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WO2023116373A1 - 一种生成标记的核酸分子群的方法及其试剂盒 - Google Patents

一种生成标记的核酸分子群的方法及其试剂盒 Download PDF

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WO2023116373A1
WO2023116373A1 PCT/CN2022/135363 CN2022135363W WO2023116373A1 WO 2023116373 A1 WO2023116373 A1 WO 2023116373A1 CN 2022135363 W CN2022135363 W CN 2022135363W WO 2023116373 A1 WO2023116373 A1 WO 2023116373A1
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sequence
primer
nucleic acid
oligonucleotide
region
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French (fr)
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徐讯
陈奥
章文蔚
廖莎
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Priority to CN202280085230.5A priority Critical patent/CN118434882A/zh
Priority to US18/722,541 priority patent/US20250163492A1/en
Publication of WO2023116373A1 publication Critical patent/WO2023116373A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

Definitions

  • This application relates to the technical field of transcriptome sequencing and biomolecular spatial information detection. Specifically, the present application relates to a method for positionally marking nucleic acid molecules, and a method for constructing a library of nucleic acid molecules for transcriptome sequencing. In addition, the present application also relates to a library of nucleic acid molecules constructed by the method, and a kit for implementing the method.
  • the present application provides a new method for generating labeled nucleic acid molecule groups, and a method for constructing a nucleic acid molecule library and performing high-throughput sequencing based on the method.
  • the application provides a method of generating a population of labeled nucleic acid molecules, comprising the steps of:
  • nucleic acid array includes a solid support, and the solid support is coupled with multiple oligonucleotide probes; each oligonucleotide probe comprises At least one copy; and, the oligonucleotide probe comprises or consists of: a consensus sequence X1, a tag sequence Y and a consensus sequence X2 from a 5' to a 3' direction, wherein,
  • oligonucleotide probes have different tag sequences Y, and the tag sequence Y has a unique nucleotide sequence corresponding to the position of the oligonucleotide probe on the solid support;
  • RNA for example, mRNA
  • pretreatment comprising the following steps:
  • RNA for example, mRNA
  • the cDNA chain includes the RNA (for example, mRNA) complementary cDNA sequence, and 3' terminal overhang
  • the primer A contains a capture sequence A
  • the capture sequence A can anneal with the RNA to be captured (for example, mRNA) and initiate an extension reaction
  • primer B annealing primer B to the cDNA strand generated in (a), and performing an extension reaction to generate a first extension product, which is the first nucleic acid molecule to be labeled, thereby generating a first population of nucleic acid molecules
  • the primer B comprises a consensus sequence B, a 3' end overhang complementary sequence, and an optional tag sequence B; the 3' end overhang complementary sequence is located at 3 of the primer B ' end; said consensus sequence B is located upstream of said 3' end overhanging complementary sequence (e.g., at the 5' end
  • RNA for example, mRNA
  • primer A' contains a consensus sequence A and a capture sequence A, and the capture sequence A can anneal to the RNA (eg, mRNA) to be captured and initiate an extension reaction; the consensus sequence A is located upstream of the capture sequence A (for example, at the 5' end of the primer A');
  • primer B' combine primer B' with the cDNA generated in (a) The strands are annealed and extended to generate a first extension product; wherein, the primer B' comprises a consensus sequence B, a 3' end overhang complementary sequence, and an optional tag sequence B; the 3' end overhang A complementary sequence is located at the 3' end of the primer B'; the consensus sequence B is located upstream of the complementary sequence overhanging the 3' end (eg, at the
  • step (2) (i) implementing annealing conditions to the product of step (2), such that the oligonucleotide probe anneals to the first nucleic acid molecule to be labeled at the corresponding position of the oligonucleotide probe (for example annealing in situ), And carry out an extension reaction to generate an extension product, which is a second nucleic acid molecule with a position marker, thereby generating a second nucleic acid molecule population; wherein, the consensus sequence X2 of the oligonucleotide probe or a partial sequence thereof (a) capable of annealing to the complementary sequence of the consensus sequence B or a partial sequence thereof of the first extension product obtained in step (2)(i), or, (b) capable of annealing to the first extension product obtained in step (2)(ii); Annealing to the complementary sequence of said consensus sequence A or a partial sequence thereof of two extension products; or,
  • the bridging oligonucleotide pair is composed of a first bridging oligonucleotide and a second bridging oligonucleotide, and the first bridging oligonucleotide and the second bridging oligonucleotide are each independently comprising: a first region and a second region, and optionally a third region located between the first region and the second region, the first region being located upstream (e.g., the 5' end) of the second region; wherein ,
  • the first region of the first bridging oligonucleotide is capable of annealing to the first region of the second bridging oligonucleotide; the second region of the first bridging oligonucleotide is capable of annealing to the oligonucleotide Annealing to the consensus sequence X2 of the acid probe or a partial sequence thereof;
  • the second region (a) of the second bridging oligonucleotide can anneal to the complementary sequence of the consensus sequence B of the first extension product obtained in step (2)(i) or a partial sequence thereof, or, (b ) capable of annealing to the complementary sequence of the consensus sequence A or a partial sequence thereof of the second extension product obtained in step (2)(ii);
  • the first bridging oligonucleotide and the second bridging oligonucleotide of the bridging oligonucleotide pair each of the bridging oligonucleotides is in single-stranded form, or the first bridging oligonucleotide and the second bridging oligonucleotide of the pair of bridging oligonucleotides are in a partially double-stranded form by annealing to each other;
  • ligation reaction ligation of nucleic acid molecules that hybridize to the first and second regions of the same first bridging oligonucleotide, and/or, hybridizing to the first and second regions of the same second bridging oligonucleotide
  • the nucleic acid molecules of the region are connected; and an extension reaction is carried out; wherein, the connection reaction and the extension reaction are carried out in any order;
  • the obtained reaction product is the second nucleic acid molecule with a position marker, thereby generating the second nucleic acid molecule population.
  • step (3)(ii) of the method in step (3)(ii) of the method:
  • the step comprises: using nucleic acid ligase to hybridize to the nucleic acid molecules of the first region and the second region of the same first bridging oligonucleotide; or,
  • the first bridging oligonucleotide comprises a first region, a second region and a third region therebetween, the first region and the second region that will hybridize to the same first bridging oligonucleotide
  • the step of ligating the nucleic acid molecule of the region includes: using a nucleic acid polymerase (for example, without 5' to 3' end exonuclease activity or strand displacement activity) to carry out a polymerization reaction using the third region as a template, and using a nucleic acid ligase to hybridize linked to the nucleic acid molecules of the first region, the third region and the second region of the same first bridging oligonucleotide;
  • the step comprising: using nucleic acid ligase to hybridize to the nucleic acid molecules of the first region and the second region of the same second bridging oligonucleotide; or,
  • the second bridging oligonucleotide comprises a first region, a second region and a third region therebetween, the first region and the second region that will hybridize to the same second bridging oligonucleotide
  • the step of ligating the nucleic acid molecule of the region includes: using a nucleic acid polymerase (for example, without 5' to 3' end exonuclease activity or strand displacement activity) to carry out a polymerization reaction using the third region as a template, and using a nucleic acid ligase to hybridize The nucleic acid molecules at the first region, the third region and the second region of the same second bridging oligonucleotide are linked.
  • each oligonucleotide probe comprises one copy.
  • each oligonucleotide probe comprises multiple copies.
  • the region where each of the oligonucleotide probes is coupled to the solid support is referred to as a microspot. It is easy to understand that when each oligonucleotide probe is one copy, each micro-dot is coupled with a probe, and the oligonucleotide probes of different micro-dots have different label sequences Y; when each oligonucleotide When the nucleotide probe contains multiple copies, each micro-dot is coupled with multiple probes, the oligonucleotide probes in the same micro-dot have the same label sequence Y, and the oligonucleotide probes in different micro-dots have Different label sequences Y.
  • the solid support comprises a plurality of microdots, each microdot is coupled to an oligonucleotide probe, and each oligonucleotide probe may comprise one or more copies.
  • the solid support comprises a plurality (eg, at least 10, at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , or more) microdots; in certain embodiments, the solid support comprises at least 10 4 (eg, at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12 ) microdots/square millimeter.
  • the solid support comprises at least 10 4 (eg, at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12 ) microdots/square millimeter.
  • the interval between adjacent microdots is less than 100 ⁇ m, less than 50 ⁇ m, less than 10 ⁇ m, less than 5 ⁇ m, less than 1 ⁇ m, less than 0.5 ⁇ m, less than 0.1 ⁇ m, less than 0.05 ⁇ m, or less than 0.01 ⁇ m.
  • the microdots have a size (e.g., equivalent diameter) of less than 100 ⁇ m, less than 50 ⁇ m, less than 10 ⁇ m, less than 5 ⁇ m, less than 1 ⁇ m, less than 0.5 ⁇ m, less than 0.1 ⁇ m, less than 0.05 ⁇ m, or less than 0.01 ⁇ m .
  • the method comprises step (1), step (2)(i) and step (3); wherein, in step (2)(i)(b), the primer B contains a consensus sequence B, Complementary sequence of 3' end overhang, and tag sequence B.
  • the first extension product described in step (2)(i)(b) sequentially comprises from the 5' end to the 3' end: the primer A formed by using the primer A as the reverse transcription primer and the RNA complementary cDNA sequence, the 3' end overhang sequence, the complementary sequence of the tag sequence B, the complementary sequence of the consensus sequence B.
  • each copy of the second nucleic acid molecule derived from the same oligonucleotide probe has a different tag sequence B as UMI.
  • Embodiments comprising step (1), step (2)(i) and step (3)(i)
  • the method comprises step (1), step (2)(i) and step (3)(i); wherein, the consensus sequence X2 or a partial sequence thereof can be combined with the consensus sequence B
  • the complementary sequence or partial sequence thereof is annealed;
  • the extension product obtained in step (3)(i) is a labeled nucleic acid molecule, which comprises: the first strand containing the first nucleic acid molecule sequence to be labeled, and/or , the second strand containing the oligonucleotide probe sequence.
  • partial sequence of XX (sequence) or “partial sequence of XX (sequence)" means the nucleotide sequence of at least one segment of "XX (sequence)".
  • the entire nucleotide sequence of the consensus sequence X2 can anneal to the complementary sequence of the consensus sequence B or the nucleotide sequence of a partial segment of the complementary sequence of the consensus sequence B, and the consensus sequence X2 It is also possible to anneal with the complementary sequence of the consensus sequence B or the nucleotide sequence of a partial segment of the complementary sequence of the consensus sequence B with the nucleotide sequence of a partial segment thereof.
  • annealing means that in the two nucleotide sequences that are annealed to each other, each base in one nucleotide sequence can pair with the base in the other nucleotide sequence without mismatching or a gap; or, in two nucleotide sequences that anneal to each other, most of the bases in one nucleotide sequence can pair with the bases in the other nucleotide sequence, which allows mismatches or gaps ( For example, a mismatch or gap of one or several nucleotides). That is, the two nucleotide sequences that can be annealed can be either completely complementary or partially complementary. Unless otherwise indicated herein or clearly contradicted by the context, the description of "annealing" here applies to the entire text.
  • the first strand comprises from the 5' end to the 3' end: a cDNA sequence complementary to the RNA formed by using the primer A as a reverse transcription primer, and the overhanging sequence at the 3' end , the complementary sequence of the tag sequence B, the complementary sequence of the consensus sequence B, the complementary sequence of the tag sequence Y, the complementary sequence of the consensus sequence X1.
  • the second strand comprises from the 5' end to the 3' end: the consensus sequence X1, the tag sequence Y, the consensus sequence X2, the tag sequence B, the 3' The complementary sequence of the terminal overhang sequence, the complementary sequence of the cDNA sequence complementary to the RNA formed by using the primer A as a reverse transcription primer.
  • Embodiment comprising step (1), step (2)(i) and step (3)(i): a chain
  • the consensus sequence X2 or a partial sequence thereof can anneal to the complementary sequence of the consensus sequence B or a partial sequence thereof (for example, a 3' end partial sequence), and in step (2)(i) The complementary sequence of said consensus sequence B of the first extension product has a 3' free end.
  • the extension product obtained in step (3)(i) is a labeled nucleic acid molecule comprising the first strand.
  • the first strand comprises from the 5' end to the 3' end: a cDNA sequence complementary to the RNA formed by using the primer A as a reverse transcription primer, and the overhanging sequence at the 3' end , the complementary sequence of the tag sequence B, the complementary sequence of the consensus sequence B, the complementary sequence of the tag sequence Y, the complementary sequence of the consensus sequence X1.
  • step (3)(i) the oligonucleotide probe cannot initiate an extension reaction (eg, the 3' end is blocked).
  • the capture sequence A of the primer A is a random oligonucleotide sequence.
  • the first extension product described in step (2)(i)(b) sequentially comprises from the 5' end to the 3' end: the primer A formed by using the primer A as the reverse transcription primer and the RNA complementary cDNA sequence, the 3' end overhang sequence, the complementary sequence of the tag sequence B, the complementary sequence of the consensus sequence B.
  • the first strand comprises from the 5' end to the 3' end: a cDNA sequence complementary to the RNA formed by using the primer A as a reverse transcription primer, and the overhanging sequence at the 3' end , the complementary sequence of the tag sequence B, the complementary sequence of the consensus sequence B, the complementary sequence of the tag sequence Y, the complementary sequence of the consensus sequence X1.
  • the capture sequence A of the primer A is a poly(T) sequence or a specific sequence for a specific target nucleic acid.
  • the primer A further contains a consensus sequence A, and an optional tag sequence A, such as a random oligonucleotide sequence.
  • the capture sequence A is located at the 3' end of the primer A.
  • the consensus sequence A is located upstream of the capture sequence A (eg, at the 5' end of the primer A).
  • the first extension product in step (2)(i)(b) sequentially comprises from the 5' end to the 3' end: the consensus sequence A, the optional tag sequence A, and the
  • the primer A is the cDNA sequence complementary to the RNA formed by the reverse transcription primer, the overhang sequence at the 3' end, the complementary sequence of the tag sequence B, and the complementary sequence of the consensus sequence B.
  • the first strand comprises from the 5' end to the 3' end: the consensus sequence A, optionally the tag sequence A, the primer A formed with the primer A as a reverse transcription primer and the The cDNA sequence complementary to the RNA, the 3' end overhang sequence, the complementary sequence of the tag sequence B, the complementary sequence of the consensus sequence B, the complementary sequence of the tag sequence Y, the complementary sequence of the consensus sequence X1 sequence.
  • Embodiment comprising step (1), step (2)(i) and step (3)(i): two chains
  • the consensus sequence X2 or a partial sequence thereof can anneal to the complementary sequence of the consensus sequence B or a partial sequence thereof, and the oligonucleotide probe
  • the consensus sequence X2 of has a 3' free end.
  • the extension product obtained in step (3)(i) is a labeled nucleic acid molecule comprising the second strand.
  • the second strand comprises from the 5' end to the 3' end: the consensus sequence X1, the tag sequence Y, the consensus sequence X2, the tag sequence B, the 3' The complementary sequence of the terminal overhang sequence, the complementary sequence of the cDNA sequence complementary to the RNA formed by using the primer A as a reverse transcription primer.
  • the first extension product obtained in step (2)(i) cannot initiate an extension reaction (eg, the 3' end is blocked).
  • the capture sequence A of the primer A is a random oligonucleotide sequence.
  • the first extension product described in step (2)(i)(b) sequentially comprises from the 5' end to the 3' end: the primer A formed by using the primer A as the reverse transcription primer and the RNA complementary cDNA sequence, the 3' end overhang sequence, the complementary sequence of the tag sequence B, the complementary sequence of the consensus sequence B.
  • the second strand comprises from the 5' end to the 3' end: the consensus sequence X1, the tag sequence Y, the consensus sequence X2, the tag sequence B, the 3' The complementary sequence of the terminal overhang sequence, the complementary sequence of the cDNA sequence complementary to the RNA formed by using the primer A as a reverse transcription primer.
  • the capture sequence A of the primer A is a poly(T) sequence or a specific sequence for a specific target nucleic acid.
  • the primer A further contains a consensus sequence A, and an optional tag sequence A, such as a random oligonucleotide sequence.
  • the capture sequence A is located at the 3' end of the primer A.
  • the consensus sequence A is located upstream of the capture sequence A (eg, at the 5' end of the primer A).
  • the first extension product in step (2)(i)(b) sequentially comprises from the 5' end to the 3' end: the consensus sequence A, optionally the tag sequence A, The cDNA sequence complementary to the RNA formed by using the primer A as a reverse transcription primer, the overhang sequence at the 3' end, the complementary sequence of the tag sequence B, and the complementary sequence of the consensus sequence B.
  • the second strand comprises from the 5' end to the 3' end: the consensus sequence X1, the tag sequence Y, the consensus sequence X2, the tag sequence B, the 3' The complementary sequence of the terminal overhang sequence, the complementary sequence of the cDNA sequence complementary to the RNA formed by using the primer A as a reverse transcription primer, the optional complementary sequence of the tag sequence A, the complementary sequence of the consensus sequence A sequence.
  • the term "UMI” refers to "Unique Molecular Identifier, a unique molecular label", which can be used to perform qualitative and/or quantitative nucleic acid molecules. Unless otherwise indicated herein or clearly contradicted by the context, the present application does not limit the position and quantity of the UMI or its complementary sequence in the nucleic acid molecule. For example, when the cDNA chain contains the UMI or its complementary sequence, the UMI or its complementary sequence can be located at the 3' end of the cDNA sequence in the cDNA chain, or at the 5' end of the cDNA sequence, or The UMI or its complement is contained at both the 3' end and the 5' end.
  • the UMI or its complementary sequence can be located at the 3' end of the complementary sequence of the cDNA sequence in the complementary strand of the cDNA strand, or at the end of the complementary sequence of the cDNA sequence.
  • the 5' end may also contain the UMI or its complementary sequence at both the 3' end and the 5' end.
  • the UMI can be introduced via primer A, and/or via primer B.
  • the UMI can be introduced via primer A', and/or via primer B'.
  • step (1) An exemplary embodiment of the present application comprising step (1), step (2)(i) and step (3)(i) is described in detail as follows:
  • An exemplary scheme for preparing a cDNA strand containing a complementary sequence of UMI at the 3' end using RNA (such as mRNA) in the sample as a template comprises the following steps (as shown in Figure 2):
  • RNA molecules (for example, mRNA molecules) in the permeabilized sample are reverse-transcribed using reverse transcriptase (for example, reverse transcriptase with terminal transfer activity) and primer A to generate cDNA, and 3 An overhang (eg, an overhang comprising 3 cytosine nucleotides) is added to the ' end.
  • reverse transcriptase for example, reverse transcriptase with terminal transfer activity
  • primer A primer A to generate cDNA
  • 3 An overhang eg, an overhang comprising 3 cytosine nucleotides
  • 3 reverse transcriptases having terminal transfer activity can be used for the reverse transcription reaction.
  • the reverse transcriptase used does not have RNaseH activity.
  • the primer A comprises a poly(T) sequence and a consensus sequence A (CA).
  • a poly(T) sequence is located at the 3' end of the primer A to initiate reverse transcription.
  • the primer A comprises a random oligonucleotide sequence that can be used to capture RNA without a poly(A) tail.
  • the random oligonucleotide sequence is located at the 3' end of the primer A to initiate reverse transcription.
  • primer B to anneal or hybridize with the cDNA strand, said primer B comprising a consensus sequence B (CB), a unique molecular tag sequence (UMI) and the complementary sequence of the 3' end overhang of the cDNA.
  • CB consensus sequence B
  • UMI unique molecular tag sequence
  • the nucleic acid fragment hybridized or annealed to the primer B can be extended using the UMI sequence and the consensus sequence B as templates, thereby generating a complementary primer carrying the UMI sequence at the 3' end. sequence, the nucleic acid molecule of the complementary sequence of the consensus sequence B.
  • the consensus sequence B is located upstream of the UMI sequence (for example, the 5' end), and the sequence complementary to the 3' end overhang of the cDNA strand is located at the 3' end of the primer B.
  • the primer B may include GGG at its 3' end.
  • the nucleotides of the primer B can also be modified (for example, using locked nucleic acid) to enhance the complementary pairing between the primer B and the 3' end overhang of the cDNA strand.
  • nucleic acid polymerases for example, DNA polymerase or reverse transcriptase
  • DNA polymerase or reverse transcriptase can be used to carry out the extension reaction, as long as it can use the sequence of the primer B or a partial sequence thereof as a template to extend the captured Nucleic acid fragments (reverse transcription products) are sufficient.
  • reverse transcriptase enzyme as in the previous reverse transcription step can be used to extend the captured nucleic acid fragment (reverse transcription product).
  • this step is performed simultaneously with step (1) (eg, in the same reaction system).
  • the method optionally further comprises step (3): adding RNaseH to digest the RNA strand in the RNA/cDNA hybrid duplex to form a cDNA single strand.
  • said method does not comprise said step (3).
  • the exemplary structure of the cDNA strand prepared by the above exemplary embodiment comprises: consensus sequence A, cDNA sequence, 3' end overhang sequence, complementary sequence of UMI sequence, and complementary sequence of consensus sequence B.
  • the performance scheme includes the following steps (as shown in Figure 4):
  • the consensus sequence X2 of the ChIP-seq or a partial sequence thereof can anneal to the complementary sequence of the consensus sequence B of the cDNA strand obtained in the above step 1 or a partial sequence thereof.
  • the cDNA strand is annealed or hybridized with ChIP-seq, and under the action of polymerase, a new nucleic acid molecule containing ChIP-seq information (ie, a nucleic acid molecule marked with ChIP-seq) is formed.
  • the exemplary structure of the new nucleic acid molecule containing chip sequence information formed by the above exemplary embodiment comprises: a consensus sequence A from the 5' end to the 3' end, a cDNA sequence, an overhang sequence at the 3' end, and the complement of the UMI sequence sequence, the complementary sequence of the consensus sequence B, the complementary sequence of the tag sequence Y, and the nucleic acid strand of the complementary sequence of the consensus sequence X1 and/or its complementary nucleic acid strand.
  • Embodiment comprising step (1), step (2)(i) and step (3)(ii)
  • the method comprises step (1), step (2)(i) and step (3)(ii); wherein the second region of the second bridging oligonucleotide is capable of combining with step (2) Annealing to the complementary sequence of the consensus sequence B of the first extension product obtained in (i) or a partial sequence thereof;
  • the reaction product obtained in step (3)(ii) is a labeled nucleic acid molecule, which comprises: The first strand of the first nucleic acid molecule sequence to be labeled, and/or, the second strand containing the oligonucleotide probe sequence.
  • the second region of the second bridging oligonucleotide can be compatible with the complementary sequence of the consensus sequence B or the complementary sequence of the consensus sequence B of the first extension product obtained in step (2)(i).
  • the nucleotide sequences of the partial segments are annealed.
  • the first strand comprises from the 5' end to the 3' end: a cDNA sequence complementary to the RNA formed by using the primer A as a reverse transcription primer, and the overhanging sequence at the 3' end , the complementary sequence of the tag sequence B, the complementary sequence of the consensus sequence B, optionally the complementary sequence of the third region of the second bridging oligonucleotide, the first bridging oligonucleotide sequence, The complementary sequence of the tag sequence Y, the complementary sequence of the consensus sequence X1.
  • the second strand comprises from the 5' end to the 3' end: the consensus sequence X1, the tag sequence Y, the consensus sequence X2, optionally the first bridging oligo
  • the complementary sequence of the third region of nucleotides, the second bridging oligonucleotide sequence, the tag sequence B, the complementary sequence of the 3' end overhang sequence, formed by using the primer A as a reverse transcription primer The complement of the cDNA sequence complementary to said RNA.
  • Embodiment comprising step (1), step (2)(i) and step (3)(ii): a chain
  • the second region of the second bridging oligonucleotide can be complementary to the consensus sequence B of the first extension product obtained in step (2)(i) or a partial sequence thereof (such as , 3' end partial sequence) annealing, and the second region of the first bridging oligonucleotide has a 3' free end.
  • the reaction product obtained in step (3)(ii) is a labeled nucleic acid molecule comprising the first strand.
  • the first strand comprises from the 5' end to the 3' end: a cDNA sequence complementary to the RNA formed by using the primer A as a reverse transcription primer, and the overhanging sequence at the 3' end , the complementary sequence of the tag sequence B, the complementary sequence of the consensus sequence B, optionally the complementary sequence of the third region of the second bridging oligonucleotide, the first bridging oligonucleotide sequence, The complementary sequence of the tag sequence Y, the complementary sequence of the consensus sequence X1.
  • the second region of the first bridging oligonucleotide is located at the 3' end of the first bridging oligonucleotide.
  • the first region of the first bridging oligonucleotide is located at the 5' end of the first bridging oligonucleotide.
  • said first bridging oligonucleotide does not contain said third region, and/or said second bridging oligonucleotide does not contain said third region.
  • the 5' end of the first bridging oligonucleotide contains a phosphorylation modification.
  • the 3' end of the first bridging oligonucleotide contains a free -OH.
  • step (3)(ii) the second bridging oligonucleotide is unable to initiate an extension reaction (eg, the 3' end is blocked), and/or, the oligonucleotide The probe cannot initiate an extension reaction (eg, the 3' end is blocked).
  • the capture sequence A of the primer A is a random oligonucleotide sequence.
  • the first extension product described in step (2)(i)(b) of the method sequentially comprises from the 5' end to the 3' end: The cDNA sequence complementary to the RNA, the 3' end overhang sequence, the complementary sequence of the tag sequence B, the complementary sequence of the consensus sequence B.
  • the first strand comprises from the 5' end to the 3' end: a cDNA sequence complementary to the RNA formed by using the primer A as a reverse transcription primer, and the overhanging sequence at the 3' end , the complementary sequence of the tag sequence B, the complementary sequence of the consensus sequence B, optionally the complementary sequence of the third region of the second bridging oligonucleotide, the first bridging oligonucleotide sequence, The complementary sequence of the tag sequence Y, the complementary sequence of the consensus sequence X1.
  • the capture sequence A of the primer A is a poly(T) sequence or a specific sequence for a specific target nucleic acid.
  • the primer A further contains a consensus sequence A, and an optional tag sequence A, such as a random oligonucleotide sequence.
  • the capture sequence A is located at the 3' end of the primer A.
  • the first extension product in step (2)(i)(b) sequentially comprises from the 5' end to the 3' end: the consensus sequence A, optionally the tag sequence A, The cDNA sequence complementary to the RNA formed by using the primer A as a reverse transcription primer, the overhang sequence at the 3' end, the complementary sequence of the tag sequence B, and the complementary sequence of the consensus sequence B.
  • the first strand comprises from the 5' end to the 3' end: the consensus sequence A, optionally the tag sequence A, the primer A formed with the primer A as a reverse transcription primer and the The cDNA sequence complementary to the RNA, the 3' end overhang sequence, the complementary sequence of the tag sequence B, the complementary sequence of the consensus sequence B, optionally the third region of the second bridging oligonucleotide
  • step (3)(ii) at the positions corresponding to the first bridging oligonucleotide, the second bridging oligonucleotide, the oligonucleotide probe and the oligonucleotide probe
  • the nucleic acid molecules hybridized to the first region and the second region of the same first bridging oligonucleotide are ligated, and/or, the nucleic acid molecules hybridized to the same second bridging oligonucleotide
  • the ligation reaction process of connecting the nucleic acid molecules of the first region and the second region and the extension reaction described in step (3)(ii) can be carried out in any order, as long as the second nucleic acid molecule with a position marker can be obtained.
  • nucleic acid molecules that hybridize to the first region and the second region of the same second bridging oligonucleotide can be ligated, and the first The bridging oligonucleotide initiates the extension reaction resulting in the first strand being obtained.
  • the polymerase used in the extension reaction preferably does not have strand displacement activity or 5' to 3' excision activity.
  • the first strand can be obtained in the following exemplary ways:
  • the polymerase used in the extension reaction preferably has strand displacement activity or 5' to 3' excision activity.
  • said first bridging oligonucleotide can be obtained by initiating an extension reaction with said first bridging oligonucleotide and then ligating nucleic acid molecules hybridizing to the first and second regions of the same second bridging oligonucleotide. first chain.
  • the polymerase used in the extension reaction preferably does not have strand displacement activity or 5' to 3' excision activity.
  • Embodiment comprising step (1), step (2)(i) and step (3)(ii): two strands
  • the second region of the second bridging oligonucleotide is capable of annealing to the consensus sequence B complementary sequence or a partial sequence thereof of the first extension product obtained in step (2)(i), and
  • the second region of the second bridging oligonucleotide has a 3' free end.
  • the reaction product obtained in step (3)(ii) is a labeled nucleic acid molecule comprising said second strand.
  • the second strand comprises from the 5' end to the 3' end: the consensus sequence X1, the tag sequence Y, the consensus sequence X2, optionally the first bridging oligo
  • the complementary sequence of the third region of nucleotides, the second bridging oligonucleotide sequence, the tag sequence B, the complementary sequence of the 3' end overhang sequence, formed by using the primer A as a reverse transcription primer The complement of the cDNA sequence complementary to said RNA.
  • the second region of the second bridging oligonucleotide is located at the 3' end of the second bridging oligonucleotide.
  • the first region of the second bridging oligonucleotide is located at the 5' end of the second bridging oligonucleotide.
  • said first bridging oligonucleotide does not contain said third region, and/or said second bridging oligonucleotide does not contain said third region.
  • the 5' end of the second bridging oligonucleotide contains a phosphorylation modification.
  • the 3' end of the second bridging oligonucleotide contains a free -OH.
  • step (3)(ii) the first bridging oligonucleotide is unable to initiate an extension reaction (eg, the 3' end is blocked), and/or, step (2)(i ) The first extension product obtained cannot initiate the extension reaction (for example, the 3' end is blocked).
  • the capture sequence A of the primer A is a random oligonucleotide sequence.
  • the first extension product described in step (2)(i)(b) sequentially comprises from the 5' end to the 3' end: the primer A formed by using the primer A as the reverse transcription primer and the RNA complementary cDNA sequence, the 3' end overhang sequence, the complementary sequence of the tag sequence B, the complementary sequence of the consensus sequence B.
  • the second strand comprises from the 5' end to the 3' end: the consensus sequence X1, the tag sequence Y, the consensus sequence X2, optionally the first bridging oligo
  • the complementary sequence of the third region of nucleotides, the second bridging oligonucleotide sequence, the tag sequence B, the complementary sequence of the 3' end overhang sequence, formed by using the primer A as a reverse transcription primer The complement of the cDNA sequence complementary to said RNA.
  • the capture sequence A of the primer A is a poly(T) sequence or a specific sequence for a specific target nucleic acid.
  • the primer A further contains a consensus sequence A, and an optional tag sequence A, such as a random oligonucleotide sequence.
  • the capture sequence A is located at the 3' end of the primer A.
  • the first extension product in step (2)(i)(b) sequentially comprises from the 5' end to the 3' end: the consensus sequence A, optionally the tag sequence A, The cDNA sequence complementary to the RNA formed by using the primer A as a reverse transcription primer, the overhang sequence at the 3' end, the complementary sequence of the tag sequence B, and the complementary sequence of the consensus sequence B.
  • the second strand comprises from the 5' end to the 3' end: the consensus sequence X1, the tag sequence Y, the consensus sequence X2, optionally the first bridging oligo
  • the complementary sequence of the third region of nucleotides, the second bridging oligonucleotide sequence, the tag sequence B, the complementary sequence of the 3' end overhang sequence, formed by using the primer A as a reverse transcription primer The complementary sequence of the cDNA sequence complementary to the RNA, optionally the complementary sequence of the tag sequence A, the complementary sequence of the consensus sequence A.
  • step (3)(ii) at the positions corresponding to the first bridging oligonucleotide, the second bridging oligonucleotide, the oligonucleotide probe and the oligonucleotide probe
  • the nucleic acid molecules hybridized to the first region and the second region of the same first bridging oligonucleotide are ligated, and/or, the nucleic acid molecules hybridized to the same second bridging oligonucleotide
  • the ligation reaction process of connecting the nucleic acid molecules of the first region and the second region and the extension reaction described in step (3)(ii) can be carried out in any order, as long as the second nucleic acid molecule with a position marker can be obtained.
  • nucleic acid molecules hybridized to the first region and the second region of the same first bridging oligonucleotide can be connected, and the second The bridging oligonucleotide initiates the extension reaction, resulting in the second strand.
  • the polymerase used in the extension reaction preferably does not have strand displacement activity or 5' to 3' excision activity.
  • the second chain can be obtained in the following exemplary ways:
  • said second bridging oligonucleotide can be obtained by initiating an extension reaction with said second bridging oligonucleotide and then ligating nucleic acid molecules hybridizing to the first and second regions of the same first bridging oligonucleotide. second chain.
  • the polymerase used in the extension reaction preferably does not have strand displacement activity or 5' to 3' excision activity.
  • step (1) An exemplary embodiment of the present application comprising step (1), step (2)(i) and step (3)(ii) is described in detail as follows:
  • An exemplary scheme for preparing a cDNA chain using RNA (such as mRNA) in a sample as a template comprises the following steps (as shown in Figure 2):
  • RNA molecules (for example, mRNA molecules) in the permeabilized sample are reverse-transcribed using reverse transcriptase (for example, reverse transcriptase with terminal transfer activity) and primer A to generate cDNA, and 3 An overhang (eg, an overhang comprising 3 cytosine nucleotides) is added to the ' end.
  • reverse transcriptase for example, reverse transcriptase with terminal transfer activity
  • primer A primer A to generate cDNA
  • 3 An overhang eg, an overhang comprising 3 cytosine nucleotides
  • 3 reverse transcriptases having terminal transfer activity can be used for the reverse transcription reaction.
  • the reverse transcriptase used does not have RNaseH activity.
  • the primer A comprises a poly(T) sequence and a consensus sequence A (CA).
  • a poly(T) sequence is located at the 3' end of the primer A to initiate reverse transcription.
  • the primer A comprises a random oligonucleotide sequence that can be used to capture RNA without a poly(A) tail.
  • the random oligonucleotide sequence is located at the 3' end of the primer A to initiate reverse transcription.
  • primer B to anneal or hybridize with the cDNA strand, said primer B comprising a consensus sequence B (CB), a unique molecular tag sequence (UMI) and the complementary sequence of the 3' end overhang of the cDNA.
  • CB consensus sequence B
  • UMI unique molecular tag sequence
  • the nucleic acid fragment hybridized or annealed to the primer B can be extended using the UMI sequence and the consensus sequence B as templates, thereby generating a complementary primer carrying the UMI sequence at the 3' end. sequence, the nucleic acid molecule of the complementary sequence of the consensus sequence B.
  • the consensus sequence B is located upstream of the UMI sequence (for example, the 5' end), and the sequence complementary to the 3' end overhang of the cDNA strand is located at the 3' end of the primer B.
  • the primer B may include GGG at its 3' end.
  • the nucleotides of the primer B can also be modified (for example, using locked nucleic acid) to enhance the complementary pairing between the primer B and the 3' end overhang of the cDNA strand.
  • nucleic acid polymerases for example, DNA polymerase or reverse transcriptase
  • DNA polymerase or reverse transcriptase can be used to carry out the extension reaction, as long as it can use the sequence of the primer B or a partial sequence thereof as a template to extend the captured Nucleic acid fragments (reverse transcription products) are sufficient.
  • reverse transcriptase enzyme as in the previous reverse transcription step can be used to extend the captured nucleic acid fragment (reverse transcription product).
  • this step is performed simultaneously with step (1) (eg, in the same reaction system).
  • the method optionally further comprises step (3): adding RNaseH to digest the RNA strand in the RNA/cDNA hybrid duplex to form a cDNA single strand.
  • said method does not comprise said step (3).
  • the exemplary structure of the cDNA strand prepared by the above exemplary embodiment comprises: consensus sequence A, cDNA sequence, 3' end overhang sequence, complementary sequence of UMI sequence, and complementary sequence of consensus sequence B.
  • the sexual scheme includes the following steps (as shown in Figure 3):
  • a bridging oligonucleotide pair consisting of a first bridging oligonucleotide and a second bridging oligonucleotide, wherein each of the first bridging oligonucleotide and the second bridging oligonucleotide is independently Including: a first region (P1) and a second region (P2), the first region is located upstream of the second region (for example, the 5' end); wherein,
  • the first region of the first bridging oligonucleotide is capable of annealing to the first region of the second bridging oligonucleotide; the second region of the first bridging oligonucleotide is capable of annealing to the oligonucleotide Annealing to the consensus sequence X2 of the acid probe or a partial sequence thereof;
  • the second region of the second bridging oligonucleotide can anneal to the complementary sequence of the consensus sequence B or a partial sequence thereof in the cDNA strand obtained in step 1 above.
  • the first bridging oligonucleotide contains spacer nucleotides between the first region and the second region, such as 1-5nt or 5-10nt spacer nucleotides, that is, the first bridging oligonucleotide
  • a bridging oligonucleotide sequence contains a third region located between the first region and the second region.
  • the first region and the second region in the first bridging oligonucleotide are adjacently connected without redundant nucleotides, that is, the first bridging oligonucleotide
  • the nucleotide sequence does not contain a third region located between the first region and the second region.
  • the second bridging oligonucleotide contains spacer nucleotides between the first region and the second region, such as 1-5nt or 5-10nt spacer nucleotides, that is, the second bridging oligonucleotide
  • the second bridging oligonucleotide sequence contains a third region located between the first region and the second region.
  • the first region and the second region in the second bridging oligonucleotide are adjacently connected without redundant nucleotides, that is, the second bridging oligonucleotide
  • the nucleotide sequence does not contain a third region located between the first region and the second region.
  • the exemplary structure of the new nucleic acid molecule containing chip sequence information formed by the above exemplary embodiment comprises: a consensus sequence A from the 5' end to the 3' end, a cDNA sequence, an overhang sequence at the 3' end, and the complement of the UMI sequence sequence, the complementary sequence of the consensus sequence B, the first bridging oligonucleotide sequence, the complementary sequence of the tag sequence Y, and the nucleic acid strand of the complementary sequence of the consensus sequence X1 and/or its complementary nucleic acid strand.
  • the method comprises step (1), step (2)(ii) and step (3).
  • the first extension product comprises from the 5' end to the 3' end: the consensus sequence A, reverse transcribed by the primer A' The cDNA sequence complementary to the RNA formed by the primers, the overhang sequence at the 3' end, the optional complementary sequence of the tag sequence B, and the complementary sequence of the consensus sequence B.
  • the extension primer is the primer B' or primer B" or a random primer, wherein the primer B" can be combined with the consensus sequence The complementary sequence of B, or a portion thereof, anneals and is able to initiate an extension reaction.
  • the second extension product comprises from the 5' end to the 3' end: a cDNA sequence complementary to the cDNA sequence formed by extending the extension primer Sequence, the complementary sequence of the consensus sequence A.
  • Embodiments comprising step (1), step (2)(ii) and step (3)(i)
  • the method comprises step (1), step (2)(ii) and step (3)(i); wherein, the consensus sequence X2 or a partial sequence thereof can be combined with the consensus sequence A
  • the complementary sequence or partial sequence thereof is annealed;
  • the extension product obtained in step (3)(i) is a labeled nucleic acid molecule, which comprises: the first strand containing the first nucleic acid molecule sequence to be labeled, and/or , the second strand containing the oligonucleotide probe sequence.
  • the consensus sequence X2 can be annealed with the complementary sequence of the consensus sequence A or the nucleotide sequence of a partial segment of the complementary sequence of the consensus sequence A with its overall nucleotide sequence, the consensus sequence X2 can also anneal to the complementary sequence of the consensus sequence A or the nucleotide sequence of a partial segment of the complementary sequence of the consensus sequence A with the nucleotide sequence of its partial segment.
  • the first strand comprises from the 5' end to the 3' end: the sequence of the first nucleic acid molecule to be labeled, the complementary sequence of the tag sequence Y, the complementary sequence of the consensus sequence X1 .
  • the second strand comprises from the 5' end to the 3' end: the consensus sequence X1, the tag sequence Y, the consensus sequence X2, and the first nucleic acid molecule to be labeled Sequence complementary cDNA sequences.
  • Embodiment comprising step (1), step (2)(ii) and step (3)(i): a chain
  • the consensus sequence X2 or a partial sequence thereof can anneal to the complementary sequence of the consensus sequence A or a partial sequence thereof (for example, a partial sequence at the 3' end); obtained in step (3)(i)
  • the extension product is a labeled nucleic acid molecule, which includes a first strand containing the sequence of the first nucleic acid molecule to be labeled.
  • step (3)(i) the oligonucleotide probe cannot initiate an extension reaction (eg, the 3' end is blocked).
  • the capture sequence A of the primer A' is a random oligonucleotide sequence.
  • the extension primer is the primer B'.
  • the second extension product comprises from the 5' end to the 3' end: the consensus sequence B, optionally the tag sequence B, The complementary sequence of the overhanging sequence at the 3' end, the complementary sequence of the cDNA sequence complementary to the RNA formed by using the primer A' as a reverse transcription primer, and the complementary sequence of the consensus sequence A.
  • the first strand comprises from the 5' end to the 3' end: the consensus sequence B, optionally the tag sequence B, the complementary sequence of the overhang sequence at the 3' end, and
  • the primer A' is the complementary sequence of the cDNA sequence complementary to the RNA formed by the reverse transcription primer, the complementary sequence of the consensus sequence A, the complementary sequence of the tag sequence Y, and the complementary sequence of the consensus sequence X1.
  • each copy of the first strand derived from the same oligonucleotide probe has a different complementary sequence of capture sequence A as the UMI.
  • the capture sequence A of the primer A' is a poly(T) sequence or a specific sequence for a specific target nucleic acid.
  • the primer A' also contains a tag sequence A, such as a random oligonucleotide sequence.
  • the capture sequence A is located at the 3' end of the primer A.
  • the extension primer is the primer B'.
  • the second extension product comprises from the 5' end to the 3' end: the consensus sequence B, optionally the tag sequence B, The complementary sequence of the overhang sequence at the 3' end, the complementary sequence of the cDNA sequence complementary to the RNA formed by using the primer A' as a reverse transcription primer, the complementary sequence of the tag sequence A, the consensus sequence A complementary sequence.
  • the first strand comprises from the 5' end to the 3' end: the consensus sequence B, optionally the tag sequence B, the complementary sequence of the overhang sequence at the 3' end, and
  • the primer A' is the complementary sequence of the cDNA sequence complementary to the RNA formed by the reverse transcription primer, the complementary sequence of the tag sequence A, the complementary sequence of the consensus sequence A, the complementary sequence of the tag sequence Y, The complementary sequence of said consensus sequence X1.
  • each copy of the first strand derived from the same oligonucleotide probe has a different complementary sequence of the tag sequence A as the UMI.
  • Embodiment comprising step (1), step (2)(ii) and step (3)(i): two strands
  • the consensus sequence X2 or a partial sequence thereof can anneal to the complementary sequence of the consensus sequence A or a partial sequence thereof; obtained in step (3)(i)
  • the extension product of is a labeled nucleic acid molecule comprising a second strand comprising the oligonucleotide probe sequence.
  • the second extension product obtained in step (2)(ii) cannot initiate an extension reaction (eg, the 3' end is blocked).
  • the capture sequence A of the primer A' is a random oligonucleotide sequence.
  • the extension primer is the primer B'.
  • the second extension product comprises from the 5' end to the 3' end: the consensus sequence B, optionally the tag sequence B, The complementary sequence of the overhanging sequence at the 3' end, the complementary sequence of the cDNA sequence complementary to the RNA formed by using the primer A' as a reverse transcription primer, and the complementary sequence of the consensus sequence A.
  • the second strand comprises from the 5' end to the 3' end: the consensus sequence X1, the tag sequence Y, the consensus sequence X2, and the first nucleic acid molecule to be labeled cDNA sequence complementary to the sequence, the 3' end overhang sequence, optionally the complementary sequence of the tag sequence B, the complementary sequence of the consensus sequence B.
  • each copy of the second strand derived from the same oligonucleotide probe has a different capture sequence A as UMI.
  • the capture sequence A of the primer A' is a poly(T) sequence or a specific sequence for a specific target nucleic acid.
  • the primer A' also contains a tag sequence A, such as a random oligonucleotide sequence.
  • the capture sequence A is located at the 3' end of the primer A.
  • the extension primer is the primer B'.
  • the second extension product comprises from the 5' end to the 3' end: the consensus sequence B, optionally the tag sequence B, The complementary sequence of the overhang sequence at the 3' end, the complementary sequence of the cDNA sequence complementary to the RNA formed by using the primer A' as a reverse transcription primer, the complementary sequence of the tag sequence A, the consensus sequence A complementary sequence.
  • the second strand comprises from the 5' end to the 3' end: the consensus sequence X1, the tag sequence Y, the consensus sequence X2, the tag sequence A, and the to-be
  • each copy of the second strand derived from the same oligonucleotide probe has a different tag sequence A as UMI.
  • step (1) An exemplary embodiment of the present application comprising step (1), step (2)(ii) and step (3)(i) is described in detail as follows:
  • An exemplary scheme for preparing a cDNA strand complementary strand containing a complementary sequence of UMI at the 3' end using RNA (such as mRNA) in the sample as a template comprises the following steps (as shown in Figure 5):
  • RNA molecules (for example, mRNA molecules) in the permeabilized sample are reverse-transcribed using reverse transcriptase (for example, reverse transcriptase with terminal transfer activity) and primer A' to generate cDNA, and An overhang (eg, an overhang comprising 3 cytosine nucleotides) is added at the 3' end.
  • reverse transcriptase for example, reverse transcriptase with terminal transfer activity
  • primer A' primer A' to generate cDNA
  • An overhang eg, an overhang comprising 3 cytosine nucleotides
  • Various reverse transcriptases having terminal transfer activity can be used for the reverse transcription reaction.
  • the reverse transcriptase used does not have RNaseH activity.
  • the primer A' comprises a poly(T) sequence, a UMI sequence, and a consensus sequence A(CA).
  • a poly(T) sequence is located at the 3' end of the primer A' to initiate reverse transcription, and the consensus sequence A is located upstream (eg 5' end) of the UMI sequence.
  • the primer A' comprises a random oligonucleotide sequence and a consensus sequence A, which can be used to capture RNA without a ploy A tail.
  • the random oligonucleotide sequence is located at the 3' end of the primer A' to initiate reverse transcription.
  • sequence complementary to the 3' end overhang of the cDNA strand is located at the 3' end of the primer B'.
  • the primer B' may include GGG at its 3' end.
  • the nucleotides of the primer B' can also be modified (for example, using a locked nucleic acid) to enhance the complementary pairing between the primer B' and the 3' end overhang of the cDNA strand.
  • nucleic acid polymerases for example, DNA polymerase or reverse transcriptase
  • DNA polymerase or reverse transcriptase can be used to carry out the extension reaction, as long as it can be captured using the sequence of the primer B' or a partial sequence thereof as a template extension Nucleic acid fragments (reverse transcription products) can be.
  • reverse transcriptase enzyme as in the previous reverse transcription step can be used to extend the captured nucleic acid fragment (reverse transcription product).
  • this step is performed simultaneously with step (1) (eg, in the same reaction system).
  • the method optionally further comprises step (3): adding RNaseH to digest the RNA strand in the RNA/cDNA hybrid duplex to form a cDNA single strand.
  • said method does not comprise said step (3).
  • extension primer the cDNA strand obtained in the previous step is used as a template for an extension reaction to obtain an extension product;
  • the extension primer is the primer B', a random primer, or a primer B", and the primer B" can be combined with The consensus sequence B or a partial sequence thereof anneals and is capable of initiating an extension reaction.
  • the exemplary structure of the cDNA strand complementary chain prepared by the above exemplary embodiment comprises: consensus sequence B, complementary sequence of 3' end overhang, complementary sequence of cDNA sequence, complementary sequence of UMI sequence, and complementary sequence of consensus sequence A sequence.
  • the consensus sequence X2 of the ChIP-seq or a partial sequence thereof can anneal to the complementary sequence of the consensus sequence A or a partial sequence thereof of the complementary strand of the cDNA strand obtained in step 1 above.
  • the complementary strand of the cDNA chain is annealed or hybridized with the ChIP-seq, and under the action of the polymerase, a new nucleic acid molecule containing the ChIP-seq information (that is, a nucleic acid molecule labeled with the ChIP-seq) is formed.
  • the exemplary structure of the new nucleic acid molecule containing chip sequence information formed by the above exemplary embodiment comprises: from the 5' end to the 3' end containing the consensus sequence B, the complementary sequence of the 3' end overhang, the cDNA sequence Complementary sequence, the complementary sequence of the UMI sequence, the complementary sequence of the consensus sequence A, the complementary sequence of the tag sequence Y, and the nucleic acid strand of the complementary sequence of the consensus sequence X1 and/or its complementary nucleic acid strand.
  • Embodiments comprising step (1), step (2)(ii) and step (3)(ii)
  • the method comprises step (1), step (2)(ii) and step (3)(ii); wherein the second region of the second bridging oligonucleotide is capable of combining with step (2) Annealing of the complementary sequence of the consensus sequence A of the second extension product obtained in (ii) or a partial sequence thereof;
  • the reaction product obtained in step (3)(ii) is a labeled nucleic acid molecule, which comprises: The first strand of the first nucleic acid molecule sequence to be labeled, and/or, the second strand containing the oligonucleotide probe sequence.
  • the second region of the second bridging oligonucleotide can be the complementary sequence of the consensus sequence A of the second extension product obtained in step (2)(ii) or a partial region of the complementary sequence of the consensus sequence A
  • the nucleotide sequences of the segments are annealed.
  • the first strand comprises from the 5' end to the 3' end: the sequence of the first nucleic acid molecule to be labeled, optionally the third region of the second bridging oligonucleotide complementary sequence, the first bridging oligonucleotide sequence, the complementary sequence of the tag sequence Y, the complementary sequence of the consensus sequence X1.
  • the second strand comprises from the 5' end to the 3' end: the consensus sequence X1, the tag sequence Y, the consensus sequence X2, optionally the first bridging oligo
  • the consensus sequence X1 the tag sequence Y
  • the consensus sequence X2 optionally the first bridging oligo
  • Embodiment comprising step (1), step (2)(ii) and step (3)(ii): a chain
  • the second region of the second bridging oligonucleotide can be compatible with the complementary sequence of the consensus sequence A of the second extension product obtained in step (2)(ii) or the 3' end portion thereof The sequences anneal and the second region of the first bridging oligonucleotide has a 3' free end.
  • the reaction product obtained in step (3)(ii) is a labeled nucleic acid molecule comprising the first strand.
  • the second region of the first bridging oligonucleotide is located at the 3' end of the first bridging oligonucleotide.
  • the first region of the first bridging oligonucleotide is located at the 5' end of the first bridging oligonucleotide. In certain embodiments, said first bridging oligonucleotide does not contain said third region, and/or said second bridging oligonucleotide does not contain said third region.
  • the 5' end of the first bridging oligonucleotide contains a phosphorylation modification.
  • the 3' end of the first bridging oligonucleotide contains a free -OH.
  • step (3)(ii) the second bridging oligonucleotide is unable to initiate an extension reaction (eg, the 3' end is blocked), and/or, the oligonucleotide The probe cannot initiate an extension reaction (eg, the 3' end is blocked).
  • the capture sequence A of the primer A' is a random oligonucleotide sequence.
  • the extension primer is the primer B'.
  • the second extension product comprises from the 5' end to the 3' end: the consensus sequence B, optionally the tag sequence B, The complementary sequence of the overhanging sequence at the 3' end, the complementary sequence of the cDNA sequence complementary to the RNA formed by using the primer A' as a reverse transcription primer, and the complementary sequence of the consensus sequence A.
  • the first strand comprises from the 5' end to the 3' end: the consensus sequence B, optionally the tag sequence B, the complementary sequence of the overhang sequence at the 3' end, and the Primer A' is the complementary sequence of the cDNA sequence complementary to the RNA formed by the reverse transcription primer, the complementary sequence of the consensus sequence A, and optionally the complementary sequence of the third region of the second bridging oligonucleotide, The first bridging oligonucleotide sequence, the complementary sequence of the tag sequence Y, the complementary sequence of the consensus sequence X1.
  • each copy of the first strand derived from the same oligonucleotide probe has a different complementary sequence of capture sequence A as the UMI.
  • the capture sequence A of the primer A' is a poly(T) sequence or a specific sequence for a specific target nucleic acid.
  • the primer A' also contains a tag sequence A, such as a random oligonucleotide sequence.
  • the capture sequence A is located at the 3' end of the primer A.
  • the extension primer is the primer B'.
  • the second extension product comprises from the 5' end to the 3' end: the consensus sequence B, optionally the tag sequence B, The complementary sequence of the overhang sequence at the 3' end, the complementary sequence of the cDNA sequence complementary to the RNA formed by using the primer A' as a reverse transcription primer, the complementary sequence of the tag sequence A, the consensus sequence A complementary sequence.
  • the first strand comprises from the 5' end to the 3' end: the consensus sequence B, optionally the tag sequence B, the complementary sequence of the overhang sequence at the 3' end, and the Primer A' is the complementary sequence of the cDNA sequence complementary to the RNA formed by the reverse transcription primer, the complementary sequence of the tag sequence A, the complementary sequence of the consensus sequence A, and optionally the second bridging oligonucleotide
  • each copy of the first strand derived from the same oligonucleotide probe has a different complementary sequence of the tag sequence A as the UMI.
  • step (3)(ii) at the positions corresponding to the first bridging oligonucleotide, the second bridging oligonucleotide, the oligonucleotide probe and the oligonucleotide probe
  • the nucleic acid molecules hybridized to the first region and the second region of the same first bridging oligonucleotide are ligated, and/or, the nucleic acid molecules hybridized to the same second bridging oligonucleotide
  • the ligation reaction process of connecting the nucleic acid molecules of the first region and the second region and the extension reaction described in step (3)(ii) can be carried out in any order, as long as the second nucleic acid molecule with a position marker can be obtained.
  • nucleic acid molecules that hybridize to the first region and the second region of the same second bridging oligonucleotide can be ligated, and the first The bridging oligonucleotide initiates the extension reaction resulting in the first strand being obtained.
  • the polymerase used in the extension reaction preferably does not have strand displacement activity or 5' to 3' excision activity.
  • the first strand can be obtained in the following exemplary ways:
  • the polymerase used in the extension reaction preferably has strand displacement activity or 5' to 3' excision activity.
  • said first bridging oligonucleotide can be obtained by initiating an extension reaction with said first bridging oligonucleotide and then ligating nucleic acid molecules hybridizing to the first and second regions of the same second bridging oligonucleotide. first chain.
  • the polymerase used in the extension reaction preferably does not have strand displacement activity or 5' to 3' excision activity.
  • Embodiment comprising step (1), step (2)(ii) and step (3)(ii): two chains
  • the second region of the second bridging oligonucleotide is capable of annealing to the complementary sequence of the consensus sequence A or a partial sequence thereof of the second extension product obtained in step (2)(ii), And the second region of the second bridging oligonucleotide has a 3' free end.
  • the reaction product obtained in step (3)(ii) is a labeled nucleic acid molecule comprising said second strand.
  • the second region of the second bridging oligonucleotide is located at the 3' end of the second bridging oligonucleotide.
  • the first region of the second bridging oligonucleotide is located at the 5' end of the second bridging oligonucleotide.
  • said first bridging oligonucleotide does not contain said third region, and/or said second bridging oligonucleotide does not contain said third region.
  • the 5' end of the second bridging oligonucleotide contains a phosphorylation modification.
  • the 3' end of the second bridging oligonucleotide contains a free -OH.
  • step (3)(ii) the first bridging oligonucleotide cannot initiate an extension reaction (eg, the 3' end is blocked), and/or, step (2)(ii) ) The second extension product obtained cannot initiate the extension reaction (for example, the 3' end is blocked).
  • the capture sequence A of the primer A' is a random oligonucleotide sequence.
  • the extension primer is the primer B'.
  • the second extension product comprises from the 5' end to the 3' end: the consensus sequence B, optionally the tag sequence B, The complementary sequence of the overhanging sequence at the 3' end, the complementary sequence of the cDNA sequence complementary to the RNA formed by using the primer A' as a reverse transcription primer, and the complementary sequence of the consensus sequence A.
  • the second strand comprises from the 5' end to the 3' end: the consensus sequence X1, the tag sequence Y, the consensus sequence X2, optionally the first bridging oligo
  • each copy of the second strand derived from the same oligonucleotide probe has a different capture sequence A as UMI.
  • the capture sequence A of the primer A' is a poly(T) sequence or a specific sequence for a specific target nucleic acid.
  • the primer A' also contains a tag sequence A, such as a random oligonucleotide sequence.
  • the capture sequence A is located at the 3' end of the primer A.
  • the extension primer is the primer B'.
  • the second extension product comprises from the 5' end to the 3' end: the consensus sequence B, optionally the tag sequence B, The complementary sequence of the overhang sequence at the 3' end, the complementary sequence of the cDNA sequence complementary to the RNA formed by using the primer A' as a reverse transcription primer, the complementary sequence of the tag sequence A, the consensus sequence A complementary sequence.
  • the second strand comprises from the 5' end to the 3' end: the consensus sequence X1, the tag sequence Y, the consensus sequence X2, optionally the first bridging oligo
  • each copy of the second strand derived from the same oligonucleotide probe has a different tag sequence A as UMI.
  • step (3)(ii) at the positions corresponding to the first bridging oligonucleotide, the second bridging oligonucleotide, the oligonucleotide probe and the oligonucleotide probe
  • the nucleic acid molecules hybridized to the first region and the second region of the same first bridging oligonucleotide are ligated, and/or, the nucleic acid molecules hybridized to the same second bridging oligonucleotide
  • the ligation reaction process of connecting the nucleic acid molecules of the first region and the second region and the extension reaction described in step (3)(ii) can be carried out in any order, as long as the second nucleic acid molecule with a position marker can be obtained.
  • nucleic acid molecules hybridized to the first region and the second region of the same first bridging oligonucleotide can be connected, and the second The bridging oligonucleotide initiates the extension reaction, resulting in the second strand.
  • the polymerase used in the extension reaction preferably does not have strand displacement activity or 5' to 3' excision activity.
  • the second chain can be obtained in the following exemplary ways:
  • said second bridging oligonucleotide can be obtained by initiating an extension reaction with said second bridging oligonucleotide and then ligating nucleic acid molecules hybridizing to the first and second regions of the same first bridging oligonucleotide. second chain.
  • the polymerase used in the extension reaction preferably does not have strand displacement activity or 5' to 3' excision activity.
  • step (1) An exemplary embodiment of the present application comprising step (1), step (2)(ii) and step (3)(ii) is described in detail as follows:
  • An exemplary scheme for preparing a cDNA strand complementary chain using RNA (such as mRNA) in a sample as a template comprises the following steps (as shown in FIG. 5 ):
  • RNA molecules (for example, mRNA molecules) in the permeabilized sample are reverse-transcribed using reverse transcriptase (for example, reverse transcriptase with terminal transfer activity) and primer A' to generate cDNA, and An overhang (eg, an overhang comprising 3 cytosine nucleotides) is added at the 3' end.
  • reverse transcriptase for example, reverse transcriptase with terminal transfer activity
  • primer A' primer A' to generate cDNA
  • An overhang eg, an overhang comprising 3 cytosine nucleotides
  • Various reverse transcriptases having terminal transfer activity can be used for the reverse transcription reaction.
  • the reverse transcriptase used does not have RNaseH activity.
  • the primer A' comprises a poly(T) sequence, a UMI sequence, and a consensus sequence A(CA).
  • a poly(T) sequence is located at the 3' end of the primer A' to initiate reverse transcription, and the consensus sequence A is located upstream (e.g., at the 5' end) of the UMI sequence.
  • the primer A' comprises a random oligonucleotide sequence and a consensus sequence A, which can be used to capture RNA without a ploy A tail.
  • the random oligonucleotide sequence is located at the 3' end of the primer A' to initiate reverse transcription.
  • sequence complementary to the 3' end overhang of the cDNA strand is located at the 3' end of the primer B'.
  • the primer B' may include GGG at its 3' end.
  • the nucleotides of the primer B' can also be modified (for example, using a locked nucleic acid) to enhance the complementary pairing between the primer B' and the 3' end overhang of the cDNA strand.
  • nucleic acid polymerases for example, DNA polymerase or reverse transcriptase
  • DNA polymerase or reverse transcriptase can be used to carry out the extension reaction, as long as it can be captured using the sequence of the primer B' or a partial sequence thereof as a template extension Nucleic acid fragments (reverse transcription products) can be.
  • reverse transcriptase enzyme as in the previous reverse transcription step can be used to extend the captured nucleic acid fragment (reverse transcription product).
  • this step is performed simultaneously with step (1) (eg, in the same reaction system).
  • the method optionally further comprises step (3): adding RNaseH to digest the RNA strand in the RNA/cDNA hybrid duplex to form a cDNA single strand.
  • said method does not comprise said step (3).
  • extension primer the cDNA strand obtained in the previous step is used as a template for an extension reaction to obtain an extension product;
  • the extension primer is the primer B', a random primer, or a primer B", and the primer B" can be combined with The consensus sequence B or a partial sequence thereof anneals and is capable of initiating an extension reaction.
  • the exemplary structure of the cDNA strand complementary chain prepared by the above exemplary embodiment comprises: consensus sequence B, complementary sequence of 3' end overhang, complementary sequence of cDNA sequence, complementary sequence of UMI sequence, and complementary sequence of consensus sequence A sequence.
  • a bridging oligonucleotide pair consisting of a first bridging oligonucleotide and a second bridging oligonucleotide, wherein each of the first bridging oligonucleotide and the second bridging oligonucleotide is independently Including: a first region (P1) and a second region (P2), the first region is located upstream of the second region (for example, the 5' end); wherein,
  • the first region of the first bridging oligonucleotide is capable of annealing to the first region of the second bridging oligonucleotide; the second region of the first bridging oligonucleotide is capable of annealing to the oligonucleotide Annealing to the consensus sequence X2 of the acid probe or a partial sequence thereof;
  • the second region of the second bridging oligonucleotide can anneal to the complementary sequence of the consensus sequence A in the complementary strand of the cDNA strand obtained in the above step 1 or a partial sequence thereof.
  • the first bridging oligonucleotide contains spacer nucleotides between the first region and the second region, such as 1-5nt or 5-10nt spacer nucleotides, that is, the first bridging oligonucleotide
  • a bridging oligonucleotide sequence contains a third region located between the first region and the second region.
  • the first region and the second region in the first bridging oligonucleotide are adjacently connected without redundant nucleotides, that is, the first bridging oligonucleotide
  • the nucleotide sequence does not contain a third region located between the first region and the second region.
  • the first region and the second region in the second bridging oligonucleotide include spacer nucleotides, such as 1-5nt or 5-10nt spacer nucleotides, that is, the second bridging oligonucleotide
  • the second bridging oligonucleotide sequence contains a third region located between the first region and the second region.
  • the first region and the second region in the second bridging oligonucleotide are adjacently connected without redundant nucleotides, that is, the second bridging oligonucleotide
  • the nucleotide sequence does not contain a third region located between the first region and the second region.
  • the first bridging oligonucleotide, the second bridging oligonucleotide and the chip sequence are annealed or hybridized to the complementary strand of the cDNA strand obtained in step 1 above, and then hybridized to the same first bridging oligonucleotide by DNA ligase and/or, link nucleic acid molecules of the first and second regions that hybridize to the same second bridging oligonucleotide. Subsequently, under the action of DNA polymerase, new nucleic acid molecules containing ChIP-seq information (ie, ChIP-seq-labeled nucleic acid molecules) are formed. The concatenation process and polymerization process are performed in any order.
  • the exemplary structure of the new nucleic acid molecule containing chip sequence information formed by the above exemplary embodiment comprises: from the 5' end to the 3' end containing the consensus sequence B, the complementary sequence of the 3' end overhang, the cDNA sequence complementary sequence, the complementary sequence of the UMI sequence, the complementary sequence of the consensus sequence A, the first bridging oligonucleotide sequence, the complementary sequence of the tag sequence Y, and the complementary sequence of the consensus sequence X1 A nucleic acid strand and/or its complementary nucleic acid strand.
  • step (2)(i)(b) of the method the cDNA strand anneals to the primer B via its 3' end overhang, and, in the presence of a nucleic acid polymerase (e.g., Under the action of DNA polymerase or reverse transcriptase), the cDNA chain is extended using the primer B as a template to generate the first extension product.
  • a nucleic acid polymerase e.g., Under the action of DNA polymerase or reverse transcriptase
  • step (2)(ii)(b) of the method the cDNA strand is annealed to the primer B' via its 3' end overhang, and, in the presence of a nucleic acid polymerase (e.g. , DNA polymerase or reverse transcriptase), the cDNA chain is extended using the primer B' as a template to generate the first extension product.
  • a nucleic acid polymerase e.g. , DNA polymerase or reverse transcriptase
  • the 3' terminal overhang has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 , at least 10 or more nucleotides in length. In certain embodiments, the 3' terminal overhang is a 3' terminal overhang of 2-5 cytosine nucleotides (eg, a CCC overhang).
  • step (2) before performing the pretreatment, the biological sample is permeabilized.
  • the biological sample is a tissue sample.
  • the tissue sample is a tissue section.
  • the tissue sections are prepared from fixed tissue, eg, formalin-fixed paraffin-embedded (FFPE) tissue or deep-frozen tissue.
  • FFPE formalin-fixed paraffin-embedded
  • each cell of the biological sample occupies one or more microspots in the nucleic acid array (i.e., each cell is individually associated with One or more micropoint contacts in the nucleic acid array).
  • performing reverse transcription in step (2) includes using reverse transcriptase.
  • the reverse transcriptase has terminal transfer activity.
  • the reverse transcriptase is capable of synthesizing a cDNA strand using RNA (eg, mRNA) as a template, and adding an overhang at the 3' end of the cDNA strand.
  • RNA eg, mRNA
  • the reverse transcriptase is capable of adding at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 nucleotides in length to the 3' end of the cDNA strand. , an overhang of at least 8, at least 9, at least 10 or more nucleotides.
  • the reverse transcriptase is capable of adding an overhang of 2-5 cytosine nucleotides (eg, a CCC overhang) at the 3' end of the cDNA strand.
  • an overhang of 2-5 cytosine nucleotides eg, a CCC overhang
  • the reverse transcriptase is selected from the group consisting of M-MLV reverse transcriptase, HIV-1 reverse transcriptase, AMV reverse transcriptase, telomerase reverse transcriptase, and transposases having the above transposase activity variants, modifications and derivatives.
  • steps (2) and (3) have one or more features selected from:
  • the primer A, primer A', primer B, primer B', the first bridging oligonucleotide, and the first bridging oligonucleotide each independently comprise or consist of naturally occurring nucleotides (such as deoxyribose nucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof; in certain embodiments, the primer A, primer A' is capable of initiating an extension reaction ;
  • the primer B comprises a modified nucleotide (such as a locked nucleic acid); in some embodiments, the 3' end of the primer B comprises one or more modified nucleotides (such as a locked nucleic acid);
  • the primer B' comprises a modified nucleotide (such as a locked nucleic acid); in some embodiments, the 3' end of the primer B' comprises one or more modified nucleotides (such as a locked nucleic acid );
  • the tag sequence A and the tag sequence B each independently have a length of 5-200 (eg, 5-30nt, 6-15nt);
  • the consensus sequence A and the consensus sequence B each independently have 10-200nt (such as 10-100nt, 20-100nt, 25-100nt, 5-10nt, 10-15nt, 15-20nt, 20-50nt, 20 -30nt, 30-40nt, 40-50nt, 50-100nt) length;
  • the primer A, primer A', primer B, and primer B' each independently have 4-200nt (such as 5-200nt, 15-230nt, 26-115nt, 10-130nt, 10-20nt, 20-50nt , 20-30nt, 30-40nt, 40-50nt, 50-100nt, 100-150nt, 150-200nt) length;
  • the first region of the first bridging oligonucleotide, and the second region each independently have 3-100nt (such as 20-100nt, 3-10nt, 10-15nt, 15-20nt, 20-70nt, 20 -30nt, 30-40nt, 40-50nt, 50-100nt) length;
  • the first region of the second bridging oligonucleotide, the second region each independently has 3-100nt (such as 20-100nt, 3-10nt, 10-15nt, 15-20nt, 20-70nt, 20 -30nt, 30-40nt, 40-50nt, 50-100nt) length;
  • the third region of the first bridging oligonucleotide, the third region of the second bridging oligonucleotide each independently has 0-50nt (such as Ont, 0-10nt, 10-15nt, 15 -20nt, 20-30nt, 30-40nt, 40-50nt) length;
  • the first bridging oligonucleotide and the second bridging oligonucleotide each independently have 6-200nt (such as 20-100nt, 20-70nt, 6-15nt, 15-20nt, 20-30nt, 30nt -40nt, 40-50nt, 50-100nt, 100-150nt, 150-200nt) length;
  • the poly(T) sequence includes at least 5, or at least 20 (eg, 6-100, 10-50) deoxythymidine residues;
  • the random oligonucleotide sequence has a length of 5-200 (eg 5nt, 5-30nt, 6-15nt).
  • the method further comprises: (4) recovering and purifying the second population of nucleic acid molecules.
  • the obtained second population of nucleic acid molecules and/or complements thereof are used for constructing a transcriptome library or for transcriptome sequencing.
  • the oligonucleotide probes in step (1) have one or more characteristics selected from the following:
  • consensus sequence X1, tag sequence Y and consensus sequence X2 each independently comprise or consist of naturally occurring nucleotides (such as deoxyribonucleotides or ribonucleotides), modified nucleotides, non- Natural nucleotides (such as peptide nucleic acid (PNA) or locked nucleic acid), or any combination thereof;
  • naturally occurring nucleotides such as deoxyribonucleotides or ribonucleotides
  • modified nucleotides such as peptide nucleic acid (PNA) or locked nucleic acid
  • the consensus sequence X1, the tag sequence Y and the consensus sequence X2 each independently have 2-200nt (such as 10-200nt, 25-100nt, 10-30nt, 10-100nt, 5-10nt, 10-15nt, 15 -20nt, 20-30nt, 30-40nt, 40-50nt, 50-100nt) length.
  • 2-200nt such as 10-200nt, 25-100nt, 10-30nt, 10-100nt, 5-10nt, 10-15nt, 15 -20nt, 20-30nt, 30-40nt, 40-50nt, 50-100nt
  • the oligonucleotide probe is coupled to the solid support via a linker.
  • the linker is a linking group capable of reacting with an activating group, and the surface of the solid support is linked with an activating group.
  • the linker comprises -SH, -DBCO, or -NHS.
  • the nucleic acid array in step (1) has one or more characteristics selected from the following:
  • the oligonucleotide probes coupled on the same solid support have the same consensus sequence X1 and/or the same consensus sequence X2; (2) the oligonucleotide probes
  • the consensus sequence X1 of the nucleotide probes comprises a cleavage site; in some embodiments, the cleavage site can be selected from a nicking enzyme (nicking enzyme) enzyme cleavage, USER enzyme cleavage, photocleavage, chemical cleavage or CRISPR cut or fractured by means of resection.
  • nicking enzyme nicking enzyme
  • the nucleic acid array in step (1) is provided by steps comprising:
  • each vector sequence comprising at least one copy (for example, multiple copies) of the vector sequence, the vector sequence comprising from the 5' to 3' direction: the complementary sequence of the consensus sequence X2, The complementary sequence of the tag sequence Y and the fixed sequence; wherein, the complementary sequences of the tag sequence Y of each carrier sequence are different from each other;
  • extension product comprises or consists of: a consensus sequence X1, a tag sequence Composed of Y and consensus sequence X2;
  • steps (3) and (4) are performed in any order;
  • the fixed sequence of the carrier sequence also includes a cleavage site, and the cleavage can be selected from nicking enzyme enzyme cleavage, USER enzyme cleavage, light cleavage, chemical cleavage or CRISPR cleavage;
  • the cleavage site contained in the fixed sequence of the carrier sequence is cut to digest the carrier sequence, so that the extension product in step (3) is separated from the template (i.e. the carrier sequence) forming the extension product, so that the oligo Nucleotide probes are attached to the surface of a solid support such as a chip.
  • the method further includes separating the extension product in step (3) from the template forming the extension product (ie, the carrier sequence) by high temperature denaturation.
  • each vector sequence is a DNB formed from a concatemer of multiple copies of the vector sequence.
  • the various vector sequences are provided in step (1) by the following steps:
  • each vector template sequence as a template to perform a nucleic acid amplification reaction to obtain an amplification product of each vector template sequence, the amplification product comprising at least one copy of the vector sequence; in certain embodiments, Rolling circle replication is performed to obtain DNBs formed from concatemers of the vector sequences.
  • the solid phase support in step (1) has one or more characteristics selected from the following:
  • the solid support is selected from latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes and silicon wafers; In some embodiments, the solid support is a chip;
  • the solid support is planar, spherical or porous
  • the solid phase support can be used as a sequencing platform, such as a sequencing chip; in some embodiments, the solid phase support is a sequencing chip for Illumina, MGI or Thermo Fisher sequencing platforms; and
  • the solid support is capable of releasing all the compounds spontaneously or upon exposure to one or more stimuli (e.g., temperature change, pH change, exposure to a specific chemical substance or phase, exposure to light, reducing agent, etc.) oligonucleotide probes.
  • stimuli e.g., temperature change, pH change, exposure to a specific chemical substance or phase, exposure to light, reducing agent, etc.
  • the present application also provides a method for constructing a library of nucleic acid molecules, which includes,
  • step (c) optionally, amplifying and/or enriching the product of step (b);
  • a library of nucleic acid molecules is thereby obtained.
  • the library of nucleic acid molecules is used for sequencing, e.g., transcriptome sequencing, e.g., single cell transcriptome sequencing (e.g., 5' or 3' transcriptome sequencing).
  • sequencing e.g., transcriptome sequencing, e.g., single cell transcriptome sequencing (e.g., 5' or 3' transcriptome sequencing).
  • the method before performing step (b), further comprises a step (pre-b): amplifying and/or enriching the population of labeled nucleic acid molecules.
  • step (pre-b) the population of labeled nucleic acid molecules is subjected to a nucleic acid amplification reaction to generate an enriched product.
  • the amplification reaction is performed using at least primer C and/or primer D, wherein the primer C is capable of hybridizing or annealing to the complementary sequence of the consensus sequence X1 or a partial sequence thereof, and Initiate an extension reaction; the primer D can hybridize or anneal to the nucleic acid molecular chain containing the tag sequence Y in the labeled nucleic acid molecule population, and initiate an extension reaction.
  • the nucleic acid amplification reaction in step (pre-b) is performed using a nucleic acid polymerase (eg, DNA polymerase, eg, DNA polymerase with strand displacement activity and/or high fidelity).
  • a nucleic acid polymerase eg, DNA polymerase, eg, DNA polymerase with strand displacement activity and/or high fidelity.
  • step (b) of the method the nucleic acid molecule is randomly disrupted with a transposase and adapters are added.
  • the nucleic acid molecule obtained in the previous step is randomly interrupted with a transposase, and a first linker and a second linker are respectively added to both ends of the fragment.
  • the transposase is selected from Tn5 transposase, MuA transposase, Sleeping Beauty transposase, Mariner transposase, Tn7 transposase, Tn10 transposase, Ty1 transposase, Tn552 transposase, and variants, modified products and derivatives having the transposition activity of the above-mentioned transposases.
  • the transposase is a Tn5 transposase.
  • step (c) at least primer C' and/or primer D' are used to amplify the product of step (b), wherein said primer C' is capable of combining with said first adapter hybridizes or anneals and initiates an extension reaction, said primer D' is capable of hybridizing or annealing to said second adapter and initiates an extension reaction.
  • step (c) at least the product of step (b) is amplified using the primer C and/or primer D'; wherein, the primer D' can be combined with the first The adapter or second adapter hybridizes or anneals and initiates an extension reaction.
  • the present application also provides a method for performing transcriptome sequencing on cells in a sample, comprising:
  • test kit comprising:
  • nucleic acid array for labeling nucleic acids comprising a solid support coupled to a plurality of oligonucleotide probes; each oligonucleotide probe comprising at least one copy; and , the oligonucleotide probe comprises or consists of: consensus sequence X1, tag sequence Y and consensus sequence X2 from the 5' to 3' direction, wherein,
  • oligonucleotide probes have different tag sequences Y, and the tag sequence Y has a unique nucleotide sequence corresponding to the position of the oligonucleotide probe on the solid support;
  • the primer A contains a capture sequence A capable of annealing to the RNA to be captured (eg, mRNA) and initiating an extension reaction;
  • the primer B comprises a consensus sequence B, a 3' end overhang complementary sequence, and an optional tag sequence B; in some embodiments, the 3' end overhang complementary sequence is located at the 3' end of the primer B ; In some embodiments, the consensus sequence B is located upstream of the complementary sequence of the 3' end overhang (for example, at the 5' end of the primer B); wherein, the 3' end overhang refers to The RNA captured by the capture sequence A of the primer A is one or more non-template nucleotides contained in the 3' end of the cDNA chain generated by template reverse transcription;
  • the primer A' contains a consensus sequence A and a capture sequence A; in some embodiments, the capture sequence A is located at the 3' end of the primer A'; in some embodiments, the consensus sequence A is located at Upstream of the capture sequence A (for example, at the 5' end of the primer A');
  • the primer B' comprises a consensus sequence B, a 3' end overhanging complementary sequence, and an optional tag sequence B; in some embodiments, the 3' end overhanging complementary sequence is located at the 3' end of the primer B' ' end; in some embodiments, the consensus sequence B is located upstream of the complementary sequence of the 3' end overhang (for example, at the 5' end of the primer B'); wherein the 3' end overhang Refers to one or more non-template nucleotides contained in the 3' end of the cDNA chain generated by reverse transcription using the RNA captured by the capture sequence A of the primer A' as a template.
  • each oligonucleotide probe comprises one copy.
  • each oligonucleotide probe comprises multiple copies.
  • the region where each of the oligonucleotide probes is coupled to the solid support is referred to as a microspot. It is easy to understand that when each oligonucleotide probe is one copy, each micro-dot is coupled with a probe, and the oligonucleotide probes of different micro-dots have different label sequences Y; when each oligonucleotide When the nucleotide probe contains multiple copies, each micro-dot is coupled with multiple probes, the oligonucleotide probes in the same micro-dot have the same label sequence Y, and the oligonucleotide probes in different micro-dots have Different label sequences Y.
  • the solid support comprises a plurality of microdots, each microdot is coupled to an oligonucleotide probe, and each oligonucleotide probe may comprise one or more copies.
  • the solid support comprises a plurality (eg, at least 10, at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , or more) microdots; in certain embodiments, the solid support comprises at least 10 4 (eg, at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12 ) microdots/square millimeter.
  • the solid support comprises at least 10 4 (eg, at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12 ) microdots/square millimeter.
  • the interval between adjacent microdots is less than 100 ⁇ m, less than 50 ⁇ m, less than 10 ⁇ m, less than 5 ⁇ m, less than 1 ⁇ m, less than 0.5 ⁇ m, less than 0.1 ⁇ m, less than 0.05 ⁇ m, or less than 0.01 ⁇ m.
  • the microdots have a size (e.g., equivalent diameter) of less than 100 ⁇ m, less than 50 ⁇ m, less than 10 ⁇ m, less than 5 ⁇ m, less than 1 ⁇ m, less than 0.5 ⁇ m, less than 0.1 ⁇ m, less than 0.05 ⁇ m, or less than 0.01 ⁇ m .
  • the kit comprises: a nucleic acid array for labeling nucleic acids as described in (i), a primer set of primer A and primer B as described in (ii), and, (iii ) a first bridging oligonucleotide and a second bridging oligonucleotide; wherein the first bridging oligonucleotide and the second bridging oligonucleotide each independently comprise: a first region and a second region , and optionally a third region located between the first region and the second region, the first region being located upstream (e.g., the 5' end) of the second region; wherein,
  • the first region of the first bridging oligonucleotide is capable of annealing to the first region of the second bridging oligonucleotide; the second region of the first bridging oligonucleotide is capable of annealing to the oligonucleotide Annealing to the consensus sequence X2 of the acid probe or a partial sequence thereof;
  • the second region of the second bridging oligonucleotide can anneal to the complementary sequence of the consensus sequence B of the primer B or a partial sequence thereof.
  • the capture sequence A of the primer A is a random oligonucleotide sequence.
  • the capture sequence A of the primer A is a poly(T) sequence or a specific sequence for a specific target nucleic acid.
  • the primer A further comprises a consensus sequence A and an optional tag sequence A, such as a random oligonucleotide sequence.
  • the capture sequence A is located at the 3' end of the primer A, and the consensus sequence A is located upstream of the primer A (eg, the 5' end).
  • the primer B contains the consensus sequence B, the complementary sequence of the 3' end overhang, and the tag sequence B.
  • the primer B comprises modified nucleotides (eg, locked nucleic acids).
  • the 3' end of primer B comprises one or more modified nucleotides (eg, locked nucleic acids).
  • the second region of the second bridging oligonucleotide is capable of annealing to the complementary sequence of the consensus sequence B of the primer B or a partial sequence thereof (for example, a 3' end partial sequence).
  • the second region of the first bridging oligonucleotide is located at the 3' end of the first bridging oligonucleotide.
  • the first region of the first bridging oligonucleotide is located at the 5' end of the first bridging oligonucleotide.
  • the 5' end of the first bridging oligonucleotide contains a phosphorylation modification.
  • the 3' end of the first bridging oligonucleotide contains a free -OH.
  • the second bridging oligonucleotide is incapable of initiating an extension reaction (e.g., the 3' end is blocked), and/or, the oligonucleotide probe is incapable of initiating an extension reaction (e.g., 3' ends are blocked).
  • the second region of the second bridging oligonucleotide is capable of annealing to the complementary sequence of the consensus sequence B of the primer B or a partial sequence thereof.
  • the second region of the second bridging oligonucleotide is located at the 3' end of the second bridging oligonucleotide.
  • the first region of the second bridging oligonucleotide is located at the 5' end of the second bridging oligonucleotide.
  • the 5' end of the second bridging oligonucleotide contains a phosphorylation modification.
  • the 3' end of the second bridging oligonucleotide contains a free -OH.
  • the first bridging oligonucleotide is incapable of initiating an extension reaction (e.g., the 3' end is blocked).
  • the kit comprises: a nucleic acid array for labeling nucleic acids as described in (i), and a primer set of primer A and primer B as described in (ii).
  • the capture sequence A of the primer A is a random oligonucleotide sequence.
  • the capture sequence A of the primer A is a poly(T) sequence or a specific sequence for a specific target nucleic acid.
  • the primer A further comprises a consensus sequence A and an optional tag sequence A, such as a random oligonucleotide sequence.
  • the capture sequence A is located at the 3' end of the primer A, and the consensus sequence A is located upstream (eg, the 5' end) of the primer A.
  • the primer B contains the consensus sequence B, the complementary sequence of the 3' end overhang, and the tag sequence B.
  • the primer B comprises modified nucleotides (eg, locked nucleic acids).
  • the 3' end of primer B comprises one or more modified nucleotides (eg, locked nucleic acids).
  • the oligonucleotide probe is capable (e.g. 3' end contains a free -OH) or incapable of initiating an extension reaction (e.g. 3' end is blocked).
  • the kit comprises: a nucleic acid array for labeling nucleic acids as described in (i), a primer set of primer A' and primer B' as described in (ii), and, (iii) a first bridging oligonucleotide and a second bridging oligonucleotide; wherein, the first bridging oligonucleotide and the second bridging oligonucleotide each independently comprise: a first region and a second bridging oligonucleotide Two regions, and optionally a third region located between the first region and the second region, the first region being located upstream (eg 5' end) of the second region; wherein,
  • the first region of the first bridging oligonucleotide is capable of annealing to the first region of the second bridging oligonucleotide; the second region of the first bridging oligonucleotide is capable of annealing to the oligonucleotide Annealing to the consensus sequence X2 of the acid probe or a partial sequence thereof;
  • the second region of the second bridging oligonucleotide can anneal to the complementary sequence of the consensus sequence A of the primer A' or a partial sequence thereof.
  • the capture sequence A of the primer A' is a random oligonucleotide sequence.
  • the capture sequence A of the primer A' is a poly(T) sequence or a specific sequence for a specific target nucleic acid.
  • the primer A' further comprises a tag sequence A, such as a random oligonucleotide sequence.
  • the capture sequence A is located at the 3' end of the primer A', and the consensus sequence A is located upstream of the tag sequence A (for example, at the 5' end of the primer A').
  • the primer B' comprises modified nucleotides (e.g., locked nucleic acids).
  • the 3' end of primer B' comprises one or more modified nucleotides (e.g., locked nucleic acids).
  • the kit further comprises a primer B" or a random primer, the primer B" can anneal to the complementary sequence of the consensus sequence B or a partial sequence thereof, and can initiate an extension reaction.
  • the second region of the second bridging oligonucleotide is capable of annealing to the complementary sequence of the consensus sequence A of the primer A' or a partial sequence thereof (for example, a partial sequence at the 3' end).
  • the second region of the first bridging oligonucleotide is located at the 3' end of the first bridging oligonucleotide.
  • the first region of the first bridging oligonucleotide is located at the 5' end of the first bridging oligonucleotide.
  • the 5' end of the first bridging oligonucleotide contains a phosphorylation modification.
  • the 3' end of the first bridging oligonucleotide contains a free -OH.
  • the second bridging oligonucleotide is incapable of initiating an extension reaction (e.g., the 3' end is blocked), and/or, the oligonucleotide probe is incapable of initiating an extension reaction (e.g., 3' ends are blocked).
  • the second region of the second bridging oligonucleotide is capable of annealing to the complementary sequence of the consensus sequence A of the primer A' or a partial sequence thereof.
  • the second region of the second bridging oligonucleotide is located at the 3' end of the second bridging oligonucleotide.
  • the first region of the second bridging oligonucleotide is located at the 5' end of the second bridging oligonucleotide.
  • the 5' end of the second bridging oligonucleotide contains a phosphorylation modification.
  • the 3' end of the second bridging oligonucleotide contains a free -OH.
  • the first bridging oligonucleotide is incapable of initiating an extension reaction (e.g., the 3' end is blocked).
  • the kit comprises: a nucleic acid array for labeling nucleic acids as described in (i), and a primer set of primer A' and primer B' as described in (ii).
  • the capture sequence A of the primer A' is a random oligonucleotide sequence.
  • the capture sequence A of the primer A' is a poly(T) sequence or a specific sequence for a specific target nucleic acid.
  • the primer A' further comprises a tag sequence A, such as a random oligonucleotide sequence.
  • the capture sequence A is located at the 3' end of the primer A', and the consensus sequence A is located upstream of the tag sequence A (for example, at the 5' end of the primer A').
  • the primer B' contains the consensus sequence B, the complementary sequence of the 3' end overhang, and the tag sequence B.
  • the primer B' comprises modified nucleotides (e.g., locked nucleic acids).
  • the 3' end of primer B' comprises one or more modified nucleotides (e.g., locked nucleic acids).
  • the kit further comprises a primer B" or a random primer, the primer B" can anneal to the complementary sequence of the consensus sequence B or a partial sequence thereof, and can initiate an extension reaction.
  • the oligonucleotide probe is capable (eg 3' end contains a free -OH) or incapable of initiating an extension reaction (eg 3' end is blocked).
  • the kit has one or more features selected from:
  • the oligonucleotide probes, primer A, primer A', primer B, primer B', primer B", random primers, the first bridging oligonucleotide, and the second bridging oligonucleotide are independent Contains or consists of naturally occurring nucleotides (such as deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof;
  • the oligonucleotide probes each independently have 15-300nt (such as 15-200nt, 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-100nt, 100-150nt, 150- 200nt) in length;
  • primer A, primer A', primer B, primer B', primer B", random primers each independently have 4-200nt (such as 5-200nt, 15-230nt, 26-115nt, 10-130nt, 10-20nt, 20-50nt, 20-30nt, 30-40nt, 40-50nt, 50-100nt, 100-150nt, 150-200nt) length;
  • the first bridging oligonucleotide and the second bridging oligonucleotide each independently have 6-200nt (such as 20-100nt, 20-70nt, 6-15nt, 15-20nt, 20-30nt , 30-40nt, 40-50nt, 50-100nt, 100-150nt, 150-200nt) length;
  • the oligonucleotide probes coupled to the same solid support have the same consensus sequence X1 and/or the same consensus sequence X2;
  • the consensus sequence X1 of the oligonucleotide probe comprises a cleavage site; Cut or fragmented by photoablation, chemical ablation, or CRISPR ablation.
  • the kit further comprises reverse transcriptase, nucleic acid ligase, nucleic acid polymerase and/or transposase.
  • the reverse transcriptase has terminal transfer activity.
  • the reverse transcriptase is capable of synthesizing a cDNA strand using RNA (eg, mRNA) as a template, and adding the 3' end overhang at the 3' end of the cDNA strand.
  • the reverse transcriptase is capable of adding at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 nucleotides in length to the 3' end of the cDNA strand. , an overhang of at least 8, at least 9, at least 10 or more nucleotides.
  • the reverse transcriptase is capable of adding an overhang of 2-5 cytosine nucleotides (eg, a CCC overhang) at the 3' end of the cDNA strand.
  • the reverse transcriptase is selected from the group consisting of M-MLV reverse transcriptase, HIV-1 reverse transcriptase, AMV reverse transcriptase, telomerase reverse transcriptase, and transposases having the above transposase activity variants, modifications and derivatives.
  • the nucleic acid polymerase has no 5' to 3' exonucleating activity or strand displacement activity.
  • the nucleic acid polymerase has 5' to 3' exonucleation activity or strand displacement activity.
  • the transposase is selected from Tn5 transposase, MuA transposase, Sleeping Beauty transposase, Mariner transposase, Tn7 transposase, Tn10 transposase, Ty1 transposase, Tn552 transposase, and variants, modified products and derivatives having the transposition activity of the above-mentioned transposases.
  • the kit further comprises: the primer C, the primer D, the primer C' and/or the primer D'.
  • the kit further comprises the primer C, the primer D and the primer D'.
  • said kit further comprises said primer C, said primer D, said primer C' and said primer D'.
  • the kit further comprises: reagents for nucleic acid hybridization, reagents for nucleic acid extension, reagents for nucleic acid amplification, reagents for recovering or purifying nucleic acids, reagents for A reagent for constructing a transcriptome sequencing library, a reagent for sequencing (such as next-generation sequencing or third-generation sequencing), or any combination thereof.
  • the present application also provides the above-mentioned method for generating a labeled nucleic acid molecule population or the use of the above-mentioned kit for constructing a library of nucleic acid molecules or for performing transcriptome sequencing.
  • DNB DNA nanoball, DNA nanoball
  • RCA rolling circle amplification
  • the RCA product is a multi-copy single-stranded DNA sequence, which can form a similar "spherical” structure due to the interaction force between the bases of the internal DNA sequence.
  • the library molecules are circularized to form single-stranded circular DNA, and then the single-stranded circular DNA can be amplified by multiple orders of magnitude using rolling circle amplification technology, and the resulting amplification product is called DNB.
  • a "population of nucleic acid molecules” refers to, for example, nucleic acid molecules derived directly or indirectly from target nucleic acid molecules (e.g., DNA double-stranded molecules, RNA/cDNA hybrid double-stranded molecules, DNA single-stranded molecules, or RNA single-stranded molecules) groups or collections.
  • the population of nucleic acid molecules comprises a library of nucleic acid molecules comprising sequences qualitatively and/or quantitatively representative of target nucleic acid molecule sequences.
  • the population of nucleic acid molecules comprises a subset of a library of nucleic acid molecules.
  • a "library of nucleic acid molecules” refers to labeled nucleic acid molecules (e.g., labeled DNA double-stranded molecules, labeled RNA/cDNA hybrid double-stranded molecules, labeled DNA Single-stranded molecules, or labeled RNA single-stranded molecules) or a collection or population of fragments thereof, wherein the combination of labeled nucleic acid molecules or fragments thereof in the collection or population exhibits qualitative and/or quantitative representation of the resulting The sequence of the target nucleic acid molecule sequence of the labeled nucleic acid molecule.
  • the library of nucleic acid molecules is a sequencing library.
  • the library of nucleic acid molecules can be used to construct a sequencing library.
  • cDNA or "cDNA strand” refers to a primer that anneals to an RNA molecule of interest, catalyzed by RNA-dependent DNA polymerase or reverse transcriptase, using at least a portion of the RNA molecule of interest as a template
  • the "complementary DNA” synthesized by the extension of DNA (this process is also called “reverse transcription”).
  • the synthesized cDNA molecule is "homologous” or “complementary” or “base paired” or “complexed” with at least a portion of the template.
  • upstream is used to describe the relative positional relationship of two nucleic acid sequences (or two nucleic acid molecules), and has the meaning generally understood by those skilled in the art.
  • the expression “one nucleic acid sequence is located upstream of another nucleic acid sequence” means that when arranged in the 5' to 3' direction, the former is located in a more forward position (i.e., closer to the 5' end) than the latter Location).
  • downstream has the opposite meaning of "upstream”.
  • Tag Sequence Y As used herein, "Tag Sequence Y”, “Tag Sequence A”, “Tag Sequence B”, “Consensus Sequence X1”, “Consensus Sequence X2”, “Consensus Sequence A”, “Consensus Sequence B”, etc.
  • the joined nucleic acid molecule or a derivative product of the joined nucleic acid molecule provides means for identification, recognition, and/or molecular manipulation or biochemical manipulation (e.g., by providing A site for annealing an oligonucleotide, such as a primer for DNA polymerase extension or an oligonucleotide for a non-target nucleic acid component of a capture reaction or ligation reaction) glycosides.
  • the oligonucleotides may consist of consecutive at least two (preferably about 6 to 100, but there is no firm limit to the length of the oligonucleotides, the exact size depends on many factors which in turn depend on the oligonucleotide
  • the final function or use of acid) nucleotides can also be composed of multiple oligonucleotides in continuous or discontinuous arrangement.
  • the oligonucleotide sequence may be unique for each nucleic acid molecule it ligates, or it may be unique for a certain class of nucleic acid molecules it ligates.
  • the oligonucleotide sequence can be reversibly or irreversibly joined to the polynucleotide sequence to be "labeled” by any means including ligation, hybridization or other methods.
  • the process of joining the oligonucleotide sequence to a nucleic acid molecule is sometimes referred to herein as "labeling" and a nucleic acid molecule undergoing labeling or containing a labeling sequence is referred to as a "labeled nucleic acid molecule" or "labeled nucleic acid molecule”. .
  • Nucleic acids or polynucleotides of the present invention may include one or more modified nucleobases, sugar moieties or internucleoside linkages.
  • nucleic acids or polynucleotides that contain modified bases, sugar moieties, or internucleoside linkages include, but are not limited to: (1) changes in Tm; (3) providing a moiety for attaching a label; (4) providing a label or a label quencher; or (5) providing a moiety for attaching another molecule in solution or bound to a surface, such as Biotin.
  • oligonucleotides such as primers can be synthesized such that the random portion comprises one or more conformationally constrained nucleic acid analogs, such as but not limited to, wherein a ribose ring is attached 2' One or more ribonucleic acid analogues in which the -O atom is "locked" to the methylene bridge of the 4'-C atom; these modified nucleotides result in an increase in the Tm or melting temperature of each nucleotide monomer by approx. 2 degrees C to about 8 degrees C.
  • conformationally constrained nucleic acid analogs such as but not limited to, wherein a ribose ring is attached 2'
  • ribonucleic acid analogues in which the -O atom is "locked" to the methylene bridge of the 4'-C atom
  • one indicator of the use of modified nucleotides in the method may be the nucleoside comprising the modification
  • Acidic oligonucleotides can be digested by single-strand-specific RNases.
  • a nucleic acid base in a single nucleotide at one or more positions in a polynucleotide or oligonucleotide may include guanine, adenine, uracil, thymine, or cytosine.
  • one or more of the nucleic acid bases may comprise modified bases such as, but not limited to, xanthine, allyamino-uracil, allyamino-thymine Nucleosides, hypoxanthine, 2-aminoadenine, 5-propynyluracil, 5-propynylcytosine, 4-thiouracil, 6-thioguanine, nitrogen-uracil and deaza-uracil, thymidine, cytosine, adenine, or guanine.
  • modified bases such as, but not limited to, xanthine, allyamino-uracil, allyamino-thymine Nucleosides, hypoxanthine, 2-aminoadenine, 5-propynyluracil, 5-propynylcytosine, 4-thiouracil, 6-thioguanine, nitrogen-uracil and deaza-uracil, thymidine, cytosine, adenine,
  • nucleic acid bases may comprise nucleic acid bases derivatized with a biotin moiety, a digoxigenin moiety, a fluorescent or chemiluminescent moiety, a quencher moiety, or some other moiety.
  • the invention is not limited to the listed nucleic acid bases; the list given shows examples of a wide range of bases that can be used in the methods of the invention.
  • one or more of the sugar moieties may include 2'-deoxyribose, or alternatively, one or more of the sugar moieties may include some other sugar moiety, such as But not limited to: Ribose or 2'-fluoro-2'-deoxyribose or 2'-O-methyl-ribose that provide resistance to some nucleases, or can be passed with visible, fluorescent, infrared fluorescent 2'-amino 2'-deoxyribose or 2'-azido- 2'-deoxyribose.
  • internucleoside linkages of nucleic acids or polynucleotides of the invention may be phosphodiester linkages, or alternatively, one or more of the internucleoside linkages may include modified linkages such as, but not limited to: Phosphate, phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkages, which are resistant to some nucleases.
  • terminal transfer activity refers to the ability to catalyze the template-independent addition (or “tailing") of one or more deoxyribonucleoside triphosphates (dNTPs) or a single dideoxyribonucleoside triphosphate to Activity of the 3' end of the cDNA.
  • dNTPs deoxyribonucleoside triphosphates
  • Examples of reverse transcriptases having terminal transfer activity include, but are not limited to, M-MLV reverse transcriptase, HIV-1 reverse transcriptase, AMV reverse transcriptase, telomerase reverse transcriptase, and reverse transcriptases having said reverse transcriptase Variants, modified products and derivatives with recording activity and terminal transfer activity. Described reverse transcriptase does not have or has RNase activity (particularly RNase H activity).
  • the reverse transcriptase used to reverse transcribe RNA to generate cDNA does not have RNase activity (particularly RNase H activity).
  • the reverse transcriptase used to reverse transcribe RNA to generate cDNA has terminal transfer activity and does not have RNase activity (particularly RNase H activity).
  • nucleic acid polymerase with "strand displacement activity” means that, in the process of elongating a new nucleic acid strand, if it encounters a downstream nucleic acid strand complementary to the template strand, it can continue the extension reaction and replace the nucleic acid strand complementary to the template strand.
  • nucleic acid polymerase having "5' to 3' exonuclease activity” refers to a nucleic acid polymerase capable of catalyzing the hydrolysis of 3, 5- Phosphodiester bond, nucleic acid polymerase that degrades nucleotides.
  • a nucleic acid polymerase (or DNA polymerase) with "high fidelity” means that, during the process of amplifying nucleic acid, the probability of introducing a wrong nucleotide (i.e., the error rate) is lower than that of the wild-type Taq enzyme (for example, the nucleic acid polymerase (or DNA polymerase) of Taq enzyme whose sequence is shown in UniProt Accession: P19821.1).
  • annealing As used herein, the terms “annealing”, “annealing”, “annealing”, “hybridizing” or “hybridizing” and the like refer to the presence of sufficient complementarity to form a complex via Watson-Crick base pairing. Complexes are formed between nucleotide sequences.
  • nucleic acid sequences that are “complementary to” or “complementary” or “hybridize” or “anneal” to each other should be able to form or form sufficiently stable “hybrids" or “hybrids” that serve the intended purpose. "Complex".
  • every nucleic acid base within the sequence represented by one nucleic acid molecule is capable of base pairing or pairing or complexing with every nucleic acid base within the sequence represented by a second nucleic acid molecule such that the two nucleic acid molecules or one of them
  • Corresponding sequences shown are “complementary” or “anneal” or “hybridize” to each other.
  • the terms “complementary” or “complementarity” are used when referring to a sequence of nucleotides related by the base pairing rules. For example, the sequence 5'-A-G-T-3' is complementary to the sequence 3'-T-C-A-5'.
  • Complementarity can be "partial,” wherein only some of the nucleic acid bases match according to the base pairing rules. Alternatively, there may be “perfect” or “total” complementarity between nucleic acids. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that rely on hybridization of nucleic acids.
  • the term “homology” refers to the degree of complementarity of one nucleic acid sequence to another nucleic acid sequence. There may be partial or complete homology (ie, complementarity).
  • a partially complementary sequence is one that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid and is referred to using the functional term "substantially homologous". Inhibition of hybridization of a perfectly complementary sequence to a target sequence can be examined under low stringency conditions using a hybridization assay (eg, Southern or Northern blot, solution hybridization, etc.). Substantially homologous sequences or probes will compete or inhibit binding (ie, hybridization) of a fully homologous sequence to a target under conditions of low stringency. This is not to say that low stringency conditions are conditions that allow non-specific binding; low stringency conditions require that the binding of two sequences to each other is a specific (ie selective) interaction.
  • a hybridization assay eg, Southern or Northern blot, solution hybridization, etc.
  • the absence of non-specific binding can be tested by using a second target that lacks complementarity or has only a low degree of complementarity (eg, less than about 30% complementarity). In cases of little or no specific binding, the probe will not hybridize to the nucleic acid target.
  • substantially homologous when used in reference to a double-stranded nucleic acid sequence, such as a cDNA or genomic clone, means hybridizable to one or both strands of the double-stranded nucleic acid sequence under low stringency conditions as described herein any oligonucleotide or probe.
  • the terms “anneal” or “hybridize” are used when referring to the pairing of complementary nucleic acid strands.
  • Hybridization and the strength of hybridization are affected by a number of factors well known in the art, including the degree of complementarity between the nucleic acids, including the stringency of conditions affected by conditions such as salt concentration, the degree of hybridization formed The Tm (melting temperature) of the body, the presence of other components (eg, the presence or absence of polyethylene glycol or betaine), the molarity of the hybridized strands, and the G:C content of the nucleic acid strands.
  • the solid support can spontaneously or upon exposure to one or more stimuli (e.g., temperature change, pH change, exposure to a particular chemical species or phase, exposure to light, reducing agent, etc.)
  • the oligonucleotide probe is released. It will be appreciated that the oligonucleotide probe may be released by cleavage of the bond between the oligonucleotide probe and the solid support, or by degradation of the solid support itself. Oligonucleotide probes, or both, which allow or are accessible to other reagents.
  • Addition of various types of labile bonds to the solid support can result in a solid support capable of responding to different stimuli.
  • Each type of labile bond can be sensitive to relevant stimuli (eg, chemical stimuli, light, temperature, etc.), so that the release of substances attached to the solid support through each labile bond can be controlled by applying appropriate stimuli.
  • labile bonds that can be coupled to solid supports include ester bonds (for example, cleavable with acids, bases, or hydroxylamine), ortho Diol bonds (e.g., cleavable by sodium periodate), Diels-Alder bonds (e.g., cleavable by heat), sulfone bonds (e.g., cleavable by bases), silane Ether bonds (e.g., cleavable by acids), glycosidic bonds (e.g., cleavable by amylases), peptide bonds (e.g., cleavable by proteases), or phosphodiester bonds (e.g., cleavable by nucleases (e.g., DNA Enzyme) cleavage)).
  • ester bonds for example, cleavable with acids, bases, or hydroxylamine
  • ortho Diol bonds e.g., cleavable by sodium periodate
  • Diels-Alder bonds
  • the solid support can be activated spontaneously or upon exposure to one or more stimuli (e.g., temperature). degradable, destructible or soluble upon exposure to a change in pH, change in pH, exposure to a particular chemical species or phase, exposure to light, reducing agents, etc.).
  • a solid support can be soluble such that the material components of the solid support dissolve upon exposure to a particular chemical or environmental change (eg, a change in temperature or a change in pH).
  • the solid support degrades or dissolves under elevated temperature and/or alkaline conditions.
  • the solid support can be thermally degradable such that when the solid support is exposed to an appropriate temperature change (eg, heating), the solid support degrades. Degradation or dissolution of a solid support bound to a substance (eg, an oligonucleotide probe) can result in the release of the substance from the solid support.
  • an appropriate temperature change eg, heating
  • transposase and reverse transcriptase and “nucleic acid polymerase” refer to protein molecules or aggregates of protein molecules responsible for catalyzing specific chemical and biological reactions.
  • the methods, compositions or kits of the invention are not limited to the use of a particular transposase, reverse transcriptase or nucleic acid polymerase from a particular source.
  • the methods, compositions, or kits of the invention include any transposase, reverse transcriptase, or nucleic acid polymerase from any source that has equivalent enzymatic activity to the particular enzyme of the particular method, composition, or kit disclosed herein.
  • the method of the present invention also includes the following embodiment: wherein any specific enzyme provided and used in the steps of the method is replaced by a combination of two or more enzymes, the two or more enzymes When used in combination, whether used separately in a stepwise fashion or together simultaneously, the reaction mixture produces the same results as those obtained with that one particular enzyme.
  • the methods, buffers and reaction conditions provided herein, including those in the Examples, are presently preferred for embodiments of the methods, compositions and kits of the invention.
  • other enzyme storage buffers, reaction buffers and reaction conditions using some of the enzymes of the invention are known in the art and may also be suitable for use in the invention and are included herein.
  • the present application provides a new method for generating labeled nucleic acid molecule populations, and based on the method for constructing a nucleic acid molecule library and performing high-throughput sequencing, thereby realizing high-precision subcellular spatial positioning of samples.
  • the method of the present application has one or more beneficial technical effects selected from the following:
  • the probes of traditional nucleic acid arrays (such as chips) used for spatial transcriptome sequencing contain fixed capture sequences, usually specific capture sequences can only capture specific target nucleic acid molecules corresponding to them, for example, when the capture sequence is poly (T), corresponding to capturing target nucleic acid molecules containing poly(A). If the target nucleic acid molecule changes, the probe sequence containing the capture sequence needs to be modified accordingly, that is, the entire nucleic acid array (such as a chip) needs to be modified, which is costly and inefficient in practical applications.
  • the nucleic acid array (such as a chip) of the present application does not contain a capture sequence, and the capture sequence exists in a reverse transcription primer independent of the nucleic acid array (that is, the capture sequence and the probe are independent of each other). Nucleotides effectuate attachment to the probe.
  • the present application can design corresponding capture sequences for different target nucleic acid molecules without changing the probe sequence (that is, without changing the nucleic acid array (such as a chip)). Changes in acid enable capture of different target nucleic acid molecules.
  • RNA permeabilization is generally performed first to release intracellular RNA. Excessive permeabilization will cause RNA to diffuse to adjacent cells Even the periphery of the tissue sample is captured by the probe, so that the in situ capture of mRNA cannot be achieved. If the permeabilization is not complete, the capture efficiency of mRNA will be affected.
  • the nucleic acid array (such as a chip) does not contain a capture sequence (only spatial information), and the purpose of tissue permeabilization is to allow reverse transcription primers to enter cells and hybridize with mRNA in situ, without the need for intense permeabilization reagent treatment, thereby Can reduce the spread of the sample.
  • Fig. 1 shows an exemplary structure of a chip used for capturing and labeling nucleic acid molecules in this application, which includes: a chip and oligonucleotide probes (also called chip sequences) coupled to the chip.
  • oligonucleotide probes also called chip sequences
  • Each oligonucleotide probe contains a label sequence Y corresponding to its position on the chip, and the coupling area between each oligonucleotide probe and the chip can be called a micro spot.
  • Each oligonucleotide probe can be single or multiple copies.
  • FIG. 2 shows an exemplary scheme for preparing a cDNA chain using RNA (such as mRNA) in a sample as a template, and an exemplary structure of the cDNA chain.
  • RNA such as mRNA
  • Figure 3 shows an exemplary scheme 1 for marking the 3' end of a cDNA strand with the complementary sequence of ChIP-seq to form a new nucleic acid molecule (ie, a ChIP-seq-labeled nucleic acid molecule) containing ChIP-seq information, and the ChIP-seq-containing Exemplary structures of novel nucleic acid molecules of sequence information.
  • CA consensus sequence A
  • CB consensus sequence B
  • X1 consensus sequence X1
  • Y tag sequence Y
  • X2 consensus sequence X2
  • P1 first region
  • P2 second region.
  • Figure 4 shows an exemplary scheme 2 for marking the 3' end of a cDNA strand with the complementary sequence of ChIP-seq to form a new nucleic acid molecule containing ChIP-seq information (that is, a nucleic acid molecule labeled with ChIP-seq), and the ChIP-seq-containing Exemplary structures of novel nucleic acid molecules of sequence information.
  • CA consensus sequence A
  • CB consensus sequence B
  • X1 consensus sequence X1
  • Y tag sequence Y
  • X2 consensus sequence X2.
  • FIG. 5 shows an exemplary scheme for preparing a complementary strand of a cDNA chain using RNA (such as mRNA) in a sample as a template, and an exemplary structure of the complementary strand of the cDNA strand.
  • RNA such as mRNA
  • Figure 6 shows an exemplary scheme 1 for marking the 3' end of the complementary strand of the cDNA strand with the complementary sequence of ChIP-seq to form a new nucleic acid molecule (that is, a nucleic acid molecule marked by ChIP-seq) containing ChIP-seq information, and, the Exemplary structures of novel nucleic acid molecules containing ChIP-seq information.
  • CA consensus sequence A
  • CB consensus sequence B
  • X1 consensus sequence X1
  • Y tag sequence Y
  • X2 consensus sequence X2
  • P1 first region
  • P2 second region.
  • Figure 7 shows an exemplary scheme 2 for marking the 3' end of the complementary strand of the cDNA strand with the complementary sequence of the ChIP-seq to form a new nucleic acid molecule (that is, a nucleic acid molecule marked by the ChIP-seq) containing the ChIP-seq information, and, the Exemplary structures of novel nucleic acid molecules containing ChIP-seq information.
  • CA consensus sequence A
  • CB consensus sequence B
  • X1 consensus sequence X1
  • Y tag sequence Y
  • X2 consensus sequence X2.
  • Fig. 8 shows the length distribution of cDNA amplification products prepared in Example 2.
  • Fig. 9 shows the spatial expression map of the mouse brain slice obtained from the sequencing analysis in Example 3.
  • DNBSEQ sequencing kit purchased from MGI, catalog number 1000019840 was used to prepare DNA nanoballs (DNB). Specific embodiments are briefly described below.
  • reaction system 40 ⁇ L of the reaction system shown in Table 2 was configured.
  • the reaction system was placed in a PCR instrument, and the reaction was carried out according to the following reaction conditions: 95°C for 3 minutes, 40°C for 3 minutes.
  • After the reaction put the reaction product on ice, add 40 ⁇ L mixed enzyme I and 2 ⁇ L mixed enzyme II (from DNBSEQ sequencing kit), 1 ⁇ L ATP (100 mM stock solution, obtained from Thermo Fisher), and 0.1 ⁇ L T4ligase (obtained from NEB, Cat. No.: M0202S).
  • the above reaction system was placed in a PCR instrument and reacted at 30° C. for 20 minutes to generate DNB.
  • the DNB was loaded onto the BGISEQ SEQ 500 sequencing chip according to the method described in the BGISEQ 500 high-throughput sequencing reagent set (SE50) (purchased from MGI, catalog number: 1000012551).
  • the sequencing chip add the MDA reagent in the BGISEQ 500 PE50 sequencing kit (purchased from MGI, 1000012554), incubate at 37°C for 30min, and then wash the chip with 5XSSC.
  • Chip surface modified with N3-PEG3500-NHS (the modification reagent was purchased from sigma, product number: JKA5086). After incubation for 30 minutes, pump into the DBCO-modified chip sequence to synthesize primers (sequence shown in SEQ ID NO: 3), and overnight at room temperature Incubation.
  • the DNB was sequenced according to the instructions of the BGISEQ-500 high-throughput sequencing reagent kit, and the read length of SE was set to 25bp.
  • the above-mentioned DBCO-modified sequence is extended to obtain the chain grown after sequencing, and the chain is decoded to obtain the position sequence information corresponding to the DNB.
  • the chain grown after sequencing continues to extend: on the basis of the above step 3, continue to carry out the cPAS reaction of 15 bases to obtain the chip sequence (SEQ ID NO: 8, which contains the consensus sequence X1 (SEQ ID NO: 4), Tag sequence Y, consensus sequence X2 (SEQ ID NO:5)).
  • Chip dicing cut the prepared chip into several small pieces, adjust the size of the slice according to the needs of the experiment, soak the chip in 50mM Tris buffer with pH 8.0, and keep it at 4°C for use.
  • Example 2 cDNA in situ synthesis and amplification
  • the mouse tissue sections were made according to the standard method of frozen sections, and the frozen sections were pasted on the chip prepared in Example 1. After 30 min of frozen methanol fixation, the tissues were permeabilized with 0.5% Triton X-100. Use 5X SSC to wash the chip twice at room temperature, configure 200 ⁇ L of the reverse transcriptase reaction system shown in Table 3, add the reaction solution to the chip, fully cover it, and react at 42°C for 90min-180min. Reverse transcriptase will use mRNA as a template to synthesize cDNA with primers containing polyT (sequence shown in SEQ ID NO: 6, which contains consensus sequence A (CA) and polyT sequence), and add CCC overhang.
  • SEQ ID NO: 6 which contains consensus sequence A (CA) and polyT sequence
  • the reverse transcriptase will use the consensus sequence B and the UMI sequence as templates to continue to extend the cDNA chain, so that the 3' end of the cDNA will carry the complementary sequence of the consensus sequence B and the complementary sequence of the UMI sequence.
  • the synthetic cDNA strand comprises the following sequence structure: the sequence of the reverse transcription primer (SEQ ID NO:6)-cDNA sequence-c(TSO) sequence (complementary sequence of SEQ ID NO:7).
  • the chip was cleaned with 5X SSC.
  • Prepare 200 ⁇ L of Bst polymerization reaction solution (purchased from NEB, M0275S) according to the instructions, pump it into the chip, and react at 65°C for 60 minutes to obtain double-stranded nucleic acid containing position information (namely, tag sequence Y(Y) or its complementary sequence c(Y)).
  • Molecule one strand of which contains the following sequence structure: cDNA sequence - complementary sequence of TSO sequence - first bridging oligonucleotide sequence - complementary sequence of ChIP-seq partial sequence.
  • cDNA concentration take 20ng cDNA (obtained in step 4 of Example 2), add 0.5 ⁇ M Tn5 interrupting enzyme and corresponding buffer (purchased from BGI, catalog number 10000028493; Tn5 interrupting enzyme coating method according to Stereomics library preparation kit- S1 operation), mix well to form a 20 ⁇ L reaction system, react at 55°C for 10 minutes, add 5 ⁇ L 0.1% SDS and mix at room temperature for 5 minutes to end the Tn5 interruption step.
  • Tn5 interrupting enzyme and corresponding buffer purchased from BGI, catalog number 10000028493; Tn5 interrupting enzyme coating method according to Stereomics library preparation kit- S1 operation
  • the reaction conditions are as follows: 95°C for 3 minutes, 40°C for 3 minutes; after the reaction is completed, put it on ice, add 40 ⁇ L of the mixed enzyme I required for DNB preparation in the DNBSEQ sequencing kit, and 2 ⁇ L of the mixed enzyme II, and 1 ⁇ L ATP, 0.1 ⁇ L T4Ligase, after mixing, put the above reaction system in a PCR instrument at 30°C, and react for 20 minutes to form DNB.

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Abstract

提供一种对核酸分子进行定位标记的方法，构建用于转录组测序的核酸分子文库的方法以及用于实施所述方法的试剂盒。

Description

一种生成标记的核酸分子群的方法及其试剂盒 技术领域
本申请涉及转录组测序(transcriptome sequencing)和生物分子空间信息检测的技术领域。具体而言，本申请涉及用于对核酸分子进行定位标记的方法，构建用于转录组测序的核酸分子文库的方法。此外，本申请还涉及，利用所述方法构建的核酸分子文库，以及用于实施所述方法的试剂盒。
背景技术
组织中细胞的空间位置显著影响其功能，为探究这种空间异质性，需要在获知空间坐标的情况下，对细胞的基因组或转录组进行量化及分析。然而要收集较小的组织区域甚至单个细胞用于基因组或转录组分析，非常费力、费钱且精确度低。因此，开发一种能够实现在单细胞级别甚至亚细胞级别高通量检测生物分子(例如核酸)的空间信息(例如，核酸的定位、分布和/或表达)的方法是十分必要的。
发明内容
本申请提供了一种新的生成标记的核酸分子群的方法，以及基于该方法构建核酸分子文库并进行高通量测序的方法。
生成标记的核酸分子群的方法
在一方面，本申请提供了一种生成标记的核酸分子群的方法，其包括下述步骤：
(1)提供：生物样本和核酸阵列；其中，所述核酸阵列包括固相支持物，所述固相支持物偶联有多种寡核苷酸探针；每种寡核苷酸探针包含至少一个拷贝；并且，所述寡核苷酸探针从5’到3’的方向上包含或者由：共有序列X1，标签序列Y和共有序列X2组成，其中，
不同种寡核苷酸探针具有不同的标签序列Y，所述标签序列Y具有与该种寡核苷酸探针在固相支持物的位置相对应的独一无二的核苷酸序列；
(2)将所述生物样本与所述核酸阵列接触，以使得所述生物样本中的RNA(例如，mRNA)的位置被对应至核酸阵列上所述寡核苷酸探针的位置；对所述生物样本中的RNA(例如，mRNA)进行预处理以生成第一核酸分子群，所述预处理包括以下步骤：
(i)(a)用引物A对所述生物样本的RNA(例如，mRNA)进行逆转录，生成cDNA链，所述cDNA链包含以所述引物A为逆转录引物形成的与所述RNA(例如，mRNA)互补的cDNA序列，以及3’末端悬突；其中，所述引物A含有捕获序列A，所述捕获序列A能与待捕获的RNA(例如，mRNA)退火并起始延伸反应；和，(b)将引物B与(a)中生成的所述cDNA链进行退火，并进行延伸反应，生成第一延伸产物，所述第一延伸产物即为待标记的第一核酸分子，从而生成第一核酸分子群；其中，所述引物B包含共有序列B，3’末端悬突互补序列，以及任选的标签序列B；所述3’末端悬突互补序列位于所述引物B的3’末端；所述共有序列B位于所述3’末端悬突互补序列的上游(例如位于所述引物B的5’端)；或，
(ii)(a)用引物A’对所述生物样本的RNA(例如，mRNA)进行逆转录，生成cDNA链； 所述cDNA链包含以所述引物A’为逆转录引物形成的与所述RNA(例如，mRNA)互补的cDNA序列，以及3’末端悬突；其中，所述引物A’含有共有序列A和捕获序列A所述捕获序列A能与待捕获的RNA(例如，mRNA)退火并起始延伸反应；所述共有序列A位于所述捕获序列A的上游(例如位于所述引物A’的5’端)；(b)将引物B’与(a)中生成的所述cDNA链进行退火，并进行延伸反应，生成第一延伸产物；其中，所述引物B’包含共有序列B，3’末端悬突互补序列，以及任选的标签序列B；所述3’末端悬突互补序列位于所述引物B’的3’末端；所述共有序列B位于所述3’末端悬突互补序列的上游(例如位于所述引物B’的5’端)；和，(c)提供延伸引物，以第一延伸产物为模板进行延伸反应，生成第二延伸产物，所述第二延伸产物即为待标记的第一核酸分子，从而生成第一核酸分子群；
(3)将前一步骤获得的第一核酸分子群通过包含选自下列的步骤生成第二核酸分子群：
(i)向步骤(2)的产物实施退火条件，使得所述寡核苷酸探针与所述寡核苷酸探针对应位置的待标记的第一核酸分子退火(例如原位退火)，并进行延伸反应，生成延伸产物，所述延伸产物即为具有位置标记的第二核酸分子，从而生成第二核酸分子群；其中，所述寡核苷酸探针的共有序列X2或其部分序列(a)能与步骤(2)(i)获得的第一延伸产物的所述共有序列B的互补序列或其部分序列退火，或者，(b)能与步骤(2)(ii)获得的第二延伸产物的所述共有序列A的互补序列或其部分序列退火；或，
(ii)在允许退火的条件下，将桥接寡核苷酸对与所述寡核苷酸探针以及前一步骤获得的第一核酸分子群接触，使得所述桥接寡核苷酸对与所述寡核苷酸探针以及所述寡核苷酸探针对应位置的待标记的第一核酸分子退火(例如原位退火)，
其中，所述桥接寡核苷酸对由第一桥接寡核苷酸和第二桥接寡核苷酸组成，所述第一桥接寡核苷酸和所述第二桥接寡核苷酸各自独立地包括：第一区域和第二区域，以及任选的位于第一区域和第二区域之间的第三区域，所述第一区域位于所述第二区域的上游(例如5’端)；其中，
所述第一桥接寡核苷酸的第一区域能与所述第二桥接寡核苷酸的第一区域退火；所述第一桥接寡核苷酸的第二区域能与所述寡核苷酸探针的共有序列X2或其部分序列退火；
所述第二桥接寡核苷酸的第二区域(a)能与步骤(2)(i)获得的第一延伸产物的所述共有序列B的互补序列或其部分序列退火，或者，(b)能与步骤(2)(ii)获得的第二延伸产物的所述共有序列A的互补序列或其部分序列退火；
其中，将所述桥接寡核苷酸对与所述第一核酸分子群、所述寡核苷酸探针接触时，所述桥接寡核苷酸对的第一桥接寡核苷酸和第二桥接寡核苷酸各自以单链的形式存在，或者，所述桥接寡核苷酸对的第一桥接寡核苷酸和第二桥接寡核苷酸以彼此退火形成部分双链的形式存在；
进行连接反应：将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接，和/或，将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接；并进行延伸反应；其中，所述连接反应与延伸反应以任意顺序进行；
所述获得的反应产物即为具有位置标记的第二核酸分子，从而生成所述第二核酸分子群。
在某些实施方案中，所述方法步骤(3)(ii)中：
(1)当所述第一桥接寡核苷酸的第一区域和第二区域相邻时，所述将杂交于同一第一桥接寡 核苷酸的第一区域和第二区域的核酸分子连接的步骤包括：使用核酸连接酶将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接；或者，
当所述第一桥接寡核苷酸包括第一区域、第二区域以及位于两者之间的第三区域时，所述将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接的步骤包括：使用核酸聚合酶(例如，无5’至3’端外切酶活性或链置换活性)以所述第三区域为模板进行聚合反应，使用核酸连接酶将杂交于同一第一桥接寡核苷酸的第一区域、第三区域和第二区域的核酸分子连接；
和/或
(2)当所述第二桥接寡核苷酸的第一区域和第二区域相邻时，所述将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接的步骤包括：使用核酸连接酶将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接；或者，
当所述第二桥接寡核苷酸包括第一区域、第二区域以及位于两者之间的第三区域时，所述将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接的步骤包括：使用核酸聚合酶(例如，无5’至3’端外切酶活性或链置换活性)以所述第三区域为模板进行聚合反应，使用核酸连接酶将杂交于同一第二桥接寡核苷酸的第一区域、第三区域和第二区域的核酸分子连接。
在某些实施方案中，每种寡核苷酸探针包含一个拷贝。
在某些实施方案中，每种寡核苷酸探针包含多个拷贝。
在某些实施方案中，每一种所述寡核苷酸探针与所述固相支持物偶联的区域即称作一个微点。容易理解，当每种寡核苷酸探针为一个拷贝时，每个微点偶联一个探针，并且不同微点的寡核苷酸探针具有不同的标签序列Y；当每种寡核苷酸探针包含多个拷贝时，每个微点偶联多个探针，同一微点内的寡核苷酸探针具有相同的标签序列Y，不同微点的寡核苷酸探针具有不同的标签序列Y。
在某些实施方案中，所述固相支持物包含多个微点，每个微点偶联一种寡核苷酸探针，每种寡核苷酸探针可包含一个或多个拷贝。
在某些实施方案中，所述固相支持物包含多个(例如，至少10个，至少10 2个，至少10 3个，至少10 4个，至少10 5个，至少10 6个，至少10 7个，至少10 8个，或更多个)微点；在某些实施方案中，所述固相支持物包含至少10 4个(例如至少10 4个，至少10 5个，至少10 6个，至少10 7个，至少10 8个，至少10 9个，至少10 10个，至少10 11个，或至少10 12个)微点/平方毫米。
在某些实施方案中，相邻的所述微点之间的间隔小于100μm，小于50μm，小于10μm，小于5μm，小于1μm，小于0.5μm，小于0.1μm，小于0.05μm，或小于0.01μm。
在某些实施方案中，所述微点的尺寸(例如等效直径)小于100μm，小于50μm，小于10μm，小于5μm，小于1μm，小于0.5μm，小于0.1μm，小于0.05μm，或小于0.01μm。
在某些实施方案中，所述方法包括步骤(1)、步骤(2)(i)和步骤(3)；其中，步骤(2)(i)(b)中，所述引物B含有共有序列B，3’末端悬突互补序列，以及标签序列B。
在某些实施方案中，步骤(2)(i)(b)中所述的第一延伸产物从5’端至3’端依次包含：以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列。
在某些实施方案中，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第二核酸分子具有不同的所述标签序列B作为UMI。
包括步骤(1)、步骤(2)(i)和步骤(3)(i)的实施方案
在某些实施方案中，所述方法包括步骤(1)、步骤(2)(i)和步骤(3)(i)；其中，所述共有序列X2或其部分序列能与所述共有序列B的互补序列或其部分序列退火；步骤(3)(i)中获得的延伸产物即为标记的核酸分子，其包含：含有所述待标记的第一核酸分子序列的第一链，和/或，含有所述寡核苷酸探针序列的第二链。
易于理解，所述“XX(序列)的部分序列”或“XX(序列)部分序列”意指“XX(序列)”的至少一个区段的核苷酸序列。
例如，所述共有序列X2可以以其整体的核苷酸序列与所述共有序列B的互补序列或所述共有序列B的互补序列的部分区段的核苷酸序列退火，所述共有序列X2也可以以其部分区段的核苷酸序列与所述共有序列B的互补序列或所述共有序列B的互补序列的部分区段的核苷酸序列退火。
所述“退火”意指，相互退火的两段核苷酸序列中，一段核苷酸序列中的每一个碱基都能够与另一段核苷酸序列中的碱基配对，而不存在错配或缺口；或者，相互退火的两段核苷酸序列中，一段核苷酸序列中的大部分碱基都能够与另一段核苷酸序列中的碱基配对，其允许存在错配或缺口(例如，一个或数个核苷酸的错配或缺口)。也即，能够退火的两段核苷酸序列既可以是完全互补，也可以是部分互补。除非本文另外指明或根据上下文明显矛盾，否则，此处有关“退火”的描述适用于本文全文。
在某些实施方案中，所述第一链从5’端至3’端包含：以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列，所述标签序列Y的互补序列，所述共有序列X1的互补序列。
在某些实施方案中，所述第二链从5’端至3’端包含：所述共有序列X1，所述标签序列Y，所述共有序列X2，所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列。
包括步骤(1)、步骤(2)(i)和步骤(3)(i)的实施方案：一链
在某些实施方案中，所述共有序列X2或其部分序列能与所述共有序列B的互补序列或其部分序列(例如，3’端部分序列)退火，并且步骤(2)(i)中的第一延伸产物的所述共有序列B的互补序列具有3’自由端。
在某些实施方案中，步骤(3)(i)中获得的延伸产物即为标记的核酸分子，其包含所述第一链。
在某些实施方案中，所述第一链从5’端至3’端包含：以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列，所述标签序列Y的互补序列，所述共有序列X1的互补序列。
在某些实施方案中，步骤(3)(i)中，所述寡核苷酸探针不能起始延伸反应(例如3’端是封闭的)。
在某些实施方案中，所述方法步骤(2)(i)(a)中，所述引物A的捕获序列A为随机寡核苷酸序列。
在某些实施方案中，步骤(2)(i)(b)中所述的第一延伸产物从5’端至3’端依次包含：以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列。
在某些实施方案中，所述第一链从5’端至3’端包含：以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列，所述标签序列Y的互补序列，所述共有序列X1的互补序列。
在某些实施方案中，所述方法步骤(2)(i)(a)中，所述引物A的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列。
在某些实施方案中，所述引物A还含有共有序列A，以及任选的标签序列A，例如为随机寡核苷酸序列。
在某些实施方案中，所述捕获序列A位于所述引物A的3’端。
在某些实施方案中，所述共有序列A位于所述捕获序列A的上游(例如位于所述引物A的5’端)。
在某些实施方案中，步骤(2)(i)(b)中所述第一延伸产物从5’端至3’端依次包含：所述共有序列A，任选的标签序列A，以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列。
在某些实施方案中，所述第一链从5’端至3’端包含：所述共有序列A，任选的所述标签序列A，以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列，所述标签序列Y的互补序列，所述共有序列X1的互补序列。
包括步骤(1)、步骤(2)(i)和步骤(3)(i)的实施方案：二链
在某些实施方案中，所述共有序列X2或其部分序列(例如，3’端部分序列)能与所述共有序列B的互补序列或其部分序列退火，并且所述寡核苷酸探针的所述共有序列X2具有3’自由端。
在某些实施方案中，步骤(3)(i)中获得的延伸产物即为标记的核酸分子，其包含所述第二链。
在某些实施方案中，所述第二链从5’端至3’端包含：所述共有序列X1，所述标签序列Y，所述共有序列X2，所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列。
在某些实施方案中，步骤(2)(i)获得的第一延伸产物不能起始延伸反应(例如3’端是封闭的)。
在某些实施方案中，所述方法步骤(2)(i)(a)中，所述引物A的捕获序列A为随机寡核苷酸序列。
在某些实施方案中，步骤(2)(i)(b)中所述的第一延伸产物从5’端至3’端依次包含：以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签 序列B的互补序列，所述共有序列B的互补序列。
在某些实施方案中，所述第二链从5’端至3’端包含：所述共有序列X1，所述标签序列Y，所述共有序列X2，所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列。
在某些实施方案中，所述方法步骤(2)(i)(a)中，所述引物A的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列。
在某些实施方案中，所述引物A还含有共有序列A，以及任选的标签序列A，例如为随机寡核苷酸序列。
在某些实施方案中，所述捕获序列A位于所述引物A的3’端。
在某些实施方案中，所述共有序列A位于所述捕获序列A的上游(例如位于所述引物A的5’端)。
在某些实施方案中，步骤(2)(i)(b)中所述第一延伸产物从5’端至3’端依次包含：所述共有序列A，任选的所述标签序列A，以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列。
在某些实施方案中，所述第二链从5’端至3’端包含：所述共有序列X1，所述标签序列Y，所述共有序列X2，所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列，任选的所述标签序列A的互补序列，所述共有序列A的互补序列。
如本文所使用的，术语“UMI”指“Unique Molecular Identifier，独特分子标签”，其可用于进行核酸分子的定性和/或定量。除非本文另外指明或根据上下文明显矛盾，本申请对所述UMI或其互补序列在核酸分子中的位置以及数量不做限定。例如，当cDNA链含有所述UMI或其互补序列，所述UMI或其互补序列可位于所述cDNA链中的cDNA序列的3’端，也可位于所述cDNA序列的5’端，也可以在3’端和5’端均包含所述UMI或其互补序列。当cDNA链互补链含有所述UMI或其互补序列，所述UMI或其互补序列可位于所述cDNA链互补链中的cDNA序列互补序列的3’端，也可位于所述cDNA序列互补序列的5’端，也可以在3’端和5’端均包含所述UMI或其互补序列。在某些实施方案中，所述UMI可以通过引物A引入，和/或通过引物B引入。在某些实施方案中，所述UMI可以通过引物A’引入，和/或通过引物B’引入。
本申请的包含步骤(1)、步骤(2)(i)和步骤(3)(i)的一个示例性实施方案详细描述如下：
一、以样本中的RNA(例如mRNA)为模板制备3’端含有UMI的互补序列的cDNA链的示例性方案包含以下步骤(如图2所示)：
(1)用逆转录酶(例如，具有末端转移活性的逆转录酶)和引物A对透化的样本中的RNA分子(例如，mRNA分子)进行逆转录，以生成cDNA，并在cDNA的3’端添加悬突(例如，包含3个胞嘧啶核苷酸的悬突)。可使用各种具有末端转移活性的逆转录酶来进行逆转录反应。在某些优选的实施方案中，所使用的逆转录酶不具有RNaseH活性。
在某些实施方案中，所述引物A包含poly(T)序列以及共有序列A(CA)。通常情况下， poly(T)序列位于所述引物A的3’末端，以便起始逆转录。
在某些实施方案中，所述引物A包含随机寡核苷酸序列，可用于捕获无ploy(A)尾的RNA。通常情况下，所述随机寡核苷酸序列位于所述引物A的3’末端，以便起始逆转录。
(2)使用引物B与cDNA链进行退火或杂交，所述引物B包含共有序列B(CB)、独特分子标签序列(UMI)以及所述cDNA的3’端悬突的互补序列。随后，与所述引物B杂交或退火的核酸片段在核酸聚合酶的作用下，可以以所述UMI序列和所述共有序列B为模板进行延伸，从而生成3’端携带所述UMI序列的互补序列、所述共有序列B的互补序列的的核酸分子。
通常情况下，所述共有序列B位于所述UMI序列的上游(例如5’端)，所述与cDNA链的3’末端悬突互补的序列位于所述引物B的3’末端。
例如，当cDNA链的3’末端包含3个胞嘧啶核苷酸的悬突时，所述引物B可在其3’端包含GGG。此外，还可以对所述引物B的核苷酸进行修饰(例如，使用锁核酸)，以增强所述引物B与cDNA链的3’末端悬突之间的互补配对。
不受理论限制，可以使用各种合适的核酸聚合酶(例如，DNA聚合酶或逆转录酶)来进行延伸反应，只要其能够以所述引物B的序列或其部分序列为模板延伸被捕获的核酸片段(逆转录产物)即可。在某些示例性实施方案中，可使用与前述逆转录步骤相同的逆转录酶来延伸被捕获的核酸片段(逆转录产物)。
在某些实施方案中，该步骤与步骤(1)同时进行(例如，在同一反应体系中进行)。
在某些实施方案中，所述方法任选地还包含步骤(3)：加入RNaseH，消化RNA/cDNA杂合双链中的RNA链，形成cDNA单链。
在某些实施方案中，所述方法不包括所述步骤(3)。
通过上述示例性实施方案所制备的cDNA链的示例性结构包含：共有序列A，cDNA序列，3’末端悬突序列，UMI序列的互补序列,以及，共有序列B的互补序列。
二、用寡核苷酸探针(也称，芯片序列)的互补序列标记cDNA链的3’端，以形成含有芯片序列信息的新核酸分子(即，经芯片序列标记的核酸分子)的示例性方案包含以下步骤(如图4所示)：
在某些实施方案中，所述芯片序列的共有序列X2或其部分序列能与上述步骤一中获得的cDNA链的所述共有序列B的互补序列或其部分序列退火。将该cDNA链与芯片序列退火或杂交，在聚合酶的作用下，形成含有芯片序列信息的新核酸分子(即，经芯片序列标记的核酸分子)。
通过上述示例性实施方案所形成的含有芯片序列信息的新核酸分子的示例性结构包含：从5’端至3’端含有共有序列A，cDNA序列，3’末端悬突序列，UMI序列的互补序列，共有序列B的互补序列，标签序列Y的互补序列，以及，共有序列X1的互补序列的核酸链和/或其互补核酸链。
包括步骤(1)、步骤(2)(i)和步骤(3)(ii)的实施方案
在某些实施方案中，所述方法包括步骤(1)、步骤(2)(i)和步骤(3)(ii)；其中，所述第二桥接寡核苷酸的第二区域能与步骤(2)(i)获得的第一延伸产物的所述共有序列B的互补序列或其部分序列退火；步骤(3)(ii)中获得的反应产物即为标记的核酸分子，其包含：含有所述待标记的第一核酸分子序列的第一链，和/或，含有所述寡核苷酸探针序列的第二链。
易于理解，所述第二桥接寡核苷酸的第二区域能与步骤(2)(i)获得的第一延伸产物的所述共有序列B的互补序列或所述共有序列B的互补序列的部分区段的核苷酸序列退火。
在某些实施方案中，所述第一链从5’端至3’端包含：以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列，任选的所述第二桥接寡核苷酸的第三区域的互补序列，所述第一桥接寡核苷酸序列，所述标签序列Y的互补序列，所述共有序列X1的互补序列。
在某些实施方案中，所述第二链从5’端至3’端包含：所述共有序列X1，所述标签序列Y，所述共有序列X2，任选的所述第一桥接寡核苷酸的第三区域的互补序列，所述第二桥接寡核苷酸序列，所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列。
包括步骤(1)、步骤(2)(i)和步骤(3)(ii)的实施方案：一链
在某些实施方案中，所述第二桥接寡核苷酸的第二区域能与步骤(2)(i)获得的第一延伸产物的所述共有序列B的互补序列或其部分序列(例如，3’端部分序列)退火，并且所述第一桥接寡核苷酸的第二区域具有3’自由端。
在某些实施方案中，步骤(3)(ii)中获得的反应产物即为标记的核酸分子，其包含所述第一链。
在某些实施方案中，所述第一链从5’端至3’端包含：以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列，任选的所述第二桥接寡核苷酸的第三区域的互补序列，所述第一桥接寡核苷酸序列，所述标签序列Y的互补序列，所述共有序列X1的互补序列。
在某些实施方案中，所述第一桥接寡核苷酸的第二区域位于所述第一桥接寡核苷酸的3’末端。
在某些实施方案中，所述第一桥接寡核苷酸的第一区域位于所述第一桥接寡核苷酸的5’末端。
在某些实施方案中，所述第一桥接寡核苷酸不含有所述第三区域，和/或，所述第二桥接寡核苷酸不含有所述第三区域。
在某些实施方案中，所述第一桥接寡核苷酸的5’末端含有磷酸化修饰。
在某些实施方案中，所述第一桥接寡核苷酸的3’末端含有自由-OH。
在某些实施方案中，步骤(3)(ii)中，所述第二桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)，和/或，所述寡核苷酸探针不能起始延伸反应(例如3’端是封闭的)。
在某些实施方案中，所述方法步骤(2)(i)(a)中，所述引物A的捕获序列A为随机寡核苷酸序列。
在某些实施方案中，所述方法步骤(2)(i)(b)中所述的第一延伸产物从5’端至3’端依次包含：以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列。
在某些实施方案中，所述第一链从5’端至3’端包含：以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列，任选的所述第二桥接寡核苷酸的第三区域的互补序列，所述第一桥接寡核苷酸序 列，所述标签序列Y的互补序列，所述共有序列X1的互补序列。
在某些实施方案中，步骤(2)(i)(a)中，所述引物A的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列。
在某些实施方案中，所述引物A还含有共有序列A，以及任选的标签序列A，例如为随机寡核苷酸序列。
在某些实施方案中，所述捕获序列A位于所述引物A的3’端。
在某些实施方案中，步骤(2)(i)(b)中所述第一延伸产物从5’端至3’端依次包含：所述共有序列A，任选的所述标签序列A，以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列。
在某些实施方案中，所述第一链从5’端至3’端包含：所述共有序列A，任选的所述标签序列A，以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列，任选的所述第二桥接寡核苷酸的第三区域的互补序列，所述第一桥接寡核苷酸序列，所述标签序列Y的互补序列，所述共有序列X1的互补序列。
易于理解，步骤(3)(ii)中，在所述第一桥接寡核苷酸、第二桥接寡核苷酸与所述寡核苷酸探针以及所述寡核苷酸探针对应位置的待标记的第一核酸分子退火之后，将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接，和/或，将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接的连接反应过程与步骤(3)(ii)中所述的延伸反应可以任意顺序进行，只要能获得带有位置标记的第二核酸分子即可。
例如，当所述连接反应与所述延伸反应在相同体系中进行，可通过将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接，并以所述第一桥接寡核苷酸起始延伸反应，获得所述第一链。在该种情况下，所述用于延伸反应的聚合酶优选不具有链置换活性或5'至3'外切活性。
例如，当所述连接反应与所述延伸反应在不同体系中进行，并且，先进行所述连接反应，后进行所述延伸反应。在该种情况下，所述第一链可以通过以下示例性方式获得：
(A)将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接，并以所述第一桥接寡核苷酸起始延伸反应，获得所述第一链；其中，所述用于延伸反应的聚合酶优选具有或者不具有链置换活性或5'至3'外切活性；
或，
(B)将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接，并以所述待标记的第一核酸分子起始延伸反应，获得所述第一链；其中，所述用于延伸反应的聚合酶优选具有链置换活性或5'至3'外切活性。
例如，当所述连接反应与所述延伸反应在不同体系中进行，并且，先进行所述延伸反应，后进行所述连接反应。在该种情况下，可通过以所述第一桥接寡核苷酸起始延伸反应，再将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接获得所述第一链。在该种情况下，所述用于延伸反应的聚合酶优选不具有链置换活性或5'至3'外切活性。
包括步骤(1)、步骤(2)(i)和步骤(3)(ii)的实施方案：二链
在某些实施方案中，所述第二桥接寡核苷酸的第二区域能与步骤(2)(i)获得的第一延伸产物的所述共有序列B互补序列或其部分序列退火，并且所述第二桥接寡核苷酸的第二区域具有3’自由端。
在某些实施方案中，步骤(3)(ii)中获得的反应产物即为标记的核酸分子，其包含所述第二链。
在某些实施方案中，所述第二链从5’端至3’端包含：所述共有序列X1，所述标签序列Y，所述共有序列X2，任选的所述第一桥接寡核苷酸的第三区域的互补序列，所述第二桥接寡核苷酸序列，所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列。
在某些实施方案中，所述第二桥接寡核苷酸的第二区域位于所述第二桥接寡核苷酸的3’末端。
在某些实施方案中，所述第二桥接寡核苷酸的第一区域位于所述第二桥接寡核苷酸的5’末端。
在某些实施方案中，所述第一桥接寡核苷酸不含有所述第三区域，和/或，所述第二桥接寡核苷酸不含有所述第三区域。
在某些实施方案中，所述第二桥接寡核苷酸的5’末端含有磷酸化修饰。
在某些实施方案中，所述第二桥接寡核苷酸的3’末端含有自由-OH。
在某些实施方案中，步骤(3)(ii)中，所述第一桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)，和/或，步骤(2)(i)获得的第一延伸产物不能起始延伸反应(例如3’端是封闭的)。
在某些实施方案中，步骤(2)(i)(a)中，所述引物A的捕获序列A为随机寡核苷酸序列。
在某些实施方案中，步骤(2)(i)(b)中所述的第一延伸产物从5’端至3’端依次包含：以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列。
在某些实施方案中，所述第二链从5’端至3’端包含：所述共有序列X1，所述标签序列Y，所述共有序列X2，任选的所述第一桥接寡核苷酸的第三区域的互补序列，所述第二桥接寡核苷酸序列，所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列。
在某些实施方案中，步骤(2)(i)(a)中，所述引物A的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列。
在某些实施方案中，所述引物A还含有共有序列A，以及任选的标签序列A，例如为随机寡核苷酸序列。
在某些实施方案中，所述捕获序列A位于所述引物A的3’端。
在某些实施方案中，步骤(2)(i)(b)中所述第一延伸产物从5’端至3’端依次包含：所述共有序列A，任选的所述标签序列A，以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，所述标签序列B的互补序列，所述共有序列B的互补序列。
在某些实施方案中，所述第二链从5’端至3’端包含：所述共有序列X1，所述标签序列Y，所述共有序列X2，任选的所述第一桥接寡核苷酸的第三区域的互补序列，所述第二桥接寡核苷酸序 列，所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列，任选的所述标签序列A的互补序列，所述共有序列A的互补序列。
易于理解，步骤(3)(ii)中，在所述第一桥接寡核苷酸、第二桥接寡核苷酸与所述寡核苷酸探针以及所述寡核苷酸探针对应位置的待标记的第一核酸分子退火之后，将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接，和/或，将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接的连接反应过程与步骤(3)(ii)中所述的延伸反应可以任意顺序进行，只要能获得带有位置标记的第二核酸分子即可。
例如，当所述连接反应与所述延伸反应在相同体系中进行，可通过将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接，并以所述第二桥接寡核苷酸起始延伸反应，获得所述第二链。在该种情况下，所述用于延伸反应的聚合酶优选不具有链置换活性或5'至3'外切活性。
例如，当所述连接反应与所述延伸反应在不同体系中进行，并且，先进行所述连接反应，后进行所述延伸反应。在该种情况下，所述第二链可以通过以下示例性方式获得：
(A)将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接，并以所述第二桥接寡核苷酸起始延伸反应，获得所述第二链；其中，所述用于延伸反应的聚合酶优选具有或者不具有链置换活性或5'至3'外切活性；
或，
(B)将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接，并以所述寡核苷酸探针起始延伸反应，获得所述第二链；其中，所述用于延伸反应的聚合酶优选具有链置换活性或5'至3'外切活性。
例如，当所述连接反应与所述延伸反应在不同体系中进行，并且，先进行所述延伸反应，后进行所述连接反应。在该种情况下，可通过以所述第二桥接寡核苷酸起始延伸反应，再将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接获得所述第二链。在该种情况下，所述用于延伸反应的聚合酶优选不具有链置换活性或5'至3'外切活性。
本申请的包含步骤(1)、步骤(2)(i)和步骤(3)(ii)的一个示例性实施方案详细描述如下：
一、以样本中的RNA(例如mRNA)为模板制备cDNA链的示例性方案包含以下步骤(如图2所示)：
(1)用逆转录酶(例如，具有末端转移活性的逆转录酶)和引物A对透化的样本中的RNA分子(例如，mRNA分子)进行逆转录，以生成cDNA，并在cDNA的3’端添加悬突(例如，包含3个胞嘧啶核苷酸的悬突)。可使用各种具有末端转移活性的逆转录酶来进行逆转录反应。在某些优选的实施方案中，所使用的逆转录酶不具有RNaseH活性。
在某些实施方案中，所述引物A包含poly(T)序列以及共有序列A(CA)。通常情况下，poly(T)序列位于所述引物A的3’末端，以便起始逆转录。
在某些实施方案中，所述引物A包含随机寡核苷酸序列，可用于捕获无ploy(A)尾的RNA。 通常情况下，所述随机寡核苷酸序列位于所述引物A的3’末端，以便起始逆转录。
(2)使用引物B与cDNA链进行退火或杂交，所述引物B包含共有序列B(CB)、独特分子标签序列(UMI)以及所述cDNA的3’端悬突的互补序列。随后，与所述引物B杂交或退火的核酸片段在核酸聚合酶的作用下，可以以所述UMI序列和所述共有序列B为模板进行延伸，从而生成3’端携带所述UMI序列的互补序列、所述共有序列B的互补序列的的核酸分子。
通常情况下，所述共有序列B位于所述UMI序列的上游(例如5’端)，所述与cDNA链的3’末端悬突互补的序列位于所述引物B的3’末端。
例如，当cDNA链的3’末端包含3个胞嘧啶核苷酸的悬突时，所述引物B可在其3’端包含GGG。此外，还可以对所述引物B的核苷酸进行修饰(例如，使用锁核酸)，以增强所述引物B与cDNA链的3’末端悬突之间的互补配对。
不受理论限制，可以使用各种合适的核酸聚合酶(例如，DNA聚合酶或逆转录酶)来进行延伸反应，只要其能够以所述引物B的序列或其部分序列为模板延伸被捕获的核酸片段(逆转录产物)即可。在某些示例性实施方案中，可使用与前述逆转录步骤相同的逆转录酶来延伸被捕获的核酸片段(逆转录产物)。
在某些实施方案中，该步骤与步骤(1)同时进行(例如，在同一反应体系中进行)。
在某些实施方案中，所述方法任选地还包含步骤(3)：加入RNaseH，消化RNA/cDNA杂合双链中的RNA链，形成cDNA单链。
在某些实施方案中，所述方法不包括所述步骤(3)。
通过上述示例性实施方案所制备的cDNA链的示例性结构包含：共有序列A，cDNA序列，3’末端悬突序列，UMI序列的互补序列,以及，共有序列B的互补序列。
二、用寡核苷酸探针(也称，芯片序列)的互补序列标记cDNA链的3’端，以形成含有芯片序列信息的新核酸分子(即，经芯片序列标记的核酸分子)的示例性方案包含以下步骤(如图3所示)：
提供由第一桥接寡核苷酸和第二桥接寡核苷酸组成的桥接寡核苷酸对，其中，所述第一桥接寡核苷酸和所述第二桥接寡核苷酸各自独立地包括：第一区域(P1)和第二区域(P2)，所述第一区域位于所述第二区域的上游(例如5’端)；其中，
所述第一桥接寡核苷酸的第一区域能与所述第二桥接寡核苷酸的第一区域退火；所述第一桥接寡核苷酸的第二区域能与所述寡核苷酸探针的共有序列X2或其部分序列退火；
所述第二桥接寡核苷酸的第二区域能与上述步骤一中获得的cDNA链中的所述共有序列B的互补序列或其部分序列退火。
在某些实施方案中，所述第一桥接寡核苷酸中第一区域和第二区域之间包含间隔核苷酸，例如1-5nt或5-10nt的间隔核苷酸，即所述第一桥接寡核苷酸序列含有位于第一区域与第二区域之间的第三区域。在某些优选的实施方案中，所述第一桥接寡核苷酸中第一区域和第二区域是相邻连接的，二者之间没有多余核苷酸，即所述第一桥接寡核苷酸序列不含有位于第一区域与第二区域之间的第三区域。
在某些实施方案中，所述第二桥接寡核苷酸中第一区域和第二区域之间包含间隔核苷酸，例 如1-5nt或5-10nt的间隔核苷酸，即所述第二桥接寡核苷酸序列含有位于第一区域与第二区域之间的第三区域。在某些优选的实施方案中，所述第二桥接寡核苷酸中第一区域和第二区域是相邻连接的，二者之间没有多余核苷酸，即所述第二桥接寡核苷酸序列不含有位于第一区域与第二区域之间的第三区域。
将该第一桥接寡核苷酸、第二桥接寡核苷酸和芯片序列和上述步骤一获得的cDNA链退火或杂交，之后通过DNA连接酶将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接，和/或，将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接。并且，在DNA聚合酶的作用下，形成含有芯片序列信息的新核酸分子(即，经芯片序列标记的核酸分子)。所述连接过程和聚合过程以任意顺序进行。
通过上述示例性实施方案所形成的含有芯片序列信息的新核酸分子的示例性结构包含：从5’端至3’端含有共有序列A，cDNA序列，3’末端悬突序列，UMI序列的互补序列，共有序列B的互补序列，第一桥接寡核苷酸序列，标签序列Y的互补序列，以及共有序列X1的互补序列的核酸链和/或其互补核酸链。
在某些实施方案中，所述方法包括步骤(1)、步骤(2)(ii)和步骤(3)。在某些实施方案中，步骤(2)(ii)(b)中，所述第一延伸产物从5’端至3’端包含：所述共有序列A，以所述引物A’为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，任选的所述标签序列B的互补序列，所述共有序列B的互补序列。
在某些实施方案中，步骤(2)(ii)(c)中，所述延伸引物为所述引物B’或引物B”或随机引物，其中，所述引物B”能与所述共有序列B的互补序列或其部分序列退火，并且能起始延伸反应。
在某些实施方案中，步骤(2)(ii)(c)中，所述第二延伸产物从5’端至3’端包含：以所述延伸引物延伸形成的与所述cDNA序列互补的序列，所述共有序列A的互补序列。
包括步骤(1)、步骤(2)(ii)和步骤(3)(i)的实施方案
在某些实施方案中，所述方法包括步骤(1)、步骤(2)(ii)和步骤(3)(i)；其中，所述共有序列X2或其部分序列能与所述共有序列A的互补序列或其部分序列退火；步骤(3)(i)中获得的延伸产物即为标记的核酸分子，其包含：含有所述待标记的第一核酸分子序列的第一链，和/或，含有所述寡核苷酸探针序列的第二链。
易于理解，所述共有序列X2可以以其整体的核苷酸序列与所述共有序列A的互补序列或所述共有序列A的互补序列的部分区段的核苷酸序列退火，所述共有序列X2也可以以其部分区段的核苷酸序列与所述共有序列A的互补序列或所述共有序列A的互补序列的部分区段的核苷酸序列退火。
在某些实施方案中，所述第一链从5’端至3’端包含：所述待标记的第一核酸分子序列，所述标签序列Y的互补序列，所述共有序列X1的互补序列。
在某些实施方案中，所述第二链从5’端至3’端包含：所述共有序列X1，所述标签序列Y，所述共有序列X2，与所述待标记的第一核酸分子序列互补的cDNA序列。
包括步骤(1)、步骤(2)(ii)和步骤(3)(i)的实施方案：一链
在某些实施方案中，所述共有序列X2或其部分序列能与所述共有序列A的互补序列或其部分序列(例如，3’端部分序列)退火；步骤(3)(i)中获得的延伸产物即为标记的核酸分子，其包含含有所述待标记的第一核酸分子序列的第一链。
在某些实施方案中，步骤(3)(i)中，所述寡核苷酸探针不能起始延伸反应(例如3’端是封闭的)。
在某些实施方案中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为随机寡核苷酸序列。
在某些实施方案中，步骤(2)(ii)(c)中，所述延伸引物为所述引物B’。在某些实施方案中，步骤(2)(ii)(c)中，所述第二延伸产物从5’端至3’端包含：所述共有序列B，任选的所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A’为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列，所述共有序列A的互补序列。在某些实施方案中，所述第一链从5’端至3’端包含：所述共有序列B，任选的所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A’为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列，所述共有序列A的互补序列，所述标签序列Y的互补序列，所述共有序列X1的互补序列。
在某些实施方案中，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第一链具有不同的捕获序列A的互补序列作为UMI。
在某些实施方案中在某些实施方案中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列。
在某些实施方案中，所述引物A’还含有标签序列A，例如为随机寡核苷酸序列。
在某些实施方案中，所述捕获序列A位于所述引物A的3’端。
在某些实施方案中，步骤(2)(ii)(c)中，所述延伸引物为所述引物B’。在某些实施方案中，步骤(2)(ii)(c)中，所述第二延伸产物从5’端至3’端包含：所述共有序列B，任选的所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A’为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列，所述标签序列A的互补序列，所述共有序列A的互补序列。在某些实施方案中，所述第一链从5’端至3’端包含：所述共有序列B，任选的所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A’为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列，所述标签序列A的互补序列，所述共有序列A的互补序列，所述标签序列Y的互补序列，所述共有序列X1的互补序列。
在某些实施方案中，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第一链具有不同的标签序列A的互补序列作为UMI。
包括步骤(1)、步骤(2)(ii)和步骤(3)(i)的实施方案：二链
在某些实施方案中，所述共有序列X2或其部分序列(例如，3’端部分序列)能与所述共有序列A的互补序列或其部分序列退火；步骤(3)(i)中获得的延伸产物即为标记的核酸分子，其包含含有所述寡核苷酸探针序列的第二链。
在某些实施方案中，步骤(2)(ii)获得的第二延伸产物不能起始延伸反应(例如3’端是封闭的)。
在某些实施方案中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为随机寡核苷酸序列。
在某些实施方案中，步骤(2)(ii)(c)中，所述延伸引物为所述引物B’。在某些实施方案中，步骤(2)(ii)(c)中，所述第二延伸产物从5’端至3’端包含：所述共有序列B，任选的所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A’为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列，所述共有序列A的互补序列。在某些实施方案中，所述第二链从5’端至3’端包含：所述共有序列X1，所述标签序列Y，所述共有序列X2，与所述待标记的第一核酸分子序列互补的cDNA序列，所述3’末端悬突序列，任选的所述标签序列B的互补序列，所述共有序列B的互补序列。
在某些实施方案中，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第二链具有不同的捕获序列A作为UMI。
在某些实施方案中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列。
在某些实施方案中，所述引物A’还含有标签序列A，例如为随机寡核苷酸序列。
在某些实施方案中，所述捕获序列A位于所述引物A的3’端。
在某些实施方案中，步骤(2)(ii)(c)中，所述延伸引物为所述引物B’。在某些实施方案中，步骤(2)(ii)(c)中，所述第二延伸产物从5’端至3’端包含：所述共有序列B，任选的所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A’为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列，所述标签序列A的互补序列，所述共有序列A的互补序列。在某些实施方案中，所述第二链从5’端至3’端包含：所述共有序列X1，所述标签序列Y，所述共有序列X2，所述标签序列A，与所述待标记的第一核酸分子序列互补的cDNA序列，所述3’末端悬突序列，任选的所述标签序列B的互补序列，所述共有序列B的互补序列。
在某些实施方案中，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第二链具有不同的标签序列A作为UMI。
本申请的包含步骤(1)、步骤(2)(ii)和步骤(3)(i)的一个示例性实施方案详细描述如下：
一、以样本中的RNA(例如mRNA)为模板制备3’端含有UMI的互补序列的cDNA链互补链的示例性方案包含以下步骤(如图5所示)：
(1)用逆转录酶(例如，具有末端转移活性的逆转录酶)和引物A’对透化的样本中的RNA分子(例如，mRNA分子)进行逆转录，以生成cDNA，并在cDNA的3’端添加悬突(例如，包含3个胞嘧啶核苷酸的悬突)。可使用各种具有末端转移活性的逆转录酶来进行逆转录反应。在某些优选的实施方案中，所使用的逆转录酶不具有RNaseH活性。
在某些实施方案中，所述引物A’包含poly(T)序列，UMI序列，以及共有序列A(CA)。通常情况下，poly(T)序列位于所述引物A’的3’末端以便起始逆转录，所述共有序列A位于所述UMI序列的上游(例如5’端)。
在某些实施方案中，所述引物A’包含随机寡核苷酸序列以及共有序列A，可用于捕获无ploy  A尾的RNA。通常情况下，所述随机寡核苷酸序列位于所述引物A’的3’末端，以便起始逆转录。
(2)使用含引物B’与cDNA链进行退火或杂交，所述引物B’包含共有序列B(CB)、以及所述cDNA的3’端悬突的互补序列。随后，与所述引物B’杂交或退火的核酸片段在核酸聚合酶的作用下，可以以所述共有序列B为模板进行延伸，在cDNA链3’末端添加所述共有序列B的的互补序列(c(CB))，从而生成3’端携带所述共有序列B的互补序列的核酸分子。
通常情况下，所述与cDNA链的3’末端悬突互补的序列位于所述引物B’的3’末端。
例如，当cDNA链的3’末端包含3个胞嘧啶核苷酸的悬突时，所述引物B’可在其3’端包含GGG。此外，还可以对所述引物B’的核苷酸进行修饰(例如，使用锁核酸)，以增强所述引物B’与cDNA链的3’末端悬突之间的互补配对。
不受理论限制，可以使用各种合适的核酸聚合酶(例如，DNA聚合酶或逆转录酶)来进行延伸反应，只要其能够以所述引物B’的序列或其部分序列为模板延伸被捕获的核酸片段(逆转录产物)即可。在某些示例性实施方案中，可使用与前述逆转录步骤相同的逆转录酶来延伸被捕获的核酸片段(逆转录产物)。
在某些实施方案中，该步骤与步骤(1)同时进行(例如，在同一反应体系中进行)。
在某些实施方案中，所述方法任选地还包含步骤(3)：加入RNaseH，消化RNA/cDNA杂合双链中的RNA链，形成cDNA单链。
在某些实施方案中，所述方法不包括所述步骤(3)。
(4)使用延伸引物，以前一步骤获得的cDNA链为模板进行延伸反应，获得延伸产物；所述延伸引物为所述引物B’，随机引物，或者引物B”，所述引物B”能与所述共有序列B或其部分序列退火，且能起始延伸反应。
通过上述示例性实施方案所制备的cDNA链互补链的示例性结构包含：共有序列B，3’末端悬突的互补序列，cDNA序列的互补序列，UMI序列的互补序列，以及共有序列A的互补序列。
二、用寡核苷酸探针(也称，芯片序列)的互补序列标记cDNA链互补链的3’端，以形成含有芯片序列信息的新核酸分子(即，经芯片序列标记的核酸分子)的示例性方案包含以下步骤(如图7所示)：
在某些实施方案中，所述芯片序列的共有序列X2或其部分序列能与上述步骤一中获得的cDNA链互补链的所述共有序列A的互补序列或其部分序列退火。将该cDNA链互补链与芯片序列退火或杂交，在聚合酶的作用下，形成含有芯片序列信息的新核酸分子(即，经芯片序列标记的核酸分子)。
通过上述示例性实施方案所形成的含有芯片序列信息的新核酸分子的示例性结构包含：从5’端至3’端含有所述共有序列B，3’末端悬突的互补序列，cDNA序列的互补序列，所述UMI序列的互补序列，所述共有序列A的互补序列，所述标签序列Y的互补序列，以及所述共有序列X1的互补序列的核酸链和/或其互补核酸链。
包括步骤(1)、步骤(2)(ii)和步骤(3)(ii)的实施方案
在某些实施方案中，所述方法包括步骤(1)、步骤(2)(ii)和步骤(3)(ii)；其中，所述第二桥接寡核苷酸的第二区域能与步骤(2)(ii)获得的第二延伸产物的共有序列A的互补序列 或其部分序列退火；步骤(3)(ii)中获得的反应产物即为标记的核酸分子，其包含：含有所述待标记的第一核酸分子序列的第一链，和/或，含有所述寡核苷酸探针序列的第二链。
易于理解，所述第二桥接寡核苷酸的第二区域能与步骤(2)(ii)获得的第二延伸产物的共有序列A的互补序列或所述共有序列A的互补序列的部分区段的核苷酸序列退火。
在某些实施方案中，所述第一链从5’端至3’端包含：所述待标记的第一核酸分子序列，任选的所述第二桥接寡核苷酸的第三区域的互补序列，所述第一桥接寡核苷酸序列，所述标签序列Y的互补序列，所述共有序列X1的互补序列。
在某些实施方案中，所述第二链从5’端至3’端包含：所述共有序列X1，所述标签序列Y，所述共有序列X2，任选的所述第一桥接寡核苷酸的第三区域的互补序列，所述第二桥接寡核苷酸序列，与所述待标记的第一核酸分子序列互补的cDNA序列。
包括步骤(1)、步骤(2)(ii)和步骤(3)(ii)的实施方案：一链
在某些实施方案中，所述第二桥接寡核苷酸的第二区域能与步骤(2)(ii)获得的第二延伸产物的所述共有序列A的互补序列或其3’端部分序列退火，并且所述第一桥接寡核苷酸的第二区域具有3’自由端。
在某些实施方案中，步骤(3)(ii)中获得的反应产物即为标记的核酸分子，其包含所述第一链。
在某些实施方案中，所述第一桥接寡核苷酸的第二区域位于所述第一桥接寡核苷酸的3’末端。
在某些实施方案中，所述第一桥接寡核苷酸的第一区域位于所述第一桥接寡核苷酸的5’末端。在某些实施方案中，所述第一桥接寡核苷酸不含有所述第三区域，和/或，所述第二桥接寡核苷酸不含有所述第三区域。
在某些实施方案中，所述第一桥接寡核苷酸的5’末端含有磷酸化修饰。
在某些实施方案中，所述第一桥接寡核苷酸的3’末端含有自由-OH。
在某些实施方案中，步骤(3)(ii)中，所述第二桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)，和/或，所述寡核苷酸探针不能起始延伸反应(例如3’端是封闭的)。
在某些实施方案中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为随机寡核苷酸序列。
在某些实施方案中，步骤(2)(ii)(c)中，所述延伸引物为所述引物B’。在某些实施方案中，步骤(2)(ii)(c)中，所述第二延伸产物从5’端至3’端包含：所述共有序列B，任选的所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A’为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列，所述共有序列A的互补序列。在某些实施方案中，所述第一链从5’端至3’端包含：所述共有序列B，任选的所述标签序列B，3’末端悬突序列的互补序列，以所述引物A’为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列，所述共有序列A的互补序列，任选的所述第二桥接寡核苷酸的第三区域的互补序列，所述第一桥接寡核苷酸序列，所述标签序列Y的互补序列，所述共有序列X1的互补序列。
在某些实施方案中，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第一链具有不同的捕获序列A的互补序列作为UMI。
在某些实施方案中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列。
在某些实施方案中，所述引物A’还含有标签序列A，例如为随机寡核苷酸序列。
在某些实施方案中，所述捕获序列A位于所述引物A的3’端。
在某些实施方案中，步骤(2)(ii)(c)中，所述延伸引物为所述引物B’。在某些实施方案中，步骤(2)(ii)(c)中，所述第二延伸产物从5’端至3’端包含：所述共有序列B，任选的所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A’为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列，所述标签序列A的互补序列，所述共有序列A的互补序列。在某些实施方案中，所述第一链从5’端至3’端包含：所述共有序列B，任选的所述标签序列B，3’末端悬突序列的互补序列，以所述引物A’为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列，所述标签序列A的互补序列，所述共有序列A的互补序列，任选的所述第二桥接寡核苷酸的第三区域的互补序列，所述第一桥接寡核苷酸序列，所述标签序列Y的互补序列，所述共有序列X1的互补序列。
在某些实施方案中，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第一链具有不同的标签序列A的互补序列作为UMI。
易于理解，步骤(3)(ii)中，在所述第一桥接寡核苷酸、第二桥接寡核苷酸与所述寡核苷酸探针以及所述寡核苷酸探针对应位置的待标记的第一核酸分子退火之后，将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接，和/或，将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接的连接反应过程与步骤(3)(ii)中所述的延伸反应可以任意顺序进行，只要能获得带有位置标记的第二核酸分子即可。
例如，当所述连接反应与所述延伸反应在相同体系中进行，可通过将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接，并以所述第一桥接寡核苷酸起始延伸反应，获得所述第一链。在该种情况下，所述用于延伸反应的聚合酶优选不具有链置换活性或5'至3'外切活性。
例如，当所述连接反应与所述延伸反应在不同体系中进行，并且，先进行所述连接反应，后进行所述延伸反应。在该种情况下，所述第一链可以通过以下示例性方式获得：
(A)将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接，并以所述第一桥接寡核苷酸起始延伸反应，获得所述第一链；其中，所述用于延伸反应的聚合酶优选具有或者不具有链置换活性或5'至3'外切活性；
或，
(B)将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接，并以所述待标记的第一核酸分子起始延伸反应，获得所述第一链；其中，所述用于延伸反应的聚合酶优选具有链置换活性或5'至3'外切活性。
例如，当所述连接反应与所述延伸反应在不同体系中进行，并且，先进行所述延伸反应，后进行所述连接反应。在该种情况下，可通过以所述第一桥接寡核苷酸起始延伸反应，再将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接获得所述第一链。在该种情况下， 所述用于延伸反应的聚合酶优选不具有链置换活性或5'至3'外切活性。
包括步骤(1)、步骤(2)(ii)和步骤(3)(ii)的实施方案：二链
在某些实施方案中，所述第二桥接寡核苷酸的第二区域能与步骤(2)(ii)获得的第二延伸产物的所述共有序列A的互补序列或其部分序列退火，并且所述第二桥接寡核苷酸的第二区域具有3’自由端。
在某些实施方案中，步骤(3)(ii)中获得的反应产物即为标记的核酸分子，其包含所述第二链。
在某些实施方案中，所述第二桥接寡核苷酸的第二区域位于所述第二桥接寡核苷酸的3’末端。
在某些实施方案中，所述第二桥接寡核苷酸的第一区域位于所述第二桥接寡核苷酸的5’末端。
在某些实施方案中，所述第一桥接寡核苷酸不含有所述第三区域，和/或，所述第二桥接寡核苷酸不含有所述第三区域。
在某些实施方案中，所述第二桥接寡核苷酸的5’末端含有磷酸化修饰。
在某些实施方案中，所述第二桥接寡核苷酸的3’末端含有自由-OH。
在某些实施方案中，步骤(3)(ii)中，所述第一桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)，和/或，步骤(2)(ii)获得的第二延伸产物不能起始延伸反应(例如3’端是封闭的)。
在某些实施方案中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为随机寡核苷酸序列。
在某些实施方案中，步骤(2)(ii)(c)中，所述延伸引物为所述引物B’。在某些实施方案中，步骤(2)(ii)(c)中，所述第二延伸产物从5’端至3’端包含：所述共有序列B，任选的所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A’为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列，所述共有序列A的互补序列。在某些实施方案中，所述第二链从5’端至3’端包含：所述共有序列X1，所述标签序列Y，所述共有序列X2，任选的所述第一桥接寡核苷酸的第三区域的互补序列，所述第二桥接寡核苷酸序列，与所述待标记的第一核酸分子序列互补的cDNA序列，所述3’末端悬突序列，任选的所述标签序列B的互补序列，所述共有序列B的互补序列。
在某些实施方案中，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第二链具有不同的捕获序列A作为UMI。
在某些实施方案中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列。
在某些实施方案中，所述引物A’还含有标签序列A，例如为随机寡核苷酸序列。
在某些实施方案中，所述捕获序列A位于所述引物A的3’端。
在某些实施方案中，步骤(2)(ii)(c)中，所述延伸引物为所述引物B’。在某些实施方案中，步骤(2)(ii)(c)中，所述第二延伸产物从5’端至3’端包含：所述共有序列B，任选的所述标签序列B，所述3’末端悬突序列的互补序列，以所述引物A’为逆转录引物形成的与所述RNA互补的cDNA序列的互补序列，所述标签序列A的互补序列，所述共有序列A的互补序列。在某些实施方案中，所述第二链从5’端至3’端包含：所述共有序列X1，所述标签序列Y，所述共有序 列X2，任选的所述第一桥接寡核苷酸的第三区域的互补序列，所述第二桥接寡核苷酸序列，所述标签序列A，与所述待标记的第一核酸分子序列互补的cDNA序列，所述3’末端悬突序列，任选的所述标签序列B的互补序列，所述共有序列B的互补序列。
在某些实施方案中，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第二链具有不同的标签序列A作为UMI。
易于理解，步骤(3)(ii)中，在所述第一桥接寡核苷酸、第二桥接寡核苷酸与所述寡核苷酸探针以及所述寡核苷酸探针对应位置的待标记的第一核酸分子退火之后，将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接，和/或，将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接的连接反应过程与步骤(3)(ii)中所述的延伸反应可以任意顺序进行，只要能获得带有位置标记的第二核酸分子即可。
例如，当所述连接反应与所述延伸反应在相同体系中进行，可通过将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接，并以所述第二桥接寡核苷酸起始延伸反应，获得所述第二链。在该种情况下，所述用于延伸反应的聚合酶优选不具有链置换活性或5'至3'外切活性。
例如，当所述连接反应与所述延伸反应在不同体系中进行，并且，先进行所述连接反应，后进行所述延伸反应。在该种情况下，所述第二链可以通过以下示例性方式获得：
(A)将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接，并以所述第二桥接寡核苷酸起始延伸反应，获得所述第二链；其中，所述用于延伸反应的聚合酶优选具有或者不具有链置换活性或5'至3'外切活性；
或，
(B)将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接，并以所述寡核苷酸探针起始延伸反应，获得所述第二链；其中，所述用于延伸反应的聚合酶优选具有链置换活性或5'至3'外切活性。
例如，当所述连接反应与所述延伸反应在不同体系中进行，并且，先进行所述延伸反应，后进行所述连接反应。在该种情况下，可通过以所述第二桥接寡核苷酸起始延伸反应，再将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接获得所述第二链。在该种情况下，所述用于延伸反应的聚合酶优选不具有链置换活性或5'至3'外切活性。
本申请的包含步骤(1)、步骤(2)(ii)和步骤(3)(ii)的一个示例性实施方案详细描述如下：
一、以样本中的RNA(例如mRNA)为模板制备cDNA链互补链的示例性方案包含以下步骤(如图5所示)：
(1)用逆转录酶(例如，具有末端转移活性的逆转录酶)和引物A’对透化的样本中的RNA分子(例如，mRNA分子)进行逆转录，以生成cDNA，并在cDNA的3’端添加悬突(例如，包含3个胞嘧啶核苷酸的悬突)。可使用各种具有末端转移活性的逆转录酶来进行逆转录反应。在某些优选的实施方案中，所使用的逆转录酶不具有RNaseH活性。
在某些实施方案中，所述引物A’包含poly(T)序列，UMI序列，以及共有序列A(CA)。通常 情况下，poly(T)序列位于所述引物A’的3’末端以便起始逆转录，所述共有序列A位于所述UMI序列的上游(例如5’端)。
在某些实施方案中，所述引物A’包含随机寡核苷酸序列以及共有序列A，可用于捕获无ploy A尾的RNA。通常情况下，所述随机寡核苷酸序列位于所述引物A’的3’末端，以便起始逆转录。
(2)使用含引物B’与cDNA链进行退火或杂交，所述引物B’包含共有序列B(CB)、以及所述cDNA的3’端悬突的互补序列。随后，与所述引物B’杂交或退火的核酸片段在核酸聚合酶的作用下，可以以所述共有序列B为模板进行延伸，在cDNA链3’末端添加所述共有序列B的的互补序列(c(CB))，从而生成3’端携带所述共有序列B的互补序列的的核酸分子。
通常情况下，所述与cDNA链的3’末端悬突互补的序列位于所述引物B’的3’末端。
例如，当cDNA链的3’末端包含3个胞嘧啶核苷酸的悬突时，所述引物B’可在其3’端包含GGG。此外，还可以对所述引物B’的核苷酸进行修饰(例如，使用锁核酸)，以增强所述引物B’与cDNA链的3’末端悬突之间的互补配对。
不受理论限制，可以使用各种合适的核酸聚合酶(例如，DNA聚合酶或逆转录酶)来进行延伸反应，只要其能够以所述引物B’的序列或其部分序列为模板延伸被捕获的核酸片段(逆转录产物)即可。在某些示例性实施方案中，可使用与前述逆转录步骤相同的逆转录酶来延伸被捕获的核酸片段(逆转录产物)。
在某些实施方案中，该步骤与步骤(1)同时进行(例如，在同一反应体系中进行)。
在某些实施方案中，所述方法任选地还包含步骤(3)：加入RNaseH，消化RNA/cDNA杂合双链中的RNA链，形成cDNA单链。
在某些实施方案中，所述方法不包括所述步骤(3)。
(4)使用延伸引物，以前一步骤获得的cDNA链为模板进行延伸反应，获得延伸产物；所述延伸引物为所述引物B’，随机引物，或者引物B”，所述引物B”能与所述共有序列B或其部分序列退火，且能起始延伸反应。
通过上述示例性实施方案所制备的cDNA链互补链的示例性结构包含：共有序列B，3’末端悬突的互补序列，cDNA序列的互补序列，UMI序列的互补序列，以及共有序列A的互补序列。
二、用寡核苷酸探针(也称，芯片序列)的互补序列标记cDNA链互补链的3’端，以形成含有芯片序列信息的新核酸分子(即，经芯片序列标记的核酸分子)的示例性方案包含以下步骤(如图6所示)：
提供由第一桥接寡核苷酸和第二桥接寡核苷酸组成的桥接寡核苷酸对，其中，所述第一桥接寡核苷酸和所述第二桥接寡核苷酸各自独立地包括：第一区域(P1)和第二区域(P2)，所述第一区域位于所述第二区域的上游(例如5’端)；其中，
所述第一桥接寡核苷酸的第一区域能与所述第二桥接寡核苷酸的第一区域退火；所述第一桥接寡核苷酸的第二区域能与所述寡核苷酸探针的共有序列X2或其部分序列退火；
所述第二桥接寡核苷酸的第二区域能与上述步骤一中获得的cDNA链互补链中的所述共有序列A的互补序列或其部分序列退火。
在某些实施方案中，所述第一桥接寡核苷酸中第一区域和第二区域之间包含间隔核苷酸，例 如1-5nt或5-10nt的间隔核苷酸，即所述第一桥接寡核苷酸序列含有位于第一区域与第二区域之间的第三区域。在某些优选的实施方案中，所述第一桥接寡核苷酸中第一区域和第二区域是相邻连接的，二者之间没有多余核苷酸，即所述第一桥接寡核苷酸序列不含有位于第一区域与第二区域之间的第三区域。
在某些实施方案中，所述第二桥接寡核苷酸中第一区域和第二区域之间包含间隔核苷酸，例如1-5nt或5-10nt的间隔核苷酸,即所述第二桥接寡核苷酸序列含有位于第一区域与第二区域之间的第三区域。在某些优选的实施方案中，所述第二桥接寡核苷酸中第一区域和第二区域是相邻连接的，二者之间没有多余核苷酸，即所述第二桥接寡核苷酸序列不含有位于第一区域与第二区域之间的第三区域。
将该第一桥接寡核苷酸、第二桥接寡核苷酸和芯片序列和上述步骤一获得的cDNA链互补链退火或杂交，之后通过DNA连接酶将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接，和/或，将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接。随后，在DNA聚合酶的作用下，形成含有芯片序列信息的新核酸分子(即，经芯片序列标记的核酸分子)。所述连接过程和聚合过程以任意顺序进行。
通过上述示例性实施方案所形成的含有芯片序列信息的新核酸分子的示例性结构包含：从5’端至3’端含有所述共有序列B，3’末端悬突的互补序列，cDNA序列的互补序列，所述UMI序列的互补序列，所述共有序列A的互补序列，所述第一桥接寡核苷酸序列，所述标签序列Y的互补序列，以及所述共有序列X1的互补序列的核酸链和/或其互补核酸链。
在某些实施方案中，所述方法在步骤(2)(i)(b)中，所述cDNA链通过其3’末端悬突与所述引物B退火，并且，在核酸聚合酶(例如，DNA聚合酶或逆转录酶)的作用下，所述cDNA链以所述引物B为模板被延伸，生成所述第一延伸产物。
在某些实施方案中，所述方法在步骤(2)(ii)(b)中，所述cDNA链通过其3’末端悬突与所述引物B’退火，并且，在核酸聚合酶(例如，DNA聚合酶或逆转录酶)的作用下，所述cDNA链以所述引物B’为模板被延伸，生成所述第一延伸产物。
在某些实施方案中，所述3’末端悬突具有至少1个，至少2个，至少3个，至少4个，至少5个，至少6个，至少7个，至少8个，至少9个，至少10个或更多个核苷酸的长度。在某些实施方案中，所述3’末端悬突为2-5个胞嘧啶核苷酸的3’末端悬突(例如CCC悬突)。
在某些实施方案中，步骤(2)中，在进行所述预处理之前，对所述生物样本进行透化处理。
在某些实施方案中，所述生物样本是组织样品。
在某些实施方案中，所述组织样品是组织切片。
在某些实施方案中，所述组织切片从固定组织制备，例如，以福尔马林固定石蜡包埋(FFPE)的组织或深度冷冻的组织。
在某些实施方案中，当所述生物样本与所述核酸阵列接触时，所述生物样本的每个细胞各自占据所述核酸阵列中的一个或多个微点(即，每个细胞各自与所述核酸阵列中的一个或多个微点接触)。
在某些实施方案中，步骤(2)中所述进行逆转录包括使用逆转录酶。
在某些实施方案中，所述逆转录酶具有末端转移活性。
在某些实施方案中，所述逆转录酶能够以RNA(例如，mRNA)为模板，合成cDNA链，且在所述cDNA链的3’端添加悬突。
在某些实施方案中，所述逆转录酶能够在cDNA链的3’末端添加长度为至少1个，至少2个，至少3个，至少4个，至少5个，至少6个，至少7个，至少8个，至少9个，至少10个或更多个核苷酸的悬突。
在某些实施方案中，所述逆转录酶能够在cDNA链的3’末端添加2-5个胞嘧啶核苷酸的悬突(例如CCC悬突)。
在某些实施方案中，所述逆转录酶选自M-MLV逆转录酶、HIV-1逆转录酶、AMV逆转录酶，端粒酶逆转录酶，以及具有上述转座酶的转座活性的变体、修饰产物和衍生物。
在某些实施方案中，步骤(2)和(3)具有选自以下的一项或多项特征：
(1)所述引物A，引物A’，引物B，引物B’，第一桥接寡核苷酸，第一桥接寡核苷酸各自独立地包含或者由天然存在的核苷酸(例如脱氧核糖核苷酸或核糖核苷酸)，经修饰的核苷酸，非天然的核苷酸，或其任何组合组成；在某些实施方案中，所述引物A，引物A’能够起始延伸反应；
(2)所述引物B包含修饰的核苷酸(例如锁核酸)；在某些实施方案中，所述引物B的3’末端包含一个或多个修饰的核苷酸(例如锁核酸)；
(3)所述引物B’包含修饰的核苷酸(例如锁核酸)；在某些实施方案中，所述引物B’的3’末端包含一个或多个修饰的核苷酸(例如锁核酸)；
(4)所述标签序列A，标签序列B各自独立地具有5-200(例如5-30nt，6-15nt)的长度；
(5)所述共有序列A，共有序列B各自独立地具有10-200nt(例如10-100nt，20-100nt，25-100nt，5-10nt，10-15nt，15-20nt，20-50nt，20-30nt，30-40nt，40-50nt，50-100nt)的长度；
(6)所述引物A，引物A’，引物B，引物B’各自独立地具有4-200nt(例如5-200nt，15-230nt，26-115nt，10-130nt，10-20nt，20-50nt，20-30nt，30-40nt，40-50nt，50-100nt，100-150nt，150-200nt)的长度；
(7)所述第一桥接寡核苷酸的第一区域，第二区域各自独立地具有3-100nt(例如20-100nt，3-10nt，10-15nt，15-20nt，20-70nt，20-30nt，30-40nt，40-50nt，50-100nt)的长度；
(8)所述第二桥接寡核苷酸的第一区域，第二区域各自独立地具有3-100nt(例如20-100nt，3-10nt，10-15nt，15-20nt，20-70nt，20-30nt，30-40nt，40-50nt，50-100nt)的长度；
(9)所述第一桥接寡核苷酸的第三区域，所述第二桥接寡核苷酸的第三区域各自独立地具有0-50nt(例如0nt，0-10nt，10-15nt，15-20nt，20-30nt，30-40nt，40-50nt)的长度；
(10)所述第一桥接寡核苷酸、第二桥接寡核苷酸各自独立地具有6-200nt(例如20-100nt，20-70nt，6-15nt，15-20nt，20-30nt，30-40nt，40-50nt，50-100nt，100-150nt，150-200nt)的长度；
(11)所述poly(T)序列包括至少5个，或至少20个(例如6-100个，10-50个)脱氧胸腺嘧啶核苷残基；
(12)所述随机寡核苷酸序列具有5-200(例如5nt，5-30nt，6-15nt)的长度。
在某些实施方案中，所述方法还包括：(4)回收和纯化所述第二核酸分子群。
在某些实施方案中，所获得的第二核酸分子群和/或其互补物用于构建转录组文库或用于转录组测序。
在某些实施方案中，步骤(1)中所述寡核苷酸探针具有选自下列的一个或多个特征：
(1)所述共有序列X1，标签序列Y和共有序列X2各自独立地包含或者由天然存在的核苷酸(例如脱氧核糖核苷酸或核糖核苷酸)，经修饰的核苷酸，非天然的核苷酸(例如肽核酸(PNA)或锁核酸)，或其任何组合组成；
(2)所述共有序列X1，标签序列Y和共有序列X2各自独立地具有2-200nt(例如10-200nt，25-100nt，10-30nt，10-100nt，5-10nt，10-15nt，15-20nt，20-30nt，30-40nt，40-50nt，50-100nt)的长度。
在某些实施方案中，所述寡核苷酸探针通过连接子与所述固相支持物偶联。
在某些实施方案中，所述连接子是能够与活化基团反应的连接基团，且所述固相支持物表面连接有活化基团。
在某些实施方案中，所述连接子包括-SH、-DBCO或-NHS。
Figure PCTCN2022135363-appb-000001
在某些实施方案中，步骤(1)所述核酸阵列具有选自下列的一个或多个特征：
在某些实施方案中，(1)偶联在同一固相支持物上的所述寡核苷酸探针具有相同的共有序列X1和/或相同的共有序列X2；(2)所述寡核苷酸探针的共有序列X1包含切割位点；在某些实施方案中，所述切割位点可以通过选自切刻酶(nicking enzyme)酶切、USER酶切、光切除、化学切除或CRISPR切除的方式而被切割或断裂。
在某些实施方案中，步骤(1)所述核酸阵列由包含以下的步骤来提供：
(1)提供多种载体序列，每种载体序列包含至少一个拷贝(例如，多个拷贝)的载体序列，所述载体序列从5’到3’的方向上包含：共有序列X2的互补序列，标签序列Y的互补序列以及固定序列；其中，每种载体序列的标签序列Y的互补序列互不相同；
(2)将所述多种载体序列连接于固相支持物(例如芯片)表面；
(3)提供固定引物，并以所述载体序列为模板，进行引物延伸反应，生成延伸产物，所述延伸产物即为寡核苷酸探针；其中，所述固定引物包含共有序列X1的序列，并且，所述固定引物能与所述固定序列退火并起始延伸反应；在某些实施方案中，所述延伸产物从5’到3’的方向上包含或者由：共有序列X1，标签序列Y和共有序列X2组成；
(4)将所述固定引物与所述固相支持物表面连接；其中，步骤(3)与(4)以任意顺序进行；
(5)任选地，所述载体序列的固定序列还包含切割位点，所述切割可以选自切刻酶(nicking enzyme)酶切、USER酶切、光切除、化学切除或CRISPR切除；对所述载体序列的固定序列所包含的切割位点进行切割，以消化所述载体序列，使得步骤(3)中的延伸产物与形成延伸产物的模板(即载体序列)分离，从而将所述寡核苷酸探针连接于固相支持物(例如芯片)表面。在某些实施方案中，所述方法还包括通过高温变性使得步骤(3)中的延伸产物与形成延伸产物的模板(即 载体序列)分离。
在某些实施方案中，每种载体序列是由多个拷贝的载体序列的多联体所形成的DNB。
在某些实施方案中，步骤(1)中通过以下步骤提供所述多种载体序列：
(i)提供多种载体模板序列，所述载体模板序列包含所述载体序列的互补序列；
(ii)以每种载体模板序列为模板，进行核酸扩增反应，以获得每种载体模板序列的扩增产物，所述扩增产物包含至少一个拷贝的载体序列；在某些实施方案中，进行滚环复制，以获得由所述载体序列的多联体所形成的DNB。
在某些实施方案中，步骤(1)所述固相支持物具有选自下列的一个或多个特征：
(1)所述固体支持物选自乳胶珠、葡聚糖珠、聚苯乙烯表面、聚丙烯表面、聚丙烯酰胺凝胶、金表面、玻璃表面、芯片、传感器、电极和硅晶片；在某些实施方案中，所述固相支持物是芯片；
(2)所述固体支持物为平面的、球形的或多孔的；
(3)所述固相支持物能够用作测序平台，例如测序芯片；在某些实施方案中，所述固相支持物是用于Illumina、MGI或Thermo Fisher测序平台的测序芯片；和
(4)所述固相支持物能够自发地或在暴露于一种或多种刺激(例如，温度变化、pH变化、暴露于特定化学物质或相、暴露于光、还原剂等)时释放所述寡核苷酸探针。
构建核酸分子文库的方法
在另一方面，本申请还提供了一种构建核酸分子文库的方法，其包括，
(a)根据如上所述的生成标记的核酸分子群的方法生成标记的核酸分子群；
(b)将所述标记的核酸分子群中的核酸分子随机打断并添加接头；和
(c)任选地，对步骤(b)的产物进行扩增和/或富集；
从而获得核酸分子文库。
在某些实施方案中，所述核酸分子文库用于测序，例如转录组测序，例如单细胞转录组测序(例如5’端或3’端转录组测序)。
在某些实施方案中，在进行步骤(b)之前，所述方法还包括步骤(pre-b)：扩增和/或富集所述标记的核酸分子群。
在某些实施方案中，在步骤(pre-b)中，对所述标记的核酸分子群进行核酸扩增反应，以产生富集产物。
在某些实施方案中，所述扩增反应使用至少引物C和/或引物D来进行，其中，所述引物C能够与所述共有序列X1的互补序列或其部分序列杂交或退火，并起始延伸反应；所述引物D能够与所述标记的核酸分子群中含有所述标签序列Y的核酸分子链杂交或退火，并起始延伸反应。
在某些实施方案中，步骤(pre-b)中的所述核酸扩增反应使用核酸聚合酶(例如DNA聚合酶。例如具有链置换活性和/或高保真性的DNA聚合酶)来进行。
在某些实施方案中，所述方法在步骤(b)中，用转座酶将所述核酸分子随机打断并添加接头。
在某些实施方案中，所述方法在步骤(b)中，用转座酶将前一步骤获得的核酸分子随机打断并在片段两端分别添加第一接头和第二接头。
在某些实施方案中，所述转座酶选自Tn5转座酶、MuA转座酶、睡美人转座酶、Mariner转 座酶、Tn7转座酶、Tn10转座酶、Ty1转座酶、Tn552转座酶，以及具有上述转座酶的转座活性的变体、修饰产物和衍生物。
在某些实施方案中，所述转座酶为Tn5转座酶。
在某些实施方案中，在步骤(c)中，至少使用引物C’和/或引物D’对步骤(b)的产物进行扩增，其中，所述引物C’能够与所述第一接头杂交或退火，并起始延伸反应，所述引物D’能够与所述第二接头杂交或退火，并起始延伸反应。
在某些实施方案中，在步骤(c)中，至少使用所述引物C和/或引物D’对步骤(b)的产物进行扩增；其中，所述引物D’能够与所述第一接头或第二接头杂交或退火，并起始延伸反应。
进行转录组测序的方法
在另一方面，本申请还提供了一种对样品中的细胞进行转录组测序的方法，其包括：
(1)根据如上所述的构建核酸分子文库的方法构建核酸分子文库；和
(2)对所述核酸分子文库进行测序。
试剂盒
在另一方面，本申请还提供了试剂盒，其包含：
(i)用于标记核酸的核酸阵列，其包括固相支持物，所述固相支持物偶联有多个寡核苷酸探针；每种寡核苷酸探针包含至少一个拷贝；并且，所述寡核苷酸探针从5’到3’的方向上包含或者由：共有序列X1，标签序列Y和共有序列X2组成，其中，
不同种寡核苷酸探针具有不同的标签序列Y，所述标签序列Y具有与该种寡核苷酸探针在固相支持物的位置相对应的独一无二的核苷酸序列；
(ii)包含引物A和引物B或者包含引物A’和引物B’的引物组，其中：
所述引物A含有捕获序列A，所述捕获序列A能与待捕获的RNA(例如，mRNA)退火并起始延伸反应；
所述引物B包含共有序列B，3’末端悬突互补序列，以及任选的标签序列B；在某些实施方案中，所述3’末端悬突互补序列位于所述引物B的3’末端；在某些实施方案中，所述共有序列B位于所述3’末端悬突互补序列的上游(例如位于所述引物B的5’端)；其中，所述3’末端悬突是指以所述引物A的捕获序列A所捕获的RNA为模板逆转录生成的cDNA链的3’末端所包含的一个或多个非模板核苷酸；
所述引物A’含有共有序列A和捕获序列A；在某些实施方案中，所述捕获序列A位于所述引物A’的3’端；在某些实施方案中，所述共有序列A位于所述捕获序列A的上游(例如位于所述引物A’的5’端)；
所述引物B’包含共有序列B，3’末端悬突互补序列，以及任选的标签序列B；在某些实施方案中，所述3’末端悬突互补序列位于所述引物B’的3’末端；在某些实施方案中，所述共有序列B位于所述3’末端悬突互补序列的上游(例如位于所述引物B’的5’端)；其中，所述3’末端悬突是指以所述引物A’的捕获序列A所捕获的RNA为模板逆转录生成的cDNA链的3’末端所包含的一个或多个非模板核苷酸。
在某些实施方案中，每种寡核苷酸探针包含一个拷贝。
在某些实施方案中，每种寡核苷酸探针包含多个拷贝。
在某些实施方案中，每一种所述寡核苷酸探针与所述固相支持物偶联的区域即称作一个微点。容易理解，当每种寡核苷酸探针为一个拷贝时，每个微点偶联一个探针，并且不同微点的寡核苷酸探针具有不同的标签序列Y；当每种寡核苷酸探针包含多个拷贝时，每个微点偶联多个探针，同一微点内的寡核苷酸探针具有相同的标签序列Y，不同微点的寡核苷酸探针具有不同的标签序列Y。
在某些实施方案中，所述固相支持物包含多个微点，每个微点偶联一种寡核苷酸探针，每种寡核苷酸探针可包含一个或多个拷贝。
在某些实施方案中，所述固相支持物包含多个(例如，至少10个，至少10 2个，至少10 3个，至少10 4个，至少10 5个，至少10 6个，至少10 7个，至少10 8个，或更多个)微点；在某些实施方案中，所述固相支持物包含至少10 4个(例如至少10 4个，至少10 5个，至少10 6个，至少10 7个，至少10 8个，至少10 9个，至少10 10个，至少10 11个，或至少10 12个)微点/平方毫米。
在某些实施方案中，相邻的所述微点之间的间隔小于100μm，小于50μm，小于10μm，小于5μm，小于1μm，小于0.5μm，小于0.1μm，小于0.05μm，或小于0.01μm。
在某些实施方案中，所述微点的尺寸(例如等效直径)小于100μm，小于50μm，小于10μm，小于5μm，小于1μm，小于0.5μm，小于0.1μm，小于0.05μm，或小于0.01μm。
在某些实施方案中，所述试剂盒包含：如(i)中所述的用于标记核酸的核酸阵列，如(ii)中所述的引物A和引物B的引物组，以及，(iii)第一桥接寡核苷酸和第二桥接寡核苷酸；其中，所述第一桥接寡核苷酸和所述第二桥接寡核苷酸各自独立地包括：第一区域和第二区域，以及任选的位于第一区域和第二区域之间的第三区域，所述第一区域位于所述第二区域的上游(例如5’端)；其中，
所述第一桥接寡核苷酸的第一区域能与所述第二桥接寡核苷酸的第一区域退火；所述第一桥接寡核苷酸的第二区域能与所述寡核苷酸探针的共有序列X2或其部分序列退火；
所述第二桥接寡核苷酸的第二区域能与所述引物B的共有序列B的互补序列或其部分序列退火。
在某些实施方案中，所述引物A的捕获序列A是随机寡核苷酸序列。
在某些实施方案中，所述引物A的捕获序列A是poly(T)序列或针对特定靶核酸的特异性序列。在某些实施方案中，所述引物A进一步包含共有序列A和任选的标签序列A，例如为随机寡核苷酸序列。在某些实施方案中，所述捕获序列A位于所述引物A的3’端，所述共有序列A位于所述引物A的上游(例如5’端)。
在某些实施方案中，所述引物B含有所述共有序列B，3’末端悬突互补序列，以及标签序列B。
在某些实施方案中，所述引物B包含修饰的核苷酸(例如锁核酸)。在某些实施方案中，所述引物B的3’末端包含一个或多个修饰的核苷酸(例如锁核酸)。
在某些实施方案中，所述第二桥接寡核苷酸的第二区域能与所述引物B的共有序列B的互补序列或其部分序列(例如，3’端部分序列)退火。
在某些实施方案中，所述第一桥接寡核苷酸的第二区域位于所述第一桥接寡核苷酸的3’末端。
在某些实施方案中，所述第一桥接寡核苷酸的第一区域位于所述第一桥接寡核苷酸的5’末端。
在某些实施方案中，所述第一桥接寡核苷酸的5’末端含有磷酸化修饰。
在某些实施方案中，所述第一桥接寡核苷酸的3’末端含有自由-OH。
在某些实施方案中，所述第二桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)，和/或，所述寡核苷酸探针不能起始延伸反应(例如3’端是封闭的)。
在某些实施方案中，所述第二桥接寡核苷酸的第二区域能与所述引物B的共有序列B互补序列或其部分序列退火。
在某些实施方案中，所述第二桥接寡核苷酸的第二区域位于所述第二桥接寡核苷酸的3’末端。
在某些实施方案中，所述第二桥接寡核苷酸的第一区域位于所述第二桥接寡核苷酸的5’末端。
在某些实施方案中，所述第二桥接寡核苷酸的5’末端含有磷酸化修饰。
在某些实施方案中，所述第二桥接寡核苷酸的3’末端含有自由-OH。
在某些实施方案中，所述第一桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)。
在某些实施方案中，所述试剂盒包含：如(i)中所述的用于标记核酸的核酸阵列，以及，如(ii)中所述的引物A和引物B的引物组。
在某些实施方案中，所述引物A的捕获序列A是随机寡核苷酸序列。
在某些实施方案中，所述引物A的捕获序列A是poly(T)序列或针对特定靶核酸的特异性序列。在某些实施方案中，所述引物A进一步包含共有序列A和任选的标签序列A，例如为随机寡核苷酸序列。在某些实施方案中，所述捕获序列A位于所述引物A的3’端，所述共有序列A位于所述引物A的上游(例如，5’端)。
在某些实施方案中，所述引物B含有所述共有序列B，3’末端悬突互补序列，以及标签序列B。
在某些实施方案中，所述引物B包含修饰的核苷酸(例如锁核酸)。在某些实施方案中，所述引物B的3’末端包含一个或多个修饰的核苷酸(例如锁核酸)。
任选地，所述寡核苷酸探针能够(例如3’末端含自由-OH)或者不能够起始延伸反应(例如3’端是封闭的)。
在某些实施方案中，所述试剂盒包含：如(i)中所述的用于标记核酸的核酸阵列，如(ii)中所述的引物A’和引物B’的引物组，以及，(iii)第一桥接寡核苷酸和第二桥接寡核苷酸；其中，所述第一桥接寡核苷酸和所述第二桥接寡核苷酸各自独立地包括：第一区域和第二区域，以及任选的位于第一区域和第二区域之间的第三区域，所述第一区域位于所述第二区域的上游(例如5’端)；其中，
所述第一桥接寡核苷酸的第一区域能与所述第二桥接寡核苷酸的第一区域退火；所述第一桥接寡核苷酸的第二区域能与所述寡核苷酸探针的共有序列X2或其部分序列退火；
所述第二桥接寡核苷酸的第二区域能与所述引物A’的共有序列A互补序列或其部分序列退火。
在某些实施方案中，所述引物A’的捕获序列A是随机寡核苷酸序列。
在某些实施方案中，所述引物A’的捕获序列A是poly(T)序列或针对特定靶核酸的特异性序 列。在某些实施方案中，所述引物A’进一步包含标签序列A，例如为随机寡核苷酸序列。在某些实施方案中，所述捕获序列A位于所述引物A’的3’端，所述共有序列A位于所述标签序列A的上游(例如位于所述引物A’的5’端)。
在某些实施方案中，所述引物B’包含修饰的核苷酸(例如锁核酸)。在某些实施方案中，所述引物B’的3’末端包含一个或多个修饰的核苷酸(例如锁核酸)。
在某些实施方案中，所述试剂盒进一步包含引物B”或随机引物，所述引物B”能与所述共有序列B的互补序列或其部分序列退火，并且能起始延伸反应。
在某些实施方案中，所述第二桥接寡核苷酸的第二区域能与所述引物A’的共有序列A的互补序列或其部分序列(例如，3’端部分序列)退火。
在某些实施方案中，所述第一桥接寡核苷酸的第二区域位于所述第一桥接寡核苷酸的3’末端。
在某些实施方案中，所述第一桥接寡核苷酸的第一区域位于所述第一桥接寡核苷酸的5’末端。
在某些实施方案中，所述第一桥接寡核苷酸的5’末端含有磷酸化修饰。
在某些实施方案中，所述第一桥接寡核苷酸的3’末端含有自由-OH。
在某些实施方案中，所述第二桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)，和/或，所述寡核苷酸探针不能起始延伸反应(例如3’端是封闭的)。
在某些实施方案中，所述第二桥接寡核苷酸的第二区域能与所述引物A’的共有序列A的互补序列或其部分序列退火。
在某些实施方案中，所述第二桥接寡核苷酸的第二区域位于所述第二桥接寡核苷酸的3’末端。
在某些实施方案中，所述第二桥接寡核苷酸的第一区域位于所述第二桥接寡核苷酸的5’末端。
在某些实施方案中，所述第二桥接寡核苷酸的5’末端含有磷酸化修饰。
在某些实施方案中，所述第二桥接寡核苷酸的3’末端含有自由-OH。
在某些实施方案中，所述第一桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)。
在某些实施方案中，所述试剂盒包含：如(i)中所述的用于标记核酸的核酸阵列，以及，如(ii)中所述的引物A’和引物B’的引物组。
在某些实施方案中，所述引物A’的捕获序列A是随机寡核苷酸序列。
在某些实施方案中，所述引物A’的捕获序列A是poly(T)序列或针对特定靶核酸的特异性序列。在某些实施方案中，所述引物A’进一步包含标签序列A，例如为随机寡核苷酸序列。在某些实施方案中，所述捕获序列A位于所述引物A’的3’端，所述共有序列A位于所述标签序列A的上游(例如位于所述引物A’的5’端)。
在某些实施方案中，所述引物B’含有所述共有序列B，3’末端悬突互补序列，以及标签序列B。
在某些实施方案中，所述引物B’包含修饰的核苷酸(例如锁核酸)。在某些实施方案中，所述引物B’的3’末端包含一个或多个修饰的核苷酸(例如锁核酸)。
在某些实施方案中，所述试剂盒进一步包含引物B”或随机引物，所述引物B”能与所述共有序列B的互补序列或其部分序列退火，并且能起始延伸反应。
任选地，所述寡核苷酸探针能够(例如3’末端含自由-OH)或者不能够起始延伸反应(例如3’ 端是封闭的)。
在某些实施方案中，所述试剂盒具有选自以下的一项或多项特征：
(1)所述寡核苷酸探针，引物A，引物A’，引物B，引物B’，引物B”，随机引物，第一桥接寡核苷酸，第二桥接寡核苷酸各自独立地包含或者由天然存在的核苷酸(例如脱氧核糖核苷酸或核糖核苷酸)，经修饰的核苷酸，非天然的核苷酸，或其任何组合组成；
(2)所述寡核苷酸探针各自独立地具有15-300nt(例如15-200nt，15-20nt，20-30nt，30-40nt，40-50nt，50-100nt，100-150nt，150-200nt)的长度；
(3)所述引物A，引物A’，引物B，引物B’，引物B”，随机引物各自独立地具有4-200nt(例如5-200nt，15-230nt，26-115nt，10-130nt，10-20nt，20-50nt，20-30nt，30-40nt，40-50nt，50-100nt，100-150nt，150-200nt)的长度；
(4)所述第一桥接寡核苷酸和所述第二桥接寡核苷酸各自独立地具有6-200nt(例如20-100nt，20-70nt，6-15nt，15-20nt，20-30nt，30-40nt，40-50nt，50-100nt，100-150nt，150-200nt)的长度；
(5)偶联在同一固相支持物上的所述寡核苷酸探针具有相同的共有序列X1和/或相同的共有序列X2；
(6)所述寡核苷酸探针的共有序列X1包含切割位点；在某些实施方案中，所述切割位点可以通过选自切刻酶(nicking enzyme)酶切、USER酶切、光切除、化学切除或CRISPR切除的方式而被切割或断裂。
在某些实施方案中，所述试剂盒进一步包含逆转录酶，核酸连接酶，核酸聚合酶和/或转座酶。
在某些实施方案中，所述逆转录酶具有末端转移活性。在某些实施方案中，所述逆转录酶能够以RNA(例如，mRNA)为模板，合成cDNA链，且在所述cDNA链的3’端添加所述3’末端悬突。在某些实施方案中，所述逆转录酶能够在cDNA链的3’末端添加长度为至少1个，至少2个，至少3个，至少4个，至少5个，至少6个，至少7个，至少8个，至少9个，至少10个或更多个核苷酸的悬突。在某些实施方案中，所述逆转录酶能够在cDNA链的3’末端添加2-5个胞嘧啶核苷酸的悬突(例如CCC悬突)。在某些实施方案中，所述逆转录酶选自M-MLV逆转录酶、HIV-1逆转录酶、AMV逆转录酶，端粒酶逆转录酶，以及具有上述转座酶的转座活性的变体、修饰产物和衍生物。
在某些实施方案中，所述核酸聚合酶无5’至3’端外切活性或链置换活性。
在某些实施方案中，所述核酸聚合酶具有5’至3’端外切活性或链置换活性。
在某些实施方案中，所述转座酶选自Tn5转座酶、MuA转座酶、睡美人转座酶、Mariner转座酶、Tn7转座酶、Tn10转座酶、Ty1转座酶、Tn552转座酶，以及具有上述转座酶的转座活性的变体、修饰产物和衍生物。
在某些实施方案中，所述试剂盒进一步包含：所述引物C，所述引物D，所述引物C’和/或所述引物D’。例如，所述试剂盒进一步包含所述引物C，所述引物D和所述引物D’。例如，所述试剂盒进一步包含所述引物C，所述引物D，所述引物C’和所述引物D’。
在某些实施方案中，所述试剂盒进一步包含：用于进行核酸杂交的试剂、用于进行核酸延伸 的试剂、用于进行核酸扩增的试剂、用于回收或纯化核酸的试剂、用于构建转录组测序文库的试剂、用于测序(例如二代测序或三代测序)的试剂、或其任何组合。
用途
在另一方面，本申请还提供了如上的生成标记的核酸分子群的方法或如上所述的试剂盒用于构建核酸分子文库或用于进行转录组测序的用途。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子生物学、生物化学、核酸化学、细胞培养等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
当本文使用术语“例如”、“如”、“诸如”、“包括”、“包含”或其变体时，这些术语将不被认为是限制性术语，而将被解释为表示“但不限于”或“不限于”。
除非本文另外指明或根据上下文明显矛盾，否则术语“一个”和“一种”以及“该”和类似指称物在描述本发明的上下文中(尤其在以下权利要求的上下文中)应被解释成覆盖单数和复数。
如本文所用，“DNB”(DNA nano ball，DNA纳米球)是一种典型的RCA(rolling circle amplification，RCA)产物，其具有RCA产物的特征。其中，所述RCA产物是一种多拷贝的单链DNA序列，因内部DNA序列的碱基间的相互作用力，可以形成类似“球形“结构。典型地，文库分子环化形成单链环状DNA，随后使用滚环扩增技术可将单链环状DNA扩增多个数量级，所产生的扩增产物称为DNB。
如本文所用，“核酸分子群”是指例如直接或间接来源于靶核酸分子(例如DNA双链分子、RNA/cDNA杂合双链分子、DNA单链分子、或RNA单链分子)的核酸分子的群体或集合。在一些实施方案中，核酸分子群包括核酸分子文库，所述核酸分子文库包含性质上和/或数量上代表靶核酸分子序列的序列。在另一些实施方案中，核酸分子群包含核酸分子文库的子集。
如本文所用，“核酸分子文库”表示直接或间接从靶核酸分子产生的经标记的核酸分子(例如经标记的DNA双链分子、经标记的RNA/cDNA杂合双链分子、经标记的DNA单链分子、或经标记的RNA单链分子)或其片段的集合或群体，其中，在该集合或群体中经标记的核酸分子或其片段的组合显示在性质上和/或数量上代表产生经标记的核酸分子的靶核酸分子序列的序列。在某些实施方案中，所述核酸分子文库是测序文库。在某些实施方案中，所述核酸分子文库可用于构建测序文库。
如本文所用，“cDNA”或“cDNA链”是指使用感兴趣的RNA分子的至少一部分作为模板，通过RNA依赖性DNA聚合酶或反转录酶催化的与该感兴趣的RNA分子退火的引物的延伸而合成的“互补的DNA”(该过程也称为“反转录”)。所合成的cDNA分子与该模板的至少一部分“同源”或“互补”或“碱基配对”或“形成复合物”。
如本文中所使用的，术语“上游”用于描述两条核酸序列(或两个核酸分子)的相对位置关系，并且具有本领域技术人员通常理解的含义。例如，表述“一条核酸序列位于另一条核酸 序列的上游”意指，当以5'至3'方向排列时，与后者相比，前者位于更靠前的位置(即，更接近5'端的位置)。如本文中所使用的，术语“下游”具有与“上游”相反的含义。
如本文所用，“标签序列Y”、“标签序列A”、“标签序列B”、“共有序列X1”、“共有序列X2”、“共有序列A”、“共有序列B”等，是指向它所接合的核酸分子或其接合的核酸分子的衍生产物(例如，核酸分子的互补片段、核酸分子的断裂短片段等)提供鉴定、识别和/或分子操作或生物化学操作手段(例如，通过提供用于使寡核苷酸退火的位点，所述寡核苷酸诸如用于DNA聚合酶延伸的引物或者用于捕获反应或连接反应的寡核苷酸)的非靶核酸组分的寡核苷酸。所述寡核苷酸可以由连续的至少两个(优选大约6到100个，但是对寡核苷酸的长度没有确定的限制，确切大小取决于许多因素，而这些因素又取决于寡核苷酸的最终功能或用途)核苷酸组成，也可以由多段寡核苷酸连续或非连续排列组合而成。所述寡核苷酸序列可以对于其接合的每个核酸分子是唯一的，也可以对于其接合的某一类核酸分子是唯一的。所述寡核苷酸序列可以通过任何方法包括连接、杂交或其他方法与待“标记”的多核苷酸序列可逆或不可逆地接合。将所述寡核苷酸序列与核酸分子接合的过程有时在本文称为“添加标记”并且经历添加标记或含标记序列的核酸分子称为“经标记的核酸分子”或“标记的核酸分子”。
出于多种原因，本发明的核酸或多核苷酸(例如“标签序列Y”、“标签序列A”、“标签序列B”、“共有序列X1”、“共有序列X2”、“共有序列A”、“共有序列B”、“引物A”、“引物A’”、“引物B”、“引物B’”、“引物B””、“引物C”、“引物D”、“引物D’”、“随机引物”、“第一桥接寡核苷酸”、“第二桥接寡核苷酸”等)可包括一种或多种修饰的核酸碱基、糖部分或核苷间连接。例如，使用包含修饰的碱基、糖部分或核苷间连接的核酸或多核苷酸的一些原因包括但不限于：(1)Tm的改变；(2)改变多核苷酸对一种或多种核酸酶的易感性；(3)提供用于连接标记的部分；(4)提供标记或标记猝灭剂；或(5)提供用于连接溶液中或结合于表面的另一种分子的部分，诸如生物素。例如，在一些实施方案中，可将寡核苷酸诸如引物合成为使得随机部分包含一种或多种构象受限制的核酸类似物，诸如但不限于其中的核糖环被连接2’-O原子与4’-C原子的亚甲基桥“锁定”的一种或多种核糖核酸类似物；这些修饰的核苷酸导致每个核苷酸单体的Tm或解链温度提高大约2摄氏度到大约8摄氏度。例如，在其中使用包含核糖核苷酸的寡核苷酸引物的一些实施方案中，在该方法中使用修饰的核苷酸的一个指标可以是包含该修饰的核苷酸的寡核苷酸可以被单链特异性RNA酶消化。
在本发明的方法中，例如，在多核苷酸或寡核苷酸中的一个或多个位置的单核苷酸中的核酸碱基可包括鸟嘌呤、腺嘌呤、尿嘧啶、胸腺嘧啶或胞嘧啶，或者可选地，所述核酸碱基中的一种或多种可包含修饰的碱基，诸如但不限于黄嘌呤、烯丙氨基(al lyamino)-尿嘧啶、烯丙氨基-胸腺嘧啶核苷、次黄嘌呤、2-氨基腺嘌呤、5-丙炔基尿嘧啶、5-丙炔基胞嘧啶、4-硫尿嘧啶、6-硫鸟嘌呤、氮尿嘧啶和脱氮尿嘧啶、胸腺嘧啶核苷、胞嘧啶、腺嘌呤或鸟嘌呤。此外，它们可包含用如下部分衍生的核酸碱基：生物素部分、洋地黄毒苷部分、荧光部分或化学发光部分、猝灭部分或某种其他部分。本发明不限于列出的核酸碱基；给出的这份名单示出了可用于本发明方法中的范围广泛的碱基的实例。
就本发明的核酸或多核苷酸来说，糖部分中的一个或多个可包括2′-脱氧核糖，或者可选 地，糖部分中的一个或多个可包括某种其他糖部分，诸如但不限于：提供对一些核酸酶的抵抗力的核糖或2’-氟代-2’-脱氧核糖或2’-O-甲基-核糖，或可通过与可见的、荧光的、红外荧光的或其他可检测的染料或具有亲电子的、光反应性的、炔基或其他反应性化学部分的化学物质进行反应而标记的2’-氨基2’-脱氧核糖或2’-叠氮基-2’-脱氧核糖。
本发明的核酸或多核苷酸的核苷间连接可以是磷酸二酯键连接，或者可选地，核苷间连接中的一种或多种可包括修饰的连接，诸如但不限于：硫代磷酸酯、二硫代磷酸酯、硒代磷酸酯(phosphoroselenate)、或二硒代磷酸酯(phosphorodiselenate)连接，它们对一些核酸酶具有抵抗力。
如本文所用，术语“末端转移活性”是指，能催化一个或多个脱氧核糖核苷三磷酸(dNTP)或单个双脱氧核糖核苷三磷酸不依赖模板地添加(或“加尾”)至cDNA的3’末端的活性。具有末端转移活性的逆转录酶的实例包括但不限于，M-MLV逆转录酶、HIV-1逆转录酶、AMV逆转录酶、端粒酶逆转录酶，以及具有所述逆转录酶的逆转录活性和末端转移活性的变体、修饰产物和衍生物。所述逆转录酶不具有或者具有RNase活性(特别是RNase H活性)。在优选的实施方案中，用于逆转录RNA以生成cDNA的逆转录酶不具有RNase活性(特别是RNase H活性)。因此，在优选的实施方案中，用于逆转录RNA以生成cDNA的逆转录酶具有末端转移活性，且不具有RNase活性(特别是RNase H活性)。
如本文所用，具有“链置换活性”的核酸聚合酶是指，在延伸新核酸链的过程中，如果遇到下游与模板链互补的核酸链，可以继续延伸反应并将所述与模板链互补的核酸链剥离(而非降解)的核酸聚合酶。
如本文所用，具有“5’至3’端外切酶活性”的核酸聚合酶是指，能从多核苷酸链的5’端开始按5’端至3’端的次序催化水解3、5-磷酸二酯键，降解核苷酸的核酸聚合酶。
如本文所用，具有“高保真性”的核酸聚合酶(或DNA聚合酶)是指，在扩增核酸的过程中，引入错误核苷酸的概率(即，错误率)低于野生型Taq酶(例如其序列如UniProt Acession:P19821.1所示的Taq酶)的核酸聚合酶(或DNA聚合酶)。
如本文所用，术语“发生退火”、“进行退火”、“退火”、“使杂交”或“杂交”等是指，具有经由沃森-克里克碱基配对形成复合物的充分互补性的核苷酸序列之间形成复合物。就本发明来说，彼此之间“对其互补”或“与之互补”或与其“杂交”或“退火”的核酸序列应该能形成或形成服务于预定目的的足够稳定的“杂交体”或“复合物”。不要求由一个核酸分子显示的序列内的每个核酸碱基能够与由第二核酸分子显示的序列内的每个核酸碱基进行碱基配对或配对或复合，以便这两个核酸分子或其中显示的相应序列与彼此“互补”或“退火”或“杂交”。如本文所述，在提及按碱基配对法则联系的核苷酸的序列时使用术语“互补的”或“互补性”。例如，序列5’-A-G-T-3’与序列3’-T-C-A-5’互补。互补性可以是“部分的”，其中核酸碱基中只有一些根据碱基配对法则匹配。或者，在核酸之间可具有“完全的”或“全部的”互补性。核酸链之间的互补性的程度对核酸链之间的杂交的效率和强度具有显著影响。这在扩增反应以及依赖于核酸的杂交的检测方法中是特别重要的。术语“同源性”是指一条核酸序列与另一条核酸序列的互补性程度。可具有部分同源性或完全同源性(即，互补性)。部分互补的 序列是至少部分地抑制完全互补的序列与靶核酸的杂交的序列并且使用功能术语“基本上同源的”称呼。完全互补的序列与靶序列的杂交的抑制可使用杂交测定(例如，DNA印迹或RNA印迹，溶液杂交等)在低严格度条件下来检查。基本上同源的序列或探针将竞争或抑制完全同源的序列与靶在低严格度条件下的结合(即杂交)。这并不是说低严格度条件是允许非特异性结合的条件；低严格度条件要求两条序列与彼此的结合是特异性(即选择性)相互作用。非特异性结合的不存在可以通过使用缺乏互补性或只具有低互补性程度(例如，小于约30％的互补性)的第二靶来测试。在特异性结合很低或不存在的情况下，探针将不与核酸靶杂交。当用于提及双链核酸序列诸如cDNA或基因组克隆时，术语“基本上同源的”是指可在本文所述的低严格度条件下与双链核酸序列的一条链或两条链杂交的任何寡核苷酸或探针。如本文所用，在提及互补的核酸链的配对时使用术语“退火”或“杂交”。杂交和杂交强度(即，核酸链之间的缔合强度)受本领域中公知的许多因素影响，包括核酸之间的互补性程度，包括受诸如盐浓度影响的条件的严格度，形成的杂交体的Tm(解链温度)，其他组分的存在(如，存在或不存在聚乙二醇或甜菜碱)，杂交链的摩尔浓度以及核酸链的G:C含量。
如本文所述，所述固相支持物能够自发地或在暴露于一种或多种刺激(例如，温度变化、pH变化、暴露于特定化学物质或相、暴露于光、还原剂等)时释放所述寡核苷酸探针。应当理解的是，可以通过寡核苷酸探针与固相支持物之间的键的裂解来释放所述寡核苷酸探针，或通过固相支持物本身的降解来释放所述寡核苷酸探针，或两者兼而有之，所述寡核苷酸探针允许被其他试剂接近或可被其他试剂接近。
向所述固相支持物中添加多种类型的不稳定键可导致生成能够对不同刺激有反应的固相支持物。每种类型的不稳定键可以对相关的刺激(例如，化学刺激、光、温度等)敏感，使得通过施加适当的刺激可以控制通过每个不稳定键连接到固相支持物的物质的释放。除了可热裂解的键、二硫键和UV敏感键之外，可以与固相支持物偶合的不稳定键的其他非限制性实例包括酯键(例如，可用酸、碱或羟胺裂解)、邻位二醇键(例如，可通过高碘酸钠裂解)、狄尔斯-阿尔德(Diels-Alder)键(例如，可通过热裂解)、砜键(例如，可通过碱裂解)、甲硅烷基醚键(例如，可通过酸裂解)、糖苷键(例如，可通过淀粉酶裂解)、肽键(例如，可通过蛋白酶裂解)或磷酸二酯键(例如，可通过核酸酶(例如，DNA酶)裂解))。
除了上文所述的固相支持物与寡核苷酸之间的可裂解键之外或作为其替代，固相支持物可以在自发地或在暴露于一种或多种刺激(例如，温度变化、pH变化、暴露于特定化学物质或相、暴露于光、还原剂等)时为可降解、可破坏或可溶解的。在一些情况下，固相支持物可以是可溶解的，使得固相支持物的材料组分在暴露于特定化学物质或环境变化(例如变化温度或pH变化)时溶解。在一些情况下，固相支持物在升高的温度和/或碱性条件下降解或溶解。在一些情况下，固相支持物可以是可热降解的，使得当固相支持物暴露于适当的温度变化(例如，加热)时，固相支持物降解。与物质(例如，寡核苷酸探针)结合的固相支持物的降解或溶解可导致物质从固相支持物中释放。
如本文所用，术语“转座酶”和“逆转录酶”以及“核酸聚合酶”是指负责催化特异性化学反应和生物学反应的蛋白质分子或蛋白质分子聚集体。一般来说，本发明的方法、组合物或 试剂盒不限于使用来自特定来源的特定的转座酶、逆转录酶或核酸聚合酶。反之，本发明的方法、组合物或试剂盒包括与本文公开的特定方法、组合物或试剂盒的特定酶具有等同酶活性的来自任何来源的任何转座酶、逆转录酶或核酸聚合酶。更进一步，本发明的方法还包括如下实施方案：其中在所述方法的步骤中提供和使用的任何一种特定的酶被两种或多种酶的组合取代，所述两种或多种酶在组合使用时，不论是以分步方式分别使用还是同时一起使用，反应混合物产生的结果与使用该一种特定的酶获得的结果相同。本文提供的方法、缓冲液和反应条件，包括在实施例中的方法、缓冲液和反应条件目前对于本发明的方法、组合物和试剂盒的实施方案是优选的。然而，使用本发明的一些酶的其他的酶储存缓冲液、反应缓冲液和反应条件是本领域已知的，其也可能适于在本发明中使用并且被包括在本文中。
发明的有益效果
本申请提供了一种新的生成标记的核酸分子群的方法，以及基于该方法构建核酸分子文库并进行高通量测序，进而实现对样本进行高精度的亚细胞级空间定位。本申请的方法具有一个或多个选自下列的有益技术效果：
(1)传统的用于空间转录组测序的核酸阵列(例如芯片)的探针含有固定的捕获序列，通常特定的捕获序列只能捕获与其对应的特定靶核酸分子，例如，当捕获序列为poly(T)，则对应捕获含有poly(A)的靶核酸分子。若靶核酸分子发生改变，则需对含有捕获序列的探针序列进行相应改动，也即，需对整个核酸阵列(例如芯片)进行改动，在实际应用中成本大，效率低。而本申请的核酸阵列(例如芯片)不含有捕获序列，捕获序列存在于独立于核酸阵列的逆转录引物中(也即捕获序列与探针相互独立)，捕获序列捕获靶核酸分子后通过桥接寡核苷酸实现与探针的连接。
因此，本申请可以在不改变探针序列的情况下(也即，在不改变核酸阵列(例如芯片)的情况下)，针对不同靶核酸分子设计相应捕获序列，通过捕获序列和桥接寡核苷酸的改变实现对不同靶核酸分子的捕获。
(2)传统的空间转录组方法都采用poly(T)为捕获序列，无法捕获无poly(A)尾的RNA。而本申请可以将捕获序列中的poly(T)更换为连续的随机序列组(例如随机引物序列，如N6，N8等)以实现对不含poly(A)尾的靶核酸分子的捕获，并且，所述连续的随机序列组还可以同时作为独特分子标签(UMI)序列。
(3)传统的用于空间转录组测序的核酸阵列(例如芯片)上具有固定的捕获探针，一般先进行组织透化释放细胞内的RNA，如果过度透化将导致RNA扩散至相邻细胞甚至组织样本外围，并被探针捕获，从而无法实现mRNA的原位捕获，如果透化不完全又将影响mRNA的捕获效率。而本申请的方法，核酸阵列(例如芯片)不含有捕获序列(仅有空间信息)，组织透化的目的是让逆转录引物进入细胞与mRNA原位杂交，无需激烈的透化试剂处理，从而能够减少样本的扩散。
下面将结合附图和实施例对本发明的优选实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得可实施。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
附图说明
图1显示了本申请中用于捕获和标记核酸分子的芯片的示例性结构，其包含：芯片和偶联在芯片上的寡核苷酸探针(也称芯片序列)。每种寡核苷酸探针包含与其在芯片上的位置相对应的标签序列Y，每种寡核苷酸探针与芯片的偶联区域可称为微点。每种寡核苷酸探针可以是单拷贝的或多拷贝的。
图2显示了以样本中的RNA(例如mRNA)为模板制备cDNA链的示例性方案，以及，所述cDNA链的示例性结构。CA：共有序列A；CB：共有序列B。
图3显示了用芯片序列的互补序列标记cDNA链的3’端，形成含有芯片序列信息的新核酸分子(即，经芯片序列标记的核酸分子)的示例性方案1，以及，所述含有芯片序列信息的新核酸分子示例性结构。CA：共有序列A；CB：共有序列B；X1：共有序列X1；Y：标签序列Y；X2：共有序列X2；P1：第一区域；P2：第二区域。
图4显示了用芯片序列的互补序列标记cDNA链的3’端，形成含有芯片序列信息的新核酸分子(即，经芯片序列标记的核酸分子)的示例性方案2，以及，所述含有芯片序列信息的新核酸分子的示例性结构。CA：共有序列A；CB：共有序列B；X1：共有序列X1；Y：标签序列Y；X2：共有序列X2。
图5显示了以样本中的RNA(例如mRNA)为模板制备cDNA链的互补链的示例性方案，以及，所述cDNA链互补链的示例性结构。CA：共有序列A；CB：共有序列B；EP：延伸引物。
图6显示了用芯片序列的互补序列标记cDNA链互补链的3’端，形成含有芯片序列信息的新核酸分子(即，经芯片序列标记的核酸分子)的示例性方案1，以及，所述含有芯片序列信息的新核酸分子示例性结构。CA：共有序列A；CB：共有序列B；X1：共有序列X1；Y：标签序列Y；X2：共有序列X2；P1：第一区域；P2：第二区域。
图7显示了用芯片序列的互补序列标记cDNA链互补链的3’端，形成含有芯片序列信息的新核酸分子(即，经芯片序列标记的核酸分子)的示例性方案2，以及，所述含有芯片序列信息的新核酸分子的示例性结构。CA：共有序列A；CB：共有序列B；X1：共有序列X1；Y：标签序列Y；X2：共有序列X2。
图8显示了实施例2制备的cDNA扩增产物的长度分布。
图9显示了实施例3测序分析获得的鼠脑切片空间表达图谱。
序列信息
本申请涉及的部分序列的信息提供于下面的表1中。
表1 序列信息
Figure PCTCN2022135363-appb-000002
注：“r”表示其3’相邻位置的核苷酸为核糖核苷酸；“+”表示其3’相邻位置的核苷酸存在LNA(锁核苷酸)修饰；“*”表示硫代磷酸修饰；“p”表示磷酸化修饰；N＝A,T,C or G；V＝A,C or G。
实施例1：捕获芯片的制备
1.设计含有用于定位芯片位置的信息的DNA文库分子的序列，其从5’端到3’端包含：共有序列X1(X1)，标签序列Y(Y)和共有序列X2(X2)的编码序列。DNA文库分子的典型核苷酸序列如SEQ ID NO:1所示。委托北京六合华大基因科技有限公司(Beijing Liuhe BGI Co.,Ltd)合成DNA文库分子。
2.文库分子的扩增和装载
(1)使用DNBSEQ测序试剂盒(购自MGI，货号1000019840)来制备DNA纳米球(DNB)。具体的实施方案简要描述如下。
简言之，配置如表2所示的反应体系40μL。将该反应体系放置于PCR仪，并按照如下反应条件进行反应：95℃ 3min，40℃ 3min。反应结束后，将反应产物放于冰上，加入40μL混合酶I和2μL混合酶II(来自于DNBSEQ测序试剂盒)，1μL ATP(母液100mM，获自Thermo Fisher)，和0.1μL T4ligase(获自NEB，货号：M0202S)。混匀后，将上述反应体系置于PCR仪并在30℃反应20min，生成DNB。
表2 制备DNB的反应体系
Figure PCTCN2022135363-appb-000003
(2)随后，将DNB按照BGISEQ 500高通量测序试剂套装(SE50)(购自MGI，货号：1000012551)所述的方法将DNB装载至BGISEQ SEQ 500测序芯片上。
在测序芯片内，加入BGISEQ 500 PE50测序试剂盒(购自MGI，1000012554)中的MDA试剂，37℃孵育30min后，5XSSC清洗芯片。
(3)芯片表面修饰N3-PEG3500-NHS(修饰试剂购自sigma，货号：JKA5086)，孵育30min后，泵入DBCO修饰的芯片序列合成引物(序列如SEQ ID NO：3所示)，常温过夜孵育。
3.位置序列信息的测序解码。按照BGISEQ-500高通量测序试剂套装的说明书对DNB进行测序，SE设置读长25bp。在上述DBCO修饰的序列进行延伸获得测序后生长出来的链，对该链进行解码，获得对应DNB的位置序列信息。
4.测序后生长出来的链继续延伸：在上述步骤3基础上继续进行15个碱基的cPAS反应，得到芯片序列(SEQ ID NO:8，其含有共有序列X1(SEQ ID NO:4)，标签序列Y，共有序列X2(SEQ ID NO:5))。
5.使用限制性内切酶HaeIII切除DNB，并高温变性去除DNB上的残留片段，使芯片仅残留步骤4的芯片序列。
6.芯片切块：将制备好的芯片切成若干小片，切片大小根据实验需要进行调整，将芯片浸泡在50mM pH8.0的Tris buffer中，4℃待用。
实施例2：cDNA原位合成与扩增
1.cDNA合成
按照冰冻切片的标准方法制作小鼠组织切片，将冰冻切片贴在实施例1中准备好的芯片上，30min冰冻甲醇固定后，使用0.5％Triton X-100对组织进行透化。使用5X SSC室温清洗芯片两次，配置如表3所示的逆转录酶反应体系200μL，将反应液加到芯片上，充分覆盖，42℃反应90min-180min。逆转录酶将以mRNA为模板，以含有polyT的引物(序列如SEQ ID NO：6所示，其含有共有序列A(CA)和polyT序列)进行cDNA合成，并在cDNA链的3’末端添加CCC悬突。在TSO序列(SEQ ID NO:7，其含有共有序列B(CB)，UMI序列(NNNNNN)以及末端的 GGG序列)与cDNA链杂交退火后(通过TSO序列末端的GGG与cDNA链的CCC悬突的互补配对)，逆转录酶将以共有序列B以及UMI序列为模板，继续延伸cDNA链，使cDNA的3’端带上共有序列B的互补序列和UMI序列的互补序列。芯片上加入甲酰胺溶液，并在55℃反应5min。
表3 cDNA合成体系
Figure PCTCN2022135363-appb-000004
合成的cDNA链包含如下的序列结构：逆转录引物的序列(SEQ ID NO：6)-cDNA序列-c(TSO)的序列(SEQ ID NO:7的互补序列)。
2.测序芯片的芯片序列与cDNA链的连接
将含有5’端磷酸化修饰的两种桥接寡核苷酸(第一桥接寡核苷酸和第二桥接寡核苷酸，SEQ ID Nos:9-10)用2X SSC稀释至100μM，30℃退火后待用；
配置如表4所示的反应液1ml，向芯片中泵入合适的体积，保证芯片中充满所述连接反应液，室温反应30min。
反应结束后，5X SSC清洗芯片。按照说明书配制Bst聚合反应液(购自NEB，M0275S)200μL，泵入芯片，65℃反应60min，得到含有位置信息(即标签序列Y(Y)或其互补序列c(Y))的双链核酸分子，其中的一条链包含如下的序列结构：cDNA序列-TSO序列的互补序列–第一桥接寡核苷酸序列-芯片序列部分序列的互补序列。
表4 连接体系
Figure PCTCN2022135363-appb-000005
3.cDNA释放
使用75μL 80mM KOH室温孵育芯片5min，收集液体后加入10μL 1M pH8.0 Tris-HCl中和cDNA回收液。
4.cDNA扩增
配制如表5所示的反应体系200μL，用于转录组测序建库，分成2管PCR：
表5 cDNA扩增体系
Figure PCTCN2022135363-appb-000006
将上述反应体系至于PCR仪，设置如下反应程序，95℃ 3min，11循环(98℃ 20s，58℃20s，72℃ 3min)，72℃ 5min，4℃∞。反应结束后，用XP beads(购自AMPure)进行磁珠纯化回收。使用Qubit试剂盒对dsDNA浓度进行定量，并且，使用2100仪器(购自Agilent)检测cDNA扩增产物的长度分布。检测结果如图8示。
实施例3：cDNA建库与测序
1.Tn5打断
根据cDNA浓度，取20ng cDNA(实施例2步骤4中获得的)，加入0.5μM Tn5打断酶及相应buffer(购自BGI，货号10000028493；Tn5打断酶包被方法按照Stereomics文库制备试剂盒-S1操作)，混匀配成20μL的反应体系，在55℃反应10min后，加入5μL 0.1％SDS混匀室温5min结束Tn5打断步骤。
2.PCR扩增
配置如下反应体系100μL：
表6 建库扩增反应体系
Figure PCTCN2022135363-appb-000007
混匀后置于PCR仪，设置如下程序95℃ 3min，11循环(98℃ 20s，58℃ 20s，72℃ 3min)，72℃ 5min，4℃∞。反应结束后，用XP beads进行磁珠纯化回收。使用Qubit定量dsDNA浓度。
3.测序
取上述打断后的扩增产物80fmol，进行DNB制备。配置如下40μL反应体系：
表7 测序用DNB制备体系
Figure PCTCN2022135363-appb-000008
将上述反应体积放置于PCR仪反应，反应条件如下：95℃ 3min，40℃ 3min；反应结束后，放于冰上，加入40μL DNBSEQ测序试剂盒中DNB制备所需的混合酶I，2μL混合酶II，及1μL ATP，0.1μL T4Ligase，混匀后，将上述反应体系至于PCR仪30℃，反应20min，形成DNB。
按照测序仪MGISEQ 2000配套的PE50试剂盒所述的方法，将DNB装载至MGISEQ2000的测序芯片上，并按照相关说明书进行测序，选择PE自定义测序模型，其中一链测序分成两段测序，先测25bp后进行36个循环暗反应，再测6bp的UMI序列，二链测序设置50bp。
4、结果
(1)登录网站http://stereomap.cngb.org/Stereo-Draftsman/report/index，按照网站操作指南进行数据分析。将步骤3测序得到的read1序列(来自于一链测序)前25bp与实施例1中的芯片制备过程中的25bp位置信息进行比对，把能够比对到芯片上的位置信息的reads保留下来，并将它们对应到相应的芯片位置上。找出对应到芯片位置上的reads所对应的read2(来自于二链测序)，将reads2与进行鼠脑基因组的比对，根据UMI信息去掉重复的reads，获得鼠脑中每个基因表达的数目。
(2)利用每个基因表达的数目进一步作图，获得如图9所示的鼠脑切片空间表达图谱。
图9的结果显示，基于本申请的方法能够高通量地测定组织样本中基因的空间表达情况。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (63)

  1. 一种生成标记的核酸分子群的方法，其包括下述步骤：
    (1)提供：生物样本和核酸阵列；其中，所述核酸阵列包括固相支持物，所述固相支持物偶联有多种寡核苷酸探针；每种寡核苷酸探针包含至少一个拷贝；并且，所述寡核苷酸探针从5’到3’的方向上包含或者由：共有序列X1，标签序列Y和共有序列X2组成，其中，
    不同种寡核苷酸探针具有不同的标签序列Y，所述标签序列Y具有与该种寡核苷酸探针在固相支持物的位置相对应的独一无二的核苷酸序列；
    (2)将所述生物样本与所述核酸阵列接触，以使得所述生物样本中的RNA(例如，mRNA)的位置被对应至核酸阵列上所述寡核苷酸探针的位置；对所述生物样本中的RNA(例如，mRNA)进行预处理以生成第一核酸分子群，所述预处理包括以下步骤：
    (i)(a)用引物A对所述生物样本的RNA(例如，mRNA)进行逆转录，生成cDNA链，所述cDNA链包含以所述引物A为逆转录引物形成的与所述RNA(例如，mRNA)互补的cDNA序列，以及3’末端悬突；其中，所述引物A含有捕获序列A，所述捕获序列A能与待捕获的RNA(例如，mRNA)退火并起始延伸反应；和，(b)将引物B与(a)中生成的所述cDNA链进行退火，并进行延伸反应，生成第一延伸产物，所述第一延伸产物即为待标记的第一核酸分子，从而生成第一核酸分子群；其中，所述引物B包含共有序列B，3’末端悬突互补序列，以及任选的标签序列B；所述3’末端悬突互补序列位于所述引物B的3’末端；所述共有序列B位于所述3’末端悬突互补序列的上游(例如位于所述引物B的5’端)；或，
    (ii)(a)用引物A’对所述生物样本的RNA(例如，mRNA)进行逆转录，生成cDNA链；所述cDNA链包含以所述引物A’为逆转录引物形成的与所述RNA(例如，mRNA)互补的cDNA序列，以及3’末端悬突；其中，所述引物A’含有共有序列A和捕获序列A，所述捕获序列A能与待捕获的RNA(例如，mRNA)退火并起始延伸反应；所述共有序列A位于所述捕获序列A的上游(例如位于所述引物A’的5’端)；(b)将引物B’与(a)中生成的所述cDNA链进行退火，并进行延伸反应，生成第一延伸产物；其中，所述引物B’包含共有序列B，3’末端悬突互补序列，以及任选的标签序列B；所述3’末端悬突互补序列位于所述引物B’的3’末端；所述共有序列B位于所述3’末端悬突互补序列的上游(例如位于所述引物B’的5’端)；和，(c)提供延伸引物，以第一延伸产物为模板进行延伸反应，生成第二延伸产物，所述第二延伸产物即为待标记的第一核酸分子，从而生成第一核酸分子群；
    (3)将前一步骤获得的第一核酸分子群通过包含选自下列的步骤生成第二核酸分子群：
    (i)向步骤(2)的产物实施退火条件，使得所述寡核苷酸探针与所述寡核苷酸探针对应位置的待标记的第一核酸分子退火(例如原位退火)，并进行延伸反应，生成延伸产物，所述延伸产物即为具有位置标记的第二核酸分子，从而生成第二核酸分子群；其中，所述寡核苷酸探针的共有序列X2或其部分序列(a)能与步骤(2)(i)获得的第一延伸产物的所述共有序列B的互补序列或其部分序列退火，或者，(b)能与步骤(2)(ii)获得的第二延伸产物的所述共有序列A的互补序列或其部分序列退火；或，
    (ii)在允许退火的条件下，将桥接寡核苷酸对与所述寡核苷酸探针以及前一步骤获得的第一核酸分子群接触，使得所述桥接寡核苷酸对与所述寡核苷酸探针以及所述寡核苷酸探针对应位置的待标记的第一核酸分子退火(例如原位退火)，
    其中，所述桥接寡核苷酸对由第一桥接寡核苷酸和第二桥接寡核苷酸组成，所述第一桥接寡核苷酸和所述第二桥接寡核苷酸各自独立地包括：第一区域和第二区域，以及任选的位于第一区域和第二区域之间的第三区域，所述第一区域位于所述第二区域的上游(例如5’端)；其中，
    所述第一桥接寡核苷酸的第一区域能与所述第二桥接寡核苷酸的第一区域退火；所述第一桥接寡核苷酸的第二区域能与所述寡核苷酸探针的共有序列X2或其部分序列退火；
    所述第二桥接寡核苷酸的第二区域(a)能与步骤(2)(i)获得的第一延伸产物的所述共有序列B的互补序列或其部分序列退火，或者，(b)能与步骤(2)(ii)获得的第二延伸产物的所述共有序列A的互补序列或其部分序列退火；
    其中，将所述桥接寡核苷酸对与所述第一核酸分子群、所述寡核苷酸探针接触时，所述桥接寡核苷酸对的第一桥接寡核苷酸和第二桥接寡核苷酸各自以单链的形式存在，或者，所述桥接寡核苷酸对的第一桥接寡核苷酸和第二桥接寡核苷酸以彼此退火形成部分双链的形式存在；
    进行连接反应：将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接，和/或，将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接；并进行延伸反应；其中，所述连接反应与延伸反应以任意顺序进行；所获得的反应产物即为具有位置标记的第二核酸分子，从而生成所述第二核酸分子群。
  2. 权利要求1的方法，其中，步骤(3)(ii)中：
    (1)当所述第一桥接寡核苷酸的第一区域和第二区域相邻时，所述将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接的步骤包括：使用核酸连接酶将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接；或者，
    当所述第一桥接寡核苷酸包括第一区域、第二区域以及位于两者之间的第三区域时，所述将杂交于同一第一桥接寡核苷酸的第一区域和第二区域的核酸分子连接的步骤包括：使用核酸聚合酶(例如，无5’至3’端外切酶活性或链置换活性)以所述第三区域为模板进行聚合反应，使用核酸连接酶将杂交于同一第一桥接寡核苷酸的第一区域、第三区域和第二区域的核酸分子连接；
    和/或
    (2)当所述第二桥接寡核苷酸的第一区域和第二区域相邻时，所述将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接的步骤包括：使用核酸连接酶将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接；或者，
    当所述第二桥接寡核苷酸包括第一区域、第二区域以及位于两者之间的第三区域时，所述将杂交于同一第二桥接寡核苷酸的第一区域和第二区域的核酸分子连接的步骤包括：使用核酸聚合酶(例如，无5’至3’端外切酶活性或链置换活性)以所述第三区域为模板进行聚合反应，使用核酸连接酶将杂交于同一第二桥接寡核苷酸的第一区域、第三区域和第二区域的核酸分子连接。
  3. 权利要求1或2的方法，其包括步骤(1)、步骤(2)(i)和步骤(3)；其中，步骤(2)(i)(b)中，所述引物B含有共有序列B，3’末端悬突互补序列，以及标签序列B；
    优选地，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第二核酸分子具有不同的所述标签序列B作为UMI。
  4. 权利要求3的方法，其包括步骤(1)、步骤(2)(i)和步骤(3)(i)；其中，所述共有序列X2或其部分序列能与所述共有序列B的互补序列或其部分序列退火；步骤(3)(i)中获得的延伸产物即为标记的核酸分子，其包含：含有所述待标记的第一核酸分子序列的第一链，和/或，含有所述寡核苷酸探针序列的第二链。
  5. 权利要求4的方法，其中，所述共有序列X2或其部分序列能与所述共有序列B的互补序列或其部分序列退火，并且步骤(2)(i)中的第一延伸产物的所述共有序列B的互补序列具有3’自由端；其中，步骤(3)(i)中获得的延伸产物即为标记的核酸分子，其包含所述第一链；
    优选地，步骤(3)(i)中，所述寡核苷酸探针不能起始延伸反应(例如3’端是封闭的)。
  6. 权利要求5的方法，其中，步骤(2)(i)(a)中，所述引物A的捕获序列A为随机寡核苷酸序列。
  7. 权利要求5的方法，其中，步骤(2)(i)(a)中，所述引物A的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列；
    优选地，所述引物A还含有共有序列A，以及任选的标签序列A，例如为随机寡核苷酸序列。
  8. 权利要求4的方法，其中，所述共有序列X2或其部分序列能与所述共有序列B的互补序列或其部分序列退火，并且所述寡核苷酸探针的所述共有序列X2具有3’自由端；其中，步骤(3)(i)中获得的延伸产物即为标记的核酸分子，其包含所述第二链；
    优选地，步骤(2)(i)获得的第一延伸产物不能起始延伸反应(例如3’端是封闭的)。
  9. 权利要求8的方法，其中，步骤(2)(i)(a)中，所述引物A的捕获序列A为随机寡核苷酸序列。
  10. 权利要求8的方法，其中，步骤(2)(i)(a)中，所述引物A的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列；
    优选地，所述引物A还含有共有序列A，以及任选的标签序列A，例如为随机寡核苷酸序列。
  11. 权利要求3的方法，其包括步骤(1)、步骤(2)(i)和步骤(3)(ii)；其中，所述第二桥接寡核苷酸的第二区域能与步骤(2)(i)获得的第一延伸产物的所述共有序列B的互补序列或其部分序列退火；步骤(3)(ii)中获得的反应产物即为标记的核酸分子，其包含：含有所述待标记的第一核酸分子序列的第一链，和/或，含有所述寡核苷酸探针序列的第二链。
  12. 权利要求11的方法，其中，所述第二桥接寡核苷酸的第二区域能与步骤(2)(i)获得的第一延伸产物的所述共有序列B的互补序列或其部分序列退火，并且所述第一桥接寡核苷酸的第二区域具有3’自由端；其中，步骤(3)(ii)中获得的反应产物即为标记的核酸分子，其包含所述第一链；
    优选地，所述第一桥接寡核苷酸具备以下特征的一项或多项：i)所述第一桥接寡核苷酸的第二区域位于所述第一桥接寡核苷酸的3’末端；ii)所述第一桥接寡核苷酸的第一区域位于所述第一桥接寡核苷酸的5’末端；iii)所述第一桥接寡核苷酸的5’末端含有磷酸化修饰；iv)所述第一桥接寡核苷酸的3’末端含有自由-OH；
    优选地，所述第二桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)，和/或，所述寡核苷酸探针不能起始延伸反应(例如3’端是封闭的)。
  13. 权利要求12的方法，其中，步骤(2)(i)(a)中，所述引物A的捕获序列A为随机寡核苷酸序列。
  14. 权利要求12的方法，其中，步骤(2)(i)(a)中，所述引物A的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列；
    优选地，所述引物A还含有共有序列A，以及任选的标签序列A，例如为随机寡核苷酸序列。
  15. 权利要求11的方法，其中，所述第二桥接寡核苷酸的第二区域能与步骤(2)(i)获得的第一延伸产物的所述共有序列B互补序列或其部分序列退火，并且所述第二桥接寡核苷酸的第二区域具有3’自由端；其中，步骤(3)(ii)中获得的反应产物即为标记的核酸分子，其包含所述第二链；
    优选地，所述第二桥接寡核苷酸具备以下特征的一项或多项：i)所述第二桥接寡核苷酸的第二区域位于所述第二桥接寡核苷酸的3’末端；ii)所述第二桥接寡核苷酸的第一区域位于所述第二桥接寡核苷酸的5’末端；iii)所述第二桥接寡核苷酸的5’末端含有磷酸化修饰；iv)所述第二桥接寡核苷酸的3’末端含有自由-OH；
    优选地，所述第一桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)，和/或，步骤(2)(i)获得的第一延伸产物不能起始延伸反应(例如3’端是封闭的)。
  16. 权利要求15的方法，其中，步骤(2)(i)(a)中，所述引物A的捕获序列A为随机寡核苷酸序列。
  17. 权利要求15的方法，其中，步骤(2)(i)(a)中，所述引物A的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列；
    优选地，所述引物A还含有共有序列A，以及任选的标签序列A，例如为随机寡核苷酸序列。
  18. 权利要求1或2的方法，其包括步骤(1)、步骤(2)(ii)和步骤(3)；其中，步骤(2)(ii)(b)中，所述第一延伸产物从5’端至3’端包含：所述共有序列A，以所述引物A’为逆转录引物形成的与所述RNA互补的cDNA序列，所述3’末端悬突序列，任选的所述标签序列B的互补序列，所述共有序列B的互补序列；
    优选地，步骤(2)(ii)(c)中，所述延伸引物为所述引物B’或引物B”或随机引物，其中，所述引物B”能与所述共有序列B的互补序列或其部分序列退火，并且能起始延伸反应。
  19. 权利要求18的方法，其包括步骤(1)、步骤(2)(ii)和步骤(3)(i)；其中，所述共有序列X2或其部分序列能与所述共有序列A的互补序列或其部分序列退火；步骤(3)(i)中获得的延伸产物即为标记的核酸分子，其包含：含有所述待标记的第一核酸分子序列的第一链，和/或，含有所述寡核苷酸探针序列的第二链。
  20. 权利要求19的方法，其中，所述共有序列X2或其部分序列能与所述共有序列A的互补序列或其部分序列退火；步骤(3)(i)中获得的延伸产物即为标记的核酸分子，其包含含有所述待标记的第一核酸分子序列的第一链；
    优选地，步骤(3)(i)中，所述寡核苷酸探针不能起始延伸反应(例如3’端是封闭的)。
  21. 权利要求20的方法，其中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为随机寡核苷酸序列；
    优选地，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第一链具有不同的捕获序列A的互补序列作为UMI。
  22. 权利要求20的方法，其中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列；其中，所述引物A’还含有标签序列A，例如为随机寡核苷酸序列；
    优选地，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第一链具有不同的标签序列A的互补序列作为UMI。
  23. 权利要求19的方法，其中，所述共有序列X2或其部分序列能与所述共有序列A的互补序列或其部分序列退火；步骤(3)(i)中获得的延伸产物即为标记的核酸分子，其包含含有所述寡核苷酸探针序列的第二链；
    优选地，步骤(2)(ii)获得的第二延伸产物不能起始延伸反应(例如3’端是封闭的)。
  24. 权利要求23的方法，其中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为随机寡核苷酸序列；
    优选地，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第二链具有不同的捕获序列A作为UMI。
  25. 权利要求23的方法，其中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列；其中，所述引物A’还含有标签序列A，例如为随机寡核苷酸序列；
    优选地，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第二链具有不同的标签序列A作为UMI。
  26. 权利要求18的方法，其包括步骤(1)、步骤(2)(ii)和步骤(3)(ii)；其中，所述第二桥接寡核苷酸的第二区域能与步骤(2)(ii)获得的第二延伸产物的共有序列A的互补序列或其部分序列退火；步骤(3)(ii)中获得的反应产物即为标记的核酸分子，其包含：含有所述待标记的第一核酸分子序列的第一链，和/或，含有所述寡核苷酸探针序列的第二链。
  27. 权利要求20的方法，其中，所述第二桥接寡核苷酸的第二区域能与步骤(2)(ii)获得的第二延伸产物的所述共有序列A的互补序列或其部分序列退火，并且所述第一桥接寡核苷酸的第二区域具有3’自由端；其中，步骤(3)(ii)中获得的反应产物即为标记的核酸分子，其包含所述第一链；
    优选地，所述第一桥接寡核苷酸具备以下特征的一项或多项：i)所述第一桥接寡核苷酸的第二区域位于所述第一桥接寡核苷酸的3’末端；ii)所述第一桥接寡核苷酸的第一区域位于所述第一桥接寡核苷酸的5’末端；iii)所述第一桥接寡核苷酸的5’末端含有磷酸化修饰；iv)所述第一桥接寡核苷酸的3’末端含有自由-OH；
    优选地，所述第二桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)，和/或，所述寡核苷酸探针不能起始延伸反应(例如3’端是封闭的)。
  28. 权利要求27的方法，其中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为随机寡核苷酸序列；
    优选地，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第一链具有不同的捕获序列A的互补序列作为UMI。
  29. 权利要求27的方法，其中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列；其中，，所述引物A’还含有标签序列A，例如为随机寡核苷酸序列；
    优选地，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第一链具有不同的标签序列A的互补序列作为UMI。
  30. 权利要求26的方法，其中，所述第二桥接寡核苷酸的第二区域能与步骤(2)(ii)获得的第二延伸产物的所述共有序列A的互补序列或其部分序列退火，并且所述第二桥接寡核苷酸的第二区域具有3’自由端；其中，步骤(3)(ii)中获得的反应产物即为标记的核酸分子，其包含所述第二链；
    优选地，所述第二桥接寡核苷酸具备以下特征的一项或多项：i)所述第二桥接寡核苷酸的第二区域位于所述第二桥接寡核苷酸的3’末端；ii)所述第二桥接寡核苷酸的第一区域位于所述第二桥接寡核苷酸的5’末端；iii)所述第二桥接寡核苷酸的5’末端含有磷酸化修饰；iv)所述第二桥接寡核苷酸的3’末端含有自由-OH；
    优选地，所述第一桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)，和/或，步骤(2)(ii)获得的第二延伸产物不能起始延伸反应(例如3’端是封闭的)。
  31. 权利要求30的方法，其中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为随机寡核苷酸序列；
    优选地，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第二链具有不同的捕获序列A作为UMI。
  32. 权利要求30的方法，其中，步骤(2)(ii)(a)中，所述引物A’的捕获序列A为poly(T)序列或针对特定靶核酸的特异性序列；其中，所述引物A’还含有标签序列A，例如为随机寡核苷酸序列；
    优选地，步骤(3)中，源自同一种寡核苷酸探针的每个拷贝的所述第二链具有不同的标签序列A作为UMI。
  33. 权利要求1-17任一项的方法，其中，在步骤(2)(i)(b)中，所述cDNA链通过其3’末端悬突与所述引物B退火，并且，在核酸聚合酶(例如，DNA聚合酶或逆转录酶)的作用下，所述cDNA链以所述引物B为模板被延伸，生成所述第一延伸产物。
  34. 权利要求1-2、18-32任一项的方法，其中，在步骤(2)(ii)(b)中，所述cDNA链通过其3’末端悬突与所述引物B’退火，并且，在核酸聚合酶(例如，DNA聚合酶或逆转录酶)的作用下，所述cDNA链以所述引物B’为模板被延伸，生成所述第一延伸产物。
  35. 权利要求1-34任一项的方法，其中，所述3’末端悬突具有至少1个，至少2个，至少3个，至少4个，至少5个，至少6个，至少7个，至少8个，至少9个，至少10个或更多个核苷 酸的长度。
  36. 权利要求1-35任一项的方法，其中，步骤(2)中，在进行所述预处理之前，对所述生物样本进行透化处理。
  37. 权利要求1-36任一项的方法，其中，所述生物样本是组织样品；
    优选地，所述组织样品是组织切片。
  38. 权利要求1-37任一项的方法，其中，步骤(2)中所述进行逆转录包括使用逆转录酶；
    优选地，所述逆转录酶具有末端转移活性；
    优选地，所述逆转录酶能够以RNA(例如，mRNA)为模板，合成cDNA链，且在所述cDNA链的3’端添加悬突。
  39. 权利要求1-38任一项所述的方法，其中，步骤(2)和(3)具有选自以下的一项或多项特征：
    (1)所述引物A，引物A’，引物B，引物B’，第一桥接寡核苷酸，第二桥接寡核苷酸各自独立地包含或者由天然存在的核苷酸(例如脱氧核糖核苷酸或核糖核苷酸)，经修饰的核苷酸，非天然的核苷酸，或其任何组合组成；优选地，所述引物A，引物A’能够起始延伸反应；
    (2)所述引物B包含修饰的核苷酸(例如锁核酸)；优选地，所述引物B的3’末端包含一个或多个修饰的核苷酸(例如锁核酸)；
    (3)所述引物B’包含修饰的核苷酸(例如锁核酸)；优选地，所述引物B’的3’末端包含一个或多个修饰的核苷酸(例如锁核酸)；
    (4)所述标签序列A，标签序列B各自独立地具有5-200(例如5-30nt，6-15nt)的长度；
    (5)所述共有序列A，共有序列B各自独立地具有10-200nt(例如10-100nt，20-100nt，25-100nt，5-10nt，10-15nt，15-20nt，20-50nt，20-30nt，30-40nt，40-50nt，50-100nt)的长度；
    (6)所述引物A，引物A’，引物B，引物B’各自独立地具有4-200nt(例如5-200nt，15-230nt，26-115nt，10-130nt，10-20nt，20-50nt，20-30nt，30-40nt，40-50nt，50-100nt，100-150nt，150-200nt)的长度；
    (7)所述第一桥接寡核苷酸的第一区域，第二区域各自独立地具有3-100nt(例如20-100nt，3-10nt，10-15nt，15-20nt，20-70nt，20-30nt，30-40nt，40-50nt，50-100nt)的长度；
    (8)所述第二桥接寡核苷酸的第一区域，第二区域各自独立地具有3-100nt(例如20-100nt，3-10nt，10-15nt，15-20nt，20-70nt，20-30nt，30-40nt，40-50nt，50-100nt)的长度；
    (9)所述第一桥接寡核苷酸的第三区域，所述第二桥接寡核苷酸的第三区域各自独立地具有0-50nt(例如0nt，0-10nt，10-15nt，15-20nt，20-30nt，30-40nt，40-50nt)的长度；
    (10)所述第一桥接寡核苷酸、第二桥接寡核苷酸各自独立地具有6-200nt(例如20-100nt，20-70nt，6-15nt，15-20nt，20-30nt，30-40nt，40-50nt，50-100nt，100-150nt，150-200nt)的长度；
    (11)所述poly(T)序列包括至少5个，或至少20个(例如6-100个，10-50个)脱氧胸腺嘧啶核苷残基；
    (12)所述随机寡核苷酸序列具有5-200(例如5nt，5-30nt，6-15nt)的长度。
  40. 权利要求1-39任一项所述的方法，其中，所述方法还包括：(4)回收和纯化所述第二核酸分子群。
  41. 权利要求1-40任一项所述的方法，其中，所获得的第二核酸分子群和/或其互补物用于构建转录组文库或用于转录组测序。
  42. 权利要求1-41任一项的方法，其中，步骤(1)中所述寡核苷酸探针具有选自下列的一个或多个特征：
    (1)所述共有序列X1，标签序列Y和共有序列X2各自独立地包含或者由天然存在的核苷酸(例如脱氧核糖核苷酸或核糖核苷酸)，经修饰的核苷酸，非天然的核苷酸(例如肽核酸(PNA)或锁核酸)，或其任何组合组成；
    (2)所述共有序列X1，标签序列Y和共有序列X2各自独立地具有2-200nt(例如10-200nt，25-100nt，10-30nt，10-100nt，5-10nt，10-15nt，15-20nt，20-30nt，30-40nt，40-50nt，50-100nt)的长度。
  43. 权利要求1-43任一项的方法，其中，所述寡核苷酸探针通过连接子与所述固相支持物偶联；
    优选地，所述连接子是能够与活化基团反应的连接基团，且所述固相支持物表面连接有活化基团；
    优选地，所述连接子包括-SH、-DBCO或-NHS；
    Figure PCTCN2022135363-appb-100001
  44. 权利要求1-43任一项的方法，其中，步骤(1)所述核酸阵列具有选自下列的一个或多个特征：
    (1)偶联在同一固相支持物上的所述寡核苷酸探针具有相同的共有序列X1和/或相同的共有序列X2；
    (2)所述寡核苷酸探针的共有序列X1包含切割位点；优选地，所述切割位点可以通过选自切刻酶(nickingenzyme)酶切、USER酶切、光切除、化学切除或CRISPR切除的方式而被切割或断裂。
  45. 权利要求1-44任一项的方法，其中，步骤(1)所述核酸阵列由包含以下的步骤来提供：
    (1)提供多种载体序列，每种载体序列包含至少一个拷贝的载体序列，所述载体序列从5’到3’的方向上包含：共有序列X2的互补序列，标签序列Y的互补序列以及固定序列；其中，每种载体序列的标签序列Y的互补序列互不相同；
    (2)将所述多种载体序列连接于固相支持物(例如芯片)表面；
    (3)提供固定引物，并以所述载体序列为模板，进行引物延伸反应，生成延伸产物，所述延伸产物即为寡核苷酸探针；其中，所述固定引物包含共有序列X1的序列，并且，所述固定引物能与所述固定序列退火并起始延伸反应；优选地，所述延伸产物从5’到3’的方向上包含或者由：共有序列X1，标签序列Y和共有序列X2组成；
    (4)将所述固定引物与所述固相支持物表面连接；其中，步骤(3)与(4)以任意顺序进行；
    (5)任选地，所述载体序列的固定序列还包含切割位点，所述切割可以选自切刻酶(nicking enzyme)酶切、USER酶切、光切除、化学切除或CRISPR切除；对所述载体序列的固定序列所包含的切割位点进行切割，以消化所述载体序列，使得步骤(3)中的延伸产物与形成延伸产物的模板(即载体序列)分离，从而将所述寡核苷酸探针连接于固相支持物(例如芯片)表面；优选地，所述方法还包括通过高温变性使得步骤(3)中的延伸产物与形成延伸产物的模板(即载体序列)分离；
    优选地，每种载体序列是由多个拷贝的载体序列的多联体所形成的DNB；
    优选地，步骤(1)中通过以下步骤提供所述多种载体序列：
    (i)提供多种载体模板序列，所述载体模板序列包含所述载体序列的互补序列；
    (ii)以每种载体模板序列为模板，进行核酸扩增反应，以获得每种载体模板序列的扩增产物，所述扩增产物包含至少一个拷贝的载体序列；优选地，进行滚环复制，以获得由所述载体序列的多联体所形成的DNB。
  46. 权利要求1-45任一项的方法，其中，步骤(1)所述固相支持物具有选自下列的一个或多个特征：
    (1)所述固体支持物选自乳胶珠、葡聚糖珠、聚苯乙烯表面、聚丙烯表面、聚丙烯酰胺凝胶、金表面、玻璃表面、芯片、传感器、电极和硅晶片；优选地，所述固相支持物是芯片；
    (2)所述固体支持物为平面的、球形的或多孔的；
    (3)所述固相支持物能够用作测序平台，例如测序芯片；优选地，所述固相支持物是用于Illumina、MGI或Thermo Fisher测序平台的测序芯片；和
    (4)所述固相支持物能够自发地或在暴露于一种或多种刺激(例如，温度变化、pH变化、暴露于特定化学物质或相、暴露于光、还原剂等)时释放所述寡核苷酸探针。
  47. 一种构建核酸分子文库的方法，其包括，
    (a)根据权利要求1-46任一项的方法生成标记的核酸分子群；
    (b)将所述标记的核酸分子群中的核酸分子随机打断并添加接头；和
    (c)任选地，对步骤(b)的产物进行扩增和/或富集；
    从而获得核酸分子文库；
    优选地，所述核酸分子文库用于测序，例如转录组测序，例如单细胞转录组测序(例如5’端或3’端转录组测序)。
  48. 权利要求47的方法，其中，在进行步骤(b)之前，所述方法还包括步骤(pre-b)：扩增和/或富集所述标记的核酸分子群；
    优选地，所述扩增反应使用至少引物C和/或引物D来进行，其中，所述引物C能够与所述共有序列X1的互补序列或其部分序列杂交或退火，并起始延伸反应；所述引物D能够与所述标记的核酸分子群中含有所述标签序列Y的核酸分子链杂交或退火，并起始延伸反应。
  49. 权利要求47或48所述的方法，其中，在步骤(b)中，用转座酶将前一步骤获得的核酸分子随机打断并在片段两端分别添加接头；
    优选地，在步骤(c)中，至少使用引物C’和/或引物D’对步骤(b)的产物进行扩增，其中， 片段两端的接头分别为第一接头和第二接头，所述引物C’能够与所述第一接头杂交或退火，并起始延伸反应，所述引物D’能够与所述第二接头杂交或退火，并起始延伸反应。
  50. 一种对样品中的细胞进行转录组测序的方法，其包括：
    (1)根据权利要求47-49任一项的方法构建核酸分子文库；和
    (2)对所述核酸分子文库进行测序。
  51. 试剂盒，其包含：
    (i)用于标记核酸的核酸阵列，其包括固相支持物，所述固相支持物偶联有多个寡核苷酸探针；每种寡核苷酸探针包含至少一个拷贝；并且，所述寡核苷酸探针从5’到3’的方向上包含或者由：共有序列X1，标签序列Y和共有序列X2组成，其中，
    不同种寡核苷酸探针具有不同的标签序列Y，所述标签序列Y具有与该种寡核苷酸探针在固相支持物的位置相对应的独一无二的核苷酸序列；
    (ii)包含引物A和引物B或者包含引物A’和引物B’的引物组，其中：
    所述引物A含有捕获序列A，所述捕获序列A能与待捕获的RNA(例如，mRNA)退火并起始延伸反应；
    所述引物B包含共有序列B，3’末端悬突互补序列，以及任选的标签序列B；其中，所述3’末端悬突互补序列位于所述引物B的3’末端，所述共有序列B位于所述3’末端悬突互补序列的上游(例如位于所述引物B的5’端)；其中，所述3’末端悬突是指以所述引物A的捕获序列A所捕获的RNA为模板逆转录生成的cDNA链的3’末端所包含的一个或多个非模板核苷酸；
    所述引物A’含有共有序列A和捕获序列A；其中，所述捕获序列A位于所述引物A’的3’端，所述共有序列A位于所述捕获序列A的上游(例如位于所述引物A’的5’端)；
    所述引物B’包含共有序列B，3’末端悬突互补序列，以及任选的标签序列B；其中，所述3’末端悬突互补序列位于所述引物B’的3’末端，所述共有序列B位于所述3’末端悬突互补序列的上游(例如位于所述引物B’的5’端)；其中，所述3’末端悬突是指以所述引物A’的捕获序列A所捕获的RNA为模板逆转录生成的cDNA链的3’末端所包含的一个或多个非模板核苷酸。
  52. 权利要求51的试剂盒，其包含：如(i)中所述的用于标记核酸的核酸阵列，如(ii)中所述的引物A和引物B的引物组，以及，(iii)第一桥接寡核苷酸和第二桥接寡核苷酸；其中，所述第一桥接寡核苷酸和所述第二桥接寡核苷酸各自独立地包括：第一区域和第二区域，以及任选的位于第一区域和第二区域之间的第三区域，所述第一区域位于所述第二区域的上游(例如5’端)；其中，
    所述第一桥接寡核苷酸的第一区域能与所述第二桥接寡核苷酸的第一区域退火；所述第一桥接寡核苷酸的第二区域能与所述寡核苷酸探针的共有序列X2或其部分序列退火；
    所述第二桥接寡核苷酸的第二区域能与所述引物B的共有序列B的互补序列或其部分序列退火；
    其中，所述引物A的捕获序列A是随机寡核苷酸序列；或者，所述引物A的捕获序列A是poly(T)序列或针对特定靶核酸的特异性序列，所述引物A优选地进一步包含共有序列A和任选的标签序列A，例如为随机寡核苷酸序列；
    其中，所述引物B含有所述共有序列B，3’末端悬突互补序列，以及标签序列B；
    优选地，所述引物B包含修饰的核苷酸(例如锁核酸)；优选地，所述引物B的3’末端包含一个或多个修饰的核苷酸(例如锁核酸)。
  53. 权利要求52的试剂盒，其中，所述第二桥接寡核苷酸的第二区域能与所述引物B的共有序列B的互补序列或其部分序列退火；
    优选地，所述第一桥接寡核苷酸具备以下特征的一项或多项：i)所述第一桥接寡核苷酸的第二区域位于所述第一桥接寡核苷酸的3’末端；ii)所述第一桥接寡核苷酸的第一区域位于所述第一桥接寡核苷酸的5’末端；iii)所述第一桥接寡核苷酸的5’末端含有磷酸化修饰；iv)所述第一桥接寡核苷酸的3’末端含有自由-OH；
    优选地，所述第二桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)，和/或，所述寡核苷酸探针不能起始延伸反应(例如3’端是封闭的)。
  54. 权利要求52的试剂盒，其中，所述第二桥接寡核苷酸的第二区域能与所述引物B的共有序列B互补序列或其部分序列退火；
    优选地，所述第二桥接寡核苷酸具备以下特征的一项或多项：i)所述第二桥接寡核苷酸的第二区域位于所述第二桥接寡核苷酸的3’末端；ii)所述第二桥接寡核苷酸的第一区域位于所述第二桥接寡核苷酸的5’末端；iii)所述第二桥接寡核苷酸的5’末端含有磷酸化修饰；iv)所述第二桥接寡核苷酸的3’末端含有自由-OH；
    优选地，所述第一桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)。
  55. 权利要求51的试剂盒，其包含：如(i)中所述的用于标记核酸的核酸阵列，以及，如(ii)中所述的引物A和引物B的引物组；
    其中，所述引物A的捕获序列A是随机寡核苷酸序列；或者，所述引物A的捕获序列A是poly(T)序列或针对特定靶核酸的特异性序列，所述引物A优选地进一步包含共有序列A和任选的标签序列A，例如为随机寡核苷酸序列；
    其中，所述引物B含有所述共有序列B，3’末端悬突互补序列，以及标签序列B；
    优选地，所述引物B包含修饰的核苷酸(例如锁核酸)；优选地，所述引物B的3’末端包含一个或多个修饰的核苷酸(例如锁核酸)。
  56. 权利要求51的试剂盒，其包含：如(i)中所述的用于标记核酸的核酸阵列，如(ii)中所述的引物A’和引物B’的引物组，以及，(iii)第一桥接寡核苷酸和第二桥接寡核苷酸；其中，所述第一桥接寡核苷酸和所述第二桥接寡核苷酸各自独立地包括：第一区域和第二区域，以及任选的位于第一区域和第二区域之间的第三区域，所述第一区域位于所述第二区域的上游(例如5’端)；其中，
    所述第一桥接寡核苷酸的第一区域能与所述第二桥接寡核苷酸的第一区域退火；所述第一桥接寡核苷酸的第二区域能与所述寡核苷酸探针的共有序列X2或其部分序列退火；
    所述第二桥接寡核苷酸的第二区域能与所述引物A’的共有序列A互补序列或其部分序列退火；
    其中，所述引物A’的捕获序列A是随机寡核苷酸序列；或者，所述引物A’的捕获序列A是poly(T)序列或针对特定靶核酸的特异性序列，所述引物A’进一步包含标签序列A，例如为随机寡 核苷酸序列；
    优选地，所述引物B’包含修饰的核苷酸(例如锁核酸)；优选地，所述引物B’的3’末端包含一个或多个修饰的核苷酸(例如锁核酸)；
    优选地，所述试剂盒进一步包含引物B”或随机引物，所述引物B”能与所述共有序列B的互补序列或其部分序列退火，并且能起始延伸反应。
  57. 权利要求56的试剂盒，其中，所述第二桥接寡核苷酸的第二区域能与所述引物A’的共有序列A的互补序列或其部分序列退火；
    优选地，所述第一桥接寡核苷酸具备以下特征的一项或多项：i)所述第一桥接寡核苷酸的第二区域位于所述第一桥接寡核苷酸的3’末端；ii)所述第一桥接寡核苷酸的第一区域位于所述第一桥接寡核苷酸的5’末端；iii)所述第一桥接寡核苷酸的5’末端含有磷酸化修饰；iv)所述第一桥接寡核苷酸的3’末端含有自由-OH；
    优选地，所述第二桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)，和/或，所述寡核苷酸探针不能起始延伸反应(例如3’端是封闭的)。
  58. 权利要求56的试剂盒，其中，所述第二桥接寡核苷酸的第二区域能与所述引物A’的共有序列A的互补序列或其部分序列退火；
    优选地，所述第二桥接寡核苷酸具备以下特征的一项或多项：i)所述第二桥接寡核苷酸的第二区域位于所述第二桥接寡核苷酸的3’末端；ii)所述第二桥接寡核苷酸的第一区域位于所述第二桥接寡核苷酸的5’末端；iii)所述第二桥接寡核苷酸的5’末端含有磷酸化修饰；iii)所述第二桥接寡核苷酸的3’末端含有自由-OH；
    优选地，所述第一桥接寡核苷酸不能起始延伸反应(例如3’端是封闭的)。
  59. 权利要求51的试剂盒，其包含：如(i)中所述的用于标记核酸的核酸阵列，以及，如(ii)中所述的引物A’和引物B’的引物组；
    其中，所述引物A’的捕获序列A是随机寡核苷酸序列；或者，所述引物A’的捕获序列A是poly(T)序列或针对特定靶核酸的特异性序列，所述引物A’进一步包含标签序列A，例如为随机寡核苷酸序列；
    其中，所述引物B’含有所述共有序列B，3’末端悬突互补序列，以及标签序列B；
    优选地，所述引物B’包含修饰的核苷酸(例如锁核酸)；优选地，所述引物B’的3’末端包含一个或多个修饰的核苷酸(例如锁核酸)；
    优选地，所述试剂盒进一步包含引物B”或随机引物，所述引物B”能与所述共有序列B的互补序列或其部分序列退火，并且能起始延伸反应。
  60. 权利要求51-59任一项的试剂盒，其具有选自以下的一项或多项特征：
    (1)所述寡核苷酸探针，引物A，引物A’，引物B，引物B’，引物B”，随机引物，第一桥接寡核苷酸，第二桥接寡核苷酸各自独立地包含或者由天然存在的核苷酸(例如脱氧核糖核苷酸或核糖核苷酸)，经修饰的核苷酸，非天然的核苷酸，或其任何组合组成；
    (2)所述寡核苷酸探针各自独立地具有15-300nt(例如15-200nt，15-20nt，20-30nt，30-40nt，40-50nt，50-100nt，100-150nt，150-200nt)的长度；
    (3)所述引物A，引物A’，引物B，引物B’，引物B”，随机引物各自独立地具有4-200nt(例如5-200nt，15-230nt，26-115nt，10-130nt，10-20nt，20-50nt，20-30nt，30-40nt，40-50nt，50-100nt，100-150nt，150-200nt)的长度；
    (4)所述第一桥接寡核苷酸和所述第二桥接寡核苷酸各自独立地具有6-200nt(例如20-100nt，20-70nt，6-15nt，15-20nt，20-30nt，30-40nt，40-50nt，50-100nt，100-150nt，150-200nt)的长度；
    (5)偶联在同一固相支持物上的所述寡核苷酸探针具有相同的共有序列X1和/或相同的共有序列X2；
    (6)所述寡核苷酸探针的共有序列X1包含切割位点；优选地，所述切割位点可以通过选自切刻酶(nicking enzyme)酶切、USER酶切、光切除、化学切除或CRISPR切除的方式而被切割或断裂。
  61. 权利要求51-60任一项的试剂盒，其进一步包含逆转录酶，核酸连接酶，核酸聚合酶和/或转座酶；
    优选地，所述逆转录酶具有末端转移活性；优选地，所述逆转录酶能够以RNA(例如，mRNA)为模板，合成cDNA链，且在所述cDNA链的3’端添加所述3’末端悬突。
  62. 权利要求51-61任一项的试剂盒，其进一步包含：用于进行核酸杂交的试剂、用于进行核酸延伸的试剂、用于进行核酸扩增的试剂、用于回收或纯化核酸的试剂、用于构建转录组测序文库的试剂、用于测序(例如二代测序或三代测序)的试剂、或其任何组合。
  63. 权利要求1-46任一项的方法或权利要求51-62任一项的试剂盒用于构建核酸分子文库或用于进行转录组测序的用途。
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