RU2825578C1 - Methods and compositions for determining ligands on matrices using indices and barcodes - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a method for sequencing a target nucleic acid on a matrix, involving: (a) obtaining a first and a second population of granules, wherein: the first population of granules contains oligonucleotides containing first capture probes, first barcodes and barcode primer binding sites adjacent to the first barcodes, and the second population of granules comprises oligonucleotides containing second capture probes, second barcodes and barcode primer binding sites adjacent to the second barcodes; (b) obtaining a first and a second plurality of polynucleotides, wherein: the first plurality of polynucleotides comprises first target nucleic acids, wherein the first plurality of polynucleotides is in solution, and the second plurality of polynucleotides comprises second target nucleic acids, wherein the second plurality of polynucleotides is in solution; (c) hybridising the first target nucleic acids with the first capture probes to obtain hybridised first granules and hybridising the second target nucleic acids with the second capture probes to obtain hybridised second granules; (d) random distribution of hybridized first granules and hybridized second granules on matrix; (e) decoding the positions of the first and second beads on the array by sequencing the first and second bar codes on the array; and (f) extending the first and second capture probes to obtain nucleotide sequence data for the first and second target nucleic acids.

EFFECT: invention provides accurate genotyping.

43 cl, 15 dwg, 5 ex

Description

RELATED APPLICATIONS

[0001] This application claims priority to WIPO Provisional Application No. 62/909,014, filed October 1, 2019, entitled “METHODS AND COMPOSITIONS FOR SEQUENCE NUCLEIC ACID ON AN ARRAY”; and U.S. Provisional Application No. 62/903,108, filed September 20, 2019, entitled “METHODS AND COMPOSITIONS FOR HIGH-THROUGHPUT GENOTYPING ON ARRAYS USING INDEXES AND BARCODES,” each of which is incorporated herein by reference in its entirety.

FIELD OF APPLICATION OF THE INVENTION

[0002] Some embodiments provided herein include methods and compositions for detecting target ligands on an array. In some embodiments, a capture probe specifically binds to a target ligand from a sample, the position of beads containing the capture probe in the array is determined, and the bead is decoded to determine the capture probe and the sample. In some embodiments, a barcode indicates the presence of a capture probe attached to the bead; and an index indicates a subpopulation of beads. Some embodiments relate to sequencing target polynucleotides from several different nucleic acid samples on a bead array.

PREREQUISITES FOR THE CREATION OF THE INVENTION

[0003] Detection of specific nucleotide sequences present in a biological sample is used, for example, as a method for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying genetic predisposition to a particular disease, and assessing the response to various forms of treatment. A common technique for detecting specific nucleotide sequences in a biological sample is nucleic acid sequencing.

[0004] Nucleic acid sequencing technology has evolved significantly from the chemical digestion methods used by Maxam and Gilbert and the chain extension methods used by Sanger. Several sequencing techniques are now in use that allow parallel processing of thousands of nucleic acids, all on a single chip. Some platforms include bead-based and microarray-based configurations in which silica beads are functionalized with probes, depending on the use of such configurations in applications including sequencing, genotyping, and gene expression profiling.

[0005] Methods for genotyping different samples on existing bead-based arrays require spacers to physically subdivide different regions of the bead microarray into multiple sectors. Individual samples are then loaded into each individual section created by the spacer. However, such methods can be used with relatively small numbers of input samples, but they prove labor-intensive or difficult to control as the sample density per bead chip increases from 24 to 96, 384, 1536 or more samples per bead chip.

SUMMARY OF THE INVENTION

[0006] Some embodiments include a method for sequencing target nucleic acids on an array, comprising: (a) providing first and second populations of beads, wherein: the first population of beads comprises oligonucleotides comprising first capture probes, first barcodes, and barcode primer binding sites adjacent to the first barcodes, and a second population of beads comprises oligonucleotides comprising second capture probes, second barcodes, and barcode primer binding sites adjacent to the second barcodes; (b) providing a first and second plurality of polynucleotides, wherein: the first plurality of polynucleotides comprises first target nucleic acids, wherein the plurality of first polynucleotides are in solution, and the second plurality of polynucleotides comprises second target nucleic acids, wherein the plurality of second polynucleotides are in solution; (c) hybridizing first target nucleic acids to first capture probes to produce hybridized first beads and hybridizing second target nucleic acids to second capture probes to produce hybridized second beads; (d) randomly distributing the hybridized first beads and the hybridized second beads on an array; (e) decoding the positions of the first and second beads on the array by sequencing the first and second barcodes; and (f) extending the first and second capture probes to obtain nucleotide sequence data for the first and second target nucleic acids.

[0007] In some embodiments, the first plurality of polynucleotides comprises first indices and index primer binding sites adjacent to the first indices; and the second plurality of polynucleotides comprises second target nucleic acids, second indices, and index primer binding sites adjacent to the second indices.

[0008] In some embodiments, the first or second plurality of polynucleotides are produced by labeling a nucleic acid sample with a plurality of transposomes. In some embodiments, the plurality of transposomes comprise a first or second index. Some embodiments also include adding adapters to the labeled nucleic acid sample, wherein the adapters comprise the first or second index. Some embodiments also include amplifying the labeled nucleic acid sample with primers comprising the first or second index.

[0009] In some embodiments, extending the first and second capture probes incorporates sequences complementary to the first and second indices and the first and second index primer binding sites into the extended capture probes.

[0010] In some embodiments, the first population of beads comprises first indices and index primer binding sites adjacent to the first indices, and the second population of beads comprises second indices and index primer binding sites adjacent to the second indices. In some embodiments, the oligonucleotides of the first population of beads comprise first indices; and the oligonucleotides of the second population of beads comprise second indices. In some embodiments, the first indices indicate a source of the first target nucleic acids, and the second indices indicate a source of the second target nucleic acids. In some embodiments, the first indices match each other and the second indices match each other.

[0011] Some embodiments also include sequencing the first and second indices. In some embodiments, sequencing the first and second indices includes extension primers hybridized to index primer binding sites. In some embodiments, the index primer binding sites are the same.

[0012] In some embodiments, the first and second target nucleic acids are obtained from different nucleic acid samples. In some embodiments, the first and second target nucleic acids are obtained from different genomic DNAs.

[0013] In some embodiments, the first and second barcodes indicate a nucleotide sequence of a first or second capture probe. In some embodiments, the first barcodes are different from each other and the second barcodes are different from each other. In some embodiments, sequencing the first and second barcodes includes extension primers hybridized to barcode primer binding sites. In some embodiments, the barcode primer binding sites are the same.

[0014] In some embodiments, extending the first and second capture probes comprises polymerase extension. In some embodiments, extending the first and second capture probes comprises adding one nucleotide to the capture probe. Some embodiments also include ligating locus-specific oligonucleotides to the extended capture probes. In some embodiments, extending the first and second capture probes comprises ligating locus-specific oligonucleotides to the capture probes.

[0015] In some embodiments, step (c) is performed in solution.

[0016] In some embodiments, the matrix is located on the surface of the flow cell. In some embodiments, the first and second beads are configured to be attached to the matrix. In some embodiments, the first and second beads comprise biotin, streptavidin, or a derivative thereof; and the matrix comprises biotin, streptavidin, or a derivative thereof. In some embodiments, the first and second beads are magnetic.

[0017] Some embodiments include a method for sequencing target nucleic acids on an array, comprising: (a) obtaining first and second populations of beads, wherein: the first population of beads comprises oligonucleotides comprising first capture probes, first barcodes, and barcode primer binding sites adjacent to the first barcodes, and the second population of beads comprises oligonucleotides comprising second capture probes, second barcodes, and barcode primer binding sites adjacent to the second barcodes; (b) obtaining a first and a second plurality of polynucleotides, wherein: the first plurality of polynucleotides comprises first target nucleic acids, first indices, and index primer binding sites adjacent to the first indices, wherein the plurality of first polynucleotides is in solution, and a second plurality of polynucleotides comprises second target nucleic acids, second indices, and second primer binding sites adjacent to the second indices, wherein the plurality of second polynucleotides is in solution; (c) hybridizing the first target nucleic acids to first capture probes to obtain hybridized first beads and hybridizing the second target nucleic acids to second capture probes to obtain hybridized second beads; (d) randomly distributing the hybridized first beads and the hybridized second beads on an array; (e) decoding the positions of the first and second beads on the array by sequencing the first and second barcodes; (f) extending the first and second capture probes to obtain nucleotide sequence data for the first and second target nucleic acids; and (g) determining a source of the nucleotide sequence data of the first and second target nucleic acids by sequencing the first and second indices.

[0018] In some embodiments, the first plurality of polynucleotides comprises first indices and index primer binding sites adjacent to the first indices; and the second plurality of polynucleotides comprises second target nucleic acids, second indices, and index primer binding sites adjacent to the second indices.

[0019] In some embodiments, the first or second plurality of polynucleotides are produced by labeling a nucleic acid sample with a plurality of transposomes. In some embodiments, the plurality of transposomes comprise a first or second index. Some embodiments also include adding adapters to the labeled nucleic acid sample, wherein the adapters comprise the first or second index. Some embodiments also include amplifying the labeled nucleic acid sample with primers comprising the first or second index.

[0020] In some embodiments, extending the first and second capture probes incorporates sequences complementary to the first and second indices and the first and second index primer binding sites into the extended capture probes.

[0021] Some embodiments also include a method for sequencing target nucleic acids on an array, comprising: (a) obtaining first and second populations of beads, wherein: the first population of beads comprises oligonucleotides comprising first capture probes, first barcodes, and barcode primer binding sites adjacent to the first barcodes, and first indices and index primer binding sites adjacent to the first indices, and the second population of beads comprises oligonucleotides comprising second capture probes, second barcodes, and barcode primer binding sites adjacent to the second barcodes, and second indices and index primer binding sites adjacent to the second indices; (b) obtaining a first and a second plurality of polynucleotides, wherein: the first plurality of polynucleotides comprises first target nucleic acids, wherein the plurality of first polynucleotides is in solution, and the second plurality of polynucleotides comprises second target nucleic acids, wherein the plurality of second polynucleotides is in solution; (c) hybridizing the first target nucleic acids to first capture probes to obtain hybridized first beads and hybridizing second target nucleic acids to second capture probes to obtain hybridized second beads; (d) randomly distributing the hybridized first beads and the hybridized second beads on an array; (e) decoding the positions of the first and second beads on the array by sequencing the first and second barcodes; and (f) extending the first and second capture probes to obtain nucleotide sequence data for the first and second target nucleic acids.

[0022] In some embodiments, the oligonucleotides of the first population of beads comprise first indices; and the oligonucleotides of the second population of beads comprise second indices. In some embodiments, the first indices indicate a source of the first target nucleic acids, and the second indices indicate a source of the second target nucleic acids. In some embodiments, the first indices match each other and the second indices match each other.

[0023] Some embodiments also include sequencing the first and second indices. In some embodiments, sequencing the first and second indices includes extension primers hybridized to index primer binding sites. In some embodiments, the index primer binding sites are the same.

[0024] In some embodiments, the first and second target nucleic acids are obtained from different nucleic acid samples. In some embodiments, the first and second target nucleic acids are obtained from different genomic DNAs.

[0025] In some embodiments, the first and second barcodes indicate a nucleotide sequence of a first or second capture probe. In some embodiments, the first barcodes are different from each other and the second barcodes are different from each other. In some embodiments, sequencing the first and second barcodes includes extension primers hybridized to barcode primer binding sites. In some embodiments, the barcode primer binding sites are the same.

[0026] In some embodiments, extending the first and second capture probes comprises polymerase extension. In some embodiments, the first and second capture probes comprise adding one nucleotide to the capture probe. Some embodiments also comprise ligating locus-specific oligonucleotides to the extended capture probes. In some embodiments, extending the first and second capture probes comprises ligating locus-specific oligonucleotides to the capture probes.

[0027] In some embodiments, step (c) is performed in solution.

[0028] In some embodiments, the flow cell comprises an array. In some embodiments, the array comprises a plurality of wells. In some embodiments, the first and second beads are configured to be attached to the array. In some embodiments, the first and second beads comprise biotin, streptavidin, or a derivative thereof; and the array comprises biotin, streptavidin, or a derivative thereof. In some embodiments, the first and second beads are magnetic.

[0029] Some embodiments comprise a kit comprising: a plurality of populations of beads comprising oligonucleotides attached to the beads, wherein the oligonucleotides comprise indices, index primer binding sites adjacent to the indices, capture probes, barcodes, and barcode primer binding sites adjacent to the barcodes, wherein the indices are different between the populations of beads. In some embodiments, the index primer binding sites are the same across the plurality of populations. In some embodiments, the barcodes indicate a nucleotide sequence of the capture probes. In some embodiments, the barcodes are different within the population of beads. In some embodiments, the barcode primer binding sites are the same across the plurality of populations. Some embodiments also comprise a reagent selected from: a locus-specific oligonucleotide; a transposome for labeling a nucleic acid sample; a transposome comprising an index and an index primer binding site; an adapter comprising an index and an index primer binding site; a primer capable of hybridizing to the index primer binding site or its complement; and a primer capable of hybridizing to the barcode primer binding site or its complement. Some embodiments also include a flow cell.

[0030] Some embodiments include a method for producing an indexed population of beads, comprising: (a) producing a population of beads, wherein each bead comprises an adapter, a capture probe, and a first polynucleotide comprising a barcode and a barcode primer binding site; (b) producing a plurality of index polynucleotides, wherein each index polynucleotide comprises an index and an index primer binding site; and (c) attaching the plurality of index polynucleotides to the population of beads via adapters, thereby producing an indexed population of beads.

[0031] In some embodiments, (c) comprises extension of adapters by polymerase extension.

[0032] In some embodiments, each index polynucleotide comprises an adaptor binding site, and attachment comprises hybridization of the adaptor binding sites to adaptors.

[0033] In some embodiments, (c) comprises ligating index polynucleotides with adapters.

[0034] In some embodiments, the attachment comprises hybridizing the shunt polynucleotide to an adapter and an index polynucleotide.

[0035] In some embodiments, (c) comprises attaching a plurality of index polynucleotides to the adaptors of the bead population via a chemically reactive functional group. In some embodiments, the attachment comprises a click chemistry reaction.

[0036] In some embodiments, the first polynucleotides of the population of granules comprise capture probes that differ from each other.

[0037] In some embodiments, the index of each index polynucleotide is the same.

[0038] In some embodiments, the first polynucleotide comprises a capture probe.

[0039] Some embodiments also include contacting the indexed population of beads with a plurality of nucleic acids comprising a target nucleic acid.

[0040] Some embodiments also include mixing an indexed population of beads in contact with a plurality of nucleic acids comprising a nucleic target with an additional indexed population of beads, wherein the additional indexed population of beads comprises an index polynucleotide comprising an index different from the index of the indexed population of beads in contact with the plurality of nucleic acids.

[0041] In some embodiments, the capture probe comprises a protein.

[0042] In some embodiments, the method is performed on a flow cell.

[0043] Some embodiments include a method for detecting a target ligand, comprising: (a) obtaining a population of beads, each bead comprising a capture probe, a first polynucleotide comprising a barcode, and a barcode primer binding site; (b) obtaining an index polynucleotide comprising an index, an index primer binding site, and an adaptor capable of binding to the barcode primer binding site; (c) specifically binding the target ligand to the capture probe; (d) hybridizing the index polynucleotide to the first polynucleotide via the adaptor; (e) detecting the target ligand on the array; and (f) detecting the index and the barcode of the first polynucleotide.

[0044] In some embodiments, (e) includes distributing a population of granules on a matrix.

[0045] In some embodiments, (f) comprises hybridizing an index primer to an index primer binding site and determining the index sequence.

[0046] In some embodiments, dehybridizing an index polynucleotide from a first polynucleotide; hybridizing a barcode primer to a barcode primer binding site; and extending the barcode primer to determine a barcode sequence.

[0047] In some embodiments, the index polynucleotide further comprises a cleavable linker located between the adapter and the index, and (i) comprises: (i) cleaving the cleavable linker; and (ii) extending the adapter to determine the barcode sequence.

[0048] In some embodiments, the capture probe comprises a protein.

[0049] In some embodiments, the target ligand comprises a target nucleic acid. In some embodiments, the first polynucleotide comprises a capture probe. In some embodiments, (e) comprises extending the first polynucleotide hybridized to the target nucleic acid. In some embodiments, extending comprises adding a detectable dideoxynucleotide.

[0050] In some embodiments, the method is performed on a flow cell.

[0051] Some embodiments include a method for detecting a target ligand, comprising: (a) obtaining a population of beads, each bead comprising a capture probe, a first polynucleotide comprising a barcode, a barcode primer binding site, and a second polynucleotide; (b) obtaining an index polynucleotide comprising an index, an index primer binding site, and an adapter; (c) specifically binding the target ligand to the capture probe; (d) attaching the index polynucleotide to the second polynucleotide via the adapter; (e) detecting the target ligand on the array; and (f) detecting the index and barcode of the first polynucleotide.

[0052] In some embodiments, the second polynucleotide comprises a barcode and a barcode primer binding site.

[0053] In some embodiments, (d) comprises adding a reactive functional group to the second polynucleotide, wherein the adapter is capable of attaching to the reactive functional group. In some embodiments, adding the reactive functional group comprises a click chemistry reaction.

[0054] In some embodiments, (e) includes distributing a population of granules on a matrix.

[0055] In some embodiments, (f) comprises hybridizing an index primer to an index primer binding site and determining the index sequence.

[0056] In some embodiments, (f) comprises hybridizing a barcode primer to a barcode primer binding site and determining the barcode sequence.

[0057] In some embodiments, the capture probe comprises a protein.

[0058] In some embodiments, the target ligand comprises a target nucleic acid. In some embodiments, the first polynucleotide comprises a capture probe. In some embodiments, (e) comprises extending the first polynucleotide hybridized to the target nucleic acid. In some embodiments, extending comprises adding a detectable dideoxynucleotide.

[0059] In some embodiments, the method is performed on a flow cell.

[0060] Some embodiments include a method of detecting a target ligand on an array, comprising: (a) providing first and second populations of beads, wherein each bead comprises: a capture probe, wherein the capture probe is capable of specifically binding to the target ligand, a nucleic acid encoding a barcode and a barcode primer binding site, wherein the barcode points to the capture probe, and a nucleic acid encoding an index and an index primer binding site, wherein the index points to a source of the bead from the first population or the second population; (b) contacting the first population of beads with a first sample comprising a first target ligand, wherein the first target ligand specifically binds to the capture probe of the first population of beads, and thereby providing a target-bound first population of beads; (c) contacting the second population of beads with a second sample comprising a second target ligand, wherein the second target ligand specifically binds to a capture probe of the second population of beads, thereby producing a target-bound second population of beads; (d) randomly distributing the target-bound first population of beads and the target-bound second population of beads on the matrix; (e) detecting the position of the beads comprising the first target ligand and the second target ligand on the matrix; and (f) determining an index sequence and a barcode of the beads comprising the first target ligand and the second target ligand on the matrix.

[0061] In some embodiments, the capture probe comprises a polynucleotide. In some embodiments, the target ligand comprises a nucleic acid. In some embodiments, detecting the position of beads comprising the first target ligand and the second target ligand on the array comprises extending the capture probe by polymerase extension or ligation.

[0062] In some embodiments, the capture probe comprises a protein.

[0063] In some embodiments, step (e) is performed after step (f).

[0064] In some embodiments, the barcodes of the first population of granules comprise different barcodes from each other, and the barcodes of the second population of granules comprise different barcodes from each other.

[0065] In some embodiments, the indices of the first population of granules are identical to each other, and the indices of the second population of granules are identical to each other.

[0066] In some embodiments, the matrix is located on the surface of the flow cell. In some embodiments, the first and second populations of beads are adapted to attach to the matrix. In some embodiments, the first and second populations of beads comprise biotin, streptavidin, or a derivative thereof; and the matrix comprises biotin, streptavidin, or a derivative thereof. In some embodiments, the first and second populations of beads are magnetic.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0067] FIG. 1 shows an exemplary embodiment of a polynucleotide comprising a barcode, a primer binding site, and a capture probe attached to a bead via a 5' linker.

[0068] FIG. 2 shows an exemplary embodiment of a polynucleotide comprising a capture probe, a barcode, and a primer binding site attached to a bead via a 5' linker, with a cleavable linker present between the capture probe and the barcode.

[0069] FIG. 3 shows an exemplary embodiment of a polynucleotide comprising a barcode, a primer binding site, and a capture probe attached to a bead via a 3' linker.

[0070] FIG. 4 shows an exemplary embodiment of a polynucleotide comprising a spacer, a barcode, a primer binding site, and a capture probe attached to a bead via a 5' linker.

[0071] FIG. 5A shows an example of an embodiment of a bead with an attached first polynucleotide containing a barcode, a barcode primer binding site, and a capture probe, and with an attached second polynucleotide containing an index and an index primer binding site.

[0072] FIG. 5B shows an exemplary embodiment of a target nucleic acid comprising a single nucleotide polymorphism (SNP) hybridized to a capture probe attached to a bead.

[0073] FIG. 5C shows an example of an embodiment of a capture probe extended by a detectable marker.

[0074] FIG. 5D shows an example of an embodiment of a barcode primer hybridized to a barcode primer binding site and an extension of the barcode primer.

[0075] FIG. 5E shows an example of an embodiment of an index primer hybridized to an index primer binding site and an extension of the index primer.

[0076] FIG. 6A shows an example of an embodiment of a bead comprising a single polynucleotide comprising a capture probe (left); a bead comprising a nucleic acid capture probe, a polynucleotide comprising a barcode indicative of the capture probe, and a polynucleotide comprising an index for distinguishing a subpopulation of beads from another subpopulation of beads (middle); and a bead comprising a capture probe comprising an antibody or antigen-binding fragment of an antibody, a polynucleotide comprising a barcode indicative of the capture probe, and a polynucleotide comprising an index for distinguishing a subpopulation of beads from another subpopulation of beads (right).

[0077] FIG. 6B shows exemplary embodiments of a bead comprising a first and second capture probe hybridized to a target nucleic acid, wherein the first capture probe comprises a cleavable linker (left); and a bead comprising a protein capture probe that transiently binds a substrate to generate a signal comprising a detectable label (right).

[0078] FIG. 6C shows an example of a dual-probe assay in which a target nucleic acid is hybridized to first and second capture probes on a bead, the first capture probe is ligated to the second capture probe, and the cleavable linker is cleaved to produce a bead comprising an extended first capture probe comprising a detectable label.

[0079] FIG. 7A is a photograph of a pool of beads immobilized on a flow cell.

[0080] FIG. 7B shows a bar graph of the number of copies for certain types of granules in certain cells.

[0081] FIG. 7C is a graph comparing the average intensity of C and the average intensity of T. The average intensity of C is the fluorescence intensity coming from the labeled cytosine in the nucleotide pair, and the average intensity of T is the fluorescence intensity coming from the labeled thymidine in the nucleotide pair.

[0082] FIG. 8 shows a histogram of a number of specific types of granules in specific bins for a representative sample.

[0083] FIG. 9 shows an embodiment of a workflow for sequencing target nucleic acids on an array, wherein the polynucleotides containing the target nucleic acids also include an index.

[0084] FIG. 10 shows an embodiment of a workflow for sequencing target nucleic acids on an array, wherein the beads include an index.

[0085] FIG. 11 shows an embodiment of a workflow for obtaining a population of indexed beads and using the indexed beads in a multi-well plate, wherein each well contains a separate sample.

[0086] FIG. 12A is a schematic view of a bead with first and second polynucleotides attached to the bead. The first polynucleotide comprises a code, such as a barcode; a primer A, such as a barcode primer binding site; and a probe, such as a capture probe. The second polynucleotide comprises an index. The second polynucleotide is attached to the bead via a spacer and an X-Y linker.

[0087] FIG. 12A is a schematic view of a bead with first and second polynucleotides attached to the bead. The first polynucleotide comprises a code, such as a barcode; primer A, such as a barcode primer binding site; and a probe, such as a capture probe. The second polynucleotide comprises an index; and primer B, such as an index primer binding site. The second polynucleotide is attached to the bead via a shunt hybridized to the second polynucleotide and an adapter. The second polynucleotide can be ligated to the adapter.

[0088] FIG. 12C is a schematic view of a bead with first and second polynucleotides attached to the bead. The first polynucleotide comprises a code, such as a barcode; primer A, such as a barcode primer binding site; and a probe, such as a capture probe. The second polynucleotide comprises an index; primer B, such as an index primer binding site; and an adapter binding site. The second polynucleotide is attached to the bead by hybridization with an adapter attached to the bead. The adapter may be extended to include the complement of the index; primer B.

[0089] FIG. 13A is a schematic view of a bead with a first polynucleotide attached to the bead. The first polynucleotide comprises a code, such as a barcode; a primer, such as a barcode primer binding site; and a probe, such as a capture probe. A second polynucleotide is shown comprising an index; primer 2, such as an index primer binding site; a spacer, which may include an optional cleavage site, such as a uridine cleavage site; and an adaptor hybridized to the barcode primer binding site.

[0090] FIG. 13B shows an embodiment of a workflow for detecting a target nucleic acid on an array in which an index polynucleotide is hybridized to a first polynucleotide attached to a bead.

[0091] FIG. 14 is a schematic view of a bead with first and second polynucleotides attached to the bead. The first polynucleotide comprises a code, such as a barcode; a primer A, such as a barcode primer binding site; and a probe, such as a capture probe. The second polynucleotide comprises repeats of an index and an index primer binding site.

[0092] FIG. 15A shows an embodiment of a workflow for detecting a target nucleic acid on an array in which an index polynucleotide is attached to a bead via a reactive group.

[0093] FIG. 15B shows an embodiment of a workflow for detecting a target nucleic acid on an array and a continuation of the workflow depicted in FIG. 15A.

DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

[0094] Some embodiments provided herein include methods and compositions for detecting target ligands on an array. In some embodiments, the target ligand may comprise a nucleic acid, a protein, or another antigen. In some embodiments, a capture probe specifically binds to a target ligand from a sample, the position of beads containing the capture probe in the array is determined, and the bead is decoded to determine the capture probe and the sample. In some embodiments, a barcode indicates the presence of a capture probe attached to the bead; and an index indicates a subpopulation of beads. In some embodiments, the barcode and index are determined by sequencing. Some embodiments also include a two-probe assay in which first and second capture probes attached to a bead are ligated to each other in the presence of a target nucleic acid, the end of the ligation product is cleaved from the bead, and the bead containing the extended capture probe is detected and decoded on the array.

[0095] Some embodiments provided herein relate to high-throughput genotyping on arrays. Some embodiments relate to decoding the positions of trace elements in an array. In some embodiments, the trace elements comprise polynucleotides having barcodes and indices. Some embodiments include sequencing the barcodes and indices to determine the positions of the polynucleotides in the array. Some aspects that may be useful for the methods and compositions described herein are described in WO 2020/086746, which is incorporated herein by reference in its entirety.

[0096] Hybridization decoding involves determining the position of a capture probe in a randomly distributed array of capture probes. The method typically involves several successive cycles of hybridization of labeled hybridization probes to one or more regions of the capture probe, visualization of hybridization events, and removal of the hybridization probes. Hybridization decoding requires special reagents, special fluidic devices, and special detectors. In some embodiments, hybridization decoding may take up to 8 hours with 7 8 successive cycles.

[0097] Embodiments provided herein include randomly distributed polynucleotide arrays containing a primer binding site and a barcode. In some embodiments, the barcode can be rapidly sequenced to decode the array using a high-throughput sequencing system. Some embodiments can significantly reduce the time required to decode the array without additional reagents, hybridization probes, or specialized decoding equipment.

[0098] Some embodiments include the use of next generation sequencing (NGS) techniques and bead-based microarrays. Some such embodiments provide highly efficient, low-cost, and high-throughput genotyping assays that can be performed on a standard NGS sequencing platform with minor modifications to substrates and reagents.

[0099] In some multiplexing methods, multiple nucleic acid samples from different sources can be processed in parallel while maintaining physical separation of each sample. Some embodiments presented herein include a method for indexing nucleic acid that, by indexing each sample by an index on the corresponding bead, eliminates the need for physical barriers separating individual samples at all stages. In some embodiments, demultiplexing of the indices is performed by sequencing, and this can be achieved at the user's facility using standard sequencing by synthesis (SBS) chemistry. In addition, by adopting a decoding by sequencing (DBS) approach, which can be implemented at the customer's facility using a standard platform and SBS chemistry, the cost, space, and time constraints associated with internal decoding of the bead array are reduced.

[0100] Some embodiments include a pool of indexed enriched beads comprising the bead depicted in FIG. 5A. In some such embodiments, the bead comprises a first polynucleotide comprising a locus-specific capture probe, a barcode for identifying the position of the bound probe on the array, and a barcode primer binding site for reading the barcode using SBS; and a second polynucleotide comprising an index for multiplexing a sample and an index primer binding site for reading the index using SBS.

[0101] In some embodiments, the complexity of the bead pool is determined by the number of capture probes or the number of composites (N) and the number of supported (S) samples. Thus, a bead pool supporting the number of composites N and samples S will consist of S x N unique bead types. In some embodiments, the capture probes and/or index primers include additional 3' orthogonal blockers to prevent interference during SBS on one of the two oligonucleotides.

[0102] Some embodiments include conducting a genotyping assay in which S pools of beads are loaded into a multi-well plate containing S wells, wherein each pool of beads has a unique sample index, wherein each well contains N unique bead types. After generating a nucleic acid library from the samples such processing of the nucleic acid sample through steps including random primer amplification followed by enzymatic fragmentation and purification, each sample library is added to an index well and allowed to hybridize with capture probes. After hybridization is complete by adding an incorporation mixture that includes fluorescent nucleotides and an appropriate polymerase, a single base extension assay is performed to probe the SNP of interest. At the end of the incorporation, all of the capture bead samples on the plate are combined and loaded into a flow cell. The flow cell can be smooth or patterned, and the surface can be suitably modified to maintain immobilization of the beads at a desired density. In some embodiments, after immobilization of the beads, a SNP read is performed, which includes one scan cycle to read the signal obtained from the fluorescent incorporation at the SNP site. This cycle may include an SBS cycle on the device. A barcode read is also performed to determine the capture probe and the position of a particular bead within the flow cell, which includes 12-20 SBS cycles, depending on the number of constituent beads in the pool. In some embodiments, this step can be replaced by additional sequencing cycles after a particular SNP. In addition, a sample index read is performed, which includes 6-12 SBS cycles to read the sample index. In some embodiments, the entire flow cell assay may include less than about 30 SBS cycles and may be completed in less than 4 hours.

[0103] Some embodiments relate to methods and compositions for sequencing target polynucleotides on an array. Some embodiments relate to sequencing target polynucleotides from several different nucleic acid samples on an array of beads. In some such embodiments, an index sequence is associated with target nucleic acids from a nucleic acid sample.

[0104] In one embodiment of the present invention, spacers are not used to separate different regions of the gene array. Instead, index sequences are added to the samples to differentiate samples hybridized to the beads and support the pooling of samples with a high number of constituents. In some embodiments, indices can be added to either pools of beads or samples.

[0105] Embodiments relate to obtaining libraries of polynucleotides from a plurality of different nucleic acid samples and determining the presence of certain elements, such as single nucleotide polymorphisms, insertions, deletions, in target nucleic acids of each nucleic acid sample. The polynucleotide libraries are screened in parallel on an array for the presence of certain elements of the target nucleic acids.

[0106] In some embodiments, each nucleic acid sample is linked to a different index sequence such that a library of polynucleotides obtained from the nucleic acid sample includes the same index. A target nucleic acid is identified in the polynucleotide library by selectively hybridizing the target nucleic acid to a capture probe attached to a bead, extending the capture probe, and detecting the extension of the capture probe on the array. Each capture probe may include a barcode, and thus the capture probe can be identified by sequencing the barcode associated with the capture probe on the array. In some such embodiments, an oligonucleotide attached to a bead includes a capture probe and a barcode.

[0107] In some embodiments, an index associated with a polynucleotide can be sequenced on an array. The position of the signals on the array for the capture probe extension, the barcode sequence, and the index sequence can identify the presence of a specific element, such as a single nucleotide polymorphism, an insertion, a deletion, in a target nucleic acid from a particular nucleic acid sample. In some embodiments, a plurality of different nucleic acid samples can be tested on a single array.

[0108] As used herein, the term "array" may refer to a population of different microelements, such as microelements comprising polynucleotides that are linked or attached to a surface in such a way that different microelements can be distinguished from one another based on relative location. A single element of the array may include a single copy of a microelement, or multiple copies of a microelement may be present as a population of microelements in a single element of the array. The population of microelements in each element is typically homogeneous and has a single type of microelement. Thus, multiple copies of a single nucleotide sequence may be present on an element, such as multiple nucleic acid molecules having the same sequence.

[0109] In some embodiments, a heterogeneous population of microelements may be present on an element. Thus, an element may, but does not have to, include only one type of microelement and may instead comprise a plurality of different types of microelements, such as a mixture of nucleic acids having different sequences. Adjacent elements of the array may be separated from each other without overlapping. Accordingly, the elements may be located next to each other or separated by a gap. In embodiments in which the elements are spaced apart, adjacent sites may be spaced, for example, by a distance of less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm, 0.5 nm, 100 pm, 50 pm, 1 pm, or any distance within a range of any two of the above distances. The arrangement of elements on the matrix can also be understood in terms of the center-to-center distances between adjacent elements. The matrix used in the invention can have adjacent elements with a center-to-center distance of less than about 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm, 0.5 nm, 100 pm, 50 pm, 1 pm or any distance within the range of any two of the above distances.

[0110] In some embodiments, the distance values described above or elsewhere herein may represent an average distance between adjacent elements of the array. Thus, not all adjacent elements need to be within the stated range unless otherwise specifically stated, such as by specifically stating that the distance is a threshold distance between all adjacent elements of the array. Embodiments may be used with arrays having elements of varying density values. Examples of density ranges for certain embodiments include from about 10,000,000 elements/ ^cm2 to about 2,000,000,000 elements/ ^cm2 ; from about 100,000,000 elements/ ^cm2 to about 1,000,000,000 elements/ ^cm2 ; from about 100,000 elements/ ^cm2 to about 10,000,000 elements/ ^cm2 ; from about 1,000,000 elements/ ^cm2 to about 5,000,000 elements/ ^cm2 ; from about 10,000 elements/ ^cm2 to about 100,000 elements/ ^cm2 ; from about 20,000 elements/ ^cm2 to about 50,000 elements/ ^cm2 ; from about 1,000 elements/ ^cm2 to about 5,000 elements/ ^cm2 , or any density within the range of any two of the foregoing densities.

[0111] As used herein, the term "surface" may refer to a portion of a substrate or support structure that is accessible for contact with reagents, beads, or analytes. The surface may be substantially smooth or flat. Alternatively, the surface may have a rounded or contoured shape. Examples of contours that may be used on a surface include wells, recesses, columns, ridges, channels, or the like. Examples of materials that may be used as a substrate or support structure include glass, such as modified or functionalized glass; plastic, such as acrylic, polystyrene, or a copolymer of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane, or TEFLON; polysaccharides or cross-linked polysaccharides, such as agarose or sepharose; nylon; nitrocellulose; resin; silica or silica-based materials, including silicon and modified silicon; carbon fiber; metal; inorganic glass; a bundle of optical fibers or various other polymers. A surface suitable for use in the invention may be made of a single material or a mixture of several different materials. In some embodiments, the surface comprises wells. In some embodiments, the support structure may comprise one or more layers. Examples of support structures may include a chip, a film, a multi-well plate, and a flow cell.

[0112] As used herein, the term "bead" may refer to a small body made of a rigid or semi-rigid material. The body may have a shape described, for example, as a sphere, oval, microsphere, or other accepted particle shape with symmetrical or asymmetrical dimensions. Examples of materials suitable for beads include glass, such as modified or functionalized glass; plastic, such as acrylic, polystyrene or a copolymer of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane or TEFLON; polysaccharides or cross-linked polysaccharides, such as agarose or sepharose; nylon; nitrocellulose; resin; silica or silica-based materials, including silicon and modified silicon; carbon fiber; metal; inorganic glass; or a variety of other polymers. Examples of beads include controlled pore glass beads, paramagnetic beads, thorium dioxide sol, sepharose beads, nanocrystals, and other beads known in the art. The beads can be made from biological or non-biological materials. Magnetic beads are particularly suitable for use due to the ease of manipulation of magnetic beads with magnets at various stages of the methods described herein. The beads used in certain embodiments can have a diameter, width, or length from about 0.1 μm to about 100 μm, from about 0.1 nm to about 500 nm. In some embodiments, the beads used in certain embodiments of the invention may have a diameter, width, or length of less than about 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm, 0.5 nm, 100 pm, 50 pm, 1 pm, or any diameter, width, or length within a range of any two of the above diameter, width, or length values. The bead size may be selected such that it has a reduced size and, therefore, more elements per unit area can be provided while maintaining sufficient signal (template copies per element) for the analysis of these elements.

[0113] In some embodiments, the polynucleotides may be attached to beads. In some embodiments, the beads may be distributed into wells on the surface of a substrate. Examples of bead arrays that may be used in certain embodiments include BEADARRAY random order technology (Illumina Inc., San Diego, Calif.). Such bead arrays are described in Michael et al., Anal Chem 70, 1242-8 (1998); Walt, Science 287, 451-2 (2000); Fan et al., Cold Spring Harb Symp QuantBiol 68:69-78 (2003); Gunderson et al., Nat Genet 37:549-54 (2005); Bibikova et al. Am J Pathol 165:1799-807 (2004); Fan et al., Genome Res 14:878-85 (2004); Kuhn et al., Genome Res 14:2347-56 (2004); Yeakley et al., NatBiotechnol 20:353-8 (2002); and Bibikova et al., Genome Res 16:383-93 (2006), each of which is incorporated herein by reference in its entirety.

[0114] As used herein, the terms "polynucleotide" and "nucleic acid" may be used interchangeably and may refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes single-, double-, or multi-stranded DNA or RNA. The term "polynucleotide" also refers to both double-stranded and single-stranded molecules. Examples of polynucleotides include a gene or gene fragment, genomic DNA, a genomic DNA fragment, an exon, an intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, non-coding RNA (ncRNA) such as PIWI-interacting RNA (piRNA), small interfering RNA (siRNA), and long non-coding RNA (lncRNA), small hairpin RNA (shRNA), small nuclear RNA (snRNA), microRNA (siRNA), small nucleolar RNA (snshRNA), and viral RNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, polynucleotide probe, primer, or an amplified copy of any of the above. A polynucleotide may include modified nucleotides such as methylated nucleotides and nucleotide analogs, including nucleotides with unnatural bases, nucleotides with modified natural bases such as aza- or deazapurines. A polynucleotide may consist of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T). Uracil (U) may also be present, for example as a natural replacement for thymine if the polynucleotide is RNA. Uracil may also be used in DNA. Thus, the term "sequence" refers to the letter representation of a polynucleotide or any nucleic acid molecule, including natural and unnatural bases.

[0115] As used herein, the term "target nucleic acid" or a grammatical equivalent thereof may refer to nucleic acid molecules or sequences that are to be sequenced, analyzed, and/or further processed. In some embodiments, the target nucleic acid may be attached to an array. In some embodiments, a capture probe may be attached to the array, and the array may subsequently be used to detect the target nucleic acid in a sample that interacts with the probe. In this regard, it should be understood that in some embodiments, the terms "target" and "probe" may be used interchangeably with respect to methods for detecting nucleic acids.

[0116] As used herein, the term "capture probe" may refer to a polynucleotide that has sufficient complementarity to specifically hybridize to a target nucleic acid. The capture probe may function as an affinity binding molecule to isolate the target nucleic acid from other nucleic acids and/or components in a mixture. In some embodiments, the target nucleic acid may be specifically linked to the capture probe via intermediate molecules. Examples of intermediate molecules include linkers, adapters, and other bridging nucleic acids that have sufficient complementarity to specifically hybridize to both the target sequence and the capture probe.

[0117] As used herein, the term "hybridization," "hybridize," or a grammatical equivalent thereof may refer to a reaction in which one or more polynucleotides interact to form a complex that is at least partially formed by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding may occur through Watson-Crick base pairing, Hoogsteen bonding, or any other sequence-specific method. The complex may have two strands forming a duplex structure, three or more strands forming a multi-strand complex, one self-hybridizing strand, or any combination thereof. In addition to hydrogen bonding, the strands may also be cross-linked or linked by other forces.

[0118] As used herein, the terms "extension," "extension," or any grammatical equivalent thereof may refer to the addition of a dNTP to a primer, polynucleotide, or other nucleic acid molecule by an extension enzyme, such as a polymerase. For example, in some methods described herein, the resulting extended primer contains RNA sequence information. Although some embodiments describe performing extension using a polymerase, such as DNA polymerase or reverse transcriptase, extension may be performed by any other method well known in the art. For example, extension may be performed by ligating short lengths of randomly selected oligonucleotides to each other, such as oligonucleotides that have hybridized to the strand of interest.

[0119] As used herein, the terms "ligation," "ligate," or other grammatical equivalents thereof may refer to the joining of two nucleotide chains by a phosphodiester bond. Such a reaction may be catalyzed by a ligase. The term "ligase" refers to a class of enzymes that catalyze this reaction with the hydrolysis of ATP or an analogous triphosphate.

Decoding by sequencing

[0120] Some embodiments of the methods and compositions provided herein include decoding the position of array microfeatures using high-throughput sequencing. In some embodiments, the array microfeatures comprise polynucleotides. In some embodiments, the polynucleotides may be randomly distributed on the surface of a substrate. In some embodiments, the polynucleotide may comprise a primer binding site and a barcode. In some embodiments, the polynucleotide may comprise a capture probe, a primer binding site, and a barcode.

[0121] Some embodiments for decoding the position of polynucleotides in an array may comprise (a) providing a substrate having an array of polynucleotides distributed on the surface of the substrate, wherein each polynucleotide comprises a barcode and a primer binding site; (b) hybridizing a plurality of primers to the primer binding sites; and (c) determining the sequences of the barcodes by extending the hybridized primers. In some such embodiments, the sequence of each barcode may indicate the position of the polynucleotide in the array. For example, in some embodiments, the array may be provided using polynucleotides wherein the barcodes are known to be associated with certain capture probes, such that determining the position of the barcode on the array may indicate the position of the corresponding capture probe. In such embodiments, each polynucleotide may be linked to a capture probe via a common element. For example, each polynucleotide and the capture probe may be linked to the same microelement, such as a bead. In further such embodiments, each polynucleotide may comprise a capture probe.

[0122] In some embodiments, the barcode may comprise a nucleotide sequence that can be used to identify a polynucleotide in an array. The barcode may include a unique nucleotide sequence that is distinguishable from other barcodes. It may also be distinguishable from other nucleotide sequences in polynucleotides and target nucleic acids by the barcode sequence, as well as by the position of the barcode in the polynucleotide, such as by its position 5' of a primer binding site. For example, in some embodiments, the barcode sequence may be present more than once in a plurality of nucleic acids, but a barcode located 5' of a primer binding site may be detected. The barcode may have any desired sequence length sufficient to provide a unique nucleotide sequence within a plurality of barcodes in a population and/or within a plurality of polynucleotides and target nucleic acids being analyzed or tested. In some embodiments, the barcode is a nucleic acid or a region within a polynucleotide in the range of about 6-30 nucleotides. The barcode may comprise, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or it may be longer. For example, the barcode may comprise 35, 40, 45 or 50 nucleotides, or it may be longer. Suitable barcodes for some embodiments are described in U.S. Patent No. 8,053,192, which is incorporated herein by reference in its entirety. In some embodiments, a barcode may distinguish a polynucleotide from another polynucleotide in an array, such that each barcode is different from another barcode. In some embodiments, a barcode may distinguish a population of polynucleotides from another population of polynucleotides in an array, such that a set of barcodes is different from another set of barcodes. Some aspects applicable to the methods and compositions provided herein are described in U.S. Patents 20180334711 A1 and 20190085384 A1, each of which is incorporated herein by reference in its entirety. In some embodiments, a barcode may include a unique molecular identifier (UMI).

[0123] In some embodiments, the primer binding site may be 3' of the barcode, such that a primer hybridized to the primer binding site may be extended to form a complement of the barcode. For example, to obtain a barcode sequence, the primer may be extended. In some embodiments, the primer binding site may be immediately adjacent to the barcode in a polynucleotide. In some embodiments, each primer binding site in a population of polynucleotides may have the same sequence. In some embodiments, a subpopulation of polynucleotides may comprise primer binding sites with a first sequence, and another subpopulation of polynucleotides may comprise primer binding sites with a second sequence. In some embodiments, hybridization of different primers to a plurality of different primer binding sites may be simultaneous, sequential, or iterative.

[0124] Some embodiments include polynucleotides comprising a capture probe. In some embodiments, a capture probe may comprise a sequence that can hybridize to a target nucleic acid. In some embodiments, a population of polynucleotides may comprise capture probes that are different from one another. In some embodiments, each capture probe may be different from one another. In some embodiments, the capture probes may be similar to one another, such as having similar sequences and/or similar sequences of similar length. In some embodiments, a capture probe may differ from one other capture probe by a number of nucleotides less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2, wherein the number of nucleotides may be consecutive nucleotides, non-consecutive nucleotides, inserted nucleotides, or deleted nucleotides in the capture probe. In some embodiments, a capture probe may differ from one other capture probe by one nucleotide. In some embodiments, the primer binding site and the barcode may be 5' of the capture probe. In some embodiments, the primer binding site and the barcode may be 3' of the capture probe.

[0125] Some embodiments include polynucleotides that contain cleavable linkers. In some such embodiments, the cleavable linker can be positioned such that cleavage of the linker can separate the capture probe from the primer binding site and the barcode. In some embodiments, the cleavable linker can be positioned on the polynucleotide between the capture probe and the primer binding site and the barcode. In some embodiments, the cleavable linker can remove a polynucleotide containing a primer binding site and a barcode associated with the capture probe. For example, the polynucleotide and the capture probe can both be associated with a bead. Cleavage of the cleavable linker can result in the removal of the polynucleotide containing the primer binding site and the barcode from the bead.

[0126] In some embodiments, the cleavable linker may have a length corresponding to at least 2, 3, 5, 10, 15, 20, 25, 30, 50, 100, 500 nucleotides, or a length within a range of any two of the foregoing lengths. In some embodiments, the cleavable linker is cleavable by agents such as light, base, acid, and/or an enzyme such as a sequence-specific restriction enzyme or protease. The cleavable linker may include a specific nucleotide sequence, such as an enzyme recognition site, and/or may include specific modified nucleotides that are cleavable by the agent. In some embodiments, the cleavable linker may comprise uracil, which can be cleaved by an exogenous base cleaving agent, such as a DNA glycosylase (uracil DNA glycosylase (UDG)). In some embodiments, the cleavable linker may comprise 8-hydroxyguanine, which can be cleaved by 8-hydroxyguanine DNA glycosylase (formamidopyridine DNA glycosylase protein (FPG)). Other examples of cleavable linkers are described in U.S. Patent Application 2005/0181394, which is incorporated herein by reference in its entirety.

[0127] In some embodiments, the polynucleotides are attached to a substrate. In some embodiments, the substrate may comprise a bead. By a single-point covalent bond to the surface of the substrate at or near the 5' end or 3' end of the polynucleotide, the polynucleotides may be immobilized on a substrate, such as a bead or other surface. In some embodiments, the polynucleotide may comprise a spacer that is attached to the substrate. In some embodiments, the spacer may have a length of at least 2, 3, 5, 10, 15, 20, 25, 30, 50, 100, 500 nucleotides, or a length within a range of any two of the foregoing lengths. Any suitable covalent coupling means known in the art may be used for this purpose. The chemical reaction selected for the coupling depends on the nature of the solid support and any derivatives or functionalizing modifications applied thereto. The polynucleotide may comprise a functional group, which may be a non-nucleotide chemical modification, to facilitate attachment. In some embodiments, the polynucleotide may comprise a sulfur-containing nucleophile, such as a phosphorothioate or thiophosphate, for example, located at the 5'-end. In the case of polyacrylamide hydrogels on a solid support, this nucleophile will bind to a bromoacetamide group present in the hydrogel. An example of a means of attaching a polynucleotide to a solid support is a linkage via 5'-phosphorothioate attachment to a hydrogel consisting of polymerized acrylamide and N-(5-bromoacetamidylpentyl)acrylamide (BRAPA), which is described in U.S. Patent No. 8,168,388, which is incorporated herein by reference in its entirety.

[0128] In some embodiments, the position of the polynucleotides in the array can be decoded by sequencing the barcodes of the polynucleotides. For example, a barcode sequence can be associated with a site on the array, and the site can be associated with a particular capture probe. Some embodiments include next generation sequencing (NGS), which can refer to sequencing methods that allow for massively parallel sequencing of clonally amplified molecules and single nucleic acid molecules. Examples of NGS include sequencing by synthesis (SBS) using reversible dye terminators and sequencing by ligation. SBS monitors the extension of a nucleic acid primer along a nucleic acid template to determine the sequence of nucleic acids in the template. The underlying chemical process can be polymerization. In a specific embodiment of the SBS polymerase-based extension, fluorescently labeled nucleotides are added to the primer in a template-dependent manner so that detection of the order and type of nucleotides added to the primer can be used to determine the template sequence.

[0129] One or more amplified nucleic acids can be subjected to SBS or another detection technique that involves repeated delivery of reagents in cycles. For example, to initiate the first cycle of SBS, one or more labeled nucleotides, DNA polymerase, etc. can be allowed to flow into or through a hydrogel bead containing one or more amplified nucleic acid molecules. Sites where primer extension results in incorporation of a labeled nucleotide can be detected. The nucleotides can further have a reversible termination property that terminates additional extension of the primer after the nucleotide has been added to the primer. For example, a nucleotide analog having a reversible terminator moiety can be added to the primer such that subsequent extension cannot occur until a blocking agent is delivered to remove the moiety. Thus, for embodiments employing reversible termination, delivery of the unblocking reagent to the flow cell may occur before or after detection. Washes may be performed between the various delivery steps. The cycle may then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n.

[0130] Some embodiments of the SBS include detection of a proton released upon incorporation of a nucleotide into an extension product. For example, an electric detector and related commercially available techniques can be used for sequencing based on the detection of released protons. Examples of such sequencing systems include pyrosequencing, such as the commercially available platform from 454 Life Sciences, a subsidiary of Roche; sequencing using γ-phosphate-labeled nucleotides, such as the commercially available platform from Pacific Biosciences; and sequencing using proton detection, such as the commercially available platform from Ion Torrent, a subsidiary of Life Technologies. Some embodiments include pyrosequencing, as described in U.S. Patent Application Nos. 2005/0130173 and 2006/0134633 and U.S. Patent Nos. 4,971,903; 6,258,568; and 6,210,891, each of which is incorporated herein by reference in its entirety. Some embodiments include sequencing by ligation, which is described in U.S. Patent No. 5,599,675; and U.S. Patent No. 5,750,341, each of which is incorporated herein by reference in its entirety.

[0131] In some embodiments, methods may be used that include monitoring DNA polymerase activity in real time. For example, nucleotide incorporation may be detected by fluorescence resonance energy transfer (FRET) interactions between a fluorophore-containing polymerase and γ-phosphate-labeled nucleotides or by zero-mode waveguides (ZMW). Another useful sequencing technique is nanopore sequencing. In some nanopore embodiments, a target nucleic acid or individual nucleotides are removed as the target nucleic acid passes through a nanopore. As the nucleic acid or nucleotide passes through the nanopore, each type of nucleotide can be identified by measuring fluctuations in the electrical conductivity of the pore.

[0132] As shown in FIG. 1, in some embodiments, the array microfeature may include a polynucleotide attached to the bead 10 via a 5' linker 20. The polynucleotide may comprise a barcode 30, a primer binding site 40, and a capture probe 50. The primer 60 may hybridize to the primer binding site and may be extended to provide a barcode sequence to decode the position of the microfeature in the array. In some embodiments, the capture probe may hybridize to target nucleic acids and the capture probe may be extended, such as by a polymerase or by a ligase. In some embodiments, the capture probe may participate in bridge amplification. Bridge amplification methods are described in U.S. Patent Nos. 7,985,565 and 7,115,400, each of which is incorporated herein by reference in its entirety.

[0133] As shown in FIG. 2, in some embodiments, the array microfeature may include a polynucleotide comprising a capture probe 50, a barcode 30, and a primer binding site 40, wherein the polynucleotide is attached to the bead 10 via a 5' linker 20. In some embodiments, the polynucleotide may comprise a cleavable linker 70 between the capture probe and the barcode. In some embodiments, the primer 60 may be hybridized to the primer binding site and the barcode sequence may be determined, thereby decoding the position of the microfeature in the array. In some embodiments, the polynucleotide may be cleaved and the barcode and primer binding site removed from the microfeature comprising the bead and the capture probe. Some of such embodiments provide decoded arrays prior to hybridization of target nucleic acids to the capture probes. In some embodiments, the target nucleic acid can be hybridized to the capture probe at a decoded position in the template. In some embodiments, the hybridized capture probe can be extended, for example, by a polymerase or by a ligase. In some embodiments, the capture probe can participate in bridge amplification.

[0134] As shown in FIG. 3, in some embodiments, the array microfeature may include a polynucleotide comprising a barcode 30 and a primer binding site 40, as well as a capture probe 50 attached to the bead 10 via a 3' linker 25. In some embodiments, the primer binding site may be adjacent to a linker attached to the bead. Some embodiments may include the use of such microfeatures in assays for screening and developing specific polymerases. For example, polymerase activity may be screened for primer sites attached to beads without spacers compared to primer sites attached to beads with spacers. In some embodiments, a target nucleic acid may be hybridized to a capture probe. In some embodiments, the hybridized target nucleic acids may be extended, for example, by a polymerase or by a ligase.

[0135] As shown in FIG. 4, embodiments of the array microelement may include a polynucleotide comprising a spacer 80, a barcode 30, a primer binding site 40, and a capture probe 50 attached to a bead 10 via a 5' linker 20.

Sequencing of multiple target nucleic acids

[0136] Some embodiments include sequencing a plurality of target nucleic acids. In some embodiments, the target nucleic acids may be obtained from different sources, such as different subjects, such as genomic DNA from different subjects. In some embodiments, different target nucleic acids may be associated with different indices, such that an index may identify a specific population of target nucleic acids, such as a population obtained from a single source.

[0137] Some embodiments include obtaining at least a first and a second subpopulation of beads, wherein each bead comprises a first polynucleotide comprising a capture probe, a barcode indicating a capture probe of the same bead, and a 3' end of the barcode for binding to a barcode primer, and a second polynucleotide comprising an index and a 3' end of the index for binding to an index primer, wherein the indices of the first subpopulation are different from the indices of the second subpopulation.

[0138] In some embodiments, the nucleotide sequences of the indices of the first subpopulation of granules comprise the same nucleotide sequence, and the nucleotide sequences of the indices of the second subpopulation of granules comprise the same nucleotide sequence. In some embodiments, the nucleotide sequences of the index primer binding sites comprise the same nucleotide sequence.

[0139] In some embodiments, each of the first and/or second bead subpopulation capture probes comprises nucleotide sequences that differ from one another. For example, the first subpopulation capture probes may differ from one another; and/or the first subpopulation capture probes may differ from one another. In some embodiments, each of the first bead subpopulation capture probes comprises capture probes that have the same nucleotide sequences as the second bead subpopulation capture probes. In some embodiments, a capture probe may comprise a nucleotide sequence that is capable of hybridizing to a single nucleotide polymorphism (SNP) or its complement. In some embodiments, the barcode primer binding sites comprise the same nucleotide sequence.

[0140] Some embodiments also include hybridizing the first target nucleic acids to the first bead subpopulation capture probes and hybridizing the second target nucleic acids to the second bead subpopulation capture probes. In some such embodiments, hybridizing the first target nucleic acids to the first bead subpopulation capture probes and hybridizing the second target nucleic acids to the second bead subpopulation capture probes are performed at different positions. For example, the different positions provide for different reaction volumes, such as different wells, such as different wells in a multi-well plate. For example, in a 96-well plate, 96 different bead subpopulations can be hybridized to 96 different target nucleic acids, wherein each different bead subpopulation hybridizes to a different target nucleic acid in a different well.

[0141] In some embodiments, different subpopulations of beads containing hybridized capture probes and nucleic acids are distributed on a substrate, such as a flat substrate. In some embodiments, the distributed subpopulations of beads comprise an array. In some embodiments, the substrate comprises a plurality of discrete sites. In some embodiments, the substrate comprises a plurality of wells. In some embodiments, the substrate comprises a plurality of channels. In some embodiments, the flow cell comprises the substrate.

[0142] In some embodiments, different subpopulations of beads containing hybridized capture probes and target nucleic acids are combined prior to distribution onto the substrate. In some embodiments, different subpopulations of beads containing hybridized capture probes and target nucleic acids are sequentially distributed onto the substrate. For example, a first subpopulation of beads containing hybridized capture probes and target nucleic acids is distributed onto the substrate prior to distribution onto the substrate of a second subpopulation of beads containing hybridized capture probes and target nucleic acids.

[0143] In some embodiments, the hybridized capture probes are extended. In some embodiments, the hybridized capture probes are extended prior to dispensing the various subpopulations of beads containing the hybridized capture probes and target nucleic acids onto a substrate. In some embodiments, the hybridized capture probes are extended after dispensing the beads containing the hybridized capture probes and target nucleic acids onto a substrate. In some embodiments, the extension of the hybridized capture probes may comprise a polymerase extension. In some embodiments, the extension of the hybridized capture probes may comprise a ligase-based extension, such as ligating an extension probe to a capture probe in the presence of a ligase. In some embodiments, a detectable marker may be added to the extended capture probe during the extension step. In some embodiments, the detectable marker may comprise a fluorescent marker.

[0144] Some embodiments include decoding beads on a substrate. Some embodiments include decoding beads by determining the positions of extended capture probes, barcodes, and indices on a substrate. In some embodiments, the presence of a specific barcode at a particular position on a substrate indicates a specific capture probe at that position. In some embodiments, the presence of a specific index at a position on a substrate indicates that a target nucleic acid of a particular subpopulation of target nucleic acids is associated with a position on the substrate. In some embodiments, the presence of an extended capture probe at a position on a substrate indicates the presence of a particular target nucleic acid in subpopulations of target nucleic acids. In some embodiments, the identity of the barcode, the identity of the index, and the presence of the extended capture probe at the same position on a surface indicates the presence of a particular target nucleic acid in a particular subpopulation of target nucleic acids.

[0145] In some embodiments, decoding beads on a substrate includes detecting the positions of hybridized capture probes or extended capture probes. In some embodiments, detecting the position of hybridized capture probes or extended capture probes includes extending the hybridized capture probes with a detectable marker. In some embodiments, determining the position of the hybridized capture probes or extended capture probes includes at least one round of sequencing by synthesis.

[0146] In some embodiments, decoding the beads on the substrate comprises decoding the position of the indices of the beads containing the extended capture probe. Some embodiments comprise hybridizing a plurality of index primers to the index primer sites and extending the hybridized index primers. In some embodiments, extending the hybridized index primers comprises at least one round of sequencing by synthesis. In some embodiments, decoding the position of the indices of the beads comprises sequencing the indices on the substrate.

[0147] In some embodiments, decoding beads on a substrate comprises decoding the position of barcodes of beads comprising an extended capture probe. Some embodiments comprise hybridizing a plurality of barcode primers to barcode primer sites and extending the hybridized barcode primers. In some embodiments, decoding beads on a substrate comprises extending the hybridized barcode primers, comprising at least one round of sequencing by synthesis. In some embodiments, decoding beads on a substrate comprises decoding the position of barcodes of beads, comprising sequencing the barcodes on the substrate.

[0148] In some embodiments, each of the first and second subpopulations of beads comprises at least 50 capture probes comprising different nucleotide sequences. In some embodiments, each of the first and second subpopulations of beads comprises at least 100, 200, 300, 400, or 500 or more capture probes comprising different nucleotide sequences. In some embodiments, each of the first and second subpopulations of beads comprises at least 5,000 capture probes comprising different nucleotide sequences. In some embodiments, each of the first and second subpopulations of beads comprises at least 50,000 capture probes comprising different nucleotide sequences.

[0149] Some embodiments include at least 10 different subpopulations of granules, wherein each subpopulation comprises an index that is different from another subpopulation. Some embodiments include at least 100 different subpopulations of granules, wherein each subpopulation comprises an index that is different from another subpopulation. Some embodiments include at least 1,000 different subpopulations of granules, wherein each subpopulation comprises an index that is different from another subpopulation. Some embodiments include at least 10,000 different subpopulations of granules, wherein each subpopulation comprises an index that is different from another subpopulation.

[0150] Aspects of some embodiments are shown in FIG. 5A - FIG. 5E. As shown in FIG. 5A, a subpopulation of beads comprises a bead with a first polynucleotide attached that includes a barcode, a barcode primer binding site, and a capture probe, and with a second polynucleotide attached that includes an index and an index primer binding site. Different subpopulations of beads may include different indices. Different subpopulations of beads, each subpopulation having a distinct index, may be distributed across wells of a 96-well plate such that each well contains one subpopulation of beads.

[0151] As shown in FIG. 5B, a target nucleic acid from a population of target nucleic acids is hybridized to a capture probe, wherein the target nucleic acid comprises a SNP. In some embodiments, a subpopulation of target nucleic acids is added to each well containing a subpopulation of beads. Each subpopulation of target nucleic acids can be obtained from a different source, such as a different subject. For example, a subpopulation of target nucleic acids can be obtained by preparing a nucleic acid library from a single source of nucleic acids, such as a single nucleic acid sample from a subject. In some embodiments, the target nucleic acids do not require adapter sequencing.

[0152] As shown in FIG. 5C, a capture probe that is hybridized to a SNP-containing target nucleic acid can be extended. In some embodiments, the extension can be accomplished by adding an incorporation mixture that contains fluorescent nucleotides and an appropriate polymerase. In some embodiments, the extension is a single base extension.

[0153] In some embodiments, all of the beads are combined and distributed over the surface of the flow cell. The flow cell may have a structured surface, and the surface may be modified to maintain immobilization of the beads at a desired density.

[0154] In some embodiments, a SNP is read. In some embodiments, a scanning cycle is performed to read the signal from the incorporation of a capture probe at the SNP site. In some embodiments, this cycle may include at least one cycle of sequencing by synthesis. In some embodiments, the 3' ends of the extended capture probes are cleaved and blocked.

[0155] As shown in FIG. 5D, the barcode primer is hybridized to the barcode primer binding site and the barcode primer is extended. In some embodiments, the extension of the barcode primer comprises reading the barcode. In some embodiments, the extension may comprise at least one cycle of sequencing by synthesis. In some embodiments, the number of cycles of sequencing by synthesis may depend on the number of different barcodes in the bead subpopulation. In some embodiments, the extension may comprise 12-20 cycles of sequencing by synthesis. In some embodiments, the extension allows for identification of the capture probe and the position of a particular bead within the flow cell.

[0156] As shown in FIG. 5E, the index primer is hybridized to the index primer binding site and the index primer is extended. In some embodiments, the extension of the index primer includes reading the index. In some embodiments, the extension may include at least one cycle of sequencing by synthesis. In some embodiments, the number of cycles of sequencing by synthesis may depend on the number of different indices in the plurality of bead subpopulations. In some embodiments, the extension may include 6-12 cycles of sequencing by synthesis.

Specific methods for detecting target ligands

[0157] Some embodiments include methods for detecting target ligands. In some embodiments, target ligands may comprise nucleic acids, proteins, or other antigens. In some embodiments, target ligands are obtained from different sources, such as different samples, different individual subjects, or different populations of subjects.

[0158] In some embodiments, methods for detecting target ligands may comprise obtaining a population of beads, wherein each bead comprises a capture probe that specifically binds to a target ligand. For example, a capture probe may comprise a nucleic acid, an antibody, or an antigen-binding fragment of an antibody. In some embodiments, a bead comprises a first polynucleotide comprising a barcode indicative of a capture probe of the same bead and a 3' end of the barcode for binding to a barcode primer. In some embodiments, each bead also comprises a second polynucleotide comprising an index and a 3' end of the index for binding to an index primer. In some embodiments, the population of beads comprises first and second subpopulations of beads. In some embodiments, the indices of the first subpopulation of beads are different from the indices of the second subpopulation of beads. For example, the indices of the first subpopulation of beads can be used to determine the first subpopulation of beads from the indices of the second subpopulation of beads.

[0159] Some embodiments include contacting first target ligands with capture probes of a first subpopulation of beads and contacting second target ligands with capture probes of a second subpopulation of beads. For example, the first target ligands can be obtained from a first sample of ligands and the second target ligands can be obtained from a second sample of ligands. Some embodiments also provide for distributing on a substrate first and second subpopulations of beads containing specifically bound first and second target ligands. Some embodiments also include detecting capture probes specifically bound to the first and second target ligands distributed on the substrate. Some embodiments also include decoding the positions of the beads containing the detected capture probes on the substrate.

[0160] In some embodiments, the capture probe comprises a nucleic acid and the target ligands comprise nucleic acids. In some embodiments, the first polynucleotide comprises a capture probe. In other embodiments, the capture probe is different from the first polynucleotide. In some embodiments, the capture probes of a subpopulation of beads comprise different nucleotide sequences from each other. In some embodiments, different subpopulations of beads may comprise the same or different capture probes. In some embodiments, the capture probes comprise a nucleotide sequence capable of hybridizing to a single nucleotide polymorphism (SNP) or its complement.

[0161] In some such embodiments, detecting the capture probes specifically bound to the target ligands comprises extending the capture probes specifically bound to the target ligands. In some embodiments, extending may comprise polymerase extension and/or ligase extension. In some embodiments, extending comprises a detectable nucleotide, such as a fluorescently labeled nucleotide. In some embodiments, extending comprises a single nucleotide extension of the capture probes. In some embodiments, extending comprises extending the capture probes with multiple nucleotides.

[0162] In some such embodiments, the capture probe comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the bead subpopulation capture probes specifically bind to different target ligands. In some embodiments, different bead subpopulations may comprise the same or different capture probes. In some such embodiments, different bead subpopulation capture probes specifically bind to the same target ligands.

[0163] In some embodiments, detecting the capture probes specifically bound to the target ligands comprises an immunoassay. For example, the target ligands specifically bound to the capture probes are contacted with a secondary antibody or antigen-binding fragment thereof, wherein the secondary antibody or antigen-binding fragment thereof comprises a detectable label, such as a fluorescent label.

[0164] In some embodiments, the barcode primer binding sites comprise the same nucleotide sequence.

[0165] In some embodiments, the nucleotide sequences of the indices of a subpopulation of granules comprise the same nucleotide sequence and allow one subpopulation of granules to be distinguished from another subpopulation of granules. For example, the nucleotide sequences of the indices of a first subpopulation of granules and the nucleotide sequences of the indices of a second subpopulation of granules comprise the same nucleotide sequence.

[0166] In some embodiments, the nucleotide sequences of the index primer binding sites comprise the same nucleotide sequence.

[0167] In some embodiments, contacting the first target ligand with the capture probes of the first subpopulation of beads and contacting the second target ligand with the capture probes of the second subpopulation of beads are performed at different positions. For example, the different positions may provide for different reaction volumes, such as different volumes in different wells in a microtiter plate.

[0168] Some embodiments also provide for combining the first and second subpopulations of beads before distributing the first and second subpopulations of beads on the substrate. In other embodiments, the first subpopulation of beads is distributed on the substrate before distributing the second subpopulation of beads on the substrate. In some embodiments, the subpopulations of beads are distributed on the substrate before detecting a capture probe specifically bound to the ligand.

[0169] In some such embodiments, detecting capture probes specifically bound to target ligands may also include determining the position of the capture probes specifically bound to target ligands on the substrate.

[0170] In some embodiments, decoding the position of the detected capture probes comprises decoding the position of the indices of the beads containing the detected capture probe. Some embodiments also comprise hybridizing a plurality of index primers to the index primer sites and extending the hybridized index primers. In some embodiments, extending the hybridized index primers comprises at least one round of sequencing by synthesis. In some embodiments, decoding the position of the indices of the beads comprises sequencing the indices on the substrate.

[0171] In some embodiments, decoding the position of the detected capture probes comprises decoding the position of the barcodes of the beads containing the detected capture probe. Some such embodiments include hybridizing a plurality of barcode primers to barcode primer sites and extending the hybridized barcode primers. In some embodiments, extending the hybridized barcode primers comprises at least one round of sequencing by synthesis. In some embodiments, decoding the position of the barcodes of the beads comprises sequencing the barcodes on the substrate.

[0172] In some embodiments, the substrate comprises a plurality of discrete sites. In some embodiments, the substrate comprises a plurality of wells. In some embodiments, the substrate comprises a plurality of channels. In some embodiments, the flow cell comprises the substrate. In some embodiments, the distributed first and second subpopulations of beads comprise a matrix.

[0173] In some embodiments, each of the first and second subpopulations of granules comprises at least 50, 100, 500, 1000, or 5000 different capture probes from each other, or any number of capture probes in the range between any two of the above quantities. Some embodiments also comprise at least 5, 10, 20, 50, 100, 200, 500, 1000 different subpopulations of granules, wherein each subpopulation comprises an index that is different from another subpopulation, or any number of different subpopulations of granules between any two of the above quantities.

[0174] FIG. 6A shows, on the left, an embodiment that comprises a bead 200 having a polynucleotide 210 attached to the bead. The polynucleotide comprises a capture probe that is hybridized to a target nucleic acid 220. The capture probe is extended by a nucleotide that comprises a detectable label 230. In some embodiments, the polynucleotide may comprise a barcode indicative of the capture probe and a barcode primer binding site suitable for sequencing and identifying the barcode. In some embodiments, the polynucleotide may also include an index indicative of a subpopulation of beads from another subpopulation of beads and an index primer binding site suitable for sequencing and determining the index. In some embodiments, the bead may be dispersed in an array on a substrate and decoded. Decoding may comprise determining the position of a detectable label on the array; determining a barcode attached to the bead on the array; and/or determining the index attached to the bead on the matrix.

[0175] FIG. 6A shows, in the middle, an embodiment that comprises a bead 200 with a capture probe 240 attached to the bead. The capture probe is hybridized to a target nucleic acid 220. Also attached to the bead is a first polynucleotide 260 comprising a barcode indicative of the capture probe and a barcode primer binding site suitable for sequencing and detecting the barcode. Also attached to the bead is a second polynucleotide 250 comprising an index indicative of a subpopulation of beads from another subpopulation of beads. The capture probe is extended by a nucleotide comprising a detectable label 230. In some embodiments, the bead can be dispersed in an array on a substrate and decoded. Decoding can include determining the position of the detectable label on the array; determining a barcode attached to the bead on the array; and/or determining an index attached to the bead on the array.

[0176] FIG. 6A depicts on the right an embodiment that comprises a bead 200 with a capture probe 270 attached to the bead, wherein the capture probe is an antibody or an antigen-binding fragment of an antibody. The capture probe is specifically bound to a ligand 280. The ligand is also bound to a secondary antibody 290 that comprises a detectable label 230. Also attached to the bead is a first polynucleotide 260 comprising a barcode indicative of the capture probe and a barcode primer binding site suitable for sequencing and detecting the barcode. Also attached to the bead is a second polynucleotide 250 comprising an index indicative of a subpopulation of beads from another subpopulation of beads. In some embodiments, the bead can be dispersed in an array on a substrate and decoded. Decoding can include determining the position of a detectable label on the array; determining a barcode attached to the bead on the array; and/or determining the index attached to the bead on the matrix.

[0177] FIG. 6B shows an embodiment on the right that comprises a bead 200 with a capture probe 330 comprising a protein attached to the bead. In some embodiments, the bead can be dispersed in an array on a substrate. The protein substrate 340 is used to contact the protein to generate a signal comprising a detectable label 230. The position of the signal on the array can be determined. In some embodiments, the bead can comprise a first polynucleotide comprising a barcode indicative of the capture probe and a barcode primer binding site suitable for sequencing and detecting the barcode, also attached to the bead. In some embodiments, the bead can comprise a second polynucleotide comprising an index indicative of a subpopulation of beads from another subpopulation of beads, also attached to the bead. In some embodiments, the bead is decoded on the array. Decoding can include determining the position of the detectable label on the array; determining a barcode attached to the bead on the array; and/or determining the index attached to the bead on the matrix.

[0178] Some embodiments include methods of detecting target ligands on an array, comprising: (a) obtaining first and second populations of beads, wherein each bead comprises: a capture probe, wherein the capture probe is capable of specifically binding to a target ligand, a nucleic acid encoding a barcode and a barcode primer binding site, wherein the barcode points to the capture probe, and a nucleic acid encoding an index and an index primer binding site, wherein the index points to a source of the bead from the first population or the second population, and (b) contacting the first population of beads with a first sample comprising a first target ligand, wherein the first target ligand specifically binds to the capture probe of the first population of beads and thereby obtaining a target-bound first population of beads; (c) contacting the second population of beads with a second sample comprising a second target ligand, wherein the second target ligand specifically binds to a capture probe of the second population of beads, thereby producing a target-bound second population of beads; (d) randomly distributing the target-bound first population of beads and the target-bound second population of beads on the array; (e) detecting the location of the beads comprising the first target ligand and the second target ligand on the array; and (f) determining an index sequence and a barcode of the beads comprising the first target ligand and the second target ligand on the array. In some embodiments, the capture probe comprises a polynucleotide. In some embodiments, the target ligand comprises a nucleic acid. In some embodiments, detecting the location of the beads comprising the first target ligand and the second target ligand on the array comprises extending the capture probe by polymerase extension or ligation. In some embodiments, the capture probe comprises a protein. In some embodiments, step (e) is performed after step (f). In some embodiments, the barcodes of the first population of beads comprise different barcodes from each other, and the barcodes of the second population of beads comprise different barcodes from each other. In some embodiments, the indices of the first population of beads are identical to each other, and the indices of the second population of beads are identical to each other. In some embodiments, the matrix is located on the surface of the flow cell. In some embodiments, the first and second populations of beads are adapted to attach to the matrix. In some embodiments, the first and second populations of beads comprise biotin, streptavidin, or a derivative thereof; and the matrix comprises biotin, streptavidin, or a derivative thereof. In some embodiments, the first and second populations of beads are magnetic.

Sequencing and analysis of target nucleic acids

[0179] Some embodiments include sequencing and/or analyzing target nucleic acids. Some embodiments include decoding the positions of polynucleotides on an array according to the methods provided herein; hybridizing the target nucleic acid to capture probes; extending the capture probes; and detecting an extension of the capture probes hybridized to the target nucleic acid at a position on the array. In some embodiments, the positions of the polynucleotides on the array may be decoded before hybridizing the target nucleic acid to the polynucleotides. In some embodiments, the positions of the polynucleotides on the array may be decoded after detecting an extension of the capture probes hybridized to the target nucleic acid. In some embodiments, each polynucleotide may be linked to a capture probe via a common feature. For example, each polynucleotide and the capture probe may be linked to the same microfeature, such as a bead. In further such embodiments, each polynucleotide may comprise a capture probe.

[0180] Some embodiments include single base extension (SBE) of capture probes. In some embodiments, SBE can be used to detect alleles, mutations, or other elements of target nucleic acids. Briefly, SBE uses a capture probe that hybridizes to a target genomic fragment at a position that is proximal or adjacent to a detection position, wherein the detection position indicates a particular locus. A polymerase can be used to extend the 3' end of the capture probe with a nucleotide analog labeled with a detection label. Based on the accuracy of the enzyme, a nucleotide is incorporated into the capture probe only if it is complementary to the detection position of the target nucleic acid. If desired, the nucleotide can be derivatized such that the use of a blocking group, including reversible blocking groups, cannot result in additional extensions and thus only a single nucleotide is added. The presence of a labeled nucleotide in the extended capture probe can be detected, for example, at a specific location on the template, and the added nucleotide determined to establish the identity of the locus or allele. SBE can be carried out under known conditions, such as those described in U.S. Patent Nos. 9,441,267 and 9,045,796, each of which is incorporated herein by reference in its entirety.

[0181] Some embodiments include allele-specific primer extension (ASPE). In some embodiments, ASPE may involve the extension of capture probes that differ in the nucleotide composition at their 3' end. The ASPE method can be performed using a nucleoside or nucleotide that contains a cleavable linker so that the label can be removed after the probe is detected. This allows for further use of the probes or confirmation that the detected signal is due to the label that has now been removed. Briefly, ASPE can be performed by hybridizing a target nucleic acid to a capture probe that has a 3' sequence region that is complementary to a detection position and a 5' sequence region that is complementary to a sequence that is adjacent to the detection position. Template-directed modification of the 3' portion of the capture probe, such as by adding a labeled nucleotide using a polymerase, produces a labeled extension product, but only if the template includes the target sequence. The presence of such a labeled primer extension product can then be detected, for example, based on its position in the template, indicating the presence of a specific allele. In some embodiments, ASPE can be performed with multiple capture probes that have similar 5' ends such that they anneal adjacent to the same detection position in the target nucleic acid, but different 3' ends such that only the capture probes that have a 3' end that is complementary to the detection position are modified by the polymerase. A capture probe that has a 3' base that is complementary to a particular detection position is called a perfect match (PM) probe for the position, while capture probes that have a 3' mismatch base and are not designed to be extended during the ASPE reaction are mismatch probes (MM) for the position. It is possible to detect the presence of a labeled nucleotide in the RM probe and determine the sequence of the 3' capture probe defined to identify the specific allele at the detection position.

[0182] Some embodiments include methods for decoding the positions of polynucleotides in an array, (a) providing a substrate having an array of polynucleotides distributed on a surface of the substrate, wherein each polynucleotide comprises a 3' end of a barcode for binding to a primer of the barcode, wherein each polynucleotide is linked to a capture probe; (b) hybridizing a plurality of primers to the primer binding sites; (c) determining barcode sequences by extending the hybridized primers, wherein the sequence of each barcode indicates the position of a polynucleotide in the array. In some embodiments, each polynucleotide is linked to a capture probe by a bead. In some embodiments, each polynucleotide comprises a capture probe. In some embodiments, the capture probe is 3' of a primer binding site. In some embodiments, the capture probe is 5' of a barcode. In some embodiments, a capture probe comprises a sequence that is different from the sequence of another capture probe. In some embodiments, a capture probe differs from another capture probe by less than 5 different nucleotides. In some embodiments, each capture probe comprises a different sequence. In some embodiments, the polynucleotides are randomly distributed on the surface of the substrate. In some embodiments, the barcode comprises a different sequence from another barcode. In some embodiments, each barcode comprises a different sequence. In some embodiments, each primer binding site comprises the same sequence. In some embodiments, the polynucleotides are attached to beads. In some embodiments, the beads are distributed in wells. In some embodiments, each polynucleotide comprises a cleavable linker. In some embodiments, the cleavable linker is configured to remove the capture probe from the primer binding site and the barcode. In some embodiments, the substrate comprises wells. In some embodiments, each polynucleotide comprises a spacer. In some embodiments, the spacer is attached to the substrate. In some embodiments, the spacer is attached to the bead. Some embodiments also include hybridizing the target nucleic acid to the capture probes. In some embodiments, hybridizing the target nucleic acid to the capture probes is performed after detecting the barcode sequences. In some embodiments, hybridizing the target nucleic acid to the capture probes is performed before detecting the barcode sequences. Some embodiments also include extending the hybridized target nucleic acid or polynucleotide. In some embodiments, extending involves ligation. Some embodiments also include amplifying the target nucleic acid.

[0183] Some embodiments include methods for sequencing a target nucleic acid. Some such methods (a) decode the positions of polynucleotides in an array according to any of the above methods; (b) hybridize the target nucleic acid to capture probes; (c) extend the capture probes hybridized to the target nucleic acid; and (d) detect the position of the extended capture probes. In some embodiments, (d) detecting the position of the extended capture probes is performed prior to (a) decoding the positions of the polynucleotides in the array. In some embodiments, the capture probes are extended with a ligase. In some embodiments, the capture probes are extended with a polymerase. In some embodiments, the capture probes are extended by the addition of a single nucleotide. Some embodiments also include cleaving primer binding sites and barcodes from the capture probes prior to hybridizing the target nucleic acid to the capture probes. Some embodiments also include cleaving the primer binding sites and barcodes from the capture probes after hybridization of the target nucleic acid to the capture probes.

[0184] Some embodiments include sequencing target polynucleotides obtained from different biological sources on an array. In some such embodiments, polynucleotides comprising target nucleic acids can be obtained from a nucleic acid sample. Examples of polynucleotide samples include genomic DNA samples, cDNA samples, RNA samples, and amplicons from individuals. A polynucleotide can include a target nucleic acid, an index, and an index primer binding site adjacent to the index. The index can be used to indicate the source of the target nucleic acid as a particular nucleic acid sample. For example, polynucleotides obtained from different nucleic acid samples can include different indices. Each index can have a position designed to bind to a particular amplification primer. This index primer binding site can be used to sequence the index by extending a primer hybridized to the index primer binding site. In some embodiments, the index is a nucleic acid or a region within a polynucleotide in the range of about 3 to 30 consecutive nucleotides. The barcode may comprise, for example, 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 or 30 nucleotides, or it may be longer. Some aspects applicable to the methods and compositions provided herein are described in U.S. Patents 20180334711 A1 and 20190085384 A1, each of which is incorporated herein by reference in its entirety. In some embodiments, the barcode or index may include a unique molecular identifier (UMI).

[0185] Polynucleotides can be produced in a variety of ways. In some embodiments, a nucleic acid sample containing target nucleic acids can be labeled with transposomes to produce nucleic acid fragments with ends containing sequences from the transposomes. In some embodiments, the transposomes can include indices and index primer binding sites such that the labeling adds the indices and index primer binding sites to the nucleic acid fragments. For example, an input nucleic acid containing a target nucleic acid can be contacted with a plurality of transposomes. The transposomes can fragment the input nucleic acid and attach adapters to the ends of the nucleic acid fragments. Examples of such labeling reactions are described in U.S. Patent No. 9,040,256, which is incorporated herein by reference in its entirety.

[0186] In some embodiments, polynucleotides containing indices can be obtained by adding adapters to the ends of nucleic acid fragments containing target nucleic acids, wherein the adapters can include indices and index primer binding sites. In some embodiments, polynucleotides containing indices can be obtained by amplifying nucleic acid fragments containing target nucleic acids with primers containing indices and index primer binding sites, such that the amplification products include indices and index primer binding sites.

[0187] In some embodiments, the target nucleic acids of the polynucleotides are hybridized to capture probes. In some embodiments, the capture probes are attached to beads. In some embodiments, the oligonucleotides comprise capture probes. The oligonucleotide may include a capture probe, a barcode, and a barcode primer binding site. In some embodiments, the barcode may be sequenced by extending a primer hybridized to the primer binding site. In some embodiments, the barcode may include a nucleotide sequence that can be used to identify a polynucleotide, such as a capture probe, in an array. The barcode may include a unique nucleotide sequence that is distinguishable from other barcodes. It can also be distinguishable from other nucleotide sequences in polynucleotides and target nucleic acids by the barcode sequence, as well as by the position of the barcode in the polynucleotide, such as by its position adjacent to the barcode primer binding site. For example, in some embodiments, the barcode sequence can be present more than once in a plurality of nucleic acids, but the barcode adjacent to the barcode primer binding site can be detected. The barcode can have any desired sequence length sufficient to provide a unique nucleotide sequence within a plurality of barcodes in a population and/or within a plurality of polynucleotides and target nucleic acids being analyzed or tested. In some embodiments, the barcode is a nucleic acid or a region within a polynucleotide in the range of about 6 to 30 nucleotides. The barcode may comprise, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or it may be longer. Suitable barcodes for some embodiments are described in U.S. Patent No. 8,053,192, which is incorporated herein by reference in its entirety. In some embodiments, the barcode may distinguish a polynucleotide from another polynucleotide in an array such that each barcode is different from another barcode. In some embodiments, the barcode may be used to identify the position of a bead in an array. In some embodiments, the barcode may be used to identify a capture probe.

[0188] In some embodiments, the target nucleic acids of the polynucleotides are hybridized to the capture probes to produce hybridized beads. In some embodiments, the hybridized beads may include an oligonucleotide comprising a barcode, a barcode primer binding site, and a capture probe, wherein the capture probe may be hybridized to the target nucleic acid of the polynucleotide, and the polynucleotide may include an index and an index primer binding site. The hybridized beads may be randomly distributed across the array.

[0189] In some embodiments, sequence information of a target nucleic acid can be obtained by extending a capture probe. In some embodiments, a capture probe can be extended to include sequences complementary to the target nucleic acid. In some such embodiments, the extension can include polymerase extension. In some embodiments, the extension is a single base extension (SBE). In some embodiments, SBE can be used to detect alleles, mutations, or other elements of target nucleic acids.

[0190] In some embodiments, the capture probe can be extended by ligation. For example, a locus-specific oligonucleotide can hybridize to a target nucleic acid at a position adjacent to a site at which the capture probe hybridizes to the target nucleic acid, and then the locus-specific oligonucleotide can be ligated to the capture probe. In some embodiments, the capture probe can be extended such that the extended capture probe includes sequences complementary to an index and an index primer binding site of the polynucleotide.

[0191] In some embodiments, the index is sequenced to determine the source of the target nucleic acid attached to the bead on the array. In some embodiments, the barcode is sequenced to decode the position of the bead on the array. In some embodiments, the barcode is sequenced to identify the capture probe attached to the bead on the array.

[0192] In some embodiments, the matrix is located on the surface of the flow cell. In some embodiments, the beads are configured to be attached to the matrix. For example, the beads may include agents such as biotin, streptavidin, or a derivative thereof; and the matrix comprises biotin, streptavidin, or a derivative thereof. In some embodiments, the beads and the matrix are magnetic.

[0193] Some embodiments include obtaining a plurality of polynucleotides from different nucleic acid samples, hybridizing each plurality of polynucleotides to a population of beads containing capture probes to obtain hybridized beads, and randomly distributing the hybridized beads on an array. An example embodiment includes obtaining first and second populations of beads, wherein the first population of beads comprises oligonucleotides containing first capture probes, first barcodes, and barcode primer binding sites adjacent to the first barcodes, and a second population of beads comprises oligonucleotides containing second capture probes, second barcodes, and barcode primer binding sites adjacent to second barcodes. The first and second plurality of polynucleotides are obtained, wherein the first plurality of polynucleotides comprises first target nucleic acids, wherein the plurality of first polynucleotides are in solution, and the second plurality of polynucleotides comprises second target nucleic acids, wherein the plurality of second polynucleotides are in solution. The first target nucleic acids are hybridized to the first capture probes to produce hybridized first beads, and the second target nucleic acids are hybridized to the second capture probes to produce hybridized second beads. The hybridized first beads and the hybridized second beads on the array are randomly distributed on the array. The positions of the first and second beads on the array are decoded by sequencing the first and second barcodes to determine which beads were bound by each set of target nucleic acids. Nucleotide sequence data is obtained for the first and second target nucleic acids by extending the first and second capture probes.

[0194] In some embodiments, the polynucleotides are indexed as described herein. For example, first and second populations of beads are obtained, wherein the first population of beads comprises oligonucleotides comprising first capture probes, first barcodes, and barcode primer binding sites adjacent to the first barcodes, and the second population of beads comprises oligonucleotides comprising second capture probes, second barcodes, and barcode primer binding sites adjacent to the second barcodes. A first and a second plurality of polynucleotides are obtained, wherein the first plurality of polynucleotides comprises first target nucleic acids, first indices and index primer binding sites adjacent to the first indices, wherein the plurality of first polynucleotides is in solution, and the second plurality of polynucleotides comprises second target nucleic acids, second indices and second primer binding sites adjacent to the second indices, wherein the plurality of second polynucleotides is in solution. The first target nucleic acids are hybridized with the first capture probes to obtain hybridized first granules, and the second target nucleic acids are hybridized with the second capture probes to obtain hybridized second granules. The hybridized first granules and the hybridized second granules are randomly distributed over the matrix. The positions of the first and second granules on the matrix are decoded by sequencing the first and second barcodes. The first and second capture probes are extended to obtain nucleotide sequence data for the first and second target nucleic acids. The source of the nucleotide sequence data of the first and second target nucleic acids is determined by sequencing the first and second indices.

[0195] In some embodiments, the first plurality of polynucleotides comprise first indices and index primer binding sites adjacent to the first indices; and the second plurality of polynucleotides comprise second target nucleic acids, second indices, and index primer binding sites adjacent to the second indices. In some embodiments, the first or second plurality of polynucleotides are produced by labeling a nucleic acid sample with a plurality of transposomes. In some embodiments, the plurality of transposomes comprise the first or second indices. Some embodiments also include adding adapters to the labeled nucleic acid sample, wherein the adapters comprise the first or second indices. Some embodiments also include amplifying the labeled nucleic acid sample with primers comprising the first or second indices. In some embodiments, extending the first and second capture probes introduces sequences complementary to the first and second indices and the first and second index primer binding sites into the extended capture probes.

[0196] In some embodiments, the beads may be indexed. For example, a bead may include a nucleic acid comprising an index and an index primer binding site. In some embodiments, an oligonucleotide attached to a bead may include a barcode, a barcode primer binding site, an index, an index primer binding site. For example, a first population of beads may comprise first indices and index primer binding sites adjacent to the first indices, and a second population of beads may comprise second indices and index primer binding sites adjacent to the second indices. In some embodiments, the oligonucleotides of the first population of beads comprise first indices; and the oligonucleotides of the second population of beads comprise second indices.

[0197] In some embodiments, first and second populations of beads are obtained, wherein the first population of beads comprises oligonucleotides comprising first capture probes, first barcodes, and barcode primer binding sites adjacent to the first barcodes, as well as first indices and index primer binding sites adjacent to the first indices, and the second population of beads comprises oligonucleotides comprising second capture probes, second barcodes, and barcode primer binding sites adjacent to the second barcodes, as well as second indices and index primer binding sites adjacent to the second indices. A first and second plurality of polynucleotides are obtained, wherein the first plurality of polynucleotides comprises first target nucleic acids, wherein the plurality of first polynucleotides is in solution, and the second plurality of polynucleotides comprises second target nucleic acids, wherein the plurality of second polynucleotides is in solution. The first target nucleic acids are hybridized with the first capture probes to obtain hybridized first beads, and the second target nucleic acids are hybridized with the second capture probes to obtain hybridized second beads. The hybridized first beads and the hybridized second beads are randomly distributed on the array. The positions of the first and second beads on the array are decoded by sequencing the first and second barcodes. The first and second capture probes are extended to obtain nucleotide sequence data for the first and second target nucleic acids. In some embodiments, the oligonucleotides of the first population of beads comprise first indices; and the oligonucleotides of the second population of beads comprise second indices.

[0198] In some embodiments, the first indices indicate a source of the first target nucleic acids, and the second indices indicate a source of the second target nucleic acids. In some embodiments, the first indices match each other and the second indices match each other. Some embodiments also include sequencing the first and second indices. In some embodiments, the first and second indices comprise extension primers hybridized to index primer binding sites. In some embodiments, the index primer binding sites are the same. In some embodiments, the first and second target nucleic acids are derived from different nucleic acid samples. In some embodiments, the first and second target nucleic acids are derived from different genomic DNAs.

[0199] In some embodiments, the first and second barcodes indicate a nucleotide sequence of a first or second capture probe. In some embodiments, the first barcodes are different from each other and the second barcodes are different from each other. In some embodiments, sequencing the first and second barcodes includes extension primers hybridized to barcode primer binding sites. In some embodiments, the barcode primer binding sites are the same.

[0200] In some embodiments, extending the first and second capture probes comprises polymerase extension. In some embodiments, extending the first and second capture probes comprises adding one nucleotide to the capture probe. Some embodiments also include ligating locus-specific oligonucleotides to the extended capture probes. In some embodiments, extending the first and second capture probes comprises ligating locus-specific oligonucleotides to the capture probes.

[0201] In some embodiments, hybridization of first target nucleic acids to first capture probes to produce hybridized first beads and hybridization of second target nucleic acids to second capture probes to produce hybridized second beads is performed in solution.

[0202] In some embodiments, the flow cell comprises an array. In some embodiments, the array comprises a plurality of wells, wherein each well comprises one or more beads. In some embodiments, the first and second beads are configured to be attached to the array. In some embodiments, the first and second beads comprise biotin, streptavidin, or a derivative thereof; and the array comprises biotin, streptavidin, or a derivative thereof. In some embodiments, the beads and the array are magnetic.

[0203] An example of implementing sequencing of target nucleic acids on an array, in which polynucleotides containing the target nucleic acid also include an index, is shown in FIG. 9. A DNA sample containing a target nucleic acid containing a single nucleotide polymorphism (star) is labeled, in which the DNA sample is fragmented and amplification primer binding sites are added to each end of the fragments. The fragments are amplified by PCR with primers that bind to the amplification primer binding sites and include a sample index and a read index primer binding site. The sample index and read index primer sequences are incorporated into the amplified products. Beads are obtained by attaching oligonucleotides to the beads. Oligonucleotides contain a linker, a decoding sequence (also known as a barcode sequence), a binding site for the decoding read primer, and a capture probe. The amplified products and beads are combined. The target nucleic acids hybridize to the capture probes. Hybridization can occur in solution or on an array. Hybridized beads are randomly distributed on the array. The capture probe is extended with fully functional nucleotides (FFN) and the extension is detected. The extended capture probe is further extended to incorporate sequences complementary to the sample index and the binding site of the index read primer. The strand containing the target nucleic acid dissociates from the extended capture. The sample index of the extended capture probe is sequenced by extending the primer hybridized to the binding site of the index read primer. The decoding sequence of the extended capture probe is sequenced by extending a primer hybridized to the decoding primer binding site of the readthrough. The source of the target nucleic acid is identified from the sample index sequence. The bead is decoded and the capture probe is identified from the decoding sequence.

[0204] Another example of implementing sequencing of target nucleic acids on an array, in which the beads include an index, is shown in FIG. 10. The beads are formed by joining capture probe oligonucleotides and sample index probe oligonucleotides. The capture probe oligonucleotides include a linker, a decoding sequence, a decoding read primer binding site, and a capture probe. The sample index probe oligonucleotide includes a linker, a sample index, and a read index primer binding site. The beads are combined with fragmented nucleic acids containing target nucleic acids, such as nucleic acids containing a single nucleotide polymorphism of interest (star). The target nucleic acid hybridizes to the capture probe either in solution or on the array. The capture probe is extended with FFN. The extension is detected. The target nucleic acid is dissociated from the extended capture probe. The sample index is sequenced by extending a primer hybridized to the index primer binding site of the read. The decoding sequence of the extended capture probe is sequenced by extending a primer hybridized to the decoding primer binding site of the read. The source of the target nucleic acid is identified from the sample index sequence. The bead is decoded and the capture probe is identified from the decoding sequence.

Some two-probe methods

[0205] Some embodiments include detecting a target nucleic acid with first and second capture probes. In some embodiments, the target nucleic acid comprises a first region capable of hybridizing to a first capture probe and a second region capable of hybridizing to a second capture probe. In some embodiments, a population of beads is obtained in which each bead comprises a first capture probe and a second capture probe. In some embodiments, one of the two capture probes is attached to the bead via a cleavable linker. In some embodiments, one of the capture probes comprises a detectable label, such as a fluorescent label. In some embodiments, the target nucleic acid hybridizes to the capture probes to form a double-stranded nucleic acid comprising a single-stranded gap. The gap is filled and the cleavable linker is cleaved. The extended capture probe is detected. In some embodiments, one of the capture probes comprises a detectable label, such as a fluorescent label. In some embodiments, the detectable label is incorporated into the extended capture probe during gap filling.

[0206] In some embodiments, the second capture probe is attached to the bead via a cleavable linker, and the second capture probe comprises a detectable label, such as a fluorescent label. Examples of cleavable linkers include linkers that can be cleaved chemically, by enzymes such as endonucleases, and by certain frequencies of light. In some embodiments, each bead also comprises a first polynucleotide comprising a barcode indicating the first or second capture probe and a 3' end of the barcode for binding to a barcode primer. In some embodiments, the first capture probe comprises a first polynucleotide. In some embodiments, the first capture probe is different from the first polynucleotide.

[0207] In some embodiments, the end of the first capture probe is ligated to the end of the second capture probe. In some embodiments, the first capture probe is ligated to the second capture probe by hybridizing a target nucleic acid to the first and second capture probes of a bead of a population of beads to produce a double-stranded nucleic acid comprising a single-stranded gap between the first and second capture probes, and filling the gap between the first and second capture probes. In some embodiments, filling the gap can be accomplished using a polymerase and/or a ligase. In some embodiments, the cleavable linker is cleaved to produce a bead comprising a first capture probe comprising a detectable label. In some embodiments, the population of beads is distributed on a substrate. In some embodiments, the population of beads is distributed on a substrate after cleavage of the cleavable linker. In some embodiments, the population of beads is distributed on the substrate before the cleavable linker is cleaved or before the first capture probe is ligated to the second capture probe. In some embodiments, the position of the bead containing the first capture probe containing the detectable label on the substrate is determined.

[0208] In some embodiments, decoding the position of a bead comprising a first capture probe comprising a detectable label on a substrate comprises decoding the position of a barcode of the bead comprising the first capture probe comprising a detectable label on the substrate. Some such embodiments may comprise hybridizing a barcode primer to a barcode primer site and extending the hybridized barcode primer. In some embodiments, extending the hybridized barcode primer comprises at least one round of sequencing by synthesis. In some embodiments, decoding the position of the barcode of the bead comprises sequencing the barcode on the substrate.

[0209] In some embodiments, each bead comprises a second polynucleotide comprising an index indicating a source of a target nucleic acid and a 3' end of the index for binding to an index primer.

[0210] In some embodiments, the population of beads comprises a first and a second subpopulation of beads, each bead comprising a second polynucleotide comprising an index and a 3' end of the index for binding to an index primer, wherein the indices of the first subpopulation are different from the indices of the second subpopulation. In some embodiments, the nucleotide sequences of the indices of the first subpopulation of beads comprise the same nucleotide sequence, and the nucleotide sequences of the indices of the second subpopulation of beads comprise the same nucleotide sequence. In some embodiments, the nucleotide sequences of the index primer binding sites comprise the same nucleotide sequence.

[0211] In some embodiments, the ligation or cleavage step with the first subpopulation of beads is performed at positions that are different from the ligation or cleavage step with the second subpopulation of beads. In some embodiments, the different positions provide different reaction volumes. In some embodiments, the different positions provide different wells.

[0212] Some embodiments also provide for combining the first and second subpopulations of granules before distributing the subpopulation of granules on the substrate. In other embodiments, the first subpopulation of granules is distributed on the substrate before distributing the second subpopulation of granules on the substrate.

[0213] In some embodiments, decoding the position of a bead containing a first capture probe containing a detectable label on a substrate includes determining the position of indices of beads containing a detected capture umbrella. Some such embodiments include hybridizing a plurality of index primers to index primer sites and extending the hybridized index primers. In some embodiments, extending the hybridized index primers includes at least one round of sequencing by synthesis. In some embodiments, decoding the position of the bead indices includes sequencing the indices on a substrate.

[0214] In some embodiments, the substrate comprises a plurality of discrete sites. In some embodiments, the substrate comprises a plurality of wells. In some embodiments, the substrate comprises a plurality of channels. In some embodiments, the flow cell comprises the substrate. In some embodiments, the distributed population of beads comprises a matrix.

[0215] FIG. 6B shows on the left an embodiment in which the bead 200 comprises a first capture probe 300 attached to the bead via a cleavable linker 310. A second capture probe 320 is attached to the bead. A target nucleic acid 220 from a particular sample is hybridized to the first and second capture probes, and the first capture probe is extended to fill the gap between the first and second capture probes with nucleotides that contain detectable labels 230. After filling the gap, the target nucleic acid is removed and the cleavable linker is cleaved to obtain an extended second capture probe containing detectable labels attached to the bead. The bead can be dispersed in an array on a substrate and decoded. A polynucleotide comprising an index indicating a sample attached to the bead and a polynucleotide comprising a barcode indicating a first or second capture probe are not shown. Decoding may include determining the position of a detectable label on the array; determining a barcode attached to the bead on the array; and/or determining an index attached to the bead on the array.

[0216] FIG. 6C depicts an embodiment in which a first capture probe is attached to a bead 200 via its 5' end. A second capture probe 360 is attached to the bead via its 3' end and a cleavable linker 310. The second capture probe comprises detectable labels 230. A target nucleic acid 220 is hybridized to the first and second capture probes, the first capture probe is extended and ligated to the second capture probe. In some embodiments, the target nucleic acid may comprise structural variants (SVs). The cleavable linker is cleaved to obtain an extended first capture probe comprising a detectable label and attached to the bead. The bead can be dispersed in an array on a substrate and decoded. A polynucleotide containing an index indicative of a sample attached to the bead and a polynucleotide containing a barcode indicative of the first or second capture probes are not shown. Decoding may include determining the position of a detectable label on the array; determining a barcode attached to the bead on the array; and/or determining an index attached to the bead on the array. In the absence of a target nucleic acid, the second capture probe and the detectable label are cleaved from the bead.

Some methods of obtaining and using indexed granules

[0217] Some embodiments include obtaining indexed beads. Some such embodiments may include providing a population of index polynucleotides, wherein each index polynucleotide comprises an index, an index primer binding site, and an anchor/adapter. In some embodiments, the anchor/adapter is capable of binding to or hybridizing with an adapter binding site attached to the bead.

[0218] Some embodiments include a method of producing an indexed population of beads, comprising (a) obtaining a population of beads, wherein each bead comprises an adapter, a capture probe, and a first polynucleotide comprising a barcode and a barcode primer binding site; (b) obtaining a plurality of index polynucleotides, wherein each index polynucleotide comprises an index and an index primer binding site; and (c) attaching the plurality of index polynucleotides to the population of beads via adapters, thereby producing an indexed population of beads. In some embodiments, (c) involves extending the adapters via polymerase extension. In some embodiments, each index polynucleotide comprises an adapter binding site, and the attaching involves hybridizing the adapter binding sites to the adapters. In some embodiments, (c) involves ligating the index polynucleotides to the adapters. In some embodiments, the attaching involves hybridizing a bypass polynucleotide to an adapter and an index polynucleotide. In some embodiments, (c) involves attaching the plurality of index polynucleotides to the adapters of the population of beads via a chemically reactive functional group. In some embodiments, the first polynucleotides of the population of beads comprise different capture probes from each other. In some embodiments, the index of each index polynucleotide is the same. Aspects suitable for methods and compositions used to connect polynucleotides to each other by chemical ligation are described in U.S. Patent No. 20180127816, which is incorporated herein by reference in its entirety.

[0219] Some embodiments also include contacting the indexed population of beads with a plurality of nucleic acids comprising a target nucleic acid. Some embodiments also include mixing the indexed population of beads contacting the plurality of nucleic acids comprising a target nucleic acid with an additional indexed population of beads, wherein the additional indexed population of beads comprises an index polynucleotide comprising an index different from the index of the indexed population of beads contacting the plurality of nucleic acids.

[0220] In some embodiments, the first polynucleotide comprises a capture probe. Some embodiments also include contacting the indexed population of beads with a plurality of nucleic acids comprising a target nucleic acid. Some embodiments also include mixing the indexed population of beads contacting the plurality of nucleic acids comprising a target nucleic acid with an additional indexed population of beads, wherein the additional indexed population of beads comprises an index polynucleotide comprising an index different from the index of the indexed population of beads contacting the plurality of nucleic acids.

[0221] In some embodiments, the capture probe comprises a protein.

[0222] In some embodiments, the method is performed on a flow cell.

[0223] An example of an embodiment for producing indexed beads is shown in FIG. 11. The index polynucleotide comprises an anchor or adapter, an index, and an index primer binding site. A plurality of populations of index polynucleotides are produced and distributed in wells of a multi-well plate. Each well may comprise a population of an index polynucleotide comprising the same index. For example, a first well may comprise a population of index polynucleotides comprising a first index, and a second well may comprise a population of index polynucleotides comprising a second index. A plurality of beads may be distributed in the wells. Each bead may include a first polynucleotide comprising a capture probe, and a second polynucleotide comprising an index anchor, such as a binding site capable of binding to the anchor or adapter of the index polynucleotide. One pool of beads is added to each well. One pool of beads may comprise a population of beads in which the capture probes differ from each other. One pool of beads containing the same index can be used with one sample so that the source of subsequent processed nucleic acid products in the sample can be identified as being derived from one sample by identifying the associated index. As shown in FIG. 11, one indexed pool of beads can be added to one well in a multi-well plate in which each well contains one sample.

[0224] The index polynucleotide index can be incorporated into the bead in several different ways. In some embodiments, the index polynucleotide is hybridized to the bead via an anchor and a second polynucleotide, and the second polynucleotide is extended, thereby incorporating the complement of the index polynucleotide index into the extended second polynucleotide attached to the bead. In some embodiments, the index polynucleotide and the second polynucleotide are ligated together. In some embodiments, the index polynucleotide is attached to a polynucleotide attached to the bead by a chemical or enzymatic method. In some embodiments, the index polynucleotide is hybridized to a polynucleotide attached to the bead, and the index of the hybridized index polynucleotide is detected on the array.

[0225] Some embodiments include adding an index to the bead by a chemical or enzymatic method. An example of adding an index to the bead by a chemical or enzymatic method is shown in FIG. 12A, which depicts a bead comprising a first and a second polynucleotide. The first polynucleotide comprises a probe, such as a capture probe; a code, such as a barcode; and a primer A, such as a barcode primer binding site, which can be used to determine the sequence of the barcode. The second polynucleotide comprises an index; and a primer B, such as an index primer binding site, which can be used to determine the sequence of the index. The second polynucleotide can be attached to the bead via a spacer and X-Y functional groups that link the second polynucleotide to the spacer. In some embodiments, the capture probe can include a sequence primer binding site. In some embodiments, a blocking group can be added to the index polynucleotide. In some embodiments, the index primer binding site may also be a hairpin with a reversibly blocked 3' end.

[0226] Some embodiments include adding an index to a bead by extending an adapter attached to the bead. In some embodiments, the adapter attached to the bead can be extended by ligation or by polymerase extension. An example of an extension by ligation is shown in FIG. 12B, which depicts a bead comprising a first and a second polynucleotide. The first polynucleotide comprises a probe, such as a capture probe; a code, such as a barcode; and a primer A, such as a barcode primer binding site, which can be used to detect the sequence of the barcode. The second polynucleotide comprises an adapter that can be extended by ligation with a third polynucleotide comprising the index and primer B, using a fourth polynucleotide comprising a shunt that is capable of hybridizing to both the second and third polynucleotides. In some embodiments, the capture probe can include a sequence primer binding site. In some embodiments, a blocking group can be added to the index polynucleotide. In some embodiments, the index primer binding site may also be a hairpin with a reversibly blocked 3' end.

[0227] An example of an extension by polymerase extension is shown in FIG. 12C, which depicts a bead comprising a first and a second polynucleotide. The first polynucleotide comprises a probe, primer A, and a code. The second polynucleotide comprises an adapter. A third polynucleotide, such as an index polynucleotide, comprises an adapter binding site capable of binding to the adapter, the index, and primer B. The third polynucleotide hybridizes to the second polynucleotide such that the second polynucleotide is extended by polymerase extension to incorporate the index into the extended adapter. In some embodiments, the capture probe may include a primer binding site sequence. In some embodiments, a blocking group may be added to the index polynucleotide. In some embodiments, the index primer binding site may also be a hairpin with a reversibly blocked 3' end.

[0228] Some embodiments include using an index polynucleotide hybridized to a first nucleotide attached to a bead. Some embodiments include methods of detecting a target ligand comprising: (a) providing a population of beads, each bead comprising a capture probe, a first polynucleotide comprising a barcode, and a barcode primer binding site; (b) providing an index polynucleotide comprising an index, an index primer binding site, and an adapter capable of binding to the barcode primer binding site; (c) specifically binding the target ligand to the capture probe; (d) hybridizing the index polynucleotide to the first polynucleotide via the adapter; (e) detecting the target ligand on the array; and (f) detecting the index and the barcode of the first polynucleotide. In some embodiments, (e) involves distributing the population of beads on the array. In some embodiments, (f) comprises hybridizing an index primer to an index primer binding site and determining the sequence of the index. In some embodiments, dehybridizing an index polynucleotide from a first polynucleotide; hybridizing a barcode primer to a barcode primer binding site; and extending the barcode primer to determine the sequence of the barcode. In some embodiments, the index polynucleotide further comprises a cleavable linker located between the adaptor and the index, and (f) comprises: (i) cleaving the cleavable linker; and (ii) extending the adaptor to determine the sequence of the barcode. In some embodiments, the capture probe comprises a protein. In some embodiments, the target ligand comprises a target nucleic acid. In some embodiments, the first polynucleotide comprises a capture probe. In some embodiments, (e) comprises extending the first polynucleotide hybridized to the target nucleic acid. In some embodiments, the extension comprises adding a detectable dideoxynucleotide. In some embodiments, the method is performed on a flow cell.

[0229] An embodiment for using an index polynucleotide hybridized to a first nucleotide attached to a bead is shown in FIG. 13A and FIG. 13B. FIG. 13A depicts a bead comprising a first polynucleotide. The first polynucleotide comprises a probe, such as a capture probe; a primer, such as a primer binding site; and a code, such as a barcode. The index polynucleotide comprises an index; a primer 2, such as an index primer binding site; a spacer, such as a cleavable spacer; and an adapter capable of hybridizing to the primer binding site of the first polynucleotide. A pool of beads can be generated using the index polynucleotide as shown in FIG. 11. In FIG. 13B (first panel) shows a bead containing a first polynucleotide, in which a target nucleic acid is hybridized to the first polynucleotide via a capture probe, and an index polynucleotide is hybridized to the first polynucleotide via a primer binding site. FIG. 13B (second panel) shows an extended first polynucleotide with one fluorescent dideoxynucleotide (star), which can be detected on the bead array. The index and barcode can be determined. FIG. 13B (third panel) shows that the fluorescent dideoxynucleotide has been removed from the extended first polynucleotide. The index primer can be hybridized to the index primer binding site and extended, and the index sequence can be determined. FIG. 13B (fourth panel) shows that the target nucleic acid has been dehybridized from the first polynucleotide. The index polynucleotide is cleaved at the cleavable linker and the adapter now matches the barcode primer hybridized to the primer binding site, which can be extended, and the barcode sequence is determined.

[0230] Some embodiments include the use of an index polynucleotide comprising a plurality of index sequences and an index primer binding site. In some such embodiments, the plurality of index sequences may result in an increase in signal at a position on the template when the index is sequenced. An example embodiment is shown in FIG. 14. FIG. 14 shows a bead comprising a first polynucleotide and a second polynucleotide. The first polynucleotide comprises: a code, such as a barcode; a primer A, such as a barcode primer binding site; and a probe, such as a capture probe. The second polynucleotide comprises repeats of the index and the index primer binding sites. In some embodiments, the repeats improve the signal intensity on beads with a low amount of immobilized indices. In some embodiments, the repeats may limit the amount of surface area occupied by primers without reducing the signal.

[0231] Some embodiments include enzymatically adding an index polynucleotide to a bead. In some embodiments, the index polynucleotide may be attached to the bead by extending a polynucleotide attached to the bead via a reactive group. In some embodiments, the index polynucleotide may be attached to the bead prior to mixing the pool of beads with other pools of beads, such as prior to loading the mixture into an array. Some embodiments include a method of detecting a target ligand comprising: (a) obtaining a population of beads, each bead comprising a capture probe, a first polynucleotide comprising a barcode, a barcode primer binding site, and a second polynucleotide; (b) obtaining an index polynucleotide comprising the index, the index primer binding site, and an adaptor; (c) specifically binding the target ligand to the capture probe; (d) attaching the index polynucleotide to the second polynucleotide via an adaptor; (e) detecting the target ligand on the array; and (f) determining an index and a barcode of the first polynucleotide. In some embodiments, the second polynucleotide comprises a barcode and a barcode primer binding site. In some embodiments, (d) comprises adding a reactive functional group to the second polynucleotide, wherein the adapter is capable of attaching to the reactive functional group. Suitable aspects of methods and compositions used to add a reactive functional group to a polynucleotide are described in U.S. Patent No. 20180127816, which is incorporated herein by reference in its entirety. In some embodiments, (e) comprises distributing a population of beads on an array. In some embodiments, (f) comprises hybridizing an index primer to an index primer binding site and determining the index sequence. In some embodiments, (i) comprises hybridizing a barcode primer to a barcode primer binding site and determining the barcode sequence. In some embodiments, the capture probe comprises a protein. In some embodiments, the target ligand comprises a target nucleic acid. In some embodiments, the first polynucleotide comprises a capture probe. In some embodiments, (e) comprises extending the first polynucleotide hybridized to the target nucleic acid. In some embodiments, extending comprises adding a detectable dideoxynucleotide. In some embodiments, the method is performed on a flow cell.

[0232] An example of implementing the enzymatic addition of an index polynucleotide to a bead is shown in FIG. 15A and FIG. 15B. FIG. 15A and FIG. 15B show hybridization of a target nucleic acid to a bead via a capture probe of a first polynucleotide, addition of an index polynucleotide to the bead via a second polynucleotide, and detection of a barcode and an index attached to the bead. FIG. 15A (first panel) shows a bead comprising first and second polynucleotides. The first polynucleotide comprises: a code, such as a barcode; a primer, such as a barcode primer binding site; and a probe, such as a capture probe. FIG. 15A (second panel) shows hybridization of a target nucleic acid to the capture probe. FIG. 15A (third panel) shows single base extension (SBE) of a first polynucleotide with a fluorescent dideoxynucleotide (star). FIG. 15A (fourth panel) shows addition of a reactive group (triangle) to a second polynucleotide, such as a reactive dideoxynucleotide. The addition may include the use of a terminal deoxynucleotidyl transferase (TdT). FIG. 15B (first panel) shows addition of an index polynucleotide containing an index, an index primer binding site, and an adapter to a second polynucleotide via a reactive group and an adapter. The bead may be loaded onto a template, the position of the bead may be determined from the fluorescent dideoxynucleotide, and the fluorescent dideoxynucleotide may be removed. FIG. 15B (second panel) shows hybridization of the index primer to the index primer binding site to determine the index sequence. The index primer and target nucleic acid can be dehybridized. FIG. 15B (third panel) shows the hybridization of barcode primers to barcode primer binding sites for barcode sequencing.

Spacers

[0233] Some embodiments provided herein include the use of spacers. The spacer can include a polynucleotide having a length that is greater than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 500 consecutive nucleotides, or a length in the range of any two of the foregoing numbers. In some embodiments, a spacer can be located between two polynucleotide elements to improve the efficiency of certain processes by reducing steric hindrance. For example, a spacer can be used to improve the efficiency of enzymatic processes, such as the use of polymerases, ligases, and/or end transferases for a substrate, such as polynucleotides, attached to a bead. In some embodiments, the spacer may be located between any two elements including: a bead, such as an attached end of a polynucleotide to the bead, an index, an index primer binding site, a barcode, a barcode primer binding site, and a capture probe.

Kits and systems

[0234] Some embodiments include kits and systems for decoding microfeatures, such as polynucleotides on an array. Some such kits and systems may include a substrate, such as a chip or a fluidic cell, with an array of polynucleotides randomly distributed on the surface of the substrate. The polynucleotides may comprise a 3' primer binding site of a barcode. In some such embodiments, each polynucleotide may comprise a capture probe. In further such embodiments, each polynucleotide may be linked to the capture probe via a common element. For example, each polynucleotide and the capture probe may be linked to the same microfeature, such as a bead. Some embodiments include a detector configured to detect signals from reagents hybridized to the polynucleotides in the array; such reagents may include sequencing reagents, such as nucleotides containing detectable labels. Some embodiments include a detector configured to detect signals that may arise from the incorporation of a nucleotide into a polynucleotide, such as pyrophosphate, or changes in hydrogen ions.

[0235] Some embodiments include kits or systems comprising at least a first and a second subpopulation of beads. In some embodiments, each bead of a subpopulation may comprise a first polynucleotide comprising a capture probe, a barcode indicative of a capture probe of the same bead, and a 3' end of a barcode for binding to a barcode primer. In some embodiments, each bead of a subpopulation may also comprise a second polynucleotide comprising an index and a 3' end of an index for binding to an index primer. In some embodiments, the index of a subpopulation of beads may be indicative of that particular subpopulation of beads. In some embodiments, the indices of the first subpopulation are different from the indices of the second subpopulation. In some embodiments of the kits and systems provided herein, the first volume comprises the first subpopulation of beads and the second volume comprises the second subpopulation of beads.

[0236] In some embodiments, each of the first and second bead subpopulation capture probes comprises nucleotide sequences that differ from one another. In some embodiments, the different first and second bead subpopulation capture probes comprise the same nucleotide sequences. In some embodiments, the capture probes comprise a nucleotide sequence that is capable of hybridizing to a single nucleotide polymorphism (SNP) or its complement.

[0237] In some embodiments, the barcode primer binding sites comprise the same nucleotide sequence. Some embodiments also include a plurality of barcode primers capable of hybridizing to the barcode primer sites.

[0238] In some embodiments, the nucleotide sequences of the indices of the first subpopulation of beads comprise the same nucleotide sequence, and the nucleotide sequences of the indices of the second subpopulation of beads comprise the same nucleotide sequence. In some embodiments, the nucleotide sequences of the index primer binding sites comprise the same nucleotide sequence. Some embodiments also include a plurality of index primers capable of hybridizing to the index primer sites.

[0239] In some embodiments, the substrate comprises a plurality of discrete sites. In some embodiments, the substrate comprises a plurality of wells. In some embodiments, the substrate comprises a plurality of channels. In some embodiments, the flow cell comprises the substrate. In some embodiments, the substrate is configured to form a combination of the first and second subpopulations of beads of the bead array on the surface of the substrate and to sequence the array in a plurality of sequencing cycles via synthesis cycles.

[0240] In some embodiments, each of the first and second subpopulations of beads comprises at least 50 capture probes comprising different nucleotide sequences. In some embodiments, each of the first and second subpopulations of beads comprises at least 500 capture probes comprising different nucleotide sequences. In some embodiments, each of the first and second subpopulations of beads comprises at least 5,000 capture probes comprising different nucleotide sequences. In some embodiments, each of the first and second subpopulations of beads comprises at least 50,000 capture probes comprising different nucleotide sequences.

[0241] Some embodiments include at least 10 different subpopulations of granules, wherein each subpopulation comprises an index that is different from another subpopulation. Some embodiments include at least 100 different subpopulations of granules, wherein each subpopulation comprises an index that is different from another subpopulation. Some embodiments include at least 1,000 different subpopulations of granules, wherein each subpopulation comprises an index that is different from another subpopulation. Some embodiments include at least 10,000 different subpopulations of granules, wherein each subpopulation comprises an index that is different from another subpopulation.

[0242] Some embodiments include a kit comprising: an array of polynucleotides randomly distributed on the surface of a substrate, wherein each polynucleotide comprises a 3' primer binding site of a barcode and is linked to a capture probe, wherein each polynucleotide comprises a different barcode and is linked to a different capture probe. In some embodiments, each polynucleotide is linked to the capture probe by a bead. In some embodiments, each polynucleotide comprises a capture probe. In some embodiments, the substrate is planar. In some embodiments, the substrate comprises wells. In some embodiments, the polynucleotides are attached to the beads. In some embodiments, the beads are distributed in the wells. In some embodiments, the flow cell comprises an array.

[0243] Some embodiments include kits or systems comprising first and second subpopulations of beads. In some embodiments, each bead comprises a capture probe that specifically binds to a target ligand. Examples of target ligands include nucleic acids, proteins, or other antigens. Examples of capture probes include nucleic acids, antibodies, and antigen-binding fragments of antibodies. In some embodiments, each bead comprises a first polynucleotide comprising a barcode indicative of a capture probe of the same bead and a 3' end of the barcode for binding to a barcode primer. In some embodiments, each bead comprises a second polynucleotide comprising an index and a 3' end of the index for binding to an index primer. In some such embodiments, the indices of the first subpopulation are different from the indices of the second subpopulation. For example, the indices of the first subpopulation of beads can be used to distinguish the first subpopulation of beads from the second subpopulation of beads. In some embodiments, the first subpopulation of granules is separated from the second subpopulation of granules. For example, the first volume contains the first subpopulation of granules, and the second volume contains the second subpopulation of granules.

[0244] In some embodiments, the capture probe comprises a nucleic acid and the target ligand comprises a target nucleic acid. In some such embodiments, the first polynucleotide comprises a capture probe. In some embodiments, the capture probes comprise a nucleotide sequence capable of hybridizing to a single nucleotide polymorphism (SNP) or its complement.

[0245] In some embodiments, the capture probe comprises an antibody or an antigen-binding fragment of an antibody.

[0246] In some embodiments, each of the first and second bead subpopulation capture probes specifically binds to different target ligands from each other. For example, each first bead subpopulation capture probe specifically binds to different target ligands from each other; and each second bead subpopulation capture probe specifically binds to different target ligands from each other. In some embodiments, different first and second bead subpopulation capture probes specifically bind to the same target ligands. For example, a set of different first bead subpopulation capture probes specifically bind to the same target ligands as a set of different first bead subpopulation capture probes.

[0247] In some embodiments, the barcode primer binding sites comprise the same nucleotide sequence. Some embodiments also include a plurality of barcode primers capable of hybridizing to the barcode primer sites.

[0248] In some embodiments, the nucleotide sequences of the indices of the first subpopulation of beads comprise the same nucleotide sequence and/or the nucleotide sequences of the indices of the second subpopulation of beads comprise the same nucleotide sequence. In some embodiments, the nucleotide sequences of the index primer binding sites comprise the same nucleotide sequence. Some embodiments also include a plurality of index primers capable of hybridizing to the index primer sites.

[0249] In some embodiments, the substrate comprises a plurality of discrete sites. In some embodiments, the substrate comprises a plurality of wells. In some embodiments, the substrate comprises a plurality of channels. In some embodiments, the flow cell comprises the substrate. In some embodiments, the substrate is configured to form a combination of the first and second subpopulations of beads of the bead array on the surface of the substrate and to sequence the array in a plurality of sequencing cycles via synthesis cycles.

[0250] In some embodiments, each of the first and second subpopulations of granules comprises at least 50, 100, 500, 1000, or 5000 different capture probes from one another, or any number of capture probes in the range between any two of the above quantities. Some embodiments also comprise at least 5, 10, 20, 50, 100, 200, 500, 1000 different subpopulations of granules, wherein each subpopulation comprises an index that is different from another subpopulation, or any number of different subpopulations of granules in the range between any two of the above quantities.

[0251] Some embodiments include a kit comprising first and second subpopulations of beads, wherein each bead comprises: a capture probe that specifically binds to a target ligand, a first polynucleotide comprising a barcode indicative of the capture probe and a 3' end of the barcode for binding to a primer of the barcode, and a second polynucleotide comprising an index and a 3' end of the index for binding to a primer of the index, wherein the indices of the first subpopulation are different from the indices of the second subpopulation, wherein the first volume comprises the first subpopulation of beads and the second volume comprises the second subpopulation of beads. In some embodiments, the capture probe comprises a nucleic acid and the target ligand comprises a target nucleic acid. In some embodiments, the first polynucleotide comprises the capture probe. In some embodiments, each of the capture probes of the first and second subpopulations of beads comprises nucleotide sequences that differ from one another. In some embodiments, the different first and second bead subpopulation capture probes comprise the same nucleotide sequences. In some embodiments, the capture probe comprises an antibody or an antigen-binding fragment thereof. In some embodiments, each of the first and second bead subpopulation capture probes specifically binds to different target ligands. In some embodiments, the different first and second bead subpopulation capture probes specifically bind to the same target ligands. In some embodiments, the barcode primer binding sites comprise the same nucleotide sequence. Some embodiments also include a plurality of barcode primers capable of hybridizing to the barcode primer sites. In some embodiments, the nucleotide sequences of the first bead subpopulation indices comprise the same nucleotide sequence, and the nucleotide sequences of the second bead subpopulation indices comprise the same nucleotide sequence. In some embodiments, the nucleotide sequences of the index primer binding sites comprise the same nucleotide sequence. Some embodiments also include a plurality of index primers capable of hybridizing to the index primer sites. In some embodiments, the flow cell comprises a substrate. In some embodiments, the substrate is configured to form a combination of the first and second bead subpopulations of the bead array on the surface of the substrate and to sequence the array in a plurality of sequencing cycles via synthesis cycles. In some embodiments, each of the first and second bead subpopulations comprises at least 50 capture probes that are different from one another. In some embodiments, each of the first and second bead subpopulations comprises at least 500 capture probes that are different from one another. Some embodiments also include at least 10 different bead subpopulations, wherein each subpopulation comprises an index that is different from another subpopulation.

[0252] Additional embodiments of the kits and systems provided herein may include multiple populations of beads comprising oligonucleotides attached to the beads, wherein the oligonucleotides comprise indices, index primer binding sites adjacent to the indices, capture probes, barcodes, and barcode primer binding sites adjacent to the barcodes, wherein the indices are different between populations of beads. In some embodiments, the index primer binding sites are the same across the multiple populations. In some embodiments, the barcodes indicate the nucleotide sequence of the capture probes. In some embodiments, the barcodes are different across the population of beads. In some embodiments, the barcode primer binding sites are the same across the multiple populations. In some embodiments, the beads comprise biotin, streptavidin, or a derivative thereof. In some embodiments, the beads are magnetic.

[0253] Some embodiments also include a reagent selected from: a locus-specific oligonucleotide; a transposome for labeling a nucleic acid sample; a transposome comprising an index and an index primer binding site; an adapter comprising an index and an index primer binding site; a primer capable of hybridizing to an index primer binding site or its complement; and/or a primer capable of hybridizing to a barcode primer binding site or its complement. Some embodiments also include a template, such as an template on the surface of a flow cell. Some embodiments include a detector configured to detect signals from reagents hybridized to polynucleotides in the template; such reagents may include sequencing reagents, such as nucleotides containing detectable labels. Some embodiments include a detector configured to detect signals that may arise from the incorporation of a nucleotide into a polynucleotide, such as pyrophosphate, or changes in hydrogen ions.

EXAMPLES

Example 1. Decoding a matrix by sequencing

[0254] A plurality of polynucleotides are synthesized, each polynucleotide comprising from 5' to 3': a spacer, a unique barcode, a primer binding site, and a unique capture probe. The sequences of the barcode and the capture probe are known; the sequence of the primer binding site is the same for each polynucleotide. Each polynucleotide is attached to a bead. The beads are randomly distributed into the wells of a chip. The bead array is decoded by hybridizing the primer to the primer binding site, extending the primer, and detecting the barcode sequence. The position of the barcode determines the position of the corresponding capture probe.

Example 2: Decoding Barcodes on an Array by Sequencing

[0255] A nucleic acid library derived from human genomic DNA was prepared. A subset of beads was prepared. A first and second polynucleotide were attached to each bead. The first polynucleotide contained a capture probe, a barcode primer binding site, and a barcode indicating the capture probe. The second polynucleotide contained an index and an index primer binding site.

[0256] A pool of beads containing 18,816 different code/probe types was loaded onto a HiSeq flow cell (FIG. 7A). Following immobilization, the 20 nucleotide long codes were sequenced using SBS chemistry and the identity of each bead was determined by aligning the code sequences to a list of bead types. FIG. 7B shows a copy number histogram for specific bead types in specific columns and demonstrates that 97.5% of the expected composition was identified using the sequencing-based decoding process and that the vast majority of bead types were present at levels sufficient for genotyping studies.

Example 3. Genotyping performance on HiSeq using FFN detection

[0257] To demonstrate the genotyping performance of the HiSeq using FFN detection, target DNA oligonucleotides were hybridized to a suspension of probe-conjugated oligonucleotide beads. The beads were loaded into the HiSeq flow cell. Probes bound to the target DNA were extended one base at a time using fluorescent nucleotides. The flow cell with loaded beads was imaged to obtain bead genotyping intensity values. The fluorescent nucleotides were digested and the beads were decoded using SBS chemistry. To measure assay throughput, decoding and genotyping reads were aligned. Individual spots were colored according to the expected genotype. In FIG. Figure 7C shows a plot of C intensity versus T intensity, where each point represents the mean of all copies for a given bead type and is colored according to the expected genotype, and demonstrates that single-base extension of the probe with fluorescent nucleotides provides accurate genotyping.

Example 4: Multiplexing 12 samples with a 10,368-plex bead pool

[0258] This example demonstrates demultiplexing multiple samples on a single flow cell, and in particular the ability to multiplex 12 samples with a 10,368-plex bead pool. One bead pool was individually hybridized to 12 different index sequences. After hybridization, the samples were pooled and loaded onto a HiSeq flow cell. Two separate reads were then performed, one to identify the sample based on the hybridized index and a separate read to identify the bead type based on the read-decode. FIG. 8 shows a histogram for a representative sample of a number of specific bead types in specific bindings, and demonstrates that most bead types are present at levels sufficient for genotyping experiments for a given sample. The table in FIG. 7 shows a summary of the demultiplexing and bead decoding consistency for the 12 pooled and simultaneously loaded indexed samples, indicating that sample representation is uniform and that most probes are present in all samples.

Example 5. Large parallel SNP genotyping

[0259] This is an example of a workflow for genotyping 384 samples in a single sequencing run. Polynucleotides containing target nucleic acids that include a single nucleotide polymorphism (SNP) of interest are produced in a 384-well plate. Each well contains DNA from a different subject. The DNA is labeled with transposomes, which fragment the DNA, and primer binding sites for amplification are added to each end of the fragments. The fragments are amplified with primers that include an index and an index primer binding site to produce amplified fragments in which at least one end of each amplified fragment includes an index and an index primer binding site. The index is different for each well such that polynucleotides produced from a particular well can be identified by a particular index. The resulting polynucleotides include an index, an index primer binding site, and a target nucleic acid.

[0260] A population of beads is added to each well. The population of beads includes an oligonucleotide attached to the bead. The oligonucleotide includes a barcode, a barcode primer binding site, and a capture probe. The capture probe is 50 nucleotides in length and is specific for a particular target nucleic acid. The barcode can be used to identify the capture probe. The population of beads includes different capture probes. The target nucleic acids hybridize to the capture probes in solution in each well to produce hybridized beads. Hybridized beads from each well are pooled and loaded into a bead array on a flow cell. Hybridized beads are randomly distributed on the array.

[0261] On the array, the capture probes are extended by one nucleotide to identify SNPs. Indexes are sequenced by extending a primer hybridized to the index primer binding sites to identify the source of the target nucleic acid. Barcodes are sequenced by extending a primer hybridized to the index primer binding sites to decode the position of the bead on the array. Specific SNPs are identified and associated with a specific DNA sample obtained from a specific subject.

[0262] As used herein, the term "comprising" is synonymous with the terms "including," "including," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0263] The above description describes several methods and materials of the present invention. The present invention is subject to modifications in methods and materials, as well as changes in manufacturing methods and equipment. Such modifications will be apparent to those skilled in the art upon examination of the present description or upon practice of the invention described herein. Therefore, it is not intended that the present invention be limited to the specific embodiments described herein, but is intended to cover all modifications and alternatives within the true scope and spirit of the invention.

[0264] All references cited herein, including, without limitation, published and unpublished applications, patents, and literature references, are hereby incorporated by reference in their entirety and are therefore incorporated into this disclosure. To the extent that publications, patents, or patent applications incorporated by reference conflict with the teachings set forth in this disclosure, the present disclosure is intended to supersede and/or take precedence over any such conflicting material.

Claims (59)

1. A method for sequencing a target nucleic acid on a matrix, comprising: (a) obtaining first and second populations of granules, wherein: the first population of granules comprises oligonucleotides comprising first capture probes, first barcodes, and barcode primer binding sites adjacent to the first barcodes, and the second population of granules comprises oligonucleotides containing second capture probes, second barcodes, and barcode primer binding sites adjacent to the second barcodes; (b) obtaining a first and second plurality of polynucleotides, wherein: the first plurality of polynucleotides comprises first target nucleic acids, wherein the first plurality of polynucleotides is in solution, and the second plurality of polynucleotides comprises second target nucleic acids, wherein the second plurality of polynucleotides is in solution; (c) hybridizing first target nucleic acids to first capture probes to produce hybridized first beads and hybridizing second target nucleic acids to second capture probes to produce hybridized second beads; (d) random distribution of hybridized first granules and hybridized second granules on the matrix; (e) decoding the positions of the first and second granules on the array by sequencing the first and second barcodes on the array; and (f) extending the first and second capture probes to obtain nucleotide sequence data for the first and second target nucleic acids. 2. The method according to paragraph 1, wherein: the first plurality of polynucleotides comprises first indices and index primer binding sites adjacent to the first indices; and the second plurality of polynucleotides comprises second target nucleic acids, second indices, and index primer binding sites adjacent to the second indices. 3. The method of claim 2, wherein the first or second plurality of polynucleotides are obtained by labeling a nucleic acid sample with a plurality of transposomes. 4. The method according to claim 3, wherein the plurality of transposomes comprise the first or second indices. 5. The method of claim 4, further comprising adding adapters to the labeled nucleic acid sample, wherein the adapters comprise a first or second index. 6. The method according to claim 5, further comprising amplifying the labeled nucleic acid sample with primers containing the first or second indices. 7. The method according to any one of claims 2 to 6, wherein extending the first and second capture probes incorporates sequences complementary to the first and second indices and the first and second index primer binding sites into the extended capture probes. 8. The method according to item 1, wherein: the first population of granules contains first indices and index primer binding sites adjacent to the first indices, and the second population of granules contains the second indices and index primer binding sites adjacent to the second indices. 9. The method according to claim 8, wherein the oligonucleotides of the first population of granules comprise first indices; and the oligonucleotides of the second population of granules comprise second indices. 10. The method according to any one of claims 2 to 9, wherein the first indices indicate a source of the first target nucleic acids and the second indices indicate a source of the second target nucleic acids. 11. The method according to any one of paragraphs 2-10, in which the first indices coincide with each other, and the second indices coincide with each other. 12. The method according to any one of paragraphs 2-11, further comprising sequencing the first and second indices. 13. The method according to claim 12, wherein sequencing the first and second indices comprises extending primers hybridized to the index primer binding sites. 14. The method according to any one of claims 2 to 13, wherein the index primer binding sites are the same. 15. The method according to any one of claims 1-14, wherein the first and second target nucleic acids are obtained from different nucleic acid samples. 16. The method according to any one of claims 1-15, wherein the first and second target nucleic acids are derived from genomic DNA. 17. The method according to any one of claims 1-16, wherein the first and second barcodes indicate a nucleotide sequence of the first or second capture probe. 18. The method according to any one of paragraphs 1-17, wherein the first barcodes are different from each other and the second barcodes are different from each other. 19. The method according to any one of claims 1-18, wherein sequencing the first and second barcodes comprises extending primers hybridized to the barcode primer binding sites. 20. The method according to any one of claims 1-19, wherein the barcode primer binding sites are the same. 21. The method according to any one of claims 1-20, wherein the extension of the first and second capture probes comprises polymerase extension. 22. The method of claim 21, wherein extending the first and second capture probes comprises adding one nucleotide to the capture probe. 23. The method according to claim 21 or 22, further comprising ligating locus-specific oligonucleotides to extended capture probes. 24. The method according to any one of claims 21-23, wherein extending the first and second capture probes comprises ligating locus-specific oligonucleotides to the capture probes. 25. The method according to any one of claims 1 to 24, wherein step (c) is performed in solution. 26. The method according to any one of claims 1-25, wherein the matrix is placed on the surface of the flow cell. 27. The method according to any one of claims 1-26, wherein the first and second granules are designed to be attached to the matrix. 28. The method according to claim 27, wherein the first and second granules comprise biotin, streptavidin or a derivative thereof; and the matrix comprises biotin, streptavidin or a derivative thereof. 29. The method of claim 27, wherein the first and second granules are magnetic. 30. The method of claim 1, wherein step (a) further comprises: (i) obtaining a plurality of index polynucleotides, wherein each index polynucleotide comprises a first index or a second index and a binding site for said index primer; and (ii) attaching a plurality of index polynucleotides to the first or second population of granules via an adapter. 31. The method of claim 30, which (ii) comprises extending the adapters by polymerase extension. 32. The method according to claim 30, wherein each index polynucleotide comprises an adapter binding site, and the attachment comprises hybridization of the adapter binding sites with adapters. 33. The method of claim 30, wherein (ii) comprises ligating index polynucleotides with adapters. 34. The method according to claim 33, wherein the attachment comprises hybridizing the shunt polynucleotide with an adapter and an index polynucleotide. 35. The method of claim 30, which (ii) comprises attaching a plurality of index polynucleotides to the adaptors of the granule population via a chemically reactive functional group. 36. The method of claim 35, wherein the attachment comprises a click chemistry reaction. 37. The method according to any one of claims 30-36, wherein the first polynucleotides of the population of granules comprise capture probes that differ from each other. 38. The method according to any one of claims 30-37, wherein the index of each index polynucleotide is the same. 39. The method according to claim 30-38, wherein the first polynucleotide comprises a capture probe. 40. The method of any one of claims 30-39, further comprising contacting the indexed population of beads with a plurality of nucleic acids comprising a target nucleic acid. 41. The method of claim 40, further comprising mixing the indexed population of beads contacted with a plurality of nucleic acids comprising a target nucleic acid with an additional indexed population of beads, wherein the additional indexed population of beads comprises an index polynucleotide comprising an index different from the index of the indexed population of beads contacted with the plurality of nucleic acids. 42. The method according to any one of claims 30-38, wherein the capture probe comprises a protein. 43. The method according to any of paragraphs 30-42, performed on a flow cell.