Methods and kits for producing a population of labeled nucleic acid molecules

Abstract

The present application relates to transcriptome sequencing and biomolecular spatial information detection. In particular, the present application relates to a method for positioning and labeling nucleic acid molecules, a method for constructing a nucleic acid molecule library for transcriptome sequencing, and a kit for carrying out the method.

Description

[Technical field]

[0001] The present application relates to the technical field of transcriptome sequencing and biomolecule spatial information detection. In particular, the present application relates to a method for positionally labeling nucleic acid molecules and a method for constructing a nucleic acid molecule library for transcriptome sequencing. In addition, the present application also relates to the nucleic acid molecule library constructed by the method and a kit for carrying out the method. [Background technology]

[0002] The spatial location of cells in tissues greatly affects their function. To investigate this spatial heterogeneity, it is necessary to quantify and analyze the genome or transcriptome of cells with knowledge of their spatial coordinates. However, collecting small tissue regions or even single cells for genome or transcriptome analysis is extremely laborious, expensive, and imprecise. Therefore, there is a need to develop methods that can achieve high-throughput detection of spatial information of biomolecules (e.g., nucleic acid localization, distribution, and / or expression) at the single-cell level, or even at the subcellular level. [Brief description of the drawings]

[0003] [Figure 1] Figure 1 shows an exemplary structure of a chip used in the present application for capturing and labeling nucleic acid molecules, including a chip and oligonucleotide probes (also called chip sequences) bound to the chip. Each type of oligonucleotide probe contains a tag sequence Y corresponding to its position on the chip, and the region where each type of oligonucleotide probe is bound to the chip can be called a microdot. Each type of oligonucleotide probe can contain a single copy or multiple copies. [Diagram 2] 2 shows an exemplary scheme for preparing a cDNA strand using a sample's RNA (eg, mRNA) as a template, as well as an exemplary structure of a cDNA strand. CA: consensus sequence A, CB: consensus sequence B. [Diagram 3] 3 shows an exemplary scheme for generating a novel nucleic acid molecule containing chip sequence information (i.e., a nucleic acid molecule labeled with a chip sequence) by labeling the 5'-end of a cDNA strand with a chip sequence (i.e., ligating the 5'-end of a cDNA strand to the 3'-end of a chip sequence), as well as an exemplary structure of a novel nucleic acid molecule containing chip sequence information. CA: consensus sequence A, CB: consensus sequence B, X1: consensus sequence X1, Y: tag sequence Y, X2: consensus sequence X2. [Figure 4] 4 shows an exemplary scheme for preparing a complementary strand of a cDNA strand using a sample RNA (e.g., mRNA) as a template and an exemplary structure of the complementary strand of a cDNA strand: CA: consensus sequence A, CB: consensus sequence B, EP: extension primer. [Diagram 5] 5 shows an exemplary scheme for generating a novel nucleic acid molecule containing information of a chip sequence (i.e., a nucleic acid molecule labeled with a chip sequence) by labeling the 5'-end of the complementary strand of a cDNA strand with a chip sequence (i.e., ligating the 5'-end of the complementary strand of a cDNA strand to the 3'-end of the chip sequence), as well as an exemplary structure of a novel nucleic acid molecule containing chip sequence information. CA: consensus sequence A, CB: consensus sequence B, X1: consensus sequence X1, Y: tag sequence Y, X2: consensus sequence X2. [Figure 6] FIG. 6 shows the length distribution of the cDNA amplification products prepared in Example 2. [Figure 7] FIG. 7 shows a mouse brain gene expression map obtained by the method of the present application. [Figure 8] FIG. 8 shows an expanded mouse brain gene expression map obtained by the methods of the present application. [Figure 9] FIG. 9 shows the gene count and UMI analysis results captured by the method of the present application. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0004] The present application provides novel methods for generating populations of labeled nucleic acid molecules, as well as methods for constructing libraries of nucleic acid molecules based on these methods and for performing high-throughput sequencing.

[0005] Methods for generating a population of labeled nucleic acid molecules

[0006] In one aspect, the present application provides a method for generating a population of labeled nucleic acid molecules, comprising: (1) providing a biological sample and a nucleic acid array, the nucleic acid array comprising a solid support, the solid support having attached thereto a plurality of types of oligonucleotide probes, each type of oligonucleotide probe comprising at least one copy, the oligonucleotide probes comprising or consisting of, in a 5' to 3' direction, a consensus sequence X1, a tag sequence Y, and a consensus sequence X2; each type of oligonucleotide probe has a different tag sequence Y, the tag sequence Y having a nucleotide sequence unique to the location on the solid support of that type of oligonucleotide probe; (2) contacting the biological sample with a nucleic acid array, whereby the locations of RNA (e.g., mRNA) in the biological sample are mapped to the locations of the oligonucleotide probes on the nucleic acid array, and pre-processing the RNA (e.g., mRNA) in the biological sample to generate a first population of nucleic acid molecules, the pre-processing comprising: (i) performing reverse transcription of RNA (e.g., mRNA) of the biological sample using primer A to generate extension products as first nucleic acid molecules to be labeled, thereby generating a population of first nucleic acid molecules, where primer A comprises consensus sequence A and capture sequence A, where capture sequence A is capable of annealing to the RNA (e.g., mRNA) to be captured to initiate an extension reaction, and where consensus sequence A is located upstream of capture sequence A (e.g., located at the 5'-end of primer A); or (ii) (a) performing reverse transcription of RNA (e.g., mRNA) of the biological sample using primer A to generate a cDNA strand, the cDNA strand being generated by reverse transcription primed by primer A and comprising a cDNA sequence that is complementary to the RNA (e.g., mRNA) and a 3'-end overhang, primer A comprising consensus sequence A and capture sequence A, capture sequence A capable of annealing to the RNA (e.g., mRNA) to be captured to initiate an extension reaction, consensus sequence A being located upstream of capture sequence A (e.g., at the 5'-end of primer A); (b) annealing primer IB with the cDNA strand generated in (a) and performing an extension reaction to generate a first extension product as a first nucleic acid molecule to be labeled, thereby generating a first population of nucleic acid molecules, wherein primer B comprises consensus sequence B, a complementary sequence of the 3'-end overhang, and optionally a tag sequence B, wherein the complementary sequence of the 3'-end overhang is located at the 3'-end of primer B and consensus sequence B is located upstream of the complementary sequence of the 3'-end overhang (e.g., located at the 5'-end of primer B); or (iii) (a) performing reverse transcription of RNA (e.g., mRNA) of the biological sample using primer A' to generate a cDNA strand, the cDNA strand being generated by reverse transcription primed by primer A' and comprising a cDNA sequence that is complementary to the RNA (e.g., mRNA) and a 3'-end overhang, primer A' comprising capture sequence A, which is capable of annealing to the RNA (e.g., mRNA) to be captured to initiate an extension reaction; (b) annealing primer B to the cDNA strand generated in (a) and performing an extension reaction. (c) providing an extension primer and performing an extension reaction using the first extension product as a template to generate a second extension product as a labeled first nucleic acid molecule, thereby generating a population of first nucleic acid molecules. The steps include: (3) contacting a bridging oligonucleotide with the product of step (2) under conditions that allow annealing, allowing the bridging oligonucleotide to anneal (e.g., anneal in situ) with the oligonucleotide probe and a labeled first nucleic acid molecule at a position corresponding to the oligonucleotide probe, and ligating the first nucleic acid molecule annealed with the bridging oligonucleotide and the oligonucleotide probe on the array to obtain a ligation product as a second nucleic acid molecule having a positioning tag, thereby generating a second population of nucleic acid molecules. A method comprising: The bridging oligonucleotide comprises a first region and a second region, and optionally a third region located between the first and second regions, the first region being upstream of the second region (e.g., 5' of the second region); the first region is capable of annealing to all or a portion of consensus sequence A of primer A in step (2)(i) or step (2)(ii), or is capable of annealing to all or a portion of consensus sequence B of primer B in step (2)(iii); The second region is capable of annealing to all or part of the consensus sequence X2.

[0007] In certain embodiments, in step (3) of the method, when the first region and the second region of the bridging oligonucleotide are immediately adjacent, ligating the first nucleic acid molecule to the oligonucleotide probe comprises using a nucleic acid ligase to ligate the nucleic acid molecule hybridized with the first region and the nucleic acid molecule hybridized with the second region of the same bridging oligonucleotide to obtain a ligation product as a second nucleic acid molecule having a positioning tag; or When the bridging oligonucleotide comprises a first region, a second region and a third region located therebetween, ligating the first nucleic acid molecule to the oligonucleotide probe comprises using a nucleic acid polymerase to carry out a polymerization reaction using the third region as a template, and using a nucleic acid ligase to ligate the nucleic acid molecule hybridized to the first region of the same bridging oligonucleotide with the nucleic acid molecule hybridized to the third region and the second region to obtain a ligation product as a second nucleic acid molecule having a positioning tag. In certain embodiments, the nucleic acid polymerase does not have 5' to 3' exonuclease activity or strand displacement activity.

[0008] In certain embodiments, each type of oligonucleotide probe comprises one copy.

[0009] In certain embodiments, each type of oligonucleotide probe comprises multiple copies.

[0010] In certain embodiments, the region where various oligonucleotide probes are attached to solid support is called microdot.It is easy to understand that when various oligonucleotide probes contain one copy, each microdot is attached to one oligonucleotide probe, and the oligonucleotide probes of different microdots have different tag sequences Y; when various oligonucleotide probes contain multiple copies, each microdot is attached to multiple oligonucleotide probes, and the oligonucleotide probes of the same microdot have the same tag sequence Y, and the oligonucleotide probes of different microdots have different tag sequences Y.

[0011] In certain embodiments, the solid support comprises a plurality of microdots, each microdot being bound to one type of oligonucleotide probe, and each type of oligonucleotide probe may comprise one or multiple copies.

[0012] In certain embodiments, the solid support comprises a plurality of (e.g., at least 10, at least 10 2 Pieces, at least 10 3 Pieces, at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 In certain embodiments, the solid support comprises at least 10 4 (e.g., at least 10 4 Pieces, at least 10 5 Pieces, at least 10 6 Pieces, at least 10 7 Pieces, at least 10 8 Pieces, at least 10 9 Pieces, at least 10 10 Pieces, at least 10 11 or at least 10 12 pcs) microdots / mm 2 Includes.

[0013] In some embodiments, the spacing between adjacent microdots is less than 100 μm, less than 50 μm, less than 10 μm, less than 5 μm, less than 1 μm, less than 0.5 μm, less than 0.1 μm, less than 0.05 μm, or less than 0.01 μm.

[0014] In certain embodiments, the microdots have a size (e.g., equivalent diameter) of less than 100 μm, less than 50 μm, less than 10 μm, less than 5 μm, less than 1 μm, less than 0.5 μm, less than 0.1 μm, less than 0.05 μm, or less than 0.01 μm.

[0015] An embodiment including steps (1), (2)(i) and (3)

[0016] In some embodiments, the method includes steps (1), (2)(i) and (3), and the ligation product obtained in step (3) is taken as a second nucleic acid molecule having a positioning tag comprising, from 5' to 3', the consensus sequence X1, the tag sequence Y, the consensus sequence X2, optionally a complementary sequence of the third region of the bridging oligonucleotide, and the sequence of the first nucleic acid molecule to be labeled.

[0017] In certain embodiments, in step (2)(i) of the method, capture sequence A is a random oligonucleotide sequence.

[0018] In some embodiments, in step (3), the ligation products derived from each copy of the same type of oligonucleotide probe have a different capture sequence A, and capture sequence A serves as a unique molecular identifier (UMI) for the second nucleic acid molecule.

[0019] In certain embodiments, the extension product in step (2)(i) (the first nucleic acid molecule to be labeled) comprises, from 5' to 3', a cDNA sequence generated by reverse transcription primed by consensus sequence A, primer A, and that is complementary to the RNA.

[0020] In certain embodiments, in step (2)(i) of the method, capture sequence A is a poly(T) sequence or a specific sequence that targets the target nucleic acid.

[0021] In certain embodiments, primer A further comprises a tag sequence A, e.g., a random oligonucleotide sequence, where tag sequence A serves as a unique molecular identifier (UMI) for the second nucleic acid molecule.

[0022] In certain embodiments, capture sequence A is located at the 3'-end of primer A and consensus sequence A is located upstream of tag sequence A (eg, located at the 5'-end of primer A).

[0023] In certain embodiments, in step (3), the ligation products derived from each copy of the same type of oligonucleotide probe have a different tag sequence A as a UMI.

[0024] In some embodiments, the extension product described in step (2)(i) comprises, from 5' to 3', consensus sequence A, tag sequence A, and a cDNA sequence generated by reverse transcription primed by primer A and that is complementary to the RNA.

[0025] An embodiment including steps (1), (2)(ii) and (3)

[0026] In some embodiments, the method includes steps (1), (2)(ii) and (3), and the ligation product obtained in step (3) is taken as a second nucleic acid molecule having a positioning tag comprising, from 5' to 3', the consensus sequence X1, the tag sequence Y, the consensus sequence X2, optionally a complementary sequence of the third region of the bridging oligonucleotide, and the sequence of the first nucleic acid molecule to be labeled.

[0027] In certain embodiments, in step (2)(ii)(a) of the method, capture sequence A is a random oligonucleotide sequence.

[0028] In some embodiments, in step (3), the ligation products derived from each copy of the same type of oligonucleotide probe have a different capture sequence A, and capture sequence A serves as a unique molecular identifier (UMI) for the second nucleic acid molecule.

[0029] In certain embodiments, the first extension product in step (2)(ii) (the first nucleic acid molecule to be labeled) comprises, from 5' to 3', consensus sequence A, a cDNA sequence generated by reverse transcription primed by primer A and complementary to the RNA, a 3'-end overhang sequence, optionally a complement of tag sequence B, and a complement of consensus sequence B.

[0030] In certain embodiments, in step (2)(ii)(a), capture sequence A is a poly(T) sequence or a specific sequence that targets the target nucleic acid.

[0031] In certain embodiments, primer A further comprises a tag sequence A, e.g., a random oligonucleotide sequence, where tag sequence A serves as a unique molecular identifier (UMI) for the second nucleic acid molecule.

[0032] In certain embodiments, capture sequence A is located at the 3'-end of primer A and consensus sequence A is located upstream of tag sequence A (eg, located at the 5'-end of primer A).

[0033] In certain embodiments, in step (3), the ligation products derived from each copy of the same type of oligonucleotide probe have a different tag sequence A as a UMI.

[0034] In certain embodiments, the first extension product in step (2)(ii) (the first nucleic acid molecule to be labeled) comprises, from 5' to 3', consensus sequence A, tag sequence A, a cDNA sequence generated by reverse transcription primed by primer A and complementary to the RNA, a 3'-end overhang sequence, optionally a complement of tag sequence B, and a complement of consensus sequence B.

[0035] In certain embodiments, in the method, primer A comprises a 5' phosphate at the 5'-end.

[0036] In certain embodiments, prior to step (3), the method further comprises treating the product of step (2)(i) or step (2)(ii) to remove RNA (e.g., by heat treatment).

[0037] An exemplary embodiment of the present application, including step (1), step (2)(ii) and step (3), is described in detail as follows:

[0038] 1. An exemplary embodiment for preparing a cDNA strand using RNA (e.g., mRNA) of a sample as a template includes the following steps (as shown in FIG. 2):

[0039] (1) Using a reverse transcriptase (e.g., a reverse transcriptase having terminal deoxynucleotidyl transferase activity) and primer A, RNA molecules (e.g., mRNA molecules) of the permeabilized tissue sample are reverse transcribed to generate cDNA, and an overhang (e.g., an overhang containing three cytosine nucleotides) is added to the 3'-end of the cDNA. Various reverse transcriptases having terminal deoxynucleotidyl transferase activity can be used to carry out the reverse transcription reaction. In certain preferred embodiments, the reverse transcriptase used does not have RNase H activity. Includes.

[0040] Primer A comprises a poly(T) sequence and a consensus sequence A (denoted as CA in the figures). In certain embodiments (e.g., when the method is used to construct a 3' transcriptome library), primer A further comprises a unique molecular identifier (UMI). Typically, the poly(T) sequence is located at the 3'-end of primer A to initiate reverse transcription. In a preferred embodiment, the UMI sequence is located upstream (e.g., 5') of the poly(T) sequence and the consensus sequence A is located upstream (e.g., 5') of the UMI sequence.

[0041] (2) A template switch oligo (TSO, containing consensus sequence B (denoted as CB in the figure), which can be used as primer B) is used to anneal or hybridize with the cDNA strand, and then the nucleic acid fragment hybridized or annealed with the TSO is extended in the presence of a nucleic acid polymerase using consensus sequence B as a template, thereby adding the complementary sequence of consensus sequence B to the 3'-end of the cDNA strand, thereby generating a nucleic acid molecule that retains consensus sequence A and tag sequence A at the 5'-end and the complementary sequence of consensus sequence B at the 3'-end.

[0042] The TSO sequence may comprise a sequence that is complementary to the 3'-end overhang of the cDNA strand. For example, if the cDNA strand comprises an overhang of three cytosine nucleotides at its 3'-end, the template switch oligo may comprise GGG at its 3'-end. Furthermore, the nucleotides of the TSO sequence may also be modified to enhance the binding affinity for complementary pairing between the TSO sequence and the 3'-end overhang of the cDNA strand (e.g., the TSO sequence may be modified to comprise a locked nucleic acid).

[0043] Without being limited by any theory, the extension reaction can be carried out using a variety of suitable nucleic acid polymerases (e.g., DNA polymerases or reverse transcriptases) so long as the sequence of the TSO or a subsequence thereof can be used as a template to extend the hybridized or annealed nucleic acid fragment (the reverse transcription product). In certain exemplary embodiments, the hybridized or annealed nucleic acid fragment (the reverse transcription product) can be extended using the same reverse transcriptase used in the reverse transcription step described above.

[0044] In certain preferred embodiments, step (2) and step (1) are carried out simultaneously.

[0045] In certain embodiments, the method optionally further comprises step (3): adding RNase H to digest the RNA strand of the RNA / cDNA hybrid to form a single-stranded cDNA.

[0046] In certain preferred embodiments, the method does not include step (3).

[0047] An exemplary structure of a cDNA strand prepared by the above exemplary embodiment includes a consensus sequence A, a UMI sequence, a sequence that is complementary to a sequence of an RNA (eg, an mRNA), and a complementary sequence of consensus sequence B.

[0048] 2. An exemplary embodiment in which the 5'-end of a cDNA strand is labeled with an oligonucleotide probe (also called a chip sequence) (i.e., the 5'-end of the cDNA strand is ligated to the 3'-end of the chip sequence) to form a new nucleic acid molecule bearing chip sequence information (i.e., a nucleic acid labeled with a chip sequence) includes the following steps (shown in FIG. 3): Providing a bridging oligonucleotide comprising at its 5'-end a sequence (first region, P1) that is at least partially complementary to the 5'-end of the cDNA sequence (e.g., at least partially complementary to consensus sequence A (CA)) and at its 3'-end a sequence (second region, P2) that is at least partially complementary to the 3'-end of the chip sequence (e.g., at least partially complementary to consensus sequence X2).

[0049] In certain preferred embodiments, the P1 and P2 sequences of the bridging oligonucleotide are immediately adjacent, with no intervening nucleotides between them.

[0050] In certain preferred embodiments, the P1 sequence, the P2 sequence, the consensus sequence A, and the consensus sequence X2 each independently have a length of 20-100 nt (e.g., 20-70 nt). The bridging oligonucleotide is annealed or hybridized with the oligonucleotide probe and the cDNA strand, and then the 5'-end of the cDNA strand is ligated to the 3'-end of the oligonucleotide probe by a DNA ligase and / or a DNA polymerase, thereby generating a novel nucleic acid molecule (i.e., a nucleic acid molecule labeled with the oligonucleotide probe) that contains the sequence information of the oligonucleotide probe. In certain preferred embodiments, the DNA polymerase does not have 5' to 3' exonuclease activity or strand displacement activity.

[0051] An exemplary structure of a novel nucleic acid molecule containing chip sequence information as formed by the exemplary embodiment above includes a consensus sequence X1, a tag sequence Y, a consensus sequence X2, a consensus sequence A, a UMI sequence, a complementary sequence of an RNA (e.g., mRNA) sequence, and a complementary sequence of a consensus sequence B.

[0052] An embodiment including steps (1), (2)(iii) and (3)

[0053] In some embodiments, the method includes steps (1), (2)(iii) and (3), and the ligation product obtained in step (3) is taken as a second nucleic acid molecule having a positioning tag comprising, from 5' to 3', the consensus sequence X1, the tag sequence Y, the consensus sequence X2, optionally a complementary sequence of the third region of the bridging oligonucleotide, and the sequence of the first nucleic acid molecule to be labeled.

[0054] In some embodiments, in step (2)(iii)(c) of the method, the extension primer is primer B or primer B', and primer B is capable of annealing to all or a portion of the complementary sequence of consensus sequence B to initiate the extension reaction.

[0055] In certain embodiments, in step (2)(iii)(c), the extension primer is primer B'.

[0056] In certain embodiments, in step (2)(iii)(a) of the method, capture sequence A of primer A' is a random oligonucleotide sequence.

[0057] In certain embodiments, in step (2)(iii)(b), primer B comprises consensus sequence B, a complementary sequence of the 3' overhang and tag sequence B.

[0058] In some embodiments, the first extension product is generated from 5' to 3' by reverse transcription primed by primer A' and comprises a cDNA sequence complementary to the RNA sequence, a 3'-end overhang sequence, a complement of tag sequence B and a complement of consensus sequence B, where the complement of tag sequence B serves as a unique molecular identifier (UMI) for the second nucleic acid molecule.

[0059] In certain embodiments, in step (2)(iii)(c), the second extension product (first nucleic acid molecule to be labeled) comprises, from 5' to 3' to the first extension product, consensus sequence B or a 3'-terminal partial sequence thereof, tag sequence B, a complementary sequence of the 3'-terminal overhang sequence, and a complementary sequence of the cDNA sequence, wherein tag sequence B serves as a unique molecular identifier (UMI) of the second nucleic acid molecule.

[0060] In certain embodiments, in step (2)(iii)(a) of the method, capture sequence A of primer A' is a poly(T) sequence or a specific sequence that targets the target nucleic acid.

[0061] In certain embodiments, primer A' further comprises a tag sequence A, eg, a random oligonucleotide sequence, and a consensus sequence A.

[0062] In certain embodiments, capture sequence A is located at the 3'-end of primer A'.

[0063] In certain embodiments, consensus sequence A is located upstream of capture sequence A (eg, at the 5'-end of primer A').

[0064] In certain embodiments, primer B comprises consensus sequence B, a complementary sequence of the 3′-end overhang and tag sequence B.

[0065] In certain embodiments, in step (2)(iii)(b), the first extension product comprises, from 5' to 3', consensus sequence A, optionally tag sequence A, a cDNA sequence generated by reverse transcription primed by primer A' and that is complementary to the RNA, a 3'-end overhang sequence, a complement of tag sequence B, and a complement of consensus sequence B.

[0066] In certain embodiments, in step (2)(iii)(c), the second extension product (first nucleic acid molecule to be labeled) comprises, from 5' to 3', consensus sequence B or a 3'-terminal partial sequence thereof, tag sequence B, a complement of the 3'-terminal overhang sequence, a complement of the cDNA sequence in the first extension product, and optionally, a complement of tag sequence A and a complement of consensus sequence A.

[0067] In certain embodiments, in step (3), the ligation products derived from each copy of the same type of oligonucleotide probe have a different tag sequence B as a UMI.

[0068] In certain embodiments, the extension primer comprises a 5' phosphate at the 5'-end.

[0069] In certain embodiments, prior to step (2)(iii)(c), the method further comprises treating the product of step (2)(iii)(a) or step (2)(iii)(b) to remove RNA (e.g., by heat treatment).

[0070] In certain embodiments, in step (2)(iii)(b) of the method, the cDNA strand is annealed to primer B via its 3'-end overhang, and the cDNA strand is extended using primer B as a template in the presence of a nucleic acid polymerase (e.g., a DNA polymerase or a reverse transcriptase) to generate a first extension product.

[0071] An exemplary embodiment of the present application, including step (1), step (2)(iii) and step (3), is described in detail as follows:

[0072] 1. An exemplary embodiment for preparing a complementary strand of a cDNA strand using RNA (e.g., mRNA) in a sample as a template includes the following steps (as shown in FIG. 4):

[0073] (1) To perform reverse transcription of RNA molecules (e.g., mRNA molecules) of the permeabilized sample, a reverse transcriptase (e.g., a reverse transcriptase having terminal deoxynucleotidyl transferase activity) and primer A' are used to generate cDNA, and an overhang (e.g., an overhang containing three cytosine nucleotides) is added to the 3'-end of the cDNA. Various reverse transcriptases having terminal deoxynucleotidyl transferase activity can be used to perform the reverse transcription reaction. In certain preferred embodiments, the reverse transcriptase used does not have RNase H activity.

[0074] The reverse transcription primer A' contains a poly(T) sequence and a consensus sequence A (CA). Usually, the poly(T) sequence is located at the 3'-end of primer A to initiate reverse transcription.

[0075] (2) Primer B is annealed or hybridized to the cDNA strand, where primer B comprises consensus sequence B (CB) and a complementary sequence of the 3'-end overhang of the cDNA. In certain embodiments (e.g., when the method is used to construct a 5' transcriptome library), primer B further comprises a unique molecular identifier (UMI). The nucleic acid fragment hybridized or annealed with primer B is then extended in the presence of a nucleic acid polymerase using consensus sequence B and the UMI sequence as templates, such that the complementary sequence of consensus sequence B and the complementary sequence of the UMI sequence are added to the 3'-end of the cDNA strand, thereby generating a nucleic acid molecule that retains consensus sequence A at its 5'-end and the complementary sequence of consensus sequence B and the complementary sequence of the UMI molecule at its 3'-end.

[0076] If the cDNA strand contains an overhang of three cytosine nucleotides at its 3'-end, primer B may contain GGG at its 3'-end. Furthermore, the nucleotides of primer B may also be modified to enhance the binding affinity for complementary pairing between primer B and the 3'-end overhang of the cDNA strand (e.g., primer B may be modified to contain a locked nucleic acid).

[0077] Without being limited by theory, the extension reaction can be carried out using a variety of suitable nucleic acid polymerases (e.g., DNA polymerases or reverse transcriptases) so long as the annealed or hybridized nucleic acid fragment (reverse transcription product) can be extended using the sequence or subsequence of primer B as a template. In certain exemplary embodiments, the annealed or hybridized nucleic acid fragment (reverse transcription product) can be extended using the same reverse transcriptase used in the reverse transcription step above.

[0078] In certain preferred embodiments, step (2) and step (1) are carried out simultaneously.

[0079] In certain embodiments, the method optionally further comprises step (3): adding RNase H to digest the RNA strand of the RNA / cDNA hybrid to form a single-stranded cDNA.

[0080] In certain preferred embodiments, the method does not include step (3).

[0081] (4) Using an extension primer, an extension reaction is carried out using the single-stranded cDNA obtained in (3) as a template to obtain an extension product, and the extension primer is capable of annealing to the entire or part of the complementary sequence of consensus sequence B to initiate the extension reaction.

[0082] In certain embodiments, the extension primer is identical to the template switch oligo.

[0083] An exemplary structure comprising a complement of a cDNA strand prepared by the above exemplary embodiment comprises consensus sequence B, a UMI sequence, a complement of the cDNA 3'-end overhang sequence, a complement of the cDNA sequence, and a complement of consensus sequence A.

[0084] 2. An exemplary embodiment of using an oligonucleotide probe (also called chip sequence) to label the 5'-end of the complementary strand of a cDNA strand (i.e., the 5'-end of the complementary strand of a cDNA strand is ligated with the 3'-end of the chip sequence) to generate a new nucleic acid molecule containing the information of the chip sequence (i.e., a nucleic acid molecule labeled with the chip sequence) includes the following steps (as shown in FIG. 5): Providing a bridging oligonucleotide comprising at its 5'-end a sequence (first region, P1) that is at least partially complementary to consensus sequence B (CB) and at its 3'-end a sequence (second region, P2) that is at least partially complementary to consensus sequence X2.

[0085] In certain preferred embodiments, the P1 and P2 sequences of the bridging oligonucleotide are immediately adjacent, with no intervening nucleotides between them.

[0086] In certain preferred embodiments, the P1 and P2 sequences each independently have a length of 20 to 100 nt (eg, 20 to 70 nt).

[0087] The bridging oligonucleotide is annealed or hybridized with the oligonucleotide probe and the complementary strand of the cDNA strand, and then the 5'-end of the complementary strand of the cDNA strand is ligated to the 3'-end of the chip sequence by DNA ligase and / or DNA polymerase to form a new nucleic acid molecule containing the sequence information of the oligonucleotide probe (i.e., an oligonucleotide probe-labeled nucleic acid molecule). In certain preferred embodiments, the DNA polymerase does not have 5' to 3' exonuclease activity or strand displacement activity.

[0088] An exemplary structure of a novel nucleic acid molecule containing chip sequence information formed by the above exemplary embodiment includes a consensus sequence X1, a tag sequence Y, a consensus sequence X2, a consensus sequence B, a UMI sequence, a complementary sequence of a cDNA sequence, and a complementary sequence of a consensus sequence A.

[0089] In certain embodiments, the 3'-overhang has a length of 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 certain embodiments, the 3'-overhang is a 3'-overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).

[0090] In some embodiments, in step (2) of the method, the biological sample is permeabilized prior to pretreatment.

[0091] In certain embodiments, the biological sample is a tissue sample.

[0092] In certain embodiments, the tissue sample is a tissue section.

[0093] In certain embodiments, tissue sections are prepared from fixed tissue, such as formalin-fixed paraffin-embedded (FFPE) tissue or flash-frozen tissue.

[0094] In certain embodiments, when a biological sample is contacted with a nucleic acid array, each cell of the biological sample individually occupies one or more microdots of the nucleic acid array (i.e., each cell is individually contacted with one or more microdots of the nucleic acid array).

[0095] In some embodiments, in step (2), reverse transcription is performed by using a reverse transcriptase.

[0096] In certain embodiments, the reverse transcriptase has terminal deoxynucleotidyl transferase activity.

[0097] In certain embodiments, the reverse transcriptase is capable of synthesizing a cDNA strand using an RNA (eg, an mRNA) as a template and adding an overhang to the 3'-end of the cDNA strand.

[0098] In certain embodiments, the reverse transcriptase is capable of adding an overhang 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 to the 3'-end of the cDNA strand.

[0099] In certain embodiments, the reverse transcriptase is capable of adding an overhang of 2-5 cytosine nucleotides (eg, a CCC overhang) to the 3'-end of the cDNA strand.

[0100] In certain embodiments, 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 variants, modified products, and derivatives thereof having the reverse transcription activity of the above reverse transcriptases.

[0101] In certain embodiments, steps (2) and (3) of the method have one or more features selected from the following: (1) Primer A, Primer A', Primer B and the bridging oligonucleotide each independently comprise or consist of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain embodiments, Primer A and Primer A' are capable of initiating an extension reaction. (2) Primer B contains modified nucleotides (e.g., locked nucleic acids). In certain embodiments, primer B contains one or more modified nucleotides (e.g., locked nucleic acids) at its 3'-end. (3) tag sequence A and tag sequence B each independently have a length of 5 to 200 nt (e.g., 5 to 30 nt, 6 to 15 nt); (4) The consensus sequence A and the consensus sequence B each independently have a length of 10 to 200 nt (e.g., 10 to 100 nt, 20 to 100 nt, 25 to 100 nt, 5 to 10 nt, 10 to 15 nt, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt), (5) primer A, primer A', and primer B each independently have a length of 10 to 200 nt (e.g., 10 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt, 100 to 150 nt, 150 to 200 nt); (6) The first region and the second region of the bridging oligonucleotide each independently have a length of 3 to 100 nt (e.g., 3 to 10 nt, 10 to 15 nt, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt), (7) The third region of the bridging oligonucleotide has a length of 0 to 100 nt (e.g., 0 to 10 nt, 10 to 15 nt, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt), (8) The bridging oligonucleotide has a length of 6 to 200 nt (e.g., 20 to 70 nt, 6 to 15 nt, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt, 100 to 150 nt, 150 to 200 nt), (9) The poly(T) sequence contains at least 10 or at least 20 (e.g., at least 30) deoxythymidine residues; (10) The random oligonucleotide sequence has a length of 5 to 200 nt (e.g., 5 to 30 nt, 6 to 15 nt).

[0102] In certain embodiments, the method further comprises the step of (4) recovering and purifying the second population of nucleic acid molecules.

[0103] In certain embodiments, in the method, the obtained second population of nucleic acid molecules and / or their complements are used to construct a transcriptome library or for transcriptome sequencing.

[0104] In certain embodiments, the oligonucleotide probe in step (1) has one or more characteristics selected from the following: (1) The consensus sequence X1, the tag sequence Y, and the consensus sequence X2 each independently comprise natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (e.g., peptide nucleic acid (PNA) or locked nucleic acid), or any combination thereof, and in certain embodiments, the consensus sequence X2 has a free hydroxyl group (-OH) at the 3'-end; (2) the consensus sequence X1, the tag sequence Y, and the consensus sequence X2 each independently have a length of 2 to 100 nt (e.g., 10 to 200 nt, 10 to 100 nt, 20 to 100 nt, 25 to 100 nt, 50 to 100 nt, 5 to 30 nt, 6 to 15 nt, 5 to 10 nt, 10 to 15 nt, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt); (3) The oligonucleotide probes each independently have a length of 15 to 200 nt (for example, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt, 100 to 150 nt, 150 to 200 nt).

[0105] In certain embodiments, the oligonucleotide probe is attached to the solid support via a linker.

[0106] In certain embodiments, the linker is a linking group capable of binding to an activating group, and the surface of the solid support is modified with an activating group.

[0107] In certain embodiments, the linker comprises -SH, -DBCO, or -NHS.

[0108] In certain embodiments, the linker is -DBCO and the surface of the solid support is [ka] (Azido-dPEG® 8-NHS ester).

[0109] In some embodiments, the nucleic acid array of step (1) has one or more features selected from the following:

[0110] In certain embodiments, oligonucleotide probes bound to the same solid support have the same consensus sequence X1 and / or the same consensus sequence X2.

[0111] In certain embodiments, the consensus sequence X1 of the oligonucleotide probe comprises a cleavage site, and in some embodiments, the cleavage site can be cleaved or destroyed by a method selected from nicking enzyme digestion, USER enzyme digestion, light-responsive excision, chemical excision, or CRISPR-mediated excision.

[0112] In certain embodiments, the nucleic acid array of step (1) is provided by the following steps: (1) providing a plurality of types of carrier sequences, each of the carrier sequences comprising at least one copy (e.g., multiple copies) of a carrier sequence, the carrier sequence comprising, in a 5' to 3' direction, a complementary sequence of a consensus sequence X2, a complementary sequence of a tag sequence Y, and an immobilization sequence, the complementary sequences of the tag sequence Y of the various carrier sequences being different from each other; (2) binding a plurality of types of carrier sequences to the surface of a solid support (e.g., a chip); (3) providing an immobilized primer and performing a primer extension reaction using the carrier sequence as a template to generate an extension product to obtain an oligonucleotide probe, wherein the immobilized primer comprises a sequence of consensus sequence X1 and is capable of annealing to the immobilized sequence of the carrier sequence to initiate an extension reaction, and in some embodiments, the extension product in the 5' to 3' direction comprises or consists of the consensus sequence X1, the tag sequence Y, and the consensus sequence X2; (4) coupling the immobilized primer to the surface of the solid support, wherein steps (3) and (4) are performed in any order; (5) Optionally, the immobilized sequence of the carrier sequence further comprises a cleavage site, and the cleavage may be selected from nicking enzyme digestion, USER enzyme digestion, light-responsive excision, chemical excision or CRISPR-mediated excision, and cleavage is performed at the cleavage site contained in the immobilized sequence of the carrier sequence to digest the carrier sequence and separate the extension product in step (3) from the template (i.e., the carrier sequence) on which the extension product is generated, thereby linking the oligonucleotide probe to the surface of a solid phase support (e.g., a chip).

[0113] In certain embodiments, the various carrier sequences are DNBs formed from concatemers of multiple copies of the carrier sequences.

[0114] In certain embodiments, the plurality of carrier sequences is subjected to the steps of: (i) providing a plurality of carrier-template sequences, the carrier-template sequences including complementary sequences of the carrier sequences; (ii) performing a nucleic acid amplification reaction using the various carrier-template sequences as templates to obtain amplification products of the various carrier-template sequences, the amplification products comprising at least one copy (e.g., multiple copies) of the carrier sequence, and in certain embodiments, rolling circle replication is performed to obtain DNBs formed from concatemers of the carrier sequences; Offered by.

[0115] In some embodiments, the solid support in step (1) has one or more characteristics selected from the following: (1) the solid support is selected from the group consisting of latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes, and silicon wafers, and in some embodiments the solid support is a chip; (2) The solid support is planar, spherical or porous; (3) The solid support can be used as a sequencing platform, e.g., a sequencing chip. In some embodiments, the solid support is a sequencing chip for an Illumina, MGI, or Thermo Fisher sequencing platform; and (4) The solid support is capable of releasing the oligonucleotide probes spontaneously or upon exposure to one or more stimuli (e.g., temperature change, pH change, exposure to certain chemicals or phases, exposure to light, exposure to a reducing agent, etc.).

[0116] Method for constructing a nucleic acid molecule library

[0117] In another aspect, the present application also provides a method for constructing a nucleic acid molecule library, comprising: (a) generating a population of labeled nucleic acid molecules according to a method as described above; and (b) randomly fragmenting the nucleic acid molecules in the population of labeled nucleic acid molecules and adding adaptors; and (c) optionally amplifying and / or enriching the product of step (b) thereby obtaining a library of nucleic acid molecules.

[0118] In certain embodiments, the nucleic acid molecule library is used for sequencing, e.g., transcriptome sequencing, e.g., single-cell transcriptome sequencing.

[0119] In certain embodiments, prior to performing step (b), the method further comprises the step (pre-b): amplifying and / or concentrating the population of labeled nucleic acid molecules.

[0120] In certain embodiments, in step (pre-b), the population of labeled nucleic acid molecules is subjected to a nucleic acid amplification reaction to generate an amplification product.

[0121] In certain embodiments, the nucleic acid amplification reaction is carried out using at least primer C and / or primer D, where primer C is capable of hybridizing or annealing to a complementary sequence of consensus sequence X1 or a complementary sequence of the 3'-terminal sequence of consensus sequence X1 to initiate an extension reaction, and primer D is capable of hybridizing or annealing to a nucleic acid molecule of the population of labeled nucleic acid molecules to initiate an extension reaction.

[0122] In certain embodiments, the nucleic acid amplification reaction in step (pre-b) is carried out using a nucleic acid polymerase (e.g., a DNA polymerase, e.g., a DNA polymerase with strand displacement activity and / or high fidelity).

[0123] In certain embodiments, in step (b), the nucleic acid molecule is randomly fragmented into fragments and adapters are added to the fragments using a transposase. In some embodiments, in step (b) of the method, the nucleic acid molecule obtained in the previous step is randomly fragmented into fragments and a first adapter and a second adapter are added to both ends of the fragments, respectively, using a transposase.

[0124] In certain embodiments, the transposase is selected from the group consisting of Tn5 transposase, MuA transposase, Sleeping Beauty transposase, Mariner transposase, Tn7 transposase, Tn10 transposase, Ty1 transposase, Tn552 transposase, and variants, modified products, and derivatives thereof that have the transposition activity of the above transposases.

[0125] In certain embodiments, the transposase is a Tn5 transposase.

[0126] In some embodiments, in step (c), the product of step (b) is amplified using at least primer C' and / or primer D', where primer C' is capable of hybridizing or annealing to a first adapter to initiate an extension reaction, and primer D' is capable of hybridizing or annealing to a second adapter to initiate an extension reaction.

[0127] In some embodiments, in step (c), the product of step (b) is amplified using at least primer C and / or primer D', where primer D' is capable of hybridizing or annealing to the first adaptor or the second adaptor to initiate an extension reaction.

[0128] Sequencing Methods

[0129] In another aspect, the present application also provides a method of sequencing a nucleic acid sample, the method comprising: (1) constructing a library of nucleic acid molecules according to the method described above; and (2) sequencing the library of nucleic acid molecules; Includes.

[0130] kit

[0131] In another aspect, the present application also provides a kit, the kit comprising: (i) a nucleic acid array for labeling nucleic acids, comprising a solid support, the solid support being bound to a plurality of types of oligonucleotide probes, each type of oligonucleotide probe comprising at least one copy, the oligonucleotide probes comprising or consisting of, in a 5' to 3' direction, a consensus sequence X1, a tag sequence Y and a consensus sequence X2; a nucleic acid array for labeling nucleic acids, wherein each type of oligonucleotide probe has a different tag sequence Y, where tag sequence Y has a nucleotide sequence that is unique to the location on the solid support of that type of oligonucleotide probe; (ii) a primer set comprising primer A, or primer A' and primer B, or a primer set comprising primer A and primer B, Primer A comprises a consensus sequence A and a capture sequence A, where the capture sequence A is capable of annealing to an RNA (e.g., an mRNA) to be captured to initiate an extension reaction, and in some embodiments, the consensus sequence A is located upstream of the capture sequence A (e.g., located at the 5'-end of primer A); Primer A' comprises a capture sequence A, which is capable of annealing to the RNA (e.g., mRNA) to be captured to initiate an extension reaction; Primer B comprises a consensus sequence B, a complementary sequence of the 3'-end overhang, and optionally a tag sequence B, and in certain embodiments, the complementary sequence of the 3'-end overhang is located at the 3'-end of primer B, and in certain embodiments, the consensus sequence B is located upstream of the complementary sequence of the 3'-end overhang (e.g., at the 5'-end of primer B), and the 3'-end overhang refers to one or more non-template nucleotides contained at the 3'-end of a cDNA strand generated by reverse transcription using the RNA captured by the capture sequence A of primer A' as a template; primer A, or a primer set comprising primer A' and primer B, or a primer set comprising primer A and primer B. and (iii) a bridging oligonucleotide comprising a first region and a second region, and optionally a third region located between the first and second regions, wherein the first region is located upstream of the second region (e.g., 5' of the second region); the first region is capable of annealing (a) to all or a portion of consensus sequence A of primer A, or (b) to all or a portion of consensus sequence B of primer B; The second region is a bridging oligonucleotide capable of annealing to all or part of the consensus sequence X2. Includes.

[0132] In certain embodiments, each type of oligonucleotide probe comprises one copy.

[0133] In certain embodiments, each type of oligonucleotide probe comprises multiple copies.

[0134] In certain embodiments, the region where various oligonucleotide probes are attached to solid support is called microdot.It is easy to understand that when various oligonucleotide probes contain one copy, each microdot is attached to one oligonucleotide probe, and the oligonucleotide probes of different microdots have different tag sequences Y; when various oligonucleotide probes contain multiple copies, each microdot is attached to multiple oligonucleotide probes, and the oligonucleotide probes of the same microdot have the same tag sequence Y, and the oligonucleotide probes of different microdots have different tag sequences Y.

[0135] In certain embodiments, the solid support comprises a plurality of microdots, each microdot being bound to one type of oligonucleotide probe, and each type of oligonucleotide probe may comprise one or multiple copies.

[0136] In certain embodiments, the solid support comprises a plurality of (e.g., 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, and in certain embodiments, the solid support comprises at least 10 4 (e.g., 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 / mm 2 Includes.

[0137] In some embodiments, the spacing between adjacent microdots is less than 100 μm, less than 50 μm, less than 10 μm, less than 5 μm, less than 1 μm, less than 0.5 μm, less than 0.1 μm, less than 0.05 μm, or less than 0.01 μm.

[0138] In certain embodiments, the microdots have a size (e.g., equivalent diameter) of less than 100 μm, less than 50 μm, less than 10 μm, less than 5 μm, less than 1 μm, less than 0.5 μm, less than 0.1 μm, less than 0.05 μm, or less than 0.01 μm.

[0139] In certain embodiments, the kit comprises a nucleic acid array for labeling a nucleic acid according to (i), a primer A according to (ii) and a bridging oligonucleotide according to (iii), wherein a first region of the bridging oligonucleotide is capable of annealing to all or a portion of the consensus sequence A of primer A and a second region of the bridging oligonucleotide is capable of annealing to all or a portion of the consensus sequence X2.

[0140] In certain embodiments, capture sequence A of primer A is a random oligonucleotide sequence.

[0141] In certain embodiments, capture sequence A of primer A is a poly(T) sequence or a specific sequence that targets a target nucleic acid; in certain embodiments, primer A further comprises a tag sequence A, e.g., a random oligonucleotide sequence; in certain embodiments, capture sequence A is located at the 3'-end of primer A and consensus sequence A is located upstream of tag sequence A (e.g., at the 5'-end of primer A).

[0142] In certain embodiments, primer A comprises a 5' phosphate at the 5'-end.

[0143] In certain embodiments, the kit comprises a nucleic acid array for labeling a nucleic acid as described in (i), a primer set comprising primer A' and primer B as described in (ii), and a bridging oligonucleotide as described in (iii), wherein a first region of the bridging oligonucleotide is capable of annealing to all or a portion of consensus sequence B of primer B, and a second region of the bridging oligonucleotide is capable of annealing to all or a portion of consensus sequence X2.

[0144] In certain embodiments, the capture sequence of primer A' is a random oligonucleotide sequence.

[0145] In certain embodiments, the capture sequence A of primer A' is a poly(T) sequence or a sequence specific for targeting a target nucleic acid, in certain embodiments, primer A' further comprises a tag sequence A and a consensus sequence A, in certain embodiments, the capture sequence A is located at the 3'-end of primer A', and in certain embodiments, the consensus sequence A is located upstream of the capture sequence A (e.g., at the 5'-end of primer A').

[0146] In certain embodiments, primer B comprises consensus sequence B, a complementary sequence of the 3′-end overhang and tag sequence B.

[0147] In certain embodiments, the kit further comprises a primer B', which is capable of annealing to all or a portion of the complementary sequence of consensus sequence B to initiate an extension reaction.

[0148] In certain embodiments, primer B or primer B' comprises a 5' phosphate at the 5'-end.

[0149] In certain embodiments, primer B comprises a modified nucleotide (e.g., a locked nucleic acid), and in certain embodiments, primer B comprises one or more modified nucleotides (e.g., one or more locked nucleic acids) at the 3'-end.

[0150] In certain embodiments, the kit comprises a nucleic acid array for labeling a nucleic acid as described in (i), a primer set comprising primer A and primer B as described in (ii), and a bridging oligonucleotide as described in (iii), wherein a first region of the bridging oligonucleotide is capable of annealing to all or a portion of consensus sequence A of primer A, and a second region of the bridging oligonucleotide is capable of annealing to all or a portion of consensus sequence X2.

[0151] In certain embodiments, capture sequence A of primer A is a random oligonucleotide sequence.

[0152] In certain embodiments, capture sequence A of primer A is a poly(T) sequence or a sequence specific for targeting a target nucleic acid, in certain embodiments, primer A further comprises a tag sequence A, e.g., a random oligonucleotide sequence, in certain embodiments, capture sequence A is located at the 3'-end of primer A and consensus sequence A is located upstream of tag sequence A (e.g., at the 5'-end of primer A).

[0153] In certain embodiments, primer A comprises a 5' phosphate at the 5'-end.

[0154] In certain embodiments, primer B comprises a modified nucleotide (e.g., a locked nucleic acid). In certain embodiments, primer B comprises one or more modified nucleotides (e.g., a locked nucleic acid) at the 3'-end.

[0155] In certain embodiments, the kit has one or more features selected from the following: (1) The oligonucleotide probe, primer A, primer A', primer B, primer B', and bridging oligonucleotide each independently comprise or consist of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides, or any combination thereof. In certain embodiments, primer A, primer A', and primer B' are capable of initiating an extension reaction. In certain embodiments, consensus sequence X2 has a free hydroxyl group (-OH) at the 3'-terminus. (2) The oligonucleotide probes each independently have a length of 15 to 300 nt (e.g., 15 to 200 nt, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt, 100 to 150 nt, 150 to 200 nt), (3) primer A, primer A', primer B and primer B' each independently have a length of 10 to 200 nt (e.g., 10 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt, 100 to 150 nt, 150 to 200 nt); (4) The bridging oligonucleotide has a length of 6 to 200 nt (e.g., 20 to 70 nt, 6 to 15 nt, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt, 100 to 150 nt, 150 to 200 nt), (5) The oligonucleotide probes bound to the same solid support have the same consensus sequence X1 and / or the same consensus sequence X2; (6) The consensus sequence X1 of the oligonucleotide probe comprises a cleavage site, and in some embodiments, the cleavage site can be cleaved or destroyed by a method selected from nicking enzyme digestion, USER enzyme digestion, light-responsive excision, chemical excision, or CRISPR-mediated excision.

[0156] In certain embodiments, the kit further comprises a reverse transcriptase, a nucleic acid ligase, a nucleic acid polymerase, and / or a transposase.

[0157] In certain embodiments, the reverse transcriptase has terminal deoxynucleotidyl transferase activity, in certain embodiments, the reverse transcriptase is capable of synthesizing a cDNA strand using RNA (e.g., mRNA) as a template and adding a 3'-end overhang to the 3'-end of the cDNA strand, in certain embodiments, the reverse transcriptase is capable of adding an overhang having a length of 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 to the 3'-end of the cDNA strand, in certain embodiments, the reverse transcriptase is capable of adding an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang) to the 3'-end of the cDNA strand, in certain embodiments, 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 variants, modified products, and derivatives thereof having the reverse transcription activity of the above reverse transcriptases.

[0158] In certain embodiments, the nucleic acid polymerase does not have 5' to 3' exonuclease activity or strand displacement activity.

[0159] In certain embodiments, the transposase is selected from the group consisting of Tn5 transposase, MuA transposase, Sleeping Beauty transposase, Marina transposase, Tn7 transposase, Tn10 transposase, Ty1 transposase, Tn552 transposase, and variants, modified products, and derivatives thereof that have the transposition activity of the above transposases.

[0160] In certain embodiments, the kit further comprises primer C, primer D, primer C', and / or primer D'. For example, the kit further comprises primer C, primer D, and primer D'. For example, the kit further comprises primer C, primer D, primer C', and primer D'.

[0161] In certain embodiments, 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 constructing a transcriptome sequencing library, reagents for sequencing (e.g., second or third generation sequencing), or any combination thereof.

[0162] use

[0163] In another aspect, the present application also provides a method for generating a population of labeled nucleic acid molecules as described above, or the use of a kit as described above for constructing a nucleic acid molecule library or for performing transcriptome sequencing.

[0164] Definition of Terms

[0165] In this application, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps of molecular biology, biochemistry, nucleic acid chemistry, cell culture, etc., as used herein, are all routine steps widely used in the corresponding technical fields. Meanwhile, in order to better understand this application, the definitions and explanations of relevant terms are provided below.

[0166] When the terms "eg," "for example," "such as," "comprise," "include," or variations thereof are used herein, these terms are not intended to be limiting terms and instead are intended to mean "including but not limited to" or "not limiting."

[0167] Unless otherwise indicated herein or clearly contradicted by context, the terms "a" and "an" as well as "the" and similar referents in the context of describing this application (particularly in the context of the claims which follow) should be construed to cover the singular and plural.

[0168] As used herein, "DNB" (DNA nanoball) is a typical RCA (rolling circle amplification) product, which has the characteristics of an RCA product, where the RCA product is a single-stranded DNA sequence with multiple copies, which can form a "globular"-like structure due to the interactions between bases contained in the DNA. Typically, the library molecules are circularized to form single-stranded circular DNA, which can then be amplified by several orders of magnitude using rolling circle amplification technology, thereby generating an amplification product called DNB.

[0169] As used herein, a "nucleic acid molecule population" refers to a collection or set of nucleic acid molecules, e.g., nucleic acid molecules derived directly or indirectly from a target nucleic acid molecule (e.g., double-stranded DNA, RNA / cDNA hybrid, single-stranded DNA, or single-stranded RNA). In some embodiments, the nucleic acid molecule population comprises a nucleic acid molecule library, which comprises sequences that are qualitatively and / or quantitatively representative of the target nucleic acid molecule sequences. In other embodiments, the nucleic acid molecule population comprises a subset of the nucleic acid molecule library.

[0170] As used herein, "nucleic acid molecule library" refers to a collection or population of labeled nucleic acid molecules (e.g., labeled double-stranded DNA, labeled RNA / cDNA hybrid, labeled single-stranded DNA or labeled single-stranded RNA) or fragments thereof generated directly or indirectly from a target nucleic acid molecule, and the combination of labeled nucleic acid molecules or fragments thereof in the collection or population is shown to be qualitatively and / or quantitatively representative of the sequence of the target nucleic acid molecule sequence from which the labeled nucleic acid molecule was generated. In certain embodiments, the nucleic acid molecule library is a sequencing library. In certain embodiments, the nucleic acid molecule library can be used to construct a sequencing library.

[0171] As used herein, "cDNA" or "cDNA strand" refers to a "complementary DNA" synthesized by using at least a portion of an RNA molecule of interest as a template and extending through a primer that anneals to the RNA molecule of interest under the catalysis of an RNA-dependent DNA polymerase or reverse transcriptase (this process is also called "reverse transcription"). The synthesized cDNA molecule is "homologous" or "complementary" to at least a portion of the template, or "base-pairs" or "complexes" with at least a portion of the template.

[0172] As used herein, the term "upstream" is used to describe the relative positional relationship of two nucleic acid sequences (or two nucleic acid molecules) and has the meaning commonly understood by those skilled in the art. For example, the expression "a nucleic acid sequence is located upstream of another nucleic acid sequence" means that the former is located at a more forward position (i.e., closer to the 5'-end) than the latter when aligned in the 5' to 3' direction. As used herein, the term "downstream" has the opposite meaning to "upstream".

[0173] 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. refer to an oligonucleotide having a non-target nucleic acid component that provides a means for identification, recognition and / or molecular or biochemical manipulation of the nucleic acid molecule ligated thereto or a derivative product of the nucleic acid molecule ligated thereto (e.g., a complementary fragment of a nucleic acid molecule, a short fragment of a nucleic acid molecule, etc.) (e.g., by providing a site for annealing with an oligonucleotide. The oligonucleotide may be, for example, a primer for DNA polymerase extension or an oligonucleotide for a capture or ligation reaction.) The oligonucleotide may consist of at least 2 (preferably about 6-100, although there is no definite limit to the length of the oligonucleotide, and the exact size depends on a number of factors, which in turn depend on the ultimate function or use of the oligonucleotide) nucleotides, and may be composed of multiple oligonucleotide segments arranged contiguously or non-contiguously. The oligonucleotide sequence may be unique to each nucleic acid molecule to which it is ligated, or may be unique to a particular type of nucleic acid molecule to which it is ligated. The oligonucleotide sequence may be reversibly or irreversibly ligated to the polynucleotide sequence to be "labeled" by any method, including ligation, hybridization, or other methods. The process of ligating an oligonucleotide sequence to a nucleic acid molecule may be referred to as "labeling," and a nucleic acid molecule to which a label has been attached or which contains a label sequence is referred to as a "labeled nucleic acid molecule" or a "tagged nucleic acid molecule."

[0174] For a variety of reasons, the nucleic acids or polynucleotides of the present application (e.g., "tag sequence Y", "tag sequence A", "tag sequence B", "consensus sequence X1", "consensus sequence X2", "consensus sequence A", "consensus sequence B", "primer A", "primer A'", "primer B", "primer C", "primer D", "primer D'", "bridging oligonucleotide", etc.) may contain one or more modified nucleobases, sugar moieties, or internucleoside linkages. For example, some reasons for using nucleic acids or polynucleotides that contain modified nucleobases, sugar moieties, or internucleoside linkages include, but are not limited to, (1) altering the Tm, (2) altering the susceptibility of the polynucleotide to one or more nucleases, (3) providing a moiety for linking a label, (4) providing a label or label quencher, or (5) providing a moiety such as biotin for attachment to another molecule in solution or bound to a surface. For example, in some embodiments, oligonucleotides, e.g., primers, can be synthesized to contain one or more nucleic acid analogs with constrained conformations in the random portion, including, but not limited to, one or more ribonucleic acid analogs in which the ribose ring is "locked" by a methylene bridge connecting the 2'-O atom to the 4'-C atom. These modified nucleotides result in an increase of about 2 degrees Celsius to about 8 degrees Celsius in the Tm or melting temperature of each molecule. For example, in some embodiments in which oligonucleotide primers containing ribonucleotides are used, one indication of using modified nucleotides in the method can be that oligonucleotides containing modified nucleotides can be digested by single-strand specific RNase.

[0175] In the methods of the present application, for example, the nucleobase of a single nucleotide at one or more positions of a polynucleotide or oligonucleotide may include guanine, adenine, uracil, thymine or cytosine, or optionally, one or more of the nucleobases may include modified bases, such as, but not limited to, xanthine, allylamino-uracil, allylamino-thymine nucleoside, hypoxanthine, 2-aminoadenine, 5-propynyluracil, 5-propynylcytosine, 4-thiouracil, 6-thioguanine, azauracil, deazauracil, thymine nucleoside, cytosine, adenine or guanine. Furthermore, they may include nucleobases derivatized with the following moieties: biotin moiety, digoxigenin moiety, fluorescent or chemiluminescent moiety, quenching moiety or some other moiety. The present application is not limited to the listed nucleobases, and the given list illustrates a wide range of examples of bases that may be used in the methods of the present application.

[0176] With respect to the nucleic acids or polynucleotides of the present application, one or more of the sugar moieties may comprise 2'-deoxyribose, or optionally, one or more of the sugar moieties may comprise some other sugar moieties, such as, but not limited to, ribose or 2'-fluoro-2'-deoxyribose or 2'-O-methyl-ribose, which are resistant to some nucleases, or 2'-amino-2'-deoxyribose or 2'-azido-2'-deoxyribose, which are labeled by reaction with a visible, fluorescent, infrared fluorescent or other detectable dye or a chemical having an electrophilic, photoreactive, alkynyl or other reactive chemical moiety.

[0177] The internucleoside linkages of the nucleic acids or polynucleotides of the present application may be phosphodiester linkages, or optionally, one or more of the internucleoside linkages may comprise modified linkages, such as, but not limited to, phosphorothioate, phosphorodithioate, phosphoroselenate, or phosphorodiselenate linkages, which are resistant to some nucleases.

[0178] As used herein, the term "terminal deoxynucleotidyl transferase 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 the 3'-end of a cDNA. Examples of reverse transcriptases with terminal deoxynucleotidyl transferase activity include, but are not limited to, -MLV reverse transcriptase, HIV-1 reverse transcriptase, AMV reverse transcriptase, telomerase reverse transcriptase, and variants, modified products, and derivatives thereof that have reverse transcriptase activity and terminal deoxynucleotidyl transferase activity of reverse transcriptase. Reverse transcriptases may or may not have RNase activity (particularly RNase H activity). In a preferred embodiment, the reverse transcriptase used for reverse transcription of RNA to generate cDNA does not have RNase activity (particularly RNase H activity). Thus, in a preferred embodiment, the reverse transcriptase used for reverse transcription of RNA to produce cDNA has terminal deoxynucleotidyl transferase activity and no RNase activity (in particular RNase H activity).

[0179] As used herein, a nucleic acid polymerase having "strand displacement activity" refers to a nucleic acid polymerase that, when it encounters a downstream nucleic acid strand that is complementary to the template strand during the process of extending a new nucleic acid strand, can continue the extension reaction and replace (rather than degrade) the nucleic acid strand that is complementary to the template strand.

[0180] As used herein, a nucleic acid polymerase with "5' to 3' exonuclease activity" refers to a nucleic acid polymerase that can catalyze the hydrolysis of 3,5-phosphodiester bonds from the 5'-end to the 3'-end of a polynucleotide, thereby degrading nucleotides.

[0181] As used herein, a nucleic acid polymerase (or DNA polymerase) with "high fidelity" refers to a nucleic acid polymerase (or DNA polymerase) that is less likely to introduce an incorrect nucleotide (i.e., error rate) during amplification of a nucleic acid than a wild-type Taq enzyme (e.g., the Taq enzyme whose sequence is set forth in the UniProt Accession: P19821.1).

[0182] As used herein, the terms "annealed", "annealing", "annealing", "hybridized" or "hybridizing" and the like refer to the formation of a complex between nucleotide sequences that have sufficient complementarity to form a complex via Watson-Crick base pairing. For the purposes of this application, nucleic acid sequences that are "complementary" or "hybridize" or "anneal" to one another must be capable of forming a sufficiently stable "hybrid" or "complex" for the intended purpose. It is not necessary that both nucleic acid molecules or corresponding sequences presented therein be "complementary" or "anneal" or "hybridize" to one another by having all nucleobases in the sequence presented by a nucleic acid molecule capable of base pairing or pairing or complexing with all nucleobases in the sequence presented by another nucleic acid molecule. As used herein, the terms "complementary" or "complementarity" are used when referring to a sequence of nucleotides related by the rules of base pairing. For example, the sequence 5'-AGT-3' is complementary to the sequence 3'-TCA-5'. Complementarity can be "partial", where only a portion of the nucleic acid bases match according to the rules of base pairing. Optionally, there can be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has a large effect on the efficiency and strength of hybridization between nucleic acid strands. The degree of complementarity is particularly important in amplification reactions and detection methods that rely on nucleic acid hybridization. The term "homology" refers to the degree of complementarity of one nucleic acid sequence to another. There can be partial homology (i.e., complementarity) or complete homology (i.e., complementarity). Partially complementary sequences are sequences that at least partially inhibit the hybridization of a completely complementary sequence to a target nucleic acid and are referred to using the functional term "substantially homologous". Inhibition of hybridization of the completely complementary sequence with the target sequence can be tested under low stringency conditions using hybridization assays (eg, Southern or Northern blotting, solution hybridization, etc.).A substantially homologous sequence or probe competes for or inhibits the binding (i.e., hybridization) of a completely homologous sequence to a target under low stringency conditions. This does not mean that low stringency conditions are conditions that allow non-specific binding, but rather that low stringency conditions require that two sequences bind to each other through specific (i.e., selective) interactions. Absence of non-specific binding can be tested by using a second target that lacks complementarity or has only a low degree of complementarity (e.g., less than about 30% complementarity). If specific binding is low or absent, the probe will not hybridize to the nucleic acid target. The term "substantially homologous" when used in reference to a double-stranded nucleic acid sequence, such as a cDNA or genomic clone, means that any oligonucleotide or probe can hybridize to one or both strands of a double-stranded nucleic acid sequence under low stringency conditions as described herein. As used herein, the term "annealing" or "hybridization" is used to refer to the pairing of complementary nucleic acid strands. Hybridization and hybridization force (i.e., the force of association between nucleic acid strands) are affected by a number of factors known in the art, including the degree of complementarity between nucleic acids, including the stringency of conditions, which is affected by factors such as salt concentration, Tm (melting temperature) for forming hybrids, the presence of other components (e.g., the presence or absence of polyethylene glycol or betaine), the molar concentration of hybridized strands, and the GC content of nucleic acid strands.

[0183] As described herein, the solid support is capable of releasing the oligonucleotide probe spontaneously or upon exposure to one or more stimuli (e.g., temperature change, pH change, exposure to certain chemicals or phases, exposure to light, reducing agents, etc.) It is understood 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, or both, and that the oligonucleotide probe may allow itself to be accessed or be accessible by other reagents.

[0184] The addition of multiple types of labile bonds to a solid support allows for the ability of the solid support to respond to different stimuli: each type of labile bond may be sensitive to a relevant stimulus (e.g., chemical stimuli, light, temperature, etc.), such that the release of the material attached to the solid support via each labile bond can be controlled by application of the appropriate stimulus. In addition to thermally cleavable bonds, disulfide bonds, and UV-sensitive bonds, other non-limiting examples of labile bonds that can be attached to a solid support include an ester bond (e.g., an ester bond that can be cleaved with acid, base, or hydroxylamine), an ortho-diol bond (e.g., an ortho-diol bond that can be cleaved by sodium periodate), a Diels-Alder bond (e.g., a Diels-Alder bond that can be cleaved thermally), a sulfone bond (e.g., a sulfone bond that can be cleaved by alkali), a silicyl ether bond (e.g., a silicyl ether bond that can be cleaved by acid), a glycosidic bond (e.g., a glycosidic bond that can be cleaved by an amylase), a peptide bond (e.g., a peptide bond that can be cleaved by a protease), or a phosphodiester bond (e.g., a phosphodiester bond that can be cleaved by a nuclease, such as a DNA enzyme).

[0185] In addition to or as an alternative to the cleavable bond between the solid support and the oligonucleotide described above, the solid support may be degradable, destructible or soluble, either spontaneously or upon exposure to one or more stimuli (e.g., temperature change, pH change, exposure to certain chemicals or phases, exposure to light, exposure to reducing agents, etc.). In some cases, the solid support may be soluble, such that the material components of the solid support dissolve upon exposure to certain chemicals or environmental changes (e.g., temperature change or pH change). In some cases, the solid support may degrade or dissolve under high temperature and / or alkaline conditions. In some cases, the solid support may be thermally degradable, such that the solid support degrades when exposed to an appropriate temperature change (e.g., heating). Decomposition or dissolution of the solid support bound to a substance (e.g., an oligonucleotide probe) may result in the release of the substance from the solid support.

[0186] As used herein, the terms "transposase" and "reverse transcriptase" and "nucleic acid polymerase" refer to a protein molecule or an aggregate of protein molecules involved in catalyzing certain chemical and biological reactions. In general, the methods, compositions, or kits of the present application are not limited to the use of a specific transposase, reverse transcriptase, or nucleic acid polymerase from a specific source. Rather, the methods, compositions, or kits of the present application may include any transposase, reverse transcriptase, or nucleic acid polymerase from any source that has an enzymatic activity equivalent to the specific enzyme of the specific method, composition, or kit disclosed herein. In addition, the methods of the present application also include the following embodiments: any one specific enzyme provided and used in the steps of the method is replaced by a combination of two or more enzymes, whether used separately, stepwise, or simultaneously together, such that the reaction mixture produces the same results as would be obtained using that specific enzyme when the two or more enzymes are used in combination. The methods, buffers, and reaction conditions provided herein, including those in the examples, are currently preferred for the embodiments of the methods, compositions, and kits of the present application. However, other enzyme storage buffers, reaction buffers and reaction conditions may be used for some of the enzymes of the present application and are known in the art and may be suitable for use in the present application and are included herein.

[0187] [Beneficial Effects of the Present Application] The present application provides a novel method for generating a labeled nucleic acid molecule population, as well as a method for constructing a nucleic acid molecule library based on the method and performing high-throughput sequencing, thereby achieving highly accurate subcellular level spatial localization of a sample. The method of the present application has one or more beneficial technical effects selected from the following:

[0188] (1) The probe of traditional nucleic acid array (e.g., chip) used for spatial transcriptome sequencing contains a fixed capture sequence. Usually, a specific capture sequence can only capture a specific target nucleic acid molecule corresponding to it. For example, if the capture sequence is poly(T), it will capture a target nucleic acid molecule containing poly(A) correspondingly. If the target nucleic acid molecule changes, the probe sequence containing the capture sequence needs to be changed to match it, i.e., the whole nucleic acid array (e.g., chip) needs to be changed, which is costly and inefficient in practical application. The nucleic acid array (e.g., chip) of the present application does not contain a capture sequence, and the capture sequence is present in a reverse transcription primer that is independent of the nucleic acid array (i.e., the capture sequence and the probe are independent of each other). After the capture sequence captures the target nucleic acid molecule, it ligates with the probe via a bridging oligonucleotide.

[0189] Thus, the present application can achieve capture of different target nucleic acid molecules by designing corresponding capture sequences for different target nucleic acid molecules and modifying the capture sequences and bridging oligonucleotides without changing the probe sequence (i.e., without changing the nucleic acid array (e.g., chip)).

[0190] (2) All traditional spatial transcriptome methods use poly(T) as a capture sequence and cannot capture RNA without a poly(A) tail, whereas the present application can achieve the capture of target nucleic acid molecules without a poly(A) tail by replacing the poly(T) of the capture sequence with a group of consecutive random sequences (e.g., random primer sequences, e.g., N6, N8, etc.), which can also simultaneously act as unique molecular identifier (UMI) sequences.

[0191] (3) Traditional nucleic acid arrays (e.g., chips) used for spatial transcriptome sequencing have immobilized capture probes. Generally, tissue permeabilization is first performed to release intracellular RNA. If permeabilization is excessive, RNA will spread to adjacent cells, even to the periphery of the tissue sample, and be captured by the probe, making it impossible to achieve in situ capture of mRNA. If permeabilization is incomplete, the capture efficiency of mRNA will be affected. With the method of the present application, the nucleic acid array (e.g., chip) does not contain capture sequences (nucleic acid arrays contain spatial information and do not contain capture sequences), and the purpose of tissue permeabilization is to allow reverse transcription primers to enter cells and hybridize in situ with mRNA without the need for strong permeabilization reagent treatment, thereby reducing the spread of the sample.

[0192] Preferred embodiments of the present application are described in detail below in conjunction with the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are merely used to illustrate the present application and do not limit the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments. EXAMPLES

[0193] The present application will now be described with reference to the following examples, which are intended to illustrate (but not limit) the present application. Unless otherwise specified, the experiments and methods described in the examples were essentially carried out according to conventional methods well known in the art and described in various references. Furthermore, if no specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. If the manufacturer of the reagents or equipment used was not specified, they were all conventional products that could be purchased commercially. Those skilled in the art will understand that the examples are intended to illustrate the present application and are not intended to limit the scope of the protection sought by the present application. All publications and other references mentioned herein are incorporated by reference in their entirety.

[0194] Sequence information

[0195] Information about some of the sequences contained in this application is provided in Table 1 below. [Table 1]

[0196] Example 1: Preparation of capture chip 1. The sequence of DNA library molecules containing the chip position information was designed, which consisted of consensus sequence X1 (X1), tag sequence (Y) and consensus sequence X2 (X2) from 5' to 3'. The general nucleotide sequence of the DNA library molecule is shown in SEQ ID NO: 1. Beijing Liuhe BGI Co., Ltd. was commissioned to synthesize the DNA library molecules.

[0197] 2. Amplification and Loading of Library Molecules (1) DNA nanoballs (DNBs) were prepared using DNBSEQ sequencing kit (purchased from MGI, catalog number 1000019840). A specific embodiment is briefly described below.

[0198] Briefly, 40 μL of reaction system as shown in Table 2 was prepared. The reaction system was placed in a PCR machine 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 was completed, the reaction product was placed on ice and 40 μL of mixed enzyme I, 2 μL of mixed enzyme II (from DNBSEQ sequencing kit), 1 μL of ATP (100 mM stock solution, obtained from Thermo Fisher) and 0.1 μL of T4 ligase (obtained from NEB, catalog number: M0202S) were added. After mixing the wells, the above reaction system was placed in a PCR machine and reacted at 30°C for 20 minutes to generate DNB.

[0199] [Table 2]

[0200] (2) The DNBs were then loaded onto a SEQ500 sequencing chip (purchased from MGI) using a SEQ500 SE50 kit set (purchased from MGI, 1000012551).

[0201] For sequencing chips, MDA reagent from the PE50 sequencing kit (purchased from MGI, 1000012554) was added and incubated at 37° C. for 30 minutes, then the chip was washed with 5× SSC.

[0202] (3) The surface of the chip was modified with N3-PEG3500-NHS (modification reagent purchased from Sigma, catalog number: JKA5086). After 30 minutes of incubation, the DBCO-modified primer (sequence shown in SEQ ID NO: 3) for chip sequence synthesis was injected thereon and incubated at room temperature overnight.

[0203] 3. Sequencing and Decoding of Positional Sequence Information: Sequencing was performed according to the instructions of the BGISEQ500 SE50 sequencing kit, and the SE read length was set to 25 bp. The fq file generated by sequencing was saved for later use.

[0204] 4. Extension of consensus sequence X2 (SEQ ID NO: 5) of chip sequence: Based on step 3 above, the cPAS reaction of 13 bases was continued to obtain a chip sequence (SEQ ID NO: 8, consisting of consensus sequence X1 (SEQ ID NO: 4), tag sequence Y and consensus sequence X2 (SEQ ID NO: 5)).

[0205] 5. Chip cutting: The prepared chip was cut into several small pieces. The size of the small pieces was adjusted according to the experimental needs. The chip was immersed in 50 mM tris buffer, pH 8.0 at 4°C for later use.

[0206] Example 2: In situ synthesis of cDNA 1. cDNA Synthesis Mouse tissue sections were prepared according to standard methods for frozen sections, and the frozen sections were attached to the chips prepared in Example 1. After fixation with frozen methanol for 30 minutes, the tissues were permeabilized using 0.5% triton x-100. The chips were washed twice at room temperature using 5X SSC, and 200 μL of a reverse transcriptase reaction system as shown in Table 3 was prepared, and the reaction solution was added to the chip so as to completely cover it, and the reaction was carried out at 42 ° C for 90 to 180 minutes. Synthesis of cDNA was carried out by reverse transcriptase using mRNA as a template and a polyT-containing primer (the sequence of which is shown in SEQ ID NO: 6 and was composed of a consensus sequence A (CA), a UMI sequence, and a polyT sequence), and a CCC overhang was added to the 3'-end of the cDNA strand. After hybridization and annealing (via complementary pairing between the GGG at the end of the TSO sequence and the CCC overhang of the cDNA strand) of the TSO sequence (SEQ ID NO: 7, composed of consensus sequence B(CB) and a GGG overhang) with the cDNA strand, the cDNA strand was continuously extended by reverse transcriptase using consensus sequence B as a template, resulting in labeling of the 3'-end of the cDNA with a c(CB) tag (the complementary sequence of consensus sequence B).

[0207] [Table 3]

[0208] The synthesized cDNA strand was composed of the following sequence: reverse transcription primer sequence (SEQ ID NO:6)-cDNA sequence-c(TSO) sequence (complementary sequence of SEQ ID NO:7).

[0209] 2. Ligation of Sequencing Chip with cDNA of ChIP Sequence After cDNA synthesis, the chip was washed twice with 5X SSC, 1 mL of the reaction system as shown in Table 4 was prepared, and the appropriate volume was injected into the chip to ensure that the chip was filled with the following ligation reaction solution, and the reaction was carried out at room temperature for 30 minutes.

[0210] By the above reaction, the 5'-end of the cDNA sequence was ligated to the 3'-end of the single-cell sequencing chip sequence (i.e., the 5'-end of the cDNA sequence was labeled with the chip sequence) to obtain a new nucleic acid molecule containing positional information (i.e., tag sequence Y), which was composed of the following sequence structure: chip sequence (sequence number 8)-reverse transcription primer sequence (sequence number 6)-cDNA sequence-c(TSO) sequence (complementary sequence of sequence number 7).

[0211] After the reaction was completed, the chip was washed with 5X SSC. 200 μL of Bst polymerization reaction solution (NEB, M0275S) was prepared according to the manufacturer's instructions, injected into the chip, and reacted at 65° C. for 60 minutes to obtain a single-stranded nucleic acid molecule having positional information.

[0212] [Table 4]

[0213] 3. Release of cDNA The chip was incubated with 75 μL of 80 mM KOH for 5 min at room temperature. After collecting the liquid, 10 μL of 1 M, pH 8.0 Tris-HCl was added to neutralize the cDNA recovery solution.

[0214] 4. Amplification of cDNA 200 μL of the reaction system shown in Table 5 was prepared for 3' transcriptome sequencing and library construction, respectively, and divided into two tubes for PCR:

[0215] [Table 5]

[0216] The above reaction system was placed in a PCR machine, and the reaction program was set as follows: 95°C for 3 min, 11 cycles (98°C for 20 sec, 58°C for 20 sec, 72°C for 3 min), 72°C for 5 min, 4°C for ∞. After the reaction was completed, XP beads (purchased from AMPure) were used for magnetic bead-based purification and recovery. The dsDNA concentration was quantified using a Qubit instrument, and the length distribution of the cDNA amplification products was detected using a 2100 Bioanalyzer (purchased from Agilent). The detection results were shown in Figure 6.

[0217] Example 3: cDNA library construction and sequencing 1.Tn5 fragmentation According to the cDNA concentration, 20ng of cDNA (obtained in step 4 of Example 2) was taken, 0.5μM of Tn5 transposase and corresponding buffer solution (purchased from BGI, Catalog No: 10000028493, coating of Tn5 transposase was operated according to the instruction of Stereomics Library Preparation Kit-S1) were added, mixed well to obtain 20μL of reaction system, and the reaction was carried out at 55℃ for 10 minutes, then 5μL of 0.1% SDS was added, mixed well at room temperature for 5 minutes to terminate Tn5 transposition.

[0218] 2. PCR Amplification 100 μL of the following reaction mixture was prepared: [Table 6]

[0219] After mixing, it was placed in a PCR machine and the following program was set: 95°C for 3 min, 11 cycles (98°C for 20 sec, 58°C for 20 sec, 72°C for 3 min), 72°C for 5 min, 4°C infinity. After the reaction was completed, XP beads were used for magnetic bead-based purification and recovery. The dsDNA concentration was quantified using a Qubit instrument.

[0220] 3. Sequencing 80 fmol of the above fragmented amplification product was taken out to prepare DNB. A 40 μL reaction system was prepared as follows: [Table 7]

[0221] The above reaction volume was placed in a PCR machine for reaction, and the reaction conditions were as follows: 95°C for 3 minutes, 40°C for 3 minutes. After the reaction was completed, the resulting reaction solution was placed on ice, and 40μL of mixed enzyme I, 2μL of mixed enzyme II, 1μL of ATP and 0.1μL of T4 ligase, which are required for DNB preparation of DNBSEQ sequencing kit, were added. After mixing, the above reaction system was placed in a PCR machine and reacted at 30°C for 20 minutes to form DNB.

[0222] According to the method described in the PE50 kit supporting MGISEQ 2000, the DNB was loaded onto the sequencing chip of MGISEQ2000, and sequencing was performed according to the related instruction manual. The PE50 sequencing model was selected, in which the first strand sequencing was divided into two sections of sequencing, 25bp was measured first, then 15 cycles of dark reaction were performed, then 10bp UMI sequence was measured, and the second strand sequencing was set up to measure 50bp.

[0223] Example 4: Data Analysis By alignment, the 25bp sequence of the first strand obtained by cDNA sequencing was matched with the fq of the chip positioning sequence (sequencing result in Example 1), and the 25bp matching reads in the two sequences were extracted for subsequent analysis. For the 25bp matching reads in the two sequences, the second strand was analyzed, and the sequence of the second strand was aligned with the mouse genome to extract unique mapping reads.

[0224] The unique mapping reads were mapped to spatial locations on the chip to obtain a mouse brain gene expression map as shown in FIG. 7, and when FIG. 7 is expanded to DNB level (i.e., nanometer level) resolution (FIG. 8), it can be seen that there is clear aggregation of single cells of the tissue sample, indicating that the method of the present application reduced RNA diffusion and enabled spatial transcriptome detection, reaching nanometer level resolution.

[0225] Statistics on the average number of genes and UMIs captured per 40,000 DNBs showed that an average of approximately 4,000 genes and 10,000 mRNA molecules were captured, indicating that the method of the present application was able to capture a sufficient number of genes and mRNA molecules for downstream analysis.

[0226] Although the specific embodiments of the present application are described in detail, those skilled in the art will understand that various modifications and changes can be made in the details based on all the technologies disclosed, and all these modifications are within the scope of protection of the present application. The full scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. 1. A method for generating a population of labeled nucleic acid molecules, comprising the steps of: (1) providing a biological sample and a nucleic acid array, the nucleic acid array comprising a solid support, the solid support being bound to a plurality of types of oligonucleotide probes, each type of oligonucleotide probe comprising at least one copy, the oligonucleotide probes comprising or consisting of, in a 5' to 3' direction, a consensus sequence X1, a tag sequence Y and a consensus sequence X2; each type of oligonucleotide probe has a different tag sequence Y, said tag sequence Y having a nucleotide sequence unique to the location on said solid support of that type of oligonucleotide probe; (2) contacting the biological sample with the nucleic acid array, whereby the locations of RNA (e.g., mRNA) in the biological sample are mapped to the locations of the oligonucleotide probes on the nucleic acid array, and pre-processing the RNA (e.g., mRNA) in the biological sample to generate a first population of nucleic acid molecules, the pre-processing comprising: (i) performing reverse transcription of the RNA (e.g., mRNA) of the biological sample using primer A to generate extension products as labeled first nucleic acid molecules, thereby generating the first population of nucleic acid molecules, wherein the primer A comprises a consensus sequence A and a capture sequence A, the capture sequence A is capable of annealing to the RNA (e.g., mRNA) to be captured to initiate an extension reaction, and the consensus sequence A is located upstream of the capture sequence A (e.g., located at the 5'-end of the primer A); or (ii) (a) performing reverse transcription of the RNA (e.g., mRNA) of the biological sample using primer A to generate a cDNA strand, the cDNA strand being generated by reverse transcription primed by primer A and comprising a cDNA sequence that is complementary to the RNA (e.g., mRNA) and a 3′-end overhang, the primer A comprising a consensus sequence A and a capture sequence A, the capture sequence A being capable of annealing to the RNA (e.g., mRNA) to be captured to initiate an extension reaction, the consensus sequence A being upstream of the capture sequence A (e.g., at the 5′-end of the primer A). and (b) annealing primer I-B with the cDNA strand generated in (a) and performing an extension reaction to generate a first extension product as a first nucleic acid molecule to be labeled, thereby generating a first population of nucleic acid molecules, wherein primer B comprises consensus sequence B, a complementary sequence of the 3'-end overhang, and optionally a tag sequence B, wherein the complementary sequence of the 3'-end overhang is located at the 3'-end of primer B and the consensus sequence B is located upstream of the complementary sequence of the 3'-end overhang (e.g., located at the 5'-end of primer B); or (iii) (a) performing reverse transcription of the RNA (e.g., mRNA) of the biological sample using primer A' to generate a cDNA strand, the cDNA strand being generated by reverse transcription primed by primer A' and comprising a cDNA sequence complementary to the RNA (e.g., mRNA) and a 3'-end overhang, the primer A' comprising a capture sequence A, the capture sequence A being capable of annealing to the RNA (e.g., mRNA) to be captured to initiate an extension reaction; (b) annealing primer B to the cDNA strand generated in (a) and performing an extension reaction to generate a first cDNA strand. generating a first population of extension products, wherein the primer B comprises a consensus sequence B, a complementary sequence of the 3'-end overhang, and optionally a tag sequence B, the complementary sequence of the 3'-end overhang being located at the 3'-end of the primer B, and the consensus sequence B being located upstream of the complementary sequence of the 3'-end overhang (e.g., at the 5'-end of the primer B); and (c) providing an extension primer and performing an extension reaction using the first extension product as a template to generate a second extension product as a labeled first nucleic acid molecule, thereby generating a first population of nucleic acid molecules. The steps include: (3) contacting a bridging oligonucleotide with the product of step (2) under conditions that allow annealing, allowing the bridging oligonucleotide to anneal (e.g., in situ anneal) with the oligonucleotide probe and the labeled first nucleic acid molecule at a position corresponding to the oligonucleotide probe, and ligating the first nucleic acid molecule annealed with the bridging oligonucleotide and the oligonucleotide probe on the array to obtain a ligation product as a second nucleic acid molecule having a positioning tag, thereby generating a second population of nucleic acid molecules. Including, the bridging oligonucleotide comprises 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 upstream of the second region (e.g., 5' of the second region); the first region is capable of annealing to all or a portion of the consensus sequence A of the primer A in step (2)(i) or step (2)(ii), or is capable of annealing to all or a portion of the consensus sequence B of the primer B in step (2)(iii); The second region is capable of annealing to all or a portion of the consensus sequence X2.

2. In step (3), when the first region and the second region of the bridging oligonucleotide are immediately adjacent, ligating the first nucleic acid molecule to the oligonucleotide probe comprises using a nucleic acid ligase to ligate a nucleic acid molecule hybridized to the first region and a nucleic acid molecule hybridized to the second region of the same bridging oligonucleotide to obtain a ligation product as a second nucleic acid molecule having a positioning tag; or 2. The method of claim 1, wherein when the bridging oligonucleotide comprises the first region, the second region and the third region located therebetween, ligating the first nucleic acid molecule to the oligonucleotide probe comprises: performing a polymerization reaction using a nucleic acid polymerase with the third region as a template; and ligating the nucleic acid molecule hybridized to the first region of the same bridging oligonucleotide with the nucleic acid molecule hybridized to the third region and the second region using a nucleic acid ligase to obtain a ligation product as the second nucleic acid molecule having a positioning tag, wherein preferably the nucleic acid polymerase does not have 5' to 3' exonuclease activity or strand displacement activity.

3. 3. The method of claim 1 or 2, comprising steps (1), (2)(i) and (3), wherein the ligation product obtained in step (3) is taken as the second nucleic acid molecule having a positioning tag comprising, from 5' to 3', the consensus sequence X1, the tag sequence Y, the consensus sequence X2, optionally a complementary sequence of the third region of the bridging oligonucleotide, and the sequence of the first nucleic acid molecule to be labeled.

4. In step (2)(i), the capture sequence A is a random oligonucleotide sequence; Preferably, in step (3), the ligation products derived from each copy of the same type of oligonucleotide probe have different capture sequences A, and the capture sequences A serve as unique molecular identifiers (UMIs) of the second nucleic acid molecules; The method of claim 3, wherein the extension product (the first nucleic acid molecule to be labeled) in step (2)(i) preferably comprises, from 5' to 3', the consensus sequence A, a cDNA sequence generated by reverse transcription primed by the primer A and complementary to an RNA.

5. In step (2)(i), the capture sequence A is a poly(T) sequence or a specific sequence of a target nucleic acid; Preferably, the primer A further comprises a tag sequence A, e.g., a random oligonucleotide sequence, wherein the tag sequence A serves as a unique molecular identifier (UMI) of the second nucleic acid molecule; Preferably, 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 (e.g., at the 5'-end of the primer A), Preferably, in step (3), the ligation products derived from each copy of the same type of oligonucleotide probe have different tag sequences A as UMIs; 4. The method of claim 3, wherein the extension product in step (2)(i) preferably comprises, from 5' to 3', the consensus sequence A, the tag sequence A, and a cDNA sequence generated by reverse transcription primed by primer A and complementary to RNA.

6. 3. The method of claim 1, comprising steps (1), (2)(ii) and (3), wherein the ligation product obtained in step (3) is taken as the second nucleic acid molecule having a positioning tag comprising, from 5' to 3', the consensus sequence X1, the tag sequence Y, the consensus sequence X2, optionally a complementary sequence of the third region of the bridging oligonucleotide, and a sequence of the first nucleic acid molecule to be labeled.

7. In step (2)(ii)(a), the capture sequence A is a random oligonucleotide sequence; Preferably, in step (3), the ligation products derived from each copy of the same type of oligonucleotide probe have different capture sequences A, and the capture sequences A serve as unique molecular identifiers (UMIs) of the second nucleic acid molecules; The method of claim 6, wherein the first extension product (first nucleic acid molecule to be labeled) in step (2)(ii) preferably comprises, from 5' to 3', the consensus sequence A, a cDNA sequence generated by reverse transcription primed by the primer A and complementary to RNA, the 3'-end overhang sequence, optionally a complementary sequence of the tag sequence B, and a complementary sequence of the consensus sequence B.

8. In step (2)(ii)(a), the capture sequence A is a poly(T) sequence or a specific sequence of a target nucleic acid; Preferably, the primer A further comprises a tag sequence A, e.g., a random oligonucleotide sequence, wherein the tag sequence A serves as a unique molecular identifier (UMI) of the second nucleic acid molecule; Preferably, 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 (e.g., at the 5'-end of the primer A), Preferably, in step (3), the ligation products derived from each copy of the same type of oligonucleotide probe have different tag sequences A as UMIs; The method of claim 6, wherein the first extension product (first nucleic acid molecule to be labeled) in step (2)(ii) preferably comprises, from 5' to 3', the consensus sequence A, the tag sequence A, a cDNA sequence generated by reverse transcription primed by the primer A and complementary to RNA, the 3'-end overhang sequence, optionally a complementary sequence of the tag sequence B, and a complementary sequence of the consensus sequence B.

9. The method of any one of claims 3 to 8, wherein said primer A comprises a 5' phosphate at the 5'-end.

10. 10. The method of any one of claims 3 to 9, further comprising the step of treating the product of step (2)(i) or step (2)(ii) to remove RNA prior to step (3).

11. 3. The method of claim 1 or 2, comprising steps (1), (2)(iii) and (3), wherein the ligation product obtained in step (3) is taken as the second nucleic acid molecule having a positioning tag comprising, from 5' to 3', the consensus sequence X1, the tag sequence Y, the consensus sequence X2, optionally a complementary sequence of the third region of the bridging oligonucleotide, and a sequence of the first nucleic acid molecule sequence to be labeled.

12. In step (2)(iii)(c), the extension primer is primer B or primer B', and primer B' is capable of annealing to all or a part of the complementary sequence of consensus sequence B to initiate an extension reaction; The method of claim 11, wherein, preferably, in step (2)(iii)(c), the extension primer is primer B'.

13. In step (2)(iii)(a), the capture sequence A of the primer A' is a random oligonucleotide sequence; Preferably, in step (2)(iii)(b), the primer B comprises the consensus sequence B, a complementary sequence of the 3'-end overhang and the tag sequence B; Preferably, the first extension product comprises, from 5' to 3', a cDNA sequence generated by reverse transcription primed by the primer A', which is complementary to an RNA sequence, the 3'-end overhang sequence, a complementary sequence of the tag sequence B, and a complementary sequence of the consensus sequence B, wherein the complementary sequence of the tag sequence B serves as a unique molecular identifier (UMI) of the second nucleic acid molecule; The method according to claim 11 or 12, wherein, in step (2)(iii)(c), the second extension product (first nucleic acid molecule to be labeled) comprises, from 5' to 3', the consensus sequence B or a 3'-terminal partial sequence thereof, the tag sequence B, a complementary sequence of the 3'-terminal overhang sequence, and a complementary sequence of the cDNA sequence in the first extension product, and the tag sequence B serves as a unique molecular identifier (UMI) of the second nucleic acid molecule.

14. In step (2)(iii)(a), the capture sequence A of the primer A' is a poly(T) sequence or a specific sequence of a target nucleic acid; Preferably, the primer A' further comprises a tag sequence A, e.g., a random oligonucleotide sequence, and a consensus sequence A; Preferably, the capture sequence A is located at the 3'-end of the primer A'; Preferably, the consensus sequence A is located upstream of the capture sequence A (e.g., at the 5'-end of the primer A'), Preferably, the primer B comprises a consensus sequence B, a complementary sequence of the 3'-end overhang and a tag sequence B; Preferably, in step (2)(iii)(b), the first extension product comprises, from 5' to 3', the consensus sequence A, optionally the tag sequence A, a cDNA sequence generated by reverse transcription primed by the primer A' and complementary to RNA, the 3'-end overhang sequence, a complementary sequence of the tag sequence B and a complementary sequence of the consensus sequence B; Preferably, in step (2)(iii)(c), the second extension product (first nucleic acid molecule to be labeled) comprises, from 5' to 3', the consensus sequence B or a 3'-terminal partial sequence thereof, the tag sequence B, a complementary sequence of the 3'-terminal overhang sequence, a complementary sequence of the cDNA sequence in the first extension product, optionally a complementary sequence of the tag sequence A, and a complementary sequence of the consensus sequence A; The method according to claim 11 or 12, wherein in step (3), the ligation products derived from each copy of the same type of oligonucleotide probe have different tag sequences B as UMIs.

15. The method of any one of claims 11 to 14, wherein the extension primer comprises a 5' phosphate at the 5'-terminus.

16. 16. The method of any one of claims 11 to 15, further comprising the step of treating the product of step (2)(iii)(a) or step (2)(iii)(b) to remove RNA prior to step (2)(iii)(c).

17. 17. The method of any one of claims 11 to 16, wherein in step (2)(iii)(b), the cDNA strand is annealed to the primer B via its 3'-end overhang, and the cDNA strand is extended using the primer B as a template in the presence of a nucleic acid polymerase (e.g., a DNA polymerase or a reverse transcriptase) to generate a first extension product.

18. 18. The method of any one of claims 11 to 17, wherein the 3'-end overhang has a length of 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, preferably wherein the 3'-end overhang is a 3'-end overhang of 2 to 5 cytosine nucleotides (e.g. a CCC overhang).

19. The method according to any one of claims 1 to 18, wherein in step (2), the biological sample is permeabilized prior to the pretreatment.

20. the biological sample is a tissue sample, Preferably, the tissue sample is a tissue section, The method according to any one of claims 1 to 19, wherein the tissue sections are preferably prepared from fixed tissue, such as formalin-fixed paraffin-embedded (FFPE) tissue or flash-frozen tissue.

21. In step (2), the reverse transcription is carried out using a reverse transcriptase; Preferably, the reverse transcriptase has terminal deoxynucleotidyl transferase activity, Preferably, the reverse transcriptase is capable of synthesizing a cDNA strand using RNA (e.g., mRNA) as a template and adding an overhang to the 3'-end of the cDNA strand; Preferably, the reverse transcriptase is capable of adding an overhang having a length of 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 to the 3'-end of the cDNA strand; Preferably, the reverse transcriptase is capable of adding an overhang of 2 to 5 cytosine nucleotides (e.g. a CCC overhang) to the 3'-end of the cDNA strand; The method according to any one of claims 1 to 20, wherein 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 variants, modified products and derivatives thereof having the reverse transcription activity of the above reverse transcriptases.

22. The method according to any one of claims 1 to 21, wherein steps (2) and (3) have one or more features selected from the following: (1) the primer A, primer A', primer B and bridging oligonucleotide each independently comprise or consist of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides or any combination thereof, and preferably the primer A and primer A' are capable of initiating an extension reaction; (2) The primer B contains a modified nucleotide (e.g., a locked nucleic acid), and preferably, the primer B contains one or more modified nucleotides (e.g., a locked nucleic acid) at the 3'-end; (3) The tag sequence A and the tag sequence B each independently have a length of 5 to 200 nt (e.g., 5 to 30 nt, 6 to 15 nt); (4) The consensus sequence A and the consensus sequence B each independently have a length of 10 to 200 nt (e.g., 10 to 100 nt, 20 to 100 nt, 25 to 100 nt, 5 to 10 nt, 10 to 15 nt, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt); (5) The primer A, primer A', and primer B each independently have a length of 10 to 200 nt (e.g., 10 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt, 100 to 150 nt, or 150 to 200 nt); (6) The first and second regions of the bridging oligonucleotide each independently have a length of 3 to 100 nt (e.g., 3 to 10 nt, 10 to 15 nt, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt); (7) The third region of the bridging oligonucleotide has a length of 0 to 100 nt (e.g., 0 to 10 nt, 10 to 15 nt, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt), (8) The bridging oligonucleotide has a length of 6 to 200 nt (e.g., 20 to 70 nt, 6 to 15 nt, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt, 100 to 150 nt, 150 to 200 nt), (9) The poly(T) sequence comprises at least 10 or at least 20 (e.g., at least 30) deoxythymidine residues; (10) The random oligonucleotide sequence has a length of 5 to 200 nt (e.g., 5 to 30 nt, 6 to 15 nt).

23. (4) The method of any one of claims 1 to 22, further comprising the step of recovering and purifying the second population of nucleic acid molecules.

24. The method of any one of claims 1 to 23, wherein the obtained second population of nucleic acid molecules and / or their complements are used for constructing a transcriptome library or for transcriptome sequencing.

25. The method of any one of claims 1 to 24, wherein the oligonucleotide probe in step (1) has one or more characteristics selected from the following: (1) The consensus sequence X1, tag sequence Y, and consensus sequence X2 each independently comprise natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides (e.g., peptide nucleic acid (PNA) or locked nucleic acid), or any combination thereof, and preferably, the consensus sequence X2 has a free hydroxyl group (—OH) at the 3′-terminus; (2) The consensus sequence X1, tag sequence Y, and consensus sequence X2 each independently have a length of 2 to 100 nt (e.g., 10 to 200 nt, 10 to 100 nt, 20 to 100 nt, 25 to 100 nt, 50 to 100 nt, 5 to 30 nt, 6 to 15 nt, 5 to 10 nt, 10 to 15 nt, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt); (3) The oligonucleotide probes each independently have a length of 15 to 200 nt (e.g., 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt, 100 to 150 nt, 150 to 200 nt).

26. the oligonucleotide probe is attached to the solid support via a linker; Preferably, the linker is a linking group capable of binding to an activating group, and the surface of the solid support is modified with the activating group; Preferably, the linker comprises -SH, -DBCO or -NHS; Preferably, the linker is -DBCO and the surface of the solid support is 【Chemistry 1】 The method according to any one of claims 1 to 25, which is modified with (Azido-dPEG®8-NHS ester).

27. 27. The method of any one of claims 1 to 26, wherein the nucleic acid array of step (1) has one or more characteristics selected from the following: (1) The oligonucleotide probes bound to the same solid support have the same consensus sequence X1 and / or the same consensus sequence X2; (2) The consensus sequence X1 of the oligonucleotide probe comprises a cleavage site, and preferably, the cleavage site can be cleaved or destroyed by a method selected from nicking enzyme digestion, USER enzyme digestion, light-responsive excision, chemical excision, or CRISPR-mediated excision.

28. The nucleic acid array of step (1) (1) providing a plurality of types of carrier sequences, each of the carrier sequences comprising at least one copy of the carrier sequence, the carrier sequence comprising, in a 5' to 3' direction, a complementary sequence of the consensus sequence X2, a complementary sequence of the tag sequence Y, and an immobilization sequence, the complementary sequences of the tag sequence Y of the various carrier sequences being different from each other; (2) binding the plurality of types of carrier sequences to the surface of the solid support (e.g., a chip); (3) providing an immobilized primer and performing a primer extension reaction using the carrier sequence as a template to generate an extension product to obtain the oligonucleotide probe, wherein the immobilized primer comprises the sequence of the consensus sequence X1 and is capable of annealing to the immobilized sequence of the carrier sequence and initiating an extension reaction, and preferably the extension product comprises or consists of, in the 5' to 3' direction, the consensus sequence X1, the tag sequence Y and the consensus sequence X2; (4) coupling the immobilized primer to the surface of the solid support, wherein steps (3) and (4) are performed in any order; (5) Optionally, the immobilized sequence of the carrier sequence further comprises a cleavage site, and the cleavage may be selected from the group consisting of nicking enzyme digestion, USER enzyme digestion, light-responsive excision, chemical excision and CRISPR-mediated excision, performing cleavage at the cleavage site contained in the immobilized sequence of the carrier sequence to digest the carrier sequence, thereby allowing the extension product in step (3) to be separated from the template (i.e., the carrier sequence) from which the extension product is generated, thereby linking the oligonucleotide probe to the surface of the solid support (e.g., a chip); The method includes the steps of: Preferably, the various carrier sequences are DNBs formed from concatemers of multiple copies of said carrier sequences, Preferably, the plurality of types of carrier sequences are prepared by the following steps: (i) providing a plurality of carrier-template sequences, the carrier-template sequences including complementary sequences of carrier sequences; (ii) performing a nucleic acid amplification reaction using the various carrier-template sequences as templates to obtain amplification products of the various carrier-template sequences, said amplification products comprising at least one copy of said carrier sequences, preferably performing rolling circle replication to obtain DNBs formed from concatemers of said carrier sequences; The method according to any one of claims 1 to 27, wherein in step (1) the

29. The method according to any one of claims 1 to 28, wherein the solid support in step (1) has one or more characteristics selected from the following: (1) The solid support is selected from the group consisting of latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes, and silicon wafers, and preferably, the solid support is a chip; (2) The solid support is flat, spherical or porous; (3) The solid support can be used as a sequencing platform, for example, a sequencing chip, and preferably, the solid support is a sequencing chip for Illumina, MGI or Thermo Fisher sequencing platforms; and (4) The solid support is capable of releasing the oligonucleotide probes spontaneously or upon exposure to one or more stimuli (e.g., temperature change, pH change, exposure to certain chemicals or phases, exposure to light, exposure to a reducing agent, etc.).

30. 1. A method for constructing a library of nucleic acid molecules, comprising: (a) generating a population of labeled nucleic acid molecules by a method according to any one of claims 1 to 29; (b) randomly fragmenting the nucleic acid molecules in the population of labeled nucleic acid molecules and adding adaptors; and (c) optionally amplifying and / or enriching the product of step (b); thereby obtaining a library of nucleic acid molecules, Preferably, the method, wherein said library of nucleic acid molecules is used for sequencing, e.g. transcriptome sequencing, e.g. single cell transcriptome sequencing.

31. Prior to carrying out step (b), the method further comprises the step (pre-b): amplifying and / or concentrating the population of labeled nucleic acid molecules; Preferably, in step (pre-b), the population of labeled nucleic acid molecules is subjected to a nucleic acid amplification reaction to generate an amplification product; Preferably, the nucleic acid amplification reaction is carried out using at least a primer C and / or a primer D, the primer C being capable of hybridizing or annealing to a complementary sequence of the consensus sequence X1 or a complementary sequence of the 3'-end sequence of the consensus sequence X1 and initiating an extension reaction, and the primer D being capable of hybridizing or annealing to the nucleic acid molecule of the labeled nucleic acid molecule population and initiating an extension reaction; 31. The method of claim 30, wherein the nucleic acid amplification reaction in step (pre-b) is preferably carried out using a nucleic acid polymerase (e.g. a DNA polymerase, e.g. a DNA polymerase with strand displacement activity and / or high fidelity).

32. In step (b), the nucleic acid molecules obtained in the previous step are randomly fragmented into fragments and adapters are added to both ends of the fragments using a transposase; Preferably, the transposase is selected from the group consisting of Tn5 transposase, MuA transposase, Sleeping Beauty transposase, Mariner transposase, Tn7 transposase, Tn10 transposase, Ty1 transposase and Tn552 transposase, as well as variants, modified products and derivatives thereof having the transposition activity of the above transposases; Preferably, the transposase is a Tn5 transposase; 32. The method of claim 31, wherein, preferably, in step (c), at least primer C' and / or primer D' are used to amplify the product of step (b), and the adapters at both ends of the fragment are a first adapter and a second adapter, respectively, and the primer C' is capable of hybridizing or annealing to the first adapter to initiate an extension reaction, and the primer D' is capable of hybridizing or annealing to the second adapter to initiate an extension reaction.

33. 1. A method for sequencing a nucleic acid sample, comprising: (1) constructing a nucleic acid molecule library by the method according to any one of claims 30 to 32; and (2) sequencing the library of nucleic acid molecules; The method includes:

34. (i) a nucleic acid array for labeling nucleic acids, comprising a solid support, the solid support being bound to a plurality of types of oligonucleotide probes, each type of oligonucleotide probe comprising at least one copy, the oligonucleotide probes comprising or consisting of, in the 5' to 3' direction, a consensus sequence X1, a tag sequence Y and a consensus sequence X2; a nucleic acid array, each type of oligonucleotide probe having a different tag sequence Y, said tag sequence Y having a nucleotide sequence unique to the location on said solid support of that type of oligonucleotide probe; (ii) a primer set comprising primer A, or primer A′ and primer B, or a primer set comprising primer A and primer B, The primer A comprises a consensus sequence A and a capture sequence A, and the capture sequence A is capable of annealing to an RNA (e.g., an mRNA) to be captured and initiating an extension reaction, and preferably the consensus sequence A is located upstream of the capture sequence A (e.g., located at the 5'-end of the primer A); the primer A' comprises a capture sequence A, which is capable of annealing to the RNA (e.g., mRNA) to be captured and initiating an extension reaction; a primer set comprising primer A, or primer A' and primer B, or primer set comprising primer A and primer B, wherein primer B comprises a consensus sequence B, a complementary sequence of a 3'-end overhang, and optionally a tag sequence B, and preferably the complementary sequence of the 3'-end overhang is located at the 3'-end of primer B, and preferably the consensus sequence B is located upstream of the complementary sequence of the 3'-end overhang (e.g., located at the 5'-end of primer B), and the 3'-end overhang refers to one or more non-template nucleotides contained at the 3'-end of a cDNA strand generated by reverse transcription using the RNA captured by the capture sequence A of primer A' as a template; and (iii) a bridging oligonucleotide comprising a first region and a second region, and optionally a third region located between the first region and the second region, wherein the first region is located upstream of the second region (e.g., 5' of the second region); the first region is capable of (a) annealing to all or a portion of the consensus sequence A of the primer A, or (b) annealing to all or a portion of the consensus sequence B of the primer B; the second region being capable of annealing to all or a portion of the consensus sequence X2; Including the kit.

35. A nucleic acid array for labeling a nucleic acid according to (i), comprising a primer A according to (ii) and a bridging oligonucleotide according to (iii), wherein the first region of the bridging oligonucleotide is capable of annealing to the whole or a part of the consensus sequence A of the primer A, and the second region of the bridging oligonucleotide is capable of annealing to the whole or a part of the consensus sequence X2; Preferably, the capture sequence A of the primer A is a random oligonucleotide sequence; Preferably, the capture sequence A of the primer A is a poly(T) sequence or a specific sequence of a target nucleic acid, preferably, the primer A further comprises a tag sequence A, e.g., a random oligonucleotide sequence, preferably, 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 (e.g., at the 5'-end of the primer A); The kit of claim 34, wherein the primer A preferably comprises a 5' phosphate at the 5'-end.

36. A nucleic acid array for labeling a nucleic acid according to (i), a primer set comprising primer A' and primer B according to (ii), and a bridging oligonucleotide according to (iii), wherein the first region of the bridging oligonucleotide is capable of annealing to the whole or a part of the consensus sequence B of primer B, and the second region of the bridging oligonucleotide is capable of annealing to the whole or a part of the consensus sequence X2; Preferably, the capture sequence A of the primer A' is a random oligonucleotide sequence; Preferably, the capture sequence A of the primer A' is a poly(T) sequence or a specific sequence of a target nucleic acid, preferably, the primer A' further comprises a tag sequence A and a consensus sequence A, preferably, the capture sequence A is located at the 3'-end of the primer A', preferably, the consensus sequence A is located upstream of the capture sequence A (e.g., at the 5'-end of the primer A'); Preferably, the primer B comprises a consensus sequence B, a complementary sequence of the 3'-end overhang and a tag sequence B; Preferably, the kit further comprises a primer B', the primer B' being capable of annealing to the whole or part of the complementary sequence of the consensus sequence B to initiate an extension reaction; Preferably, said primer B or primer B' comprises a 5' phosphate at the 5'-end, The kit of claim 34, wherein preferably said primer B comprises a modified nucleotide (e.g. a locked nucleic acid), preferably said primer B comprises one or more modified nucleotides (e.g. a locked nucleic acid) at the 3'-end.

37. A nucleic acid array for labeling a nucleic acid according to (i), a primer comprising primer A and primer B according to (ii), and a bridging oligonucleotide according to (iii), wherein the first region of the bridging oligonucleotide is capable of annealing to the whole or a part of the consensus sequence A of the primer A, and the second region of the bridging oligonucleotide is capable of annealing to the whole or a part of the consensus sequence X2; Preferably, the capture sequence A of the primer A is a random oligonucleotide sequence; Preferably, the capture sequence A of the primer A is a poly(T) sequence or a specific sequence of a target nucleic acid, preferably, the primer A further comprises a tag sequence A, e.g., a random oligonucleotide sequence, preferably, 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 (e.g., at the 5'-end of the primer A); Preferably, said primer A comprises a 5' phosphate at the 5'-end, The kit of claim 34, wherein preferably said primer B comprises a modified nucleotide (e.g. a locked nucleic acid), preferably said primer B comprises one or more modified nucleotides (e.g. a locked nucleic acid) at the 3'-end.

38. The kit according to any one of claims 34 to 37, having one or more features selected from the following: (1) the oligonucleotide probe, primer A, primer A', primer B, primer B' and bridging oligonucleotide each independently comprise or consist of natural nucleotides (e.g., deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleotides or any combination thereof, preferably the primer A, primer A' and primer B' are capable of initiating an extension reaction, and preferably the consensus sequence X2 has a free hydroxyl group (-OH) at the 3'-terminus; (2) The oligonucleotide probes each independently have a length of 15 to 300 nt (e.g., 15 to 200 nt, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt, 100 to 150 nt, 150 to 200 nt); (3) The primer A, primer A', primer B and primer B' each independently have a length of 10 to 200 nt (e.g., 10 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt, 100 to 150 nt, 150 to 200 nt); (4) The bridging oligonucleotide has a length of 6 to 200 nt (e.g., 20 to 70 nt, 6 to 15 nt, 15 to 20 nt, 20 to 30 nt, 30 to 40 nt, 40 to 50 nt, 50 to 100 nt, 100 to 150 nt, 150 to 200 nt); (5) The oligonucleotide probes bound to the same solid support have the same consensus sequence X1 and / or the same consensus sequence X2; (6) The consensus sequence X1 of the oligonucleotide probe comprises a cleavage site, and preferably, the cleavage site can be cleaved or destroyed by a method selected from nicking enzyme digestion, USER enzyme digestion, light-responsive excision, chemical excision, or CRISPR-mediated excision.

39. further comprising a reverse transcriptase, a nucleic acid ligase, a nucleic acid polymerase and / or a transposase; Preferably, said reverse transcriptase has terminal deoxynucleotidyl transferase activity, preferably said reverse transcriptase is capable of synthesizing a cDNA strand using RNA (e.g., mRNA) as a template and adding a 3'-end overhang to the 3'-end of said cDNA strand, preferably said reverse transcriptase is capable of adding an overhang having a length of 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 to the 3'-end of said cDNA strand, preferably said reverse transcriptase is capable of adding an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang) to the 3'-end of said cDNA strand, preferably said reverse transcriptase is selected from the group consisting of M-MLV reverse transcriptase, HIV-1 reverse transcriptase, AMV reverse transcriptase, telomerase reverse transcriptase, and variants, modified products and derivatives thereof having the reverse transcription activity of the above reverse transcriptases, Preferably, the nucleic acid polymerase does not have 5' to 3' exonuclease activity or strand displacement activity, The kit according to any one of claims 34 to 38, wherein the transposase is selected from the group consisting of Tn5 transposase, MuA transposase, Sleeping Beauty transposase, Mariner transposase, Tn7 transposase, Tn10 transposase, Ty1 transposase, Tn552 transposase, and variants, modified products and derivatives thereof having the transposase activity.

40. The kit of any one of claims 34 to 39, further comprising reagents for nucleic acid hybridization, for nucleic acid extension, for nucleic acid amplification and for recovering or purifying nucleic acids, for constructing a transcriptome sequencing library, for sequencing (e.g., second or third generation sequencing), or any combination thereof.

41. Use of the method according to any one of claims 1 to 29 or the kit according to any one of claims 34 to 40 for constructing a nucleic acid molecule library or for performing transcriptome sequencing.

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