleotides, wherein each splint oligonucleotide comprises: (i) ligatable ends; and (ii) a hybridization region complementary to one of the plurality of different variant sequences, wherein a splint oligonucleotide of the library of splint oligonucleotides that is complementary to the gap sequence is ligated to the circularizable probe (e.g., second probe), thereby circularizing the circularizable probe to generate a circularized probe comprising a gap filled region complementary to the gap sequence. In some embodiments, each splint oligonucleotide of the library comprises a phosphate group on the 5’-end available for ligation. [0258] In some embodiments, a system disclosed herein comprises reagents for detecting a sequence comprising the gap sequence (e.g., variant sequence) in the RCP at a location in the biological sample, thereby detecting the target RNA comprising the variant sequence at the location in the biological sample. In some embodiments, a system disclosed herein comprises reagents for base-by-base sequencing of the sequence comprising the variant sequence, and the base-by-base sequencing may comprise determining the identity of one, two, three, or more bases per cycle in sequential sequencing cycles. In some embodiments, a system disclosed herein comprises a plurality of oligonucleotide probes for detecting a sequence in the RCP. In some embodiments, a system disclosed herein comprises reagents for detecting signals associated with the oligonucleotide probes in sequential probe hybridization cycles. In some embodiments, the system comprises an optical detection system configured to detect a barcode sequence associated with a first probe and a gap filled sequence associated with a second probe. In some embodiments, the system comprises an optical detection system configured to detect the generated RCA products (e.g., as described in Section VI). [0259] In some aspects, the system comprises a plurality of oligonucleotide probes for detecting a first subset of generated RCPs and one or more reagents for sequencing a second subset of the generated RCPs. For example, each oligonucleotide probe comprises a hybridization region complementary to a sequence in a first RCA product of the first subset of the generated RCPs and a detectable region. In some instances, a plurality detectably labeled ^{MOFO-357975700} 202412023840 probes each comprises a detectable label and a sequence for hybridizing to a sequence in the first subset of the generated RCPs. In some cases, the one or more reagents for sequencing (e.g., as described in Section III.B) a second subset of the generated RCPs comprises a plurality of sequencing primers, a plurality of detectably labeled nucleotides, and a polymerase. [0260] In some embodiments, the system comprises an optical detection system configured to detect the generated RCA products. In some embodiments, the one or more reagents for sequencing is for performing sequencing by synthesis (SBS). In some instances, the one or more reagents for sequencing is for performing single nucleotide sequencing by synthesis. In some instances, the one or more reagents for sequencing is for performing sequencing by ligation (SBL). In some instances, the one or more reagents for sequencing is for performing in situ sequencing by binding (SBB). [0261] Provided herein is a system comprising: a solid support having a biological sample attached thereto; a plurality of circularizable probes, wherein the plurality of circularizable probes comprises a first probe that binds to a first target nucleic acid and a second probe that binds to a second target nucleic acid; one or more reagents for gap filling the second probe to generate an extended probe comprising a gap filled sequence; one or more reagents for circularizing the plurality of circularizable probes and extended probe to generate a plurality of circularized templates; one or more reagents for generating a plurality of rolling circle amplification products (RCPs) of the plurality of circularized template; a plurality of oligonucleotide probes for detecting a first subset of the generated RCPs; and one or more reagents for sequencing a second subset of the generated RCPs. In some instances, the system further comprises an optical detection system configured to detect the generated RCA products. [0262] In some embodiments, the systems or kits can contain reagents and/or consumables required for performing one or more steps of the provided methods. In some embodiments, the systems contain reagents for fixing, embedding, and/or permeabilizing the biological sample. In some embodiments, the systems or kits contain reagents, such as enzymes and buffers for ligation and/or amplification, such as ligases and/or polymerases. In some aspects, the system or kit can also comprise any of the reagents described herein, e.g., wash buffer and ligation buffer. In some embodiments, the systems or kits comprise reagents for detection and/or sequencing, such as detectably labeled oligonucleotides or detectable labels. In ^{MOFO-357975700} 202412023840 some embodiments, the systems or kits optionally contain other components, for example nucleic acid primers, enzymes and reagents, buffers, nucleotides, modified nucleotides, and/or reagents for additional assays. VI. OPTO-FLUIDIC INSTRUMENTS FOR ANALYSIS OF BIOLOGICAL SAMPLES [0263] Provided herein is an instrument having integrated optics and fluidics modules (an “opto-fluidic instrument” or “opto-fluidic system”) for detecting target molecules (e.g., nucleic acids, proteins, antibodies, etc.) in biological samples (e.g., one or more cells or a tissue sample) as described herein. In an opto-fluidic instrument, the fluidics module is configured to deliver one or more reagents (e.g., detectably labeled probes, labeled nucleotides for sequencing) to the biological sample and/or remove spent reagents therefrom. Additionally, the optics module is configured to illuminate the biological sample with light having one or more spectral emission curves (over a range of wavelengths) and subsequently capture one or more images of emitted light signals from the biological sample during one or more cycles (e.g., as described in Section III). In various embodiments, the captured images may be processed in real time and/or at a later time to determine the presence of the one or more target molecules in the biological sample, as well as three-dimensional position information associated with each detected target molecule. Additionally, the opto-fluidics instrument includes a sample module configured to receive (and, optionally, secure) one or more biological samples. In some instances, the sample module includes an X-Y stage configured to move the biological sample along an X-Y plane (e.g., perpendicular to an objective lens of the optics module). [0264] In various embodiments, the opto-fluidic instrument is configured to analyze one or more target molecules in their naturally occurring place (i.e., in situ) within the biological sample. For example, an opto-fluidic instrument may be an in-situ analysis system used to analyze a biological sample and detect target molecules including but not limited to DNA, RNA, proteins, antibodies, and/or the like. [0265] It is to be noted that, although the above discussion relates to an opto-fluidic instrument that can be used for in situ target molecule detection via probe hybridization and/or sequencing, the discussion herein equally applies to any opto-fluidic instrument that employs any imaging or target molecule detection technique. That is, for example, an opto-fluidic instrument ^{MOFO-357975700} 202412023840 may include a fluidics module that includes fluids needed for establishing the experimental conditions required for the probing of target molecules in the sample. Further, such an opto- fluidic instrument may also include a sample module configured to receive the sample, and an optics module including an imaging system for illuminating (e.g., exciting one or more fluorescent probes within the sample) and/or imaging light signals received from the probed sample. The in-situ analysis system may also include other ancillary modules configured to facilitate the operation of the opto-fluidic instrument, such as, but not limited to, cooling systems, motion calibration systems, etc. [0266] FIG. 2 shows an example workflow of analysis of a biological sample 210 (e.g., cell or tissue sample) using an opto-fluidic instrument 220, according to various embodiments. In various embodiments, the sample 210 can be a biological sample (e.g., a tissue) that includes molecules such as DNA, RNA, proteins, antibodies, etc. For example, the sample 210 can be a sectioned tissue that is treated to access the RNA thereof for labeling with probes and to perform sequencing as described herein. Ligation of the probes may generate a circularized probe which can be enzymatically amplified and bound with detectably labeled probes or with a sequencing primer and reagents, which can create bright signal that is convenient to image and has a high signal-to-noise ratio. [0267] In various embodiments, the sample 210 may be placed in the opto-fluidic instrument 220 for analysis and detection of the molecules in the sample 210. In various embodiments, the opto-fluidic instrument 220 can be a system configured to facilitate the experimental conditions conducive for the detection of the target molecules. For example, the opto-fluidic instrument 220 can include a fluidics module 240, an optics module 250, a sample module 260, and an ancillary module 270, and these modules may be operated by a system controller 230 to create the experimental conditions for the probing of the molecules in the sample 210 by selected probes (e.g., circularizable DNA probes), as well as to facilitate the imaging of the probed sample (e.g., by an imaging system of the optics module 250). In various embodiments, the various modules of the opto-fluidic instrument 220 may be separate components in communication with each other, or at least some of them may be integrated together. ^{MOFO-357975700} 202412023840 [0268] In various embodiments, the sample module 260 may be configured to receive the sample 210 into the opto-fluidic instrument 220. For instance, the sample module 260 may include a sample interface module (SIM) that is configured to receive a sample device (e.g., cassette) onto which the sample 210 can be deposited. That is, the sample 210 may be placed in the opto-fluidic instrument 220 by depositing the sample 210 (e.g., the sectioned tissue) on a sample device that is then inserted into the SIM of the sample module 260. In some instances, the sample module 260 may also include an X-Y stage onto which the SIM is mounted. The X-Y stage may be configured to move the SIM mounted thereon (e.g., and as such the sample device containing the sample 210 inserted therein) in perpendicular directions along the two- dimensional (2D) plane of the opto-fluidic instrument 220. Additional discussion related the SIM can be found in US Provisional Application No.: 63/348,879, filed June 3, 2022, titled “Methods, Systems, and Devices for Sample Interface,” which is incorporated herein by reference in its entirety. [0269] The experimental conditions that are conducive for the detection of the molecules in the sample 210 may depend on the target molecule detection technique that is employed by the opto-fluidic instrument 220. For example, in various embodiments, the opto- fluidic instrument 220 can be a system that is configured to detect molecules in the sample 210 via hybridization of probes as described in Section III.A. In such cases, the experimental conditions can include molecule hybridization conditions that result in the intensity of hybridization of the target molecule (e.g., nucleic acid) to a probe (e.g., oligonucleotide) being significantly higher when the probe sequence is complementary to the target molecule than when there is a single-base mismatch. The hybridization conditions include the preparation of the sample 210 using reagents such as washing/stripping reagents, hybridizing reagents, etc., and such reagents may be provided by the fluidics module 240. In some instances, the opto-fluidic instrument 220 is a system that is configured to detect molecules in the sample 210 via sequencing as described in Section III.B. [0270] In various embodiments, the fluidics module 240 may include one or more components that may be used for storing the reagents, as well as for transporting said reagents to and from the sample device containing the sample 210. For example, the fluidics module 240 may include reservoirs configured to store the reagents, as well as a waste container configured ^{MOFO-357975700} 202412023840 for collecting the reagents (e.g., and other waste) after use by the opto-fluidic instrument 220 to analyze and detect the molecules of the sample 210. Further, the fluidics module 240 may also include pumps, tubes, pipettes, etc., that are configured to facilitate the transport of the reagent to the sample device (e.g., and as such the sample 210). For instance, the fluidics module 240 may include pumps (“reagent pumps”) that are configured to pump washing/stripping reagents to the sample device for use in washing/stripping the sample 210 (e.g., as well as other washing functions such as washing an objective lens of the imaging system of the optics module 250). In some instances, the opto-fluidic instrument 220 is a system includes one or more components that may be used for storing the reagents for sequencing (e.g., nucleotide mixes comprising one or more labeled nucleotides and polymerases) as well as for transporting said reagents to and from the sample device containing the sample 210. [0271] In various embodiments, the ancillary module 270 can be a cooling system of the opto-fluidic instrument 220, and the cooling system may include a network of coolant- carrying tubes that are configured to transport coolants to various modules of the opto-fluidic instrument 220 for regulating the temperatures thereof. In such cases, the fluidics module 240 may include coolant reservoirs for storing the coolants and pumps (e.g., “coolant pumps”) for generating a pressure differential, thereby forcing the coolants to flow from the reservoirs to the various modules of the opto-fluidic instrument 220 via the coolant-carrying tubes. In some instances, the fluidics module 240 may include returning coolant reservoirs that may be configured to receive and store returning coolants, i.e., heated coolants flowing back into the returning coolant reservoirs after absorbing heat discharged by the various modules of the opto- fluidic instrument 220. In such cases, the fluidics module 240 may also include cooling fans that are configured to force air (e.g., cool and/or ambient air) into the returning coolant reservoirs to cool the heated coolants stored therein. In some instances, the fluidics module 240 may also include cooling fans that are configured to force air directly into a component of the opto-fluidic instrument 220 so as to cool said component. For example, the fluidics module 240 may include cooling fans that are configured to direct cool or ambient air into the system controller 230 to cool the same. [0272] As discussed above, the opto-fluidic instrument 220 may include an optics module 250 which include the various optical components of the opto-fluidic instrument 220, ^{MOFO-357975700} 202412023840 such as but not limited to a camera, an illumination module (e.g., LEDs), an objective lens, and/or the like. The optics module 250 may include a fluorescence imaging system that is configured to image the fluorescence emitted by the probes (e.g., oligonucleotides) in the sample 210 after the probes are excited by light from the illumination module of the optics module 250. [0273] In some instances, the optics module 250 may also include an optical frame onto which the camera, the illumination module, and/or the X-Y stage of the sample module 260 may be mounted. [0274] In various embodiments, the system controller 230 may be configured to control the operations of the opto-fluidic instrument 220 (e.g., and the operations of one or more modules thereof). In some instances, the system controller 230 may take various forms, including a processor, a single computer (or computer system), or multiple computers in communication with each other. In various embodiments, the system controller 230 may be communicatively coupled with data storage, set of input devices, display system, or a combination thereof. In some cases, some or all of these components may be considered to be part of or otherwise integrated with the system controller 230, may be separate components in communication with each other, or may be integrated together. In other examples, the system controller 230 can be, or may be in communication with, a cloud computing platform. [0275] In various embodiments, the opto-fluidic instrument 220 may analyze the sample 210 and may generate the output 290 that includes indications of the presence of the target molecules in the sample 210. For instance, with respect to the example embodiment discussed above where the opto-fluidic instrument 220 employs a hybridization technique for detecting molecules, the opto-fluidic instrument 220 may cause the sample 210 to undergo successive rounds of detectably labeled probe hybridization (e.g., using two or more sets of fluorescent probes, where each set of fluorescent probes is excited by a different color channel) and be imaged to detect target molecules in the probed sample 210. In such cases, the output 290 may include optical signatures (e.g., a codeword) specific to each gene, which allow the identification of the target molecules. [0276] In various embodiments, the optical subsystem for high-resolution in situ sequencing includes at least one objective lens, which may be an infinity-corrected objective lens. In embodiments where an infinity-corrected objective lens is used, the optical subsystem ^{MOFO-357975700} 202412023840 includes at least one tube lens configured to receive parallel rays from the infinity-corrected objective lens and focus the rays to a focal point, where an image sensor (e.g., a CMOS sensor) is positioned. In various embodiments, the optical subsystem is configured for epifluorescence microscopy (where excitation light provided to the sample in the excitation channel is filtered out from any emission light provided to the image sensor in the emission channel). An infinity- corrected objective lens may be particularly suited for epifluorescence microscopy because the parallel rays in the infinity space (i.e., the space between the objective and the tube lens in which rays from the objective travel in a parallel, collimated beam to the tube lens) allow for the insertion of additional optical components, such as beamsplitters and filters, without introducing significant optical aberrations. To achieve the high resolution necessary for imaging individual, small clusters, or amorphous/diffuse regions of target analytes, the objective lens ideally possesses a high numerical aperture (NA). For example, objectives with NAs greater than or equal to 0.9, and more preferably, greater than or equal to 1.0, are contemplated to maximize resolution and light collection efficiency from fluorescently tagged analytes. In various embodiments, to achieve a higher NA, an objective capable of immersion in a liquid having a higher refractive index than air (e.g., water with a refractive index of about 1.33 or oil with a refractive index of about 1.51) is needed. Examples of such objective lenses include water immersion objectives (e.g., for NAs as high as ~1.27) or oil immersion (e.g., for NAs as high as ~1.4). However, it is understood that objectives with lower NAs may also be utilized depending on the specific resolution requirements and/or sample characteristics. For example, the NA of the objective lens may be at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, from 0.6 to 1.4, from 0.7 to 1.4, from 0.8 to 1.4, from 0.9 to 1.4, from 1.0 to 1.4, from 0.9 to 1.1, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, or about 1.4. In various embodiments, the tube lens is selected to further optimize the imaging performance, ensuring that the combined optical system delivers sharp, high-contrast images of the target analytes throughout the field of view (FOV) in all imaging color channels (e.g., red, yellow, green, blue, nUV). In various embodiments, the objective lens includes a large FOV to maximize the image volume of a single z-stack of images (thereby reducing the number of z- stacks required to image an entire sample). For example, the FOV may have a diagonal of at least 0.50 mm, at least 0.75 mm, at least 0.80 mm, at least 0.90 mm, at least 1.00 mm, at least ^{MOFO-357975700} 202412023840 1.10 mm, at least 1.20 mm, at least 1.30 mm, at least 1.40 mm, at least 1.50 mm, at least 1.60 mm, at least 1.70 mm, at least 1.80 mm, at least 1.90 mm, at least 2.00 mm, at least 2.25mm, at least 2.50mm, at least 2.75mm, at least 3.00 mm, from 0.50 mm to 5.00 mm, from 0.75 to 4.00 mm, from 0.75 mm to 3.00 mm, from 0.75 mm to 2.00 mm, from 1.00 mm to 4.00 mm, from 1.00 mm to 3.00 mm, from 1.00 mm to 2.00 mm, about 1.00 mm, about 1.25 mm, about 1.5 mm, about 1.75 mm, about 2.00 mm, about 2.5 mm, or about 3.00 mm. [0277] In various embodiments, the optical subsystem is designed to facilitate multicolor volumetric (e.g., z-stack) imaging at a plurality of FOVs of the sample, enabling the capture of high-resolution volumetric data from the sample in a plurality of color channels. In various embodiments, the instrument and/or optical subsystem is designed such that z- repeatability of relative z-motion of the objective lens and sample is less than the depth of focus of the objective lens. In various embodiments, the objective lens moves in Z and the stage is stationary. In various embodiments, the objective lens is stationary and the stage moves in Z. In various embodiments, both the objective lens and the stage have Z-motion capability. In various embodiments, the optical subsystem is designed such that the wavefront error, chromatic shift, and/or field curvature is less than the depth of focus of the objective lens and/or less than the step size between z-slices in the z-stack. In various embodiments, the z-step size is about 0.25 µm to about 2.00 µm, about 0.50 µm to about 1.50 µm, about 0.50 µm to about 1.00 µm, about 1.00 µm, about 0.90 µm, about 0.80 µm, about 0.75 µm, about 0.70 µm, about 0.60 µm, about 0.50 µm, or about 0.25 µm. [0278] In various embodiments, the optical subsystem is designed to minimize various optical aberrations to maximize image quality across the entire z-stack of images. Specifically, the objective lens and tube lens are designed such that wavefront error, chromatic shift, and field curvature are very small. In various embodiments, the objective lens is designed such that substantially all of the illuminated FOV (which may be a smaller area than the full area of the circular FOV) is usable for decoding target analytes. In various embodiments, wavefront error, chromatic shift, and field curvature are significantly less than the depth of focus of the objective lens. By designing an optical subsystem with minimal wavefront aberration, light collected from the sample is accurately focused, preserving spatial resolution. Moreover, designing an optical subsystem with minimal chromatic shift is particularly useful for multi- ^{MOFO-357975700} 202412023840 color fluorescence imaging as misregistration of the different color channels is reduced (e.g., minimized). Lastly, designing an optical subsystem with corrected (minimal) field curvature ensures that the entire field of view remains in focus across each z-plane, allowing for greater spatial resolution in the Z-axis and potentially increasing the effective imaging area and throughput. In various embodiments, tight control of optical aberration(s) contributes to consistent and high image quality throughout the entire acquired z-stack in multicolor volumetric imaging, ultimately resulting in higher quality and reliable decoding and spatial localization of target analytes. [0279] In various embodiments, the optical subsystem is designed for high- throughput imaging, allowing for rapid in situ sequencing and/or detection workflows. In various embodiments, this optimization is achieved through several design considerations. Firstly, the optical subsystem is configured to image fluorescent dyes that require shorter exposure times to emit strong optical signals, thereby minimizing photobleaching and maximizing imaging speed. Secondly, the optical subsystem provides a large FOV, enabling the imaging of larger areas of the sample and reducing the number of z-stack acquisitions required to cover a given sample volume. Thirdly, the subsystem is engineered for rapid z-stack imaging, allowing for quick stepping between discrete z-slices in each z-stack. In various embodiments, quick z-stepping can be achieved through the integration of fast axial scanning mechanisms, which may integrate voice coil actuators, piezoelectric actuators, or other actuators to enable precise and rapid adjustment of the focal plane as well as high precision, high speed linear XY or XYZ stages (belt, screw, or electromagnetic driven), and tight feedback control loops and/or vibration control, which may integrate proportional control, proportional-integral control, or proportional-integral-derivative control, for precise and rapid switching between z-slices and/or FOVs. VII. TERMINOLOGY [0280] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not ^{MOFO-357975700} 202412023840 necessarily be construed to represent a substantial difference over what is generally understood in the art. [0281] The terms "polynucleotide" and "nucleic acid molecule", used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term comprises, but is not limited to, single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. [0282] A “primer” as used herein, in some embodiments, is an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Primers usually are extended by a DNA polymerase. [0283] In some instances, “ligation” refers to the formation of a covalent bond or linkage between the termini of two or more nucleic acids, e.g., oligonucleotides and/or polynucleotides, in a template-driven reaction. The nature of the bond or linkage may vary widely and the ligation, in some embodiments, is carried out enzymatically or chemically. As used herein, ligations are usually carried out enzymatically to form a phosphodiester linkage between a 5' carbon terminal nucleotide of one oligonucleotide with a 3' carbon of another nucleotide. [0284] The term "about" as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to "about" a value or parameter herein comprises (and describes) embodiments that are directed to that value or parameter per se. [0285] As used herein, the singular forms "a," "an," and "the" comprise plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more." ^{MOFO-357975700} 202412023840 [0286] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be comprised in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range comprises one or both of the limits, ranges excluding either or both of those comprised limits are also comprised in the claimed subject matter. This applies regardless of the breadth of the range. [0287] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a), b), etc., or i), ii), etc. does not by itself connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order. EXAMPLES [0288] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure. Example 1: Detecting a panel of analytes and variant sequences in situ using hybridization and single nucleotide sequencing readouts [0289] This example describes use of circularizable probes for detecting a panel of analytes and a variant sequence in situ. [0290] A tissue sample is obtained and sectioned (e.g., cryosectioned) onto a glass slide for processing. Thin sections, e.g., with a thickness of 10 µm, are cut with a cryostat and ^{MOFO-357975700} 202412023840 collected on glass slides. Sections are fixed (e.g., by incubating in 3.7% paraformaldehyde (PFA)), washed, and permeabilized. After permeabilization, sections are washed, and dehydrated, e.g., using an escalating ethanol series. Secure seal chambers are mounted on the slides to cover the tissue sections, and the sections are hydrated by a brief wash. To prepare for probe hybridization, a buffer is added to the tissue section. [0291] A first set of circularizable probes (e.g., padlock probes) are designed to target a panel of RNA transcripts and a second set of circularizable probes are designed to target the wildtype and the mutant allele of a single nucleotide variant (SNV). Probes of the first set of circularizable probes have two arms with hybridization regions targeting adjacent sequences of each target RNA of a plurality of RNAs. The probes of the first set of circularizable probes also comprise a barcode sequence in the non-target nucleic acid hybridizing region that corresponds to the target RNA of interest. Once hybridized to the target nucleic acid, the 5’ end and the 3’ end of the probe is positioned to be ligated to generate a circularized template (e.g., a circularized padlock probe). Probes of the second set of circularizable probes are gap fill padlock probes with arms comprising hybridization regions targeting a first sequence and a second sequence flanking an interrogatory region (e.g., comprising a nucleotide that can be a wildtype or mutant allele of a variant sequence). Once hybridized to the target nucleic acid, the 3’ of the probe is extended (e.g., using a polymerase or by ligating a splint) using the interrogatory region as template to form an extended probe comprising a gap filled sequence. In the case of ligating a splint, at least two splint oligonucleotides targeting the wildtype and the mutant variant sequence is provided to fill the gap between the binding arms. Upon binding and gap filling, probes are ligated to generate a circularized template for rolling circle amplification (RCA). The ligation is performed with a ligase in a ligation buffer at 37ºC to form circularized templates. For RCA, the cells are washed and then incubated in an RCA reaction mixture (containing Phi29 reaction buffer, dNTPs, Phi29 polymerase) to generate RCA products (RCPs) corresponding to the panel of RNA transcripts and the SNV. [0292] Sequence analysis of the first set of circularizable probes is performed by hybridization using oligonucleotide probes comprising a hybridization region that hybridizes to the RCPs and an overhang sequence that hybridizes detectably labeled detection oligonucleotides (DOs). The probes are hybridized to the barcode(s) of the RCPs in situ in a hybridization buffer. ^{MOFO-357975700} 202412023840 The cells are washed, stained with DAPI, and mounted in a mounting medium for imaging using fluorescent microscopy, and RCPs counts per unit nuclei area are detected, thereby detecting the panel of target RNAs. [0293] Next, a sequencing reaction cycle is performed to detect RCPs generated using the gap filled second set of circularized probes. The sequencing reaction uses a sequencing primer that binds to a primer binding site in the generated RCPs, a polymerase, and plurality of nucleobases, each having a dye (optionally, a nucleobase can be unlabeled). Next, the reaction is incubated to allow for incorporation of a nucleotide into the sequencing primer when the nucleobase type is complementary to the RCP nucleobase at the polymerase active site. Imaging is performed for detecting the dyes and to register the location of the RCP on the slide. The sample is washed, and then treated with a reducing agent to remove the dye and deblocked if a reversible terminator nucleobase is used. The sequencing reaction is then repeated for sufficient cycles to detect the gap filled sequence associated with the second set of circularized probes. [0294] In this manner, decoding of the barcodes associated with the RCPs generated using the first set of circularizable probes is used to detect a large panel of transcripts (e.g., 200 or more target nucleic acids) and sequencing of the gap filled sequence or complement thereof associated with the RCPs generated using the second set of circularizable probes provides sensitivity and specificity for detecting specific SNV in the biological sample. Sequencing cycles performed after hybridization cycles in some cases allow the integrity of the RNAs to be optimally preserved for identification of transcripts before the more targeted (e.g., single base) readout is performed. In some cases, an assay using single base sequencing readout in combination with a hybridization based readout allows a large number of targets to be identified (e.g., gene expression) and identification of variable sequences (e.g., SNV) or unknown sequences that may be challenging to detect using hybridization probes. Using the combination approach, in some aspects, hybridization based detection provide gene expression information that is used for cell typing and differential gene expression and additionally sequencing based detection provides sequence information from the same cells. [0295] The present disclosure is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the present disclosure. Various modifications to the compositions and methods described will ^{MOFO-357975700} 202412023840 become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure. 111 ^{MOFO-357975700}

Claims

202412023840 CLAIMS 1. A method for analyzing a biological sample, the method comprising: (a) binding a plurality of circularizable probes to a plurality of target nucleic acids in the biological sample, wherein: the plurality of circularizable probes comprises a first probe that binds to a first target nucleic acid and a second probe that binds to a second target nucleic acid, wherein the first probe comprises a 5’ end and a 3’ end; (b) ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template; (c) extending the second probe using a sequence of the second target nucleic acid as a template to generate an extended probe comprising a gap filled sequence; (d) ligating the extended probe to generate a second circularized template; (e) performing rolling circle amplification (RCA) of the first circularized template and the second circularized template in the biological sample to generate a first RCA product and a second RCA product, respectively; (f) contacting the biological sample with an oligonucleotide probe that directly or indirectly binds to a sequence in the first RCA product and detecting a first signal associated with the oligonucleotide probe, thereby detecting the first target nucleic acid, and (g) sequencing the second RCA product to determine the gap filled sequence or a portion or a complement thereof, thereby detecting the second target nucleic acid. 2. The method of claim 1, wherein (b) and (d) are performed simultaneously. 3. The method of claim 1, wherein (b) - (c) - (d) are performed in that order. 4. The method of claim 1, wherein (c) - (d) - (b) are performed in that order. 5. The method of claim 1, wherein (c) - (b) - (d) are performed in that order. 6. The method of any one of claims 1-5, wherein (g) is performed after (f). 112 ^{MOFO-357975700} 202412023840 7. The method of any one of claims 1-6, wherein the first probe comprises a barcode sequence corresponding to the first target nucleic acid or a sequence thereof. 8. The method of claim 7, wherein the barcode sequence is not complementary to the target nucleic acid or sequence thereof. 9. The method of claim 7 or claim 8, wherein the oligonucleotide probe directly or indirectly binds to the barcode sequence or a complement thereof. 10. The method of any one of claims 1-9, wherein the first probe comprises: (i) a first hybridization region complementary to a first sequence of the first target nucleic acid and (ii) a second hybridization region complementary to a second sequence of the first target nucleic acid. 11. The method of claim 10, wherein the oligonucleotide probe directly or indirectly binds to a sequence comprising: (i) the first sequence of the first target nucleic acid or a portion thereof, and/or (ii) the second sequence of the first target nucleic acid or a portion thereof. 12. The method of any one of claims 1-11 wherein the second probe does not comprise a barcode sequence corresponding to the second target nucleic acid or a sequence thereof. 13. The method of any one of claims 1-12, wherein the second probe comprises (i) a first hybridization region complementary to a first sequence of the second target nucleic acid and (ii) a second hybridization region complementary to a second sequence of the second target nucleic acid, ^{MOFO-357975700} 202412023840 wherein the first hybridization region and the second hybridization region of the second probe are common among a plurality of second probes each targeting a molecule comprising a different variant sequence of the second target nucleic acid. 14. The method of claim 13, wherein the plurality of second probes are capable of hybridizing to both a wildtype molecule and a mutant molecule of the second target nucleic acid. 15. The method of claim 13 or claim 14, wherein the first sequence and the second sequence of the second target nucleic acid are separated by a gap sequence of at least 2 nucleotides in the second target nucleic acid. 16. The method of claim 15, wherein the gap sequence is between about 2 and about 40 nucleotides in length. 17. The method of any one of claims 13-16, wherein the second target nucleic acid comprises a variant sequence at the 3’ or 5’ end of the gap sequence. 18. The method of any one of claims 13-16, wherein the second target nucleic acid comprises a variant sequence at or near the central nucleotide(s) of the gap sequence. 19. The method of any one of claims 13-16, wherein the second target nucleic acid comprises a variant sequence at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or more phosphodiester bonds from the 3’ or 5’ end of the gap sequence. 20. The method of any one of claims 10-19, wherein the first hybridization region and the second hybridization region in the first probe are equal in length. 21. The method of any one of claims 10-19, wherein the first hybridization region is shorter or longer than the second hybridization region in the first probe. ^{MOFO-357975700} 202412023840 22. The method of any one of claims 13-21, wherein the first hybridization region and the second hybridization region in the second probe are equal in length. 23. The method of any one of claims 13-21, wherein the first hybridization region is shorter or longer than the second hybridization region in the second probe. 24. The method of any one of claims 10-23, wherein the first hybridization region and/or the second hybridization region in the first probe is between about 5 and about 50 nucleotides in length. 25. The method of any one of claims 13-24, wherein the first hybridization region and/or the second hybridization region in the second probe is between about 5 and about 50 nucleotides in length. 26. The method of any one of claims 1-25, wherein the first probe is provided in two parts. 27. The method of any one of claims 1-26, wherein the second probe is provided in two parts. 28. The method of any one of claims 1-27, comprising ligating the 5’ end of the first probe to the 3’ end of the first probe using the first target nucleic acid as template. 29. The method of any one of claims 1-28, wherein the biological sample comprises different molecules of the second target nucleic acid each comprising a mutant sequence of a plurality of different mutant sequences. 30. The method of any one of claims 1-29, wherein (c) is performed using a library of splint oligonucleotides comprising a plurality of different mutant splint oligonucleotides each comprising a hybridization region complementary to sequence of a plurality of different second target nucleic acids, and a splint oligonucleotide of the library of splint oligonucleotides that is ^{MOFO-357975700} 202412023840 complementary to the second target nucleic acid is ligated to the second probe, thereby circularizing the second probe to generate the second circularized template. 31. The method of claim 30, wherein each splint oligonucleotide of the library of splint oligonucleotides comprises a 3’ hydroxyl group and a 5’ phosphate group, optionally wherein the splint oligonucleotide comprises a 5’ flap and/or one or more RNA residues at and/or near its 3’ end. 32. The method of claim 30 or claim 31, wherein the splint oligonucleotide is between about 2 and about 40 nucleotides in length. 33. The method of any one of claims 30-32, wherein the splint oligonucleotide is ligated to the second probe by a ligase using the second target nucleic acid as a template. 34. The method of any one of claims 30-33, wherein the second target nucleic acid is an RNA and the ligase has an RNA-templated ligase activity. 35. The method of any one of claims 30-34, wherein the second target nucleic acid is a DNA, and the ligase has a DNA-templated ligase activity. 36. The method of any one of claims 30-35, wherein the library of splint oligonucleotides comprises at least or about 2, at least or about 5, at least or about 10, at least or about 15, at least or about 20, at least or about 25, at least or about 30, at least or about 35, at least or about 40, at least or about 45, at least or about 50, or more splint oligonucleotides of different sequences. 37. The method of any one of claims 30-36, comprising washing the biological sample after contacting with the library of splint oligonucleotides, optionally wherein the washing is performed under less than stringent conditions. ^{MOFO-357975700} 202412023840 38. The method of any one of claims 1-37, wherein (c) comprises extending an end of the second probe by a polymerase using the second target nucleic acid as a template. 39. The method of claim 38, wherein the second target nucleic acid is an RNA and the polymerase has an RNA-templated polymerase activity. 40. The method of claim 38, wherein the second target nucleic acid is a DNA and the polymerase has a DNA-templated polymerase activity. 41. The method of claim 38, wherein the polymerase is a reverse transcriptase. 42. The method of any one of claims 38-41, wherein the polymerase incorporates two or more residues into the 3’ end of the second probe to generate the extended probe. 43. The method of any one of claims 38-42, wherein the polymerase has no or little strand displacement activity. 44. The method of any one of claims 1-43, wherein the extended probe is ligated by a ligase using the second target nucleic acid as a template, wherein the target nucleic acid is an RNA or a DNA, and the ligase has an RNA-templated ligase activity and/or a DNA-templated ligase activity. 45. The method of any one of claims 1-44, wherein the second target nucleic acid comprises a nucleotide variation, a nucleotide polymorphism, a mutation, a substitution, an insertion, a deletion, a translocation, a rearrangement, a duplication, an inversion, and/or a repetitive sequence. 46. The method of claim 45, wherein the second target nucleic acid comprises a variant sequence and the variant sequence comprises is a single nucleotide, optionally wherein the variant sequence is a single nucleotide variation (SNV), a single nucleotide polymorphism ^{MOFO-357975700} 202412023840 (SNP), a point mutation, a single nucleotide substitution, a single nucleotide insertion, or a single nucleotide deletion. 47. The method of any one of claims 1-43, wherein the second target nucleic acid comprises a sequence of an immune molecule. 48. The method of claim 47, wherein the sequence of the immune molecule is an antigen receptor transcript. 49. The method of claim 48, wherein the antigen receptor transcript is a T cell receptor (TCR) transcript, optionally wherein the TCR transcript comprises a TCRα VJ join, a TCRβ VDJ join, a TCRγ VJ join, or a TCRδ VDJ join. 50. The method of claim 48, wherein the second RCA product comprises multiple copies of a unit sequence comprising a sequence of a VDJ join or a complement thereof. 51. The method of claim 50, wherein the unit sequence comprises the D segment of the VDJ join, optionally wherein the unit sequence comprises the V segment or a portion thereof and/or the J segment or a portion thereof of the VDJ join. 52. The method of claim 48, wherein the antigen receptor transcript is an immunoglobulin (Ig) transcript, optionally wherein the Ig transcript comprises an Igκ VJ join, an Igλ VJ join, or an IgH VDJ join. 53. The method of any one of claims 47-52, comprising identifying multiple different antigen receptor transcripts present at a plurality of locations in the biological sample. 54. The method of claim 53, wherein a plurality of VDJ joins of the multiple different antigen receptor transcripts comprises at least about 50, at least about 100, at least about 500, at ^{MOFO-357975700} 202412023840 least about 1,000, at least about 5,000, at least about 10,000, or more VDJ joins of different sequences. 55. The method of any one of claims 1-43, wherein the second target nucleic acid comprises one or more exon-exon boundaries of a nucleic acid. 56. The method of any one of claims 1-43, wherein the second target nucleic acid comprises a sequence of a perturbation agent introduced to the biological sample before (a). 57. The method of claim 56, wherein the second target nucleic acid comprises a CRISPR molecule, a nucleic acid molecule edited using the CRISPR molecule, and/or a precursor or derivative thereof. 58. The method of claim 56, wherein the second target nucleic acid comprises a spacer sequence of a perturbation agent. 59. The method of claim 56, wherein the second target nucleic acid is a transcript comprising a unique barcode specific to a guide RNA. 60. The method of any one of claims 1-43, wherein the second target nucleic acid comprises a microRNA. 61. The method of any one of claims 1-60, wherein the first target nucleic acid and/or the second target nucleic acid is RNA. 62. The method of any one of claims 1-61, wherein the second target nucleic acid comprises a barcode sequence. 63. The method of claim 62, wherein the barcode sequence is for lineage tracing. ^{MOFO-357975700} 202412023840 64. The method of any one of claims 1-63, wherein the first target nucleic acid and the second target nucleic acid are RNA transcripts of different genes. 65. The method of any one of claims 1-64, wherein in (a) the method comprises: contacting the biological sample with a plurality of different first probes comprising the first probe, wherein each different first probe binds to an RNA transcript of one of a plurality of different genes. 66. The method of any one of claims 1-65, wherein in (a) the method comprises: contacting the biological sample with the second probe, wherein the second probe is capable of binding to at least two different RNA transcripts of a first gene of the plurality of different genes. 67. The method of claim 66, wherein in (a) the method comprises: contacting the biological sample with a plurality of different second probes comprising the second probe and an additional second probe, wherein the additional second probe is capable of binding to at least two different RNA transcripts of a second gene of the plurality of different genes. 68. The method of claim 67, comprising contacting the biological sample with an RNase H to cleave the first target nucleic and the second target nucleic acid to generate a first cleaved target RNA and a second cleaved target RNA. 69. The method of claim 68, wherein the RCA of the first circularized template and the second circularized template is performed using the first cleaved target RNA and the second cleaved target RNA, respectively, as a primer. 70. The method of any one of claims 1-69, wherein the first RCA product and the second RCA product is generated in situ in the biological sample or a matrix embedding the biological sample. ^{MOFO-357975700} 202412023840 71. The method of any one of claims 1-70, wherein the method comprises imaging the biological sample to detect the first RCA product and the second RCA product in situ in the biological sample or a matrix embedding the biological sample. 72. The method of any one of claims 1-71, wherein the first target nucleic acid and/or the second target nucleic acid is attached directly or indirectly to the biological sample or to a matrix embedding the biological sample. 73. The method of any one of claims 1-72, wherein the first target nucleic acid and/or the second target nucleic acid is crosslinked in the biological sample or in a matrix embedding the biological sample. 74. The method of any one of claims 1-73, wherein the first RCA product and the second RCA product is covalently linked to the first target nucleic acid and the second target nucleic acid, respectively. 75. The method of any one of claims 1-74, wherein the oligonucleotide probe hybridizes to a barcode sequence or complement thereof of the first probe. 76. The method of claim 75, wherein the barcode sequence comprises a first subunit and a second subunit, wherein a portion of the first subunit overlaps with some but not all of the second subunit. 77. The method of claim 75 or claim 76, wherein the barcode sequence is assigned a signal code sequence, wherein the first subunit or a complement thereof corresponds to a first signal code of the signal code sequence, and the second subunit or a complement thereof corresponds to a second signal code of the signal code sequence. 78. The method of any one of claims 75-77, wherein the oligonucleotide probe comprises a detectable label associated with the first signal code. ^{MOFO-357975700} 202412023840 79. The method of any one of claims 75-78, wherein (f) comprises detecting the detectable label associated with the first signal code that identifies the first target nucleic acid. 80. The method of claim 79, wherein (f) further comprises decoding the second signal code detected from a sequential cycle of hybridization, detection, and removal of a second oligonucleotide probe labeled with a second detectable label. 81. The method of any one of claims 1-75, wherein (f) comprises contacting the biological sample with an universal pool of detectably labeled probes. 82. The method of claim 81, wherein the number of different detectably labeled probes in the universal pool is four. 83. The method of claim 82, wherein the oligonucleotide probe comprises: (i) a hybridization regions complementary to the barcode sequences of the first probe or complement thereof and (ii) a reporter region complementary to a detectably labeled probe of the universal pool of detectably labeled probes. 84. The method of claim 83, wherein the detectably labeled probe comprises a sequence complementary to the reporter region and a detectable label. 85. The method of claim 84, wherein a complex formed between the barcode sequence or complement thereof, the oligonucleotide probe, and the detectably labeled probe is detected. 86. The method of any one of claims 1-85, wherein (f) comprises detecting a series of signal codes that identifies the first probe or the first target nucleic acid. ^{MOFO-357975700} 202412023840 87. The method of claim 86, wherein (f) comprises decoding a series of signal codes detected from a plurality of sequential cycles of hybridization, detection, and removal of sequential pools of oligonucleotide probes and the universal pool of detectably labeled probes. 88. The method of claim 87, wherein the series of signal codes are fluorophore sequences assigned to the corresponding first target nucleic acid. 89. The method of any one of claims 80-88, wherein the detectably labeled probes are fluorescently labeled. 90. The method of claim 89, wherein the plurality of sequential cycles comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cycles. 91. The method of any one of claims 83-90, wherein a detectable label of the detectably labeled probes corresponds to a sequence of the oligonucleotide probe. 92. The method of any one of claims 1-91, wherein a plurality of first target nucleic acids are present in the biological sample and each first target nucleic acid of the plurality of first target nucleic acids is assigned a signal code sequence, and detecting the plurality of first target nucleic acids comprises: (i) contacting the biological sample with a first oligonucleotide probe and a first detectably labeled probe to generate a first complex comprising the first oligonucleotide probe hybridized to the first RCA product and the first detectably labeled probe hybridized to the first oligonucleotide probe, (ii) imaging the biological sample to detect a first signal from the first detectably labeled probe, wherein the first signal corresponds to a first signal code in the signal code sequence assigned to the first target nucleic acid; (iii) contacting the biological sample with a second oligonucleotide probe and a second detectably labeled probe to generate a second complex comprising the second oligonucleotide ^{MOFO-357975700} 202412023840 probe hybridized to the second RCA product and the second detectably labeled probe hybridized to the second oligonucleotide probe; and (iv) imaging the biological sample to detect a second signal from the second detectably labeled probe, wherein the second signal corresponds to a second signal code in the signal code sequence assigned to the first target nucleic acid, wherein the signal code sequence comprising at least the first signal code and the second signal code is determined based on signals detected at a location in the biological sample, thereby identifying the one or more sequences of the first target nucleic acid at the location in the biological sample. 93. The method of any one of claims 1-92, wherein the gap filled sequence or the portion or complement thereof in the second RCA product is detected using sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof. 94. The method of claim 93, wherein the gap filled sequence or the portion or complement thereof in the second RCA product is sequenced using in situ sequencing by synthesis (SBS) in the biological sample. 95. The method of claim 94, wherein the gap filled sequence or the portion or complement thereof in the second RCA product is detected using single nucleotide sequencing by synthesis. 96. The method of claim 93, wherein the gap filled sequence or the portion or complement thereof in the second RCA product is sequenced using in situ sequencing by ligation (SBL) in the biological sample. 97. The method of claim 93, wherein the gap filled sequence or the portion or complement thereof in the second RCA product is sequenced using in situ sequencing by binding (SBB) in the biological sample. ^{MOFO-357975700} 202412023840 98. The method of claim 94, comprising contacting a priming strand bound to the second RCA product comprising the gap filled sequence or a complement thereof with (i) a polymerase and (ii) a first plurality of nucleotide molecules to form a complex comprising a 3’ terminus of the priming strand, the second RCA product, the polymerase, and a nucleotide molecule of the first plurality of nucleotide molecules and detecting presence of the nucleotide molecules in the complex to identify a complementary nucleotide in the second RCA product. 99. The method of claim 98, wherein the priming strand is bound to a sequencing primer binding sequence in the second RCA product, wherein at least a portion of the sequencing primer binding sequence in the second RCA product is the same as a sequence in the second target nucleic acid complementary to the second probe. 100. The method of claim 98, wherein the priming strand is bound to the second RCA product at a sequence that is not the same as a sequence of the second target nucleic acid. 101. The method of claim 99 or 100, wherein the priming strand is extended by two or more nucleotides that are not fluorescently labeled in consecutive sequencing cycles, using a sequence of the second RCA product as a template. 102. The method of any one of claims 98-101, wherein the priming strand comprises a 3’ terminal nucleotide that is reversibly blocked. 103. The method of claim 102, wherein the method further comprises removing the complex, unblocking the reversibly blocked 3’ terminal nucleotide molecule and contacting the priming strand bound to the second RCA product with a polymerase and a second plurality of nucleotide molecules. 104. The method of any one of claims 98-103, further comprising repeating contacting the priming strand with an additional plurality of nucleotide molecules to identify additional ^{MOFO-357975700} 202412023840 complementary nucleotides of the gap filled sequence in the second RCA product for at least 2, 5, 10, 20, 50, or 100 additional cycles. 105. The method of any one of claims 1-104, wherein the first target nucleic acid detected in (f) and the second target nucleic acid detected in (g) are registered to the same cell. 106. The method of any one of claims 1-105, further comprising detecting a labeling agent comprising a reporter oligonucleotide, wherein the labeling agent is bound directly or indirectly to a non-nucleic acid analyte in the biological sample. 107. The method of claim 106, wherein a sequence of the reporter oligonucleotide or a complement thereof is detected by sequencing. 108. The method of claim 107, wherein the sequence of the reporter oligonucleotide or a complement thereof is detected by using single nucleotide sequencing by synthesis. 109. The method of any one of claims 1-108, wherein the biological sample is contacted with a library of first probes to detect a panel of at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1,000, at least 1,500, at least 2,000, at least 3,000, at least 4,000, at least 5,000 or more first target nucleic acids. 110. The method of any one of claims 1-109, wherein the biological sample is a cell or tissue sample comprising cells or cellular components. 111. The method of any one of claims 1-110, wherein the biological sample is a tissue section. 112. The method of any one of claims 1-111, wherein the biological sample is a formalin- fixed, paraffin-embedded (FFPE) sample, a frozen tissue sample, or a fresh tissue sample. ^{MOFO-357975700} 202412023840 113. The method of any one of claims 1-111, wherein the biological sample is fixed and/or permeabilized. 114. The method of any one of claims 1-113, wherein the biological sample is crosslinked and/or embedded in a matrix, optionally wherein the matrix comprises a hydrogel. 115. The method of any one of claims 1-114, wherein the biological sample is cleared. 116. A system, comprising: (a) a plurality of circularizable probes, wherein: the plurality of circularizable probes comprises a first probe that binds to a first target nucleic acid and a second probe that binds to a second target nucleic acid, wherein the first probe comprises a 5’ end and a 3’ end; (b) one or more reagents for gap filling the second probe to generate an extended probe comprising a gap filled sequence; (c) one or more reagents for circularizing the plurality of circularizable probes and extended probe to generate a plurality of circularized templates; (d) one or more reagents for generating a plurality of rolling circle amplification (RCA) products of the plurality of circularized template; (e) a plurality of oligonucleotide probes for detecting a first subset of the generated RCA products; and (f) one or more reagents for sequencing a second subset of the generated RCA products. 117. The system of claim 116, wherein each oligonucleotide probe comprises: (i) a hybridization region complementary to a sequence in a first RCA product of the first subset of the generated RCA products; and (ii) a detectable region. ^{MOFO-357975700} 202412023840 118. The system of claim 116 or claim 117, comprising a plurality of detectably labeled probes each comprising a detectable label and a sequence for hybridizing to a sequence in the first subset of the generated RCA products. 119. The system of any one of claims 116-118, comprising a plurality of a sequencing primer, a plurality of detectably labeled nucleotides, and a polymerase. 120. The system of any one of claims 116-119, wherein the one or more reagents for gap filling the second probe comprises an engineered family B polymerase and dNTPs. 121. The system of claim 120, wherein the engineered family B polymerase is an engineered Tgo polymerase comprising one or more mutations that confer reverse transcriptase activity. 122. The system of any one of claims 116-121, wherein the one or more reagents for circularizing the plurality of circularizable probes comprises a ligase. 123. The system of any one of claims 116-122, the system comprises an optical detection system configured to detect the generated RCA products. 124. The system of any one of claims 116-123, comprising a solid support having a biological sample attached thereto, wherein the biological sample comprises the first target nucleic acid and the second target nucleic acid. 125. The system of claim 124, wherein the biological sample is a cell or tissue sample comprising cells or cellular components. 126. The system of claim 124, wherein the biological sample is a tissue section. 127. The system of any one of claims 124-126, wherein the biological sample is crosslinked and/or embedded in a matrix, optionally wherein the matrix comprises a hydrogel. ^{MOFO-357975700} 202412023840 128. The system of any one of claims 116-127, wherein the biological sample comprises different molecules of the second target nucleic acid each comprising a mutant sequence of a plurality of different mutant sequences. 129. The system of any one of claims 116-127, the second target nucleic acid comprises a sequence of a perturbation agent introduced to the biological sample. 130. The system of any one of claims 116-129, wherein the one or more reagents for sequencing is for performing sequencing by synthesis (SBS). 131. The system of any one of claims 116-129, wherein the one or more reagents for sequencing is for performing single nucleotide sequencing by synthesis. 132. The system of any one of claims 116-129, wherein the one or more reagents for sequencing is for performing sequencing by ligation (SBL). 133. The system of any one of claims 116-129, wherein the one or more reagents for sequencing is for performing in situ sequencing by binding (SBB). 134. The system of any one of claims 116-133, wherein the first target nucleic acid and/or the second target nucleic acid is an RNA transcript. 135. The system of any one of claims 116-134, wherein the plurality of circularizable probes is configured to detect at least 200 distinct first target nucleic acids. 136. A method, comprising: (a) binding a plurality of circularizable probes to a plurality of target mRNA analytes in a cell or tissue sample, wherein: ^{MOFO-357975700} 202412023840 the plurality of circularizable probes comprises a first probe that binds to a first target mRNA analyte and a second probe that binds to a second target nucleic acid, wherein the first probe comprises a 5’ end and a 3’ end, the plurality of circularizable probes comprises a library of first probes to detect a panel of at least 200 distinct first target nucleic acids; (b) ligating the 5’ end of the first probe to the 3’ end to generate a first circularized template; (c) extending the second probe using a sequence of the second target nucleic acid as a template to generate an extended probe comprising a gap filled sequence; (d) ligating the extended probe to generate a second circularized template; (e) performing rolling circle amplification (RCA) of the first circularized template and the second circularized template in the cell or tissue sample to generate a first RCA product and a second RCA product, respectively; (f) contacting the cell or tissue sample with an oligonucleotide probe that directly or indirectly binds to a sequence in the first RCA product and detecting a first signal associated with the oligonucleotide probe, and (g) sequencing the second RCA product to determine the gap filled sequence or a portion or a complement thereof. ^{MOFO-357975700}