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WO2025189364A1 - Adapter addition method and use thereof in preparation of high-throughput sequencing library - Google Patents

Adapter addition method and use thereof in preparation of high-throughput sequencing library

Info

Publication number
WO2025189364A1
WO2025189364A1 PCT/CN2024/081295 CN2024081295W WO2025189364A1 WO 2025189364 A1 WO2025189364 A1 WO 2025189364A1 CN 2024081295 W CN2024081295 W CN 2024081295W WO 2025189364 A1 WO2025189364 A1 WO 2025189364A1
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WO
WIPO (PCT)
Prior art keywords
enzyme
function
activity
double
stranded dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/081295
Other languages
French (fr)
Chinese (zh)
Inventor
杨林
普珺
张艳艳
杨非一
石可可
张韶红
陈恬
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MGI Tech Co Ltd
Original Assignee
MGI Tech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MGI Tech Co Ltd filed Critical MGI Tech Co Ltd
Priority to PCT/CN2024/081295 priority Critical patent/WO2025189364A1/en
Publication of WO2025189364A1 publication Critical patent/WO2025189364A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the present invention belongs to the field of nucleic acid sequencing and relates to a method for adding adapters and its application in preparing a high-throughput sequencing library, and more particularly to a method for adding adapters using an enzyme having terminal transfer activity and/or function and its application in preparing a high-throughput sequencing library.
  • the enzyme is used to add nucleotide fragment 1 to the 3' end of each strand of the double-stranded DNA fragment;
  • the nucleotide fragment 1 is reverse complementary to the nucleotide fragment 2 at the 3' end of the linker, so that the linker is bound to the double-stranded DNA fragment to obtain a double-stranded DNA fragment with the linker added.
  • the nucleotide fragment 1 consists of N1 nucleotides
  • the nucleotide fragment 2 consists of N2 nucleotides
  • N1 and N2 are each independently selected from a natural number between 1 and 10.
  • N1 and N2 are each independently selected from a natural number between 2 and 5.
  • N1 is 3 and N2 is 3.
  • the nucleotides in the nucleotide fragment 2 are ribonucleotides, deoxyribonucleotides or modified nucleotides.
  • the nucleotides in the nucleotide fragment 2 are ribonucleotides.
  • the nucleotide segment 1 is CCC; preferably, the nucleotide segment 2 is rGrGrG. It should be noted that the rGrGrG represents three consecutive ribonucleotides G.
  • the binding affinity of the RNA/DNA hybrid chain is higher than that of the RNA/RNA hybrid chain and the DNA/DNA hybrid chain.
  • the nucleotides in the nucleotide segment 2 are deoxyribonucleotides.
  • the nucleotide segment 1 is CCC; preferably, the nucleotide segment 2 is GGG.
  • the nucleotides in the nucleotide fragment 2 are modified nucleotides.
  • the modified nucleotides are locked nucleic acids.
  • the nucleotide adjacent to the 5' end of nucleotide fragment 2 is N.
  • the N represents A, T, C, or G.
  • the 5' end of the linker has a blocking structure or blocking modification for the purpose of blocking nucleic acid extension.
  • the blocking structure is a hairpin structure.
  • the blocking modification is selected from: dideoxycytidine modification, reverse dt modification, phosphate group modification, thio group modification, or spacer modification.
  • the blocking modification is a spacer modification.
  • the spacer modification is a carbon backbone modification or a sugar backbone modification.
  • the spacer modification is Spacer C3 (i.e., 3 CH 2 ), Spacer C6 (i.e., 6 CH 2 ), or Spacer C9 (i.e., 9 CH 2 ), etc.
  • the linker comprises a functional segment located downstream of the blocking structure or blocking modification.
  • the functional segment comprises a sample tag and/or a molecular tag and/or a sequencing primer.
  • the 5' end is generally considered to be upstream and the 3' end is considered to be downstream.
  • the linker comprises an enzyme cleavage site, and the enzyme cleavage site is located downstream of the blocking structure or blocking modification.
  • the enzyme cleavage site is used to remove the blocking modification or blocking structure by enzyme cleavage in a subsequent step.
  • the enzyme used for the enzyme cleavage is UDG Enzyme, USER Enzyme or RnaseH, etc.
  • the enzyme cleavage site is adjacent to the blocking structure or blocking modification.
  • the enzyme cleavage site and the blocking structure or blocking modification are separated by N3 nucleotides, and N3 is a natural number.
  • N3 is selected from a natural number of 1-10.
  • N3 is selected from a natural number of 1-5.
  • N3 is 1 or 2 or 3.
  • the enzyme cleavage site is selected from: dUTP or rNTP.
  • the enzyme having terminal transfer activity and/or function has a preference.
  • the preference refers to a preference for adding three specific nucleotides to the 3' end of a strand.
  • the preference refers to a greater than 20% probability of adding three specific nucleotides to the 3' end of a strand.
  • the three specific nucleotides are three C nucleotides.
  • linker refers to a single-stranded oligonucleotide or a single-stranded oligonucleotide derivative.
  • a single-stranded oligonucleotide refers to a single-stranded oligonucleotide of 3-100 nt in length. The 5' end of the single-stranded oligonucleotide has a blocking structure for the purpose of blocking nucleic acid extension.
  • a single-stranded oligonucleotide derivative is obtained by adding a blocking modification having the purpose of blocking nucleic acid extension to the 5' end of the single-stranded oligonucleotide.
  • the linker has various forms. According to an embodiment of the present invention, the linker has the following segments from the 5' end to the 3' end in sequence: blocking modification (inter-arm modification), U, sample tag, GGG (as shown in a of Figure 1). According to an embodiment of the present invention, the linker has the following segments from the 5' end to the 3' end in sequence: blocking modification (inter-arm modification), U, sample tag, GGG (as shown in b of Figure 1). As shown). According to an embodiment of the present invention, the linker has the following sections from the 5' end to the 3' end in sequence: blocking modification (spacer modification), sample tag, GGG (as shown in c of Figure 1).
  • the linker has the following sections from the 5' end to the 3' end in sequence: blocking modification (spacer modification), GGG (as shown in d of Figure 1). According to an embodiment of the present invention, the linker has the following sections from the 5' end to the 3' end in sequence: blocking structure (hairpin structure), U, sample tag, GGG (as shown in e of Figure 1). According to an embodiment of the present invention, the linker has the following sections from the 5' end to the 3' end in sequence: blocking structure (hairpin structure), U, GGG (as shown in f of Figure 1).
  • the linker has the following sections from the 5' end to the 3' end in sequence: blocking structure (hairpin structure), sample tag, GGG (as shown in g of Figure 1). According to an embodiment of the present invention, the linker has the following sections from the 5' end to the 3' end in sequence: blocking structure (hairpin structure), GGG (as shown in h of Figure 1).
  • the method for adding adapters to blunt-ended double-stranded DNA fragments further comprises a template extension step, wherein the template extension is to extend the double-stranded DNA fragments to which the adapters are added in a 5' to 3' direction using the adapters as a template.
  • the template extension is performed by an enzyme having DNA polymerization activity and/or function.
  • the enzyme having terminal transfer activity and/or function is the same enzyme as the enzyme having DNA polymerization activity and/or function.
  • the enzyme having terminal transfer activity and/or function is different from the enzyme having DNA polymerization activity and/or function.
  • the enzyme having DNA polymerization activity and/or function is a reverse transcriptase having terminal transfer activity.
  • the reverse transcriptase having terminal transfer activity is murine leukemia reverse transcriptase (M-MLV).
  • M-MLV murine leukemia reverse transcriptase
  • AMV avian leukemia reverse transcriptase
  • the reverse transcriptase having terminal transfer activity is SuperScript TM IV reverse transcriptase, SuperScript TM I reverse transcriptase, SuperScript TM II reverse transcriptase, or SuperScript TM III reverse transcriptase.
  • the enzyme having DNA polymerization activity and/or function is a Klenow fragment. According to an embodiment of the present invention, the enzyme having DNA polymerization activity and/or function is T4 DNA polymerase.
  • the enzyme having terminal transfer activity and/or function is a reverse transcriptase having terminal transfer activity.
  • the reverse transcriptase having terminal transfer activity is murine leukemia reverse transcriptase (M-MLV).
  • M-MLV murine leukemia reverse transcriptase
  • AMV avian leukemia reverse transcriptase
  • the reverse transcriptase having terminal transfer activity is SuperScript TM IV reverse transcriptase, SuperScript TM I reverse transcriptase, SuperScript TM II reverse transcriptase, or SuperScript TM III reverse transcriptase.
  • the connector may be connector 1 or connector 2.
  • the linker 1 is a single-stranded oligonucleotide derivative, and its structural formula is as follows: Linker 1 is obtained by performing a blocking modification on the 5' end of single-stranded oligonucleotide 1; the blocking modification is to connect three nucleotide A's via iSp6 (Spacer C6, i.e., six CH2 groups ); single-stranded oligonucleotide 1 is shown to the right of iSp6 in the structural formula (SEQ ID NO: 1); in single-stranded oligonucleotide 1, the third position from the 5' end is a modified base U, and positions 1 to 3 from the 3' end are rG (i.e., ribonucleic acid G), and the fourth position is N (N represents A ⁇ T ⁇ C ⁇ G).
  • the linker 2 is a single-stranded oligonucleotide derivative, and its structural formula is as follows:
  • the linker 2 is obtained by performing blocking modification on the 5' end of the oligonucleotide single strand 2; the blocking modification is to connect the three nucleotide A's via iSp6 (Spacer C6, i.e., 6 CH2 ); the oligonucleotide single strand Chain 2 is shown to the right of iSp6 in the structural formula (SEQ ID NO: 2).
  • the first position from the 5' end is a modified base U
  • positions 1 to 3 from the 3' end are rG (i.e., ribonucleic acid G)
  • the fourth position is N (N represents A ⁇ T ⁇ C ⁇ G).
  • the underlined portion is a sample tag (which is only an exemplary one; the sample tag is used to label the sample and can be any combination of nucleotides).
  • the connector may be connector 3 or connector 4.
  • linker 3 is a single-stranded oligonucleotide, and the sequence is as follows: The 5' end of linker 3 has a blocking structure (hairpin structure, see wavy underline mark), and the first to third positions from the 3' end are rG (i.e., ribonucleic acid G) and the fourth position is N (N represents A ⁇ T ⁇ C ⁇ G). Linker 3 is shown in SEQ ID NO: 3.
  • linker 4 is a single-stranded oligonucleotide with the following sequence:
  • the 5' end of linker 4 has a blocking structure (hairpin structure, see wavy underline mark), and the first to third positions from the 3' end are rG (i.e., ribonucleic acid G) and the fourth position is N (N represents A ⁇ T ⁇ C ⁇ G).
  • Linker 4 is shown in SEQ ID NO: 4.
  • the reaction system for adding a linker to a blunt-ended double-stranded DNA fragment contains: a blunt-ended double-stranded DNA fragment, a linker, and an enzyme having terminal transfer activity and/or function.
  • the reaction system for adding a linker to a blunt-ended double-stranded DNA fragment further contains: dNTP and a reaction buffer.
  • the reaction system for adding a linker to a blunt-ended double-stranded DNA fragment further contains: betaine and MgCl 2.
  • the linker may be linker 3 and linker 4.
  • the enzyme having terminal transfer activity and/or function may be SuperScript TM IV reverse transcriptase.
  • the reaction buffer may be 5 ⁇ Superscript IV first-strand buffer.
  • the reaction system for adding a linker to a blunt-ended double-stranded DNA fragment may be as shown in Table 8.
  • the reaction conditions for adding adapters to blunt-end double-stranded DNA fragments are: reaction at 40-45°C for 40-80 min, and then reaction at 65-75°C for 5-15 min.
  • the reaction conditions for adding adapters to blunt-end double-stranded DNA fragments are: reaction at 42°C for 60 min, and then reaction at 70°C for 10 min.
  • the reaction conditions for adding adapters to blunt-end double-stranded DNA fragments are: reaction at 40-45°C for 80-100 min.
  • the reaction conditions for adding adapters to blunt-end double-stranded DNA fragments are: reaction at 42°C for 90 min.
  • DNA is purified using XP beads.
  • DNA is purified using 30 ⁇ L of XP beads, and the purified DNA is dissolved in 20 ⁇ L of TE buffer to form the product solution.
  • the present invention also provides a method for repairing the ends of double-stranded DNA fragments and adding linkers, which comprises the following steps:
  • the method for adding a linker to a blunt-ended double-stranded DNA fragment comprises the following steps: contacting the blunt-ended double-stranded DNA fragment with an enzyme having terminal transfer activity and/or function and a linker; wherein the enzyme is used to add a nucleotide fragment 1 to the 3' end of each chain of the double-stranded DNA fragment; the nucleotide fragment 1 is reversely complementary to the nucleotide fragment 2 at the 3' end of the linker, so that the linker binds to the double-stranded DNA fragment, thereby obtaining a double-stranded DNA fragment with an added linker.
  • the method for adding a linker to a blunt-ended double-stranded DNA fragment is as described above in "Adding a linker to a blunt-ended double-stranded DNA fragment" Any of the methods described in "Methods for adding adapters to fragments".
  • processes 1 and 2 are completed in the same system.
  • the double-stranded DNA fragment is end-repaired with the aid of an enzyme having 5' ⁇ 3' polymerization and 3' ⁇ 5' exonuclease activity and/or function.
  • the enzyme having 5' ⁇ 3' polymerization and 3' ⁇ 5' exonuclease activity and/or function is a Klenow fragment.
  • the enzyme having 5' ⁇ 3' polymerization and 3' ⁇ 5' exonuclease activity and/or function is T4 DNA polymerase.
  • the present invention further provides a method for fragmenting, end-repairing and adding linkers to double-stranded DNA molecules, which comprises the following steps:
  • the method for adding a linker to a blunt-ended double-stranded DNA fragment comprises the following steps: contacting the blunt-ended double-stranded DNA fragment with an enzyme having terminal transfer activity and/or function and a linker; wherein the enzyme is used to add a nucleotide fragment 1 to the 3' end of each chain of the double-stranded DNA fragment; the nucleotide fragment 1 is reversely complementary to the nucleotide fragment 2 at the 3' end of the linker, so that the linker binds to the double-stranded DNA fragment, thereby obtaining a double-stranded DNA fragment with an added linker.
  • the method for adding a linker to a blunt-ended double-stranded DNA fragment is as described in any one of the above “methods for adding a linker to a blunt-ended double-stranded DNA fragment”.
  • processes 1, 2 and 3 are completed in the same system.
  • the double-stranded DNA fragment is end-repaired with the aid of an enzyme having 5' ⁇ 3' polymerization and 3' ⁇ 5' exonuclease activity and/or function.
  • the enzyme having 5' ⁇ 3' polymerization and 3' ⁇ 5' exonuclease activity and/or function is a Klenow fragment.
  • the enzyme having 5' ⁇ 3' polymerization and 3' ⁇ 5' exonuclease activity and/or function is T4 DNA polymerase.
  • the method for fragmenting the double-stranded DNA molecule is selected from: physical fragmentation or enzymatic fragmentation.
  • the enzymatic fragmentation is performed with the aid of an enzyme having endonuclease activity and/or function.
  • the enzyme having endonuclease activity and/or function is DNase I or Endonuclease V.
  • the reaction system of the "method for fragmenting, end-repairing, and adding adapters to double-stranded DNA molecules” contains: double-stranded DNA molecules, an enzyme with endonucleolytic activity and/or function, an enzyme with 5' ⁇ 3' polymerization and 3' ⁇ 5' exonucleolytic activity and/or function, an enzyme with terminal transfer activity and/or function, and a adapter.
  • the reaction system of the "method for fragmenting, end-repairing, and adding adapters to double-stranded DNA molecules” further contains: dNTPs and a reaction buffer.
  • the reaction system of the "method for fragmenting, end-repairing, and adding adapters to double-stranded DNA molecules" further contains: betaine and MgCl2 .
  • the adapters may be Adapter 1 and Adapter 2.
  • the adapters may be Adapter 3 and Adapter 4.
  • the enzyme with endonucleolytic activity and/or function is DNase I or Endonuclease V.
  • the enzyme with 5' ⁇ 3' polymerization and 3' ⁇ 5' exonucleolytic activity and/or function is a Klenow fragment.
  • the enzyme having terminal transfer activity and/or function may be SuperScript TM IV reverse transcriptase.
  • the reaction buffer may be 5 ⁇ Superscript IV first-strand buffer.
  • the reaction system of the “method for fragmenting, end-repairing and adding linkers to double-stranded DNA molecules” may be as shown in Table 1.
  • the “method for fragmenting, end-repairing and adding linkers to double-stranded DNA molecules” may be as shown in Table 1.
  • the reaction system of the "method for fragmenting double-stranded DNA molecules, repairing ends, and adding adapters" can be shown in Table 4.
  • the reaction conditions of the "method for fragmenting, end-repairing and adding adapters to double-stranded DNA molecules" are as follows: reaction at T1°C for 1 minute, reaction at T2°C for 2 minutes, reaction at T3°C for 3 minutes, and reaction at T4°C for 4 minutes, wherein T1: 16-40°C, T2: 55-65°C, T3: 16-55°C, T4: 55-75°C, t1 : 10-60 minutes, t2 : 10-30 minutes, t3 : 10-60 minutes, and t4 : 10-30 minutes.
  • T1 is 37°C
  • T2 is 60°C
  • T3 is 42°C
  • T4 is 70°C
  • t1 is 10 minutes
  • t2 is 10 minutes
  • t3 60 minutes
  • t4 is 10 minutes.
  • the present invention also protects the use of any of the above methods in preparing nucleic acid libraries or high-throughput sequencing.
  • the present invention also protects a method for preparing a nucleic acid library, which comprises step (1).
  • Step (1) is to obtain double-stranded DNA fragments with adapters added.
  • step (1) comprises: contacting a blunt-ended double-stranded DNA fragment with an enzyme having terminal transfer activity and/or function and a linker; wherein the enzyme is used to add a nucleotide fragment 1 to the 3' end of each chain of the double-stranded DNA fragment; wherein the nucleotide fragment 1 is reversely complementary to the nucleotide fragment 2 at the 3' end of the linker, so that the linker binds to the double-stranded DNA fragment, thereby obtaining a double-stranded DNA fragment to which a linker is added.
  • the step (1) is as described in any one of the above first aspects of the "method for adding linkers to blunt-ended double-stranded DNA fragments".
  • step (1) comprises the following process:
  • the method for adding a linker to a blunt-ended double-stranded DNA fragment comprises the following steps: contacting the blunt-ended double-stranded DNA fragment with an enzyme having terminal transfer activity and/or function and a linker; wherein the enzyme is used to add a nucleotide fragment 1 to the 3' end of each chain of the double-stranded DNA fragment; the nucleotide fragment 1 is reversely complementary to the nucleotide fragment 2 at the 3' end of the linker, so that the linker binds to the double-stranded DNA fragment, thereby obtaining a double-stranded DNA fragment with an added linker.
  • processes 1 and 2 are completed in the same system.
  • the step (1) is as described in any one of the second aspects of the above “method for repairing the ends of double-stranded DNA fragments and adding linkers”.
  • step (1) comprises the following process:
  • the method for adding a linker to a blunt-ended double-stranded DNA fragment comprises the following steps: contacting the blunt-ended double-stranded DNA fragment with an enzyme having terminal transfer activity and/or function and a linker; wherein the enzyme is used to add a nucleotide fragment 1 to the 3' end of each chain of the double-stranded DNA fragment; the nucleotide fragment 1 is reversely complementary to the nucleotide fragment 2 at the 3' end of the linker, so that the linker binds to the double-stranded DNA fragment, thereby obtaining a double-stranded DNA fragment with an added linker.
  • processes 1, 2 and 3 are completed in the same system.
  • the step (1) is as described in any one of the third aspects above, "a method for fragmenting, end-repairing and adding linkers to double-stranded DNA molecules".
  • the method for preparing a nucleic acid library further comprises step (2): taking the product of step (1), performing nick filling and blocking excision; the nick filling is performed with the aid of an enzyme having DNA ligation activity and/or function; the blocking excision is performed with the aid of an enzyme having the activity and/or function of specifically cutting the restriction site on the linker.
  • the enzyme having DNA ligation activity and/or function is T4 DNA ligase, T3 Taqligase or T7 Taqligase, etc.
  • the reaction system of step (2) contains: the product of step (1), an enzyme having DNA ligation activity and/or function, and an enzyme having activity and/or function of specifically cutting the enzyme cleavage site on the connector.
  • the reaction system of step (2) further contains: a reaction buffer.
  • the enzyme having DNA ligation activity and/or function is T4 DNA ligase.
  • the enzyme having activity and/or function of specifically cutting the enzyme cleavage site on the connector is USER Enzyme.
  • the reaction buffer is T4 DNA Ligase Reaction Buffer.
  • the reaction system of step (2) may be as shown in Table 3.
  • the reaction conditions of step (2) are 30-45° C. for 10-30 min. According to an embodiment of the present invention, the reaction conditions of step (2) are 37° C. for 15 min.
  • the method for preparing a nucleic acid library further comprises the following step (2): taking the product of step (1), performing nick filling and PCR amplification; the nick filling is performed with the aid of an enzyme having DNA ligation activity and/or function; the PCR amplification is performed with the aid of an enzyme having DNA polymerization activity and/or function; the target sequence of the primer pair used in the PCR amplification corresponds to the downstream of the blocking structure or the blocking modification.
  • the step (2) is completed in the same system.
  • the enzyme having DNA ligation activity and/or function is T4 DNA ligase, T3 Taqligase or T7 Taqligase, etc.
  • the enzyme having DNA polymerization activity and/or function is Tth DNA Polymerase.
  • the primers used in the PCR amplification comprise a functional segment, and the functional segment contains a sample tag and/or a molecular tag and/or a sequencing primer.
  • the primer pair used in the PCR amplification consists of universal primer 1 and index primer 1.
  • the sequence of universal primer 1 is as follows: 5'(phos)-GAACGACATGGCTACGATCCGACTT-3'.
  • the 5' end of universal primer 1 has a phosphorylation modification for matching the subsequent sequencing platform.
  • Universal primer 1 is shown in SEQ ID NO: 5.
  • the reaction system of step (2) contains: the product of step (1), an enzyme having DNA ligation activity and/or function, an enzyme having DNA polymerization activity and/or function, and a primer.
  • the reaction system of step (2) further contains: dNTP and reaction buffer.
  • the reaction system of step (2) further contains: Mg(OAc) 2.
  • the primers are the universal primer 1 and the tag primer 1.
  • the enzyme having DNA ligation activity and/or function is T4 DNA ligase.
  • the enzyme having DNA polymerization activity and/or function is Tth DNA Polymerase.
  • the reaction buffer is 5 ⁇ Tth RT-PCR Buffer.
  • the reaction system of step (2) may be as shown in Table 6.
  • the reaction conditions of step (2) are as follows: in the first stage, the reaction is carried out at T5°C for t5 minutes; in the second stage, the reaction is carried out at T6°C for t6 seconds, T7°C for t7 seconds, and T8°C for t8 seconds, for X cycles; in the third stage, the reaction is carried out at T9°C for t9 minutes.
  • T5 94-98°C
  • T6 94-98°C
  • T7 50-65°C
  • T8 65-75°C
  • T9 65-75°C
  • t5 0.5-2 minutes
  • t6 5-30 seconds
  • t7 10-60 seconds
  • t8 10-60 seconds
  • t9 72° C
  • t5 2 minutes
  • t6 10 seconds
  • t7 is 30 seconds
  • t8 is 30 seconds
  • t9 is 5 minutes
  • X is 10.
  • DNA is purified using XP beads.
  • DNA is purified using 40 ⁇ L of XP beads and the purified DNA is dissolved in 30 ⁇ L of TE buffer to prepare the nucleic acid library.
  • the present invention further provides a kit comprising the following components:
  • Enzymes and linkers having terminal transfer activity and/or function having terminal transfer activity and/or function
  • the enzyme is used to add nucleotide fragment 1 to the 3' end of each strand of a blunt-ended double-stranded DNA fragment;
  • the nucleotide fragment 1 is reverse complementary to the nucleotide fragment 2 at the 3' end of the linker.
  • the nucleotide fragment 1 consists of N1 nucleotides
  • the nucleotide fragment 2 consists of N2 nucleotides
  • N1 and N2 are each independently selected from a natural number between 1 and 10.
  • N1 and N2 are each independently selected from a natural number between 2 and 5.
  • N1 is 3 and N2 is 3.
  • the nucleotides in the nucleotide segment 2 are deoxyribonucleotides.
  • the nucleotide segment 1 is CCC; preferably, the nucleotide segment 2 is GGG.
  • the nucleotides in the nucleotide fragment 2 are modified nucleotides.
  • the modified nucleotides are locked nucleic acids.
  • the nucleotide adjacent to the 5' end of nucleotide fragment 2 is N.
  • the N represents A, T, C, or G.
  • the 5' end of the linker has a blocking structure or blocking modification for the purpose of blocking nucleic acid extension.
  • the blocking structure is a hairpin structure.
  • the linker comprises a functional segment, and the functional segment is located downstream of the blocking structure or blocking modification.
  • the functional segment comprises a sample tag and/or a molecular tag and/or a sequencing primer.
  • the linker comprises an enzyme cleavage site, and the enzyme cleavage site is located downstream of the blocking structure or blocking modification.
  • the enzyme cleavage site is used to remove the blocking modification or blocking structure by enzyme cleavage in a subsequent step.
  • the enzyme used for the enzyme cleavage is UDG Enzyme, USER Enzyme or RnaseH, etc.
  • the enzyme cleavage site is adjacent to the blocking structure or blocking modification.
  • the enzyme cleavage site and the blocking structure or blocking modification are separated by N3 nucleotides, and N3 is a natural number.
  • N3 is selected from a natural number of 1-10.
  • N3 is selected from a natural number of 1-5.
  • N3 is 1 or 2 or 3.
  • the enzyme cleavage site is selected from: dUTP or rNTP.
  • the enzyme having terminal transfer activity and/or function has a preference.
  • the preference refers to a preference for adding three specific nucleotides to the 3' end of a strand.
  • the preference refers to a greater than 20% probability of adding three specific nucleotides to the 3' end of a strand.
  • the three specific nucleotides are three C nucleotides.
  • the connector has various forms. According to an embodiment of the present invention, the connector has the following sections from the 5’ end to the 3’ end in sequence: blocking modification (inter-arm modification), U, sample tag, GGG (as shown in a of Figure 1). According to an embodiment of the present invention, the connector has the following sections from the 5’ end to the 3’ end in sequence: blocking modification (inter-arm modification), U, GGG (as shown in b of Figure 1). According to an embodiment of the present invention, the connector has the following sections from the 5’ end to the 3’ end in sequence: blocking modification (inter-arm modification), sample tag, GGG (as shown in c of Figure 1).
  • the connector has the following sections from the 5’ end to the 3’ end in sequence: blocking modification (inter-arm modification), GGG (as shown in d of Figure 1).
  • the connector has the following sections from the 5’ end to the 3’ end in sequence: blocking structure (hairpin structure), U, sample tag, GGG (as shown in e of Figure 1).
  • the connector has the following sections from the 5' end to the 3' end: blocking structure (hairpin structure), U, GGG (as shown in Figure 1 f).
  • the connector has the following sections from the 5' end to the 3' end: blocking structure (hairpin structure), sample tag, GGG (as shown in Figure 1 g). According to an embodiment of the present invention, the connector has the following sections from the 5' end to the 3' end: blocking structure (hairpin structure), GGG (as shown in Figure 1 h).
  • the enzyme having terminal transfer activity and/or function is a reverse transcriptase having terminal transfer activity.
  • the reverse transcriptase having terminal transfer activity is murine leukemia reverse transcriptase (M-MLV).
  • M-MLV murine leukemia reverse transcriptase
  • AMV avian leukemia reverse transcriptase
  • the reverse transcriptase having terminal transfer activity is SuperScript TM IV reverse transcriptase, SuperScript TM I reverse transcriptase, SuperScript TM II reverse transcriptase, or SuperScript TM III reverse transcriptase.
  • the connector may be connector 1 or connector 2.
  • the linker 1 is a single-stranded oligonucleotide derivative, and its structural formula is as follows: Linker 1 is obtained by performing a blocking modification on the 5' end of single-stranded oligonucleotide 1; the blocking modification is to connect three nucleotide A's via iSp6 (Spacer C6, i.e., six CH2 groups ); single-stranded oligonucleotide 1 is shown to the right of iSp6 in the structural formula (SEQ ID NO: 1); in single-stranded oligonucleotide 1, the third position from the 5' end is a modified base U, and positions 1 to 3 from the 3' end are rG (i.e., ribonucleic acid G), and the fourth position is N (N represents A ⁇ T ⁇ C ⁇ G).
  • the linker 2 is a single-stranded oligonucleotide derivative, and its structural formula is as follows: Linker 2 is obtained by performing a blocking modification on the 5' end of oligonucleotide single strand 2; the blocking modification is to connect three nucleotides A via iSp6 (Spacer C6, i.e., six CH2 groups ); oligonucleotide single strand 2 is shown to the right of iSp6 in the structural formula (SEQ ID NO: 2); in oligonucleotide single strand 2, the first position from the 5' end is a modified base U, the first to third positions from the 3' end are rG (i.e., ribonucleic acid G), and the fourth position is N (N represents A ⁇ T ⁇ C ⁇ G), and the underlined portion is a sample tag (only an exemplary one, the sample tag is used to label the sample and can be any combination of nucleotides).
  • iSp6 Spacer C
  • the connector may be connector 3 or connector 4.
  • linker 3 is a single-stranded oligonucleotide, and the sequence is as follows: The 5' end of linker 3 has a blocking structure (hairpin structure, see wavy underline mark), and the first to third positions from the 3' end are rG (i.e., ribonucleic acid G) and the fourth position is N (N represents A ⁇ T ⁇ C ⁇ G). Linker 3 is shown in SEQ ID NO: 3.
  • linker 4 is a single-stranded oligonucleotide with the following sequence:
  • the 5' end of linker 4 has a blocking structure (hairpin structure, see wavy underline mark), and the first to third positions from the 3' end are rG (i.e., ribonucleic acid G) and the fourth position is N (N represents A ⁇ T ⁇ C ⁇ G).
  • Linker 4 is shown in SEQ ID NO: 4.
  • kit further comprises the following components: a component for template extension;
  • the template extension is to extend the double-stranded DNA fragment with the adapter added in the 5' to 3' direction using the adapter as a template.
  • the component for template extension is an enzyme having DNA polymerization activity and/or function.
  • the enzyme having DNA polymerization activity and/or function is a reverse transcriptase having terminal transfer activity.
  • the reverse transcriptase having terminal transfer activity is murine leukemia reverse transcriptase (M-MLV).
  • M-MLV murine leukemia reverse transcriptase
  • AMV avian leukemia reverse transcriptase
  • the reverse transcriptase having terminal transfer activity is SuperScript TM IV reverse transcriptase, SuperScript TM I reverse transcriptase, SuperScript TM II reverse transcriptase, or SuperScript TM III reverse transcriptase.
  • the enzyme having DNA polymerization activity and/or function is a Klenow fragment. According to an embodiment of the present invention, the enzyme having DNA polymerization activity and/or function is T4 DNA polymerase.
  • the kit is used to add adapters to blunt-ended double-stranded DNA fragments.
  • the kit also includes the following components: a component for performing end repair on double-stranded DNA fragments.
  • the component for end repair of double-stranded DNA fragments is an enzyme having 5' ⁇ 3' polymerization and 3' ⁇ 5' exonucleolytic activities and/or functions.
  • the enzyme having 5' ⁇ 3' polymerization and 3' ⁇ 5' exonuclease activity and/or function is a Klenow fragment.
  • the enzyme having 5' ⁇ 3' polymerization and 3' ⁇ 5' exonuclease activity and/or function is a Klenow fragment.
  • the functional enzyme is T4 DNA polymerase.
  • the enzyme having DNA polymerization activity and/or function is a Klenow fragment. According to an embodiment of the present invention, the enzyme having DNA polymerization activity and/or function is T4 DNA polymerase.
  • the kit is used for repairing the ends of double-stranded DNA fragments and adding adapters.
  • the kit also includes the following components: a component for fragmenting double-stranded DNA molecules and a component for end-repairing double-stranded DNA fragments.
  • the components for fragmenting double-stranded DNA molecules are components required for physical shearing or components required for enzymatic shearing.
  • the component for fragmenting double-stranded DNA molecules is an enzyme having endonuclease activity and/or function.
  • the enzyme having endonuclease activity and/or function is DNase I or Endonuclease V.
  • the component for end repair of double-stranded DNA fragments is an enzyme having 5' ⁇ 3' polymerization and 3' ⁇ 5' exonucleolytic activity and/or function.
  • the enzyme having 5' ⁇ 3' polymerization and 3' ⁇ 5' exonucleolytic activity and/or function is a Klenow fragment.
  • the enzyme having 5' ⁇ 3' polymerization and 3' ⁇ 5' exonucleolytic activity and/or function is T4 DNA polymerase.
  • the kit is used for fragmenting, end-repairing and adding linkers to double-stranded DNA molecules.
  • the kit also includes the following components: a component for nick filling and a component for blocking excision.
  • the component for nick filling is an enzyme having DNA ligation activity and/or function.
  • the enzyme having DNA ligation activity and/or function is T4 DNA ligase, T3 Taqligase, or T7 Taqligase.
  • the component for blocking excision is an enzyme having the activity and/or function of specifically cleaving the cleavage site on the linker.
  • the enzyme having the activity and/or function of specifically cleaving the cleavage site on the linker is USER Enzyme or UDG enzyme, etc.
  • the kit also includes the following components: a component for nick filling and a component for PCR amplification.
  • the component for PCR amplification includes an enzyme having DNA polymerization activity and/or function.
  • the enzyme having DNA polymerization activity and/or function is Tth DNA Polymerase.
  • the component for PCR amplification further comprises a primer pair for PCR amplification.
  • the target sequence of the primer pair for PCR amplification corresponds to the downstream of the blocking structure or the blocking modification.
  • the primers for PCR amplification comprise a functional segment containing a sample tag and/or a molecular tag and/or a sequencing primer.
  • the primer pair for PCR amplification consists of a universal primer 1 and a tag primer 1.
  • the sequence of universal primer 1 is as follows: 5'(phos)-GAACGACATGGCTACGATCCGACTT-3'.
  • the 5' end of universal primer 1 has a phosphorylated Modified to match the subsequent sequencing platform.
  • Universal primer 1 is shown in SEQ ID NO: 5.
  • the sequence of the tag primer 1 is as follows: In the tag primer 1, the underlined portion is the sample tag (which is only an exemplary one, and the sample tag is used to label the sample and can be any nucleotide combination).
  • the tag primer 1 is shown in SEQ ID NO: 6.
  • the kit is used for preparing a nucleic acid library.
  • the present invention also protects a nucleic acid library prepared by any of the above methods for preparing a nucleic acid library.
  • the present invention also protects the use of the nucleic acid library in high-throughput sequencing.
  • the present invention discloses a method for connecting adapters using an enzyme having terminal transfer activity and/or function and its application in preparing high-throughput sequencing libraries.
  • the present invention provides a method for adding adapters to blunt-end double-stranded DNA fragments, contacting the blunt-end double-stranded DNA fragments with an enzyme having terminal transfer activity and/or function and an adapter; the enzyme is used to add a nucleotide fragment 1 to the 3' end of each chain of the double-stranded DNA fragment; the nucleotide fragment 1 is reversely complementary to the nucleotide fragment 2 at the 3' end of the adapter, so that the adapter is bound to the double-stranded DNA fragment to obtain a double-stranded DNA fragment with an added adapter.
  • FIG2 is a schematic flow diagram of Example 1.
  • FIG3 is a schematic flow diagram of Example 2.
  • FIG4 is a schematic flow diagram of Example 3.
  • FIG5 is a schematic diagram showing the process comparison of Example 1 and Example 2 and the comparative example.
  • the following examples are for better understanding of the present invention, but they do not limit the present invention.
  • the experimental methods in the following examples are all conventional methods unless otherwise specified.
  • the test materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.
  • the quantitative tests in the following examples were repeated three times, and the results were averaged.
  • the linkers in the examples are single-stranded oligonucleotides or single-stranded oligonucleotide derivatives. Single-stranded oligonucleotides refer to single-stranded oligonucleotides with a length of 3-100nt.
  • XP beads Agencourt AMPure XP magnetic beads from Beckman, catalog number A63881.
  • DNBSEQ-G400 DNBSEQ-G400 (also known as MGISEQ-2000) high-throughput gene sequencing platform manufactured by BGI.
  • dNTP solution Deoxynucleotide (dNTP) Solution Mix; NEB, product number N0447V.
  • SuperScript TM IV reverse transcriptase (liquid reagent; specification: 200 U/ ⁇ L): ThermoFisher, catalog number: 18090010.
  • 5 ⁇ Superscript IV first-strand buffer is the 5 ⁇ RT buffer sold in conjunction with SuperScript TM IV reverse transcriptase.
  • DNase I solution is prepared by diluting DNase I to 1000 times the volume with DNase I Reaction Buffer.
  • DNase I liquid reagent; specification: 2000 units/ml
  • DNase I Reaction Buffer NEB, catalog number M0303S.
  • Klenow fragment (liquid reagent; specification: 10 U/ ⁇ L): ThermoFisher, product number: EP0051.
  • T4 DNA Ligase liquid reagent; specification: 400,000 units/ml
  • Tth DNA Polymerase liquid reagent; specification: 5U/ ⁇ L
  • 5 ⁇ Tth RT-PCR Buffer Yishen Company, product number 14607ES72.
  • Linker 1 is a single-stranded oligonucleotide derivative with the following structural formula:
  • Linker 2 is a single-stranded oligonucleotide derivative with the following structural formula:
  • Linker 1 is obtained by performing a blocking modification on the 5' end of single-stranded oligonucleotide 1; the blocking modification is to connect three nucleotide A's via iSp6 (Spacer C6, i.e., six CH2 groups ); single-stranded oligonucleotide 1 is shown to the right of iSp6 in the structural formula (SEQ ID NO: 1); in single-stranded oligonucleotide 1, the third position from the 5' end is a modified base U, and positions 1 to 3 from the 3' end are rG (i.e., ribonucleic acid G), and the fourth position is N (N represents A ⁇ T ⁇ C ⁇ G).
  • Linker 2 is obtained by performing a blocking modification on the 5' end of oligonucleotide single strand 2; the blocking modification is to connect three nucleotides A via iSp6 (Spacer C6, i.e., six CH2 groups ); oligonucleotide single strand 2 is shown to the right of iSp6 in the structural formula (SEQ ID NO: 2); in oligonucleotide single strand 2, the first position from the 5' end is a modified base U, the first to third positions from the 3' end are rG (i.e., ribonucleic acid G), and the fourth position is N (N represents A ⁇ T ⁇ C ⁇ G), and the underlined portion is a sample tag (only an exemplary one, the sample tag is used to label the sample and can be any combination of nucleotides).
  • iSp6 Spacer C6, i.e., six CH2 groups
  • oligonucleotide single strand 2 is shown to the right
  • the reaction system in this step contains DNase I, Klenow fragment, and reverse transcriptase.
  • DNase I performs endonucleolytic functions.
  • Reverse transcriptase performs terminal transfer functions and DNA polymerization.
  • Klenow fragment performs 5' ⁇ 3' polymerization, 3' ⁇ 5' exonucleolytic functions, and strand displacement functions.
  • the template DNA double-stranded DNA molecule
  • double-stranded DNA fragments double-stranded DNA fragments
  • Klenow fragment double-stranded DNA fragments with blunt ends at both ends
  • reverse transcriptase terminal transfer function
  • three nucleotide Cs are added to the 3' end of each chain of the blunt-end fragment to form a sticky-end fragment (double-stranded DNA fragment with 3' protruding ends at both ends); with the help of the reverse complementary relationship with the sticky ends, the oligonucleotide single-stranded 1 derivative and the oligonucleotide single-stranded 2 derivative are bound to the double-stranded DNA fragment (the target is a double-stranded DNA fragment with the oligonucleotide single-stranded 1 derivative added to one end and the oligonucleot
  • the reaction system in this step contains both T4 DNA ligase and Under the action of T4 DNA ligase, the nicks in the above steps are repaired. Under the action of , the U base is removed along with its upstream blocking modification.
  • the library solution obtained in step 2 was taken for DNB preparation (using MGISEQ-2000RS High-throughput Rapid Sequencing Reagent Set, according to the instructions) and loaded on DNBSEQ-G400 for sequencing (sequencing type PE150).
  • Linker 3 is a single-stranded oligonucleotide with the following sequence:
  • Linker 4 is a single-stranded oligonucleotide with the following sequence:
  • the 5' end of adapter 3 has a blocking structure (hairpin structure, see wavy underline mark), and the first to third positions from the 3' end are rG (i.e., ribonucleic acid G) and the fourth position is N (N represents A ⁇ T ⁇ C ⁇ G).
  • Adapter 3 is shown in SEQ ID NO: 3.
  • linker 4 has a blocking structure (hairpin structure, see wavy underline mark), and the first to third positions from the 3' end are rG (i.e., ribonucleic acid G) and the fourth position is N (N represents A ⁇ T ⁇ C ⁇ G).
  • Linker 4 is shown in SEQ ID NO: 4.
  • Universal Primer 1 has a phosphorylation modification at its 5' end to facilitate compatibility with subsequent sequencing platforms. Universal Primer 1 is shown in SEQ ID NO: 5.
  • tag primer 1 the underlined portion is the sample tag (this is only an example; the sample tag is used to label the sample and can be any combination of nucleotides).
  • Tag primer 1 is shown in SEQ ID NO: 6.
  • the reaction system in this step contains both T4 DNA ligase and Tth DNA Polymerase.
  • the nick created in the previous step was repaired by ligase.
  • PCR amplification was then performed using Universal Primer 1 and Index Primer 1 as the primer pair. Due to the large amount of amplified product generated by PCR, the template strand with a hairpin structure at its 5' end was significantly diluted and thus removed.
  • the library solution obtained in step 2 was taken for DNB preparation (using MGISEQ-2000RS High-throughput Rapid Sequencing Reagent Set, according to the instructions) and loaded on DNBSEQ-G400 for sequencing (sequencing type PE150).
  • Blunt-ended double-stranded DNA fragments are obtained by fragmenting the template DNA using DNase I and then performing end repair using T4 DNA polymerase.
  • the reaction system of this step contains reverse transcriptase.
  • Reverse transcriptase performs terminal transfer function and DNA polymerization function.
  • terminal transfer function three nucleotide Cs are added to the 3' end of each chain of the blunt-ended double-stranded DNA fragment to form a sticky-end fragment (a double-stranded DNA fragment with 3' protruding ends at both ends); with the help of the reverse complementary relationship with the sticky end, oligonucleotide single strand 3 and oligonucleotide single strand 4 are bound to the double-stranded DNA fragment (the target is a double-stranded DNA fragment with oligonucleotide single strand 3 added to one end and oligonucleotide single strand 4 added to the other end), and then under the action of reverse transcriptase (DNA polymerization function), the two 3' protruding ends of the double-stranded DNA fragment are respectively converted into two The single-stranded oligonucle
  • the library solution obtained in step 2 was taken for DNB preparation (using MGISEQ-2000RS High-throughput Rapid Sequencing Reagent Set, according to the instructions) and loaded on DNBSEQ-G400 for sequencing (sequencing type PE150).
  • Control kit Prepare the library using the control kit according to the manufacturer's instructions (use 200 ng of template DNA) to obtain the library solution.
  • Control kit VAHTS Universal DNA Library Prep Kit for MGI; Novozymes, China, Cat. No. NDM607.
  • step 2 Take the library solution obtained in step 1, prepare DNB and load it on DNBSEQ-G400 for sequencing (sequencing type PE150).
  • the data sources were the sequencing results obtained in Example 1, the sequencing results obtained in Example 2, the sequencing results obtained in Example 3, and the sequencing results obtained in the comparative example.
  • the analysis was performed according to the same analysis process. In the first step, low-quality reads were filtered out and adapter sequences were removed. The data were aligned to the human reference genome hg19 using BWA software with default parameters. Finally, the obtained data were statistically analyzed using Samtools.
  • the method and kit disclosed in the present invention can be used to prepare a library for high-throughput sequencing, and have the advantages of simple operation steps and high library quality.

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Abstract

Provided are an adapter addition method, and a use thereof in the preparation of a high-throughput sequencing library. First, provided is a method for adding an adapter to a blunt-ended double-stranded DNA fragment, comprising: contacting the blunt-ended double-stranded DNA fragment with an enzyme having a terminal transferase activity and/or function and an adapter, wherein the enzyme is used for adding a nucleotide fragment 1 to the 3'-end of each strand of the double-stranded DNA fragment, and the nucleotide fragment 1 is reversely complementary to a nucleotide fragment 2 at the 3'-end of the adapter, so that the adapter is combined with the double-stranded DNA fragment, to obtain a double-stranded DNA fragment to which the adapter has been added. Furthermore, fragmentation, end repair, and adapter addition are carried out in one reaction system, thereby greatly reducing the operation complexity of library preparation. The problems of excessive steps, a high technical use threshold, a low template utilization rate, etc., in high-throughput sequencing library construction are solved.

Description

添加接头的方法及其在制备高通量测序文库中的应用Method for adding adapters and its application in preparing high-throughput sequencing libraries 技术领域Technical Field

本发明属于核酸测序领域，涉及添加接头的方法及其在制备高通量测序文库中的应用，具体涉及利用具有末端转移活性和/功能的酶进行添加接头的方法及其在制备高通量测序文库中的应用。The present invention belongs to the field of nucleic acid sequencing and relates to a method for adding adapters and its application in preparing a high-throughput sequencing library, and more particularly to a method for adding adapters using an enzyme having terminal transfer activity and/or function and its application in preparing a high-throughput sequencing library.

背景技术Background Art

在过去的几十年中，基因组学领域一直在努力研究各种生物的基因组结构和功能，以及它们与健康和疾病之间的关系。然而，由于传统测序技术的局限性，这些研究一直受到限制。高通量测序技术(NGS，Next Generation Sequencing)的出现打破了这种局面，使得大规模基因组测序变得可能。与传统测序技术相比，高通量测序技术具有更高的准确性、更高的速度和更低的成本，可以在很短的时间内获取大量的基因组序列信息。这些基因组序列数据的积累和分析，为基因组学研究提供了更深入的了解。研究人员可以通过对大规模基因组数据进行比较和分析，发现新的基因、功能和相互作用，从而对生物体系和疾病的研究带来了突破性进展。此外，高通量测序技术也被广泛应用于个体化医学研究。基于个体的基因组序列信息，研究人员可以更好地了解疾病的基因遗传风险、药物敏感性和治疗反应，从而为精准医疗提供更有力的支持。Over the past few decades, the field of genomics has been striving to study the genome structure and function of various organisms, as well as their relationship with health and disease. However, these studies have been restricted due to the limitations of traditional sequencing technologies. The emergence of high-throughput sequencing technology (NGS, Next Generation Sequencing) has broken this situation, making large-scale genome sequencing possible. Compared with traditional sequencing technologies, high-throughput sequencing technology has higher accuracy, higher speed and lower cost, and can obtain a large amount of genome sequence information in a very short time. The accumulation and analysis of these genome sequence data provide a deeper understanding of genomic research. By comparing and analyzing large-scale genomic data, researchers can discover new genes, functions and interactions, thus bringing breakthroughs to the study of biological systems and diseases. In addition, high-throughput sequencing technology is also widely used in personalized medicine research. Based on individual genome sequence information, researchers can better understand the genetic risk of disease, drug sensitivity and treatment response, thereby providing stronger support for precision medicine.

高通量测序中，文库构建是非常重要的一环，文库质量的高低直接影响后续测序数据的质量和准确性，而文库操作复杂度则会影响其应用的范围。近年来，随着高通量测序技术的不断发展，文库构建技术也在不断地进步和改进。高通量测序的文库构建还存在一些共性问题，例如步骤过多(包括片段化、末端修复、加A、接头连接、PCR过程以及中间的各个纯化步骤)，技术使用门槛高、模板利用率偏低等，限制了高通量测序的应用。In high-throughput sequencing, library construction is a very important step. The quality of the library directly affects the quality and accuracy of the subsequent sequencing data, while the complexity of library operation affects the scope of its application. In recent years, with the continuous development of high-throughput sequencing technology, library construction technology has also been continuously improved. There are still some common problems in the construction of libraries for high-throughput sequencing, such as too many steps (including fragmentation, end repair, A addition, adapter ligation, PCR process and various purification steps in between), high technical barriers to use, low template utilization, etc., which limit the application of high-throughput sequencing.

发明内容Summary of the Invention

本发明旨在至少在一定程度上解决现有技术中存在的技术问题之一。为此，本发明提出了利用具有末端转移活性和/功能的酶进行添加接头的方法及其在制备高通量测序文库中的应用。The present invention aims to at least partially address one of the technical problems existing in the prior art. To this end, the present invention provides a method for adding adapters using an enzyme having terminal transfer activity and/or function and its use in preparing high-throughput sequencing libraries.

在本发明的第一方面，本发明提供了一种给平末端双链DNA片段添加接头的方法，其包括如下步骤：将平末端双链DNA片段与具有末端转移活性和/或功能的酶和接头接触；In a first aspect of the present invention, the present invention provides a method for adding a linker to a blunt-ended double-stranded DNA fragment, comprising the steps of: contacting the blunt-ended double-stranded DNA fragment with an enzyme having terminal transfer activity and/or function and a linker;

其中，所述酶用于在所述双链DNA片段的每条链的3’末端增加核苷酸片段1；wherein the enzyme is used to add nucleotide fragment 1 to the 3' end of each strand of the double-stranded DNA fragment;

其中，所述核苷酸片段1与所述接头的3’末端的核苷酸片段2反向互补，使得所述接头结合于所述双链DNA片段，获得添加有接头的双链DNA片段。The nucleotide fragment 1 is reverse complementary to the nucleotide fragment 2 at the 3' end of the linker, so that the linker is bound to the double-stranded DNA fragment to obtain a double-stranded DNA fragment with the linker added.

根据本发明的实施例，所述核苷酸片段1由N1个核苷酸组成，所述核苷酸片段2由N2个核苷酸组成；N1和N2分别独立地选自1-10的自然数。优选地，N1和N2分别独立地选自2-5的自然数。优选地，N1为3，N2为3。According to an embodiment of the present invention, the nucleotide fragment 1 consists of N1 nucleotides, and the nucleotide fragment 2 consists of N2 nucleotides; N1 and N2 are each independently selected from a natural number between 1 and 10. Preferably, N1 and N2 are each independently selected from a natural number between 2 and 5. Preferably, N1 is 3 and N2 is 3.

根据本发明的实施例，所述核苷酸片段2中的核苷酸为核糖核苷酸、脱氧核糖核苷酸或经修饰的核苷酸。According to an embodiment of the present invention, the nucleotides in the nucleotide fragment 2 are ribonucleotides, deoxyribonucleotides or modified nucleotides.

根据本发明的实施例，所述核苷酸片段2中的核苷酸为核糖核苷酸。优选地， 所述核苷酸片段1为CCC；优选地，所述核苷酸片段2为rGrGrG。需要说明的是，所述rGrGrG代表3个连续的核糖核苷酸G。在一些实施例中，RNA/DNA杂交链的结合性高于RNA/RNA杂交链以及DNA/DNA杂交链。According to an embodiment of the present invention, the nucleotides in the nucleotide fragment 2 are ribonucleotides. Preferably, The nucleotide segment 1 is CCC; preferably, the nucleotide segment 2 is rGrGrG. It should be noted that the rGrGrG represents three consecutive ribonucleotides G. In some embodiments, the binding affinity of the RNA/DNA hybrid chain is higher than that of the RNA/RNA hybrid chain and the DNA/DNA hybrid chain.

根据本发明的实施例，所述核苷酸片段2中的核苷酸为脱氧核糖核苷酸。优选地，所述核苷酸片段1为CCC；优选地，所述核苷酸片段2为GGG。According to an embodiment of the present invention, the nucleotides in the nucleotide segment 2 are deoxyribonucleotides. Preferably, the nucleotide segment 1 is CCC; preferably, the nucleotide segment 2 is GGG.

根据本发明的实施例，所述核苷酸片段2中的核苷酸为经修饰的核苷酸。优选地，所述经修饰的核苷酸为锁核酸。According to an embodiment of the present invention, the nucleotides in the nucleotide fragment 2 are modified nucleotides. Preferably, the modified nucleotides are locked nucleic acids.

根据本发明的实施例，在所述接头中，与核苷酸片段2的5’端相邻的核苷酸为N。在一些实施例中，所述N代表A或T或C或G。According to an embodiment of the present invention, in the linker, the nucleotide adjacent to the 5' end of nucleotide fragment 2 is N. In some embodiments, the N represents A, T, C, or G.

根据本发明的实施例，所述接头的5’末端具有以阻断核酸延伸为目的的阻断结构或阻断修饰。According to an embodiment of the present invention, the 5' end of the linker has a blocking structure or blocking modification for the purpose of blocking nucleic acid extension.

根据本发明的实施例，所述阻断结构为发卡结构。According to an embodiment of the present invention, the blocking structure is a hairpin structure.

根据本发明的实施例，所述阻断修饰选自：双脱氧胞苷修饰、反向dt修饰、磷酸基团修饰、硫代基团修饰或间臂修饰。优选地，所述阻断修饰为间臂修饰。优选地，所述间臂修饰为碳骨架修饰或糖骨架修饰。优选地，所述间臂修饰为Spacer C3(即3个CH2)、Spacer C6(即6个CH2)或Spacer C9(即9个CH2)等。According to an embodiment of the present invention, the blocking modification is selected from: dideoxycytidine modification, reverse dt modification, phosphate group modification, thio group modification, or spacer modification. Preferably, the blocking modification is a spacer modification. Preferably, the spacer modification is a carbon backbone modification or a sugar backbone modification. Preferably, the spacer modification is Spacer C3 (i.e., 3 CH 2 ), Spacer C6 (i.e., 6 CH 2 ), or Spacer C9 (i.e., 9 CH 2 ), etc.

根据本发明的实施例，所述接头包含功能区段，所述功能区段位于阻断结构或阻断修饰的下游。优选地，所述功能区段包含样本标签和/或分子标签和/或测序引物。需要说明的是，对于核酸分子来说，除非另有说明，否则通常认为5’端为上游，3端为下游’。According to an embodiment of the present invention, the linker comprises a functional segment located downstream of the blocking structure or blocking modification. Preferably, the functional segment comprises a sample tag and/or a molecular tag and/or a sequencing primer. It should be noted that, for nucleic acid molecules, unless otherwise specified, the 5' end is generally considered to be upstream and the 3' end is considered to be downstream.

根据本发明的实施例，所述接头包含酶切位点，所述酶切位点位于阻断结构或阻断修饰的下游。所述酶切位点用于在后续步骤中通过酶切去除阻断修饰或阻断结构。根据本发明的实施例，所述酶切所用的酶为UDG Enzyme、USER Enzyme或RnaseH等。根据本发明的实施例，所述酶切位点与阻断结构或阻断修饰相邻。根据本发明的实施例，所述酶切位点与阻断结构或阻断修饰间隔N3个核苷酸，N3为自然数。优选地，N3选自1-10的自然数。优选地，N3选自1-5的自然数。优选地，N3为1或2或3。根据本发明的实施例，所述酶切位点选自：dUTP或rNTP。According to an embodiment of the present invention, the linker comprises an enzyme cleavage site, and the enzyme cleavage site is located downstream of the blocking structure or blocking modification. The enzyme cleavage site is used to remove the blocking modification or blocking structure by enzyme cleavage in a subsequent step. According to an embodiment of the present invention, the enzyme used for the enzyme cleavage is UDG Enzyme, USER Enzyme or RnaseH, etc. According to an embodiment of the present invention, the enzyme cleavage site is adjacent to the blocking structure or blocking modification. According to an embodiment of the present invention, the enzyme cleavage site and the blocking structure or blocking modification are separated by N3 nucleotides, and N3 is a natural number. Preferably, N3 is selected from a natural number of 1-10. Preferably, N3 is selected from a natural number of 1-5. Preferably, N3 is 1 or 2 or 3. According to an embodiment of the present invention, the enzyme cleavage site is selected from: dUTP or rNTP.

根据本发明的实施例，所述具有末端转移活性和/或功能的酶具有偏好性。如本文所用，所述偏好性指的是偏好于在链的3’末端增加3个特定的核苷酸。如本文所用，偏好性指的是存在20％以上概率在链的3’末端增加3个特定的核苷酸。根据本发明的实施例，所述3个特定的核苷酸为3个核苷酸C。According to embodiments of the present invention, the enzyme having terminal transfer activity and/or function has a preference. As used herein, the preference refers to a preference for adding three specific nucleotides to the 3' end of a strand. As used herein, the preference refers to a greater than 20% probability of adding three specific nucleotides to the 3' end of a strand. According to embodiments of the present invention, the three specific nucleotides are three C nucleotides.

如本文所用，术语“接头”是指寡聚核苷酸单链或寡聚核苷酸单链衍生物。寡聚核苷酸单链指的是长度为3-100nt的单链寡聚核苷酸。寡聚核苷酸单链的5’末端具有以阻断核酸延伸为目的的阻断结构。寡聚核苷酸单链衍生物是在寡聚核苷酸单链的5’末端添加具有以阻断核酸延伸为目的的阻断修饰得到的。As used herein, the term "linker" refers to a single-stranded oligonucleotide or a single-stranded oligonucleotide derivative. A single-stranded oligonucleotide refers to a single-stranded oligonucleotide of 3-100 nt in length. The 5' end of the single-stranded oligonucleotide has a blocking structure for the purpose of blocking nucleic acid extension. A single-stranded oligonucleotide derivative is obtained by adding a blocking modification having the purpose of blocking nucleic acid extension to the 5' end of the single-stranded oligonucleotide.

根据本发明的实施例，接头具有各种形式。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：阻断修饰(间臂修饰)、U、样本标签、GGG(如图1的a所示)。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：自5’末端至3’末端依次具有如下区段：阻断修饰(间臂修饰)、U、GGG(如图1的b 所示)。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：阻断修饰(间臂修饰)、样本标签、GGG(如图1的c所示)。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：阻断修饰(间臂修饰)、GGG(如图1的d所示)。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：阻断结构(发卡结构)、U、样本标签、GGG(如图1的e所示)。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：阻断结构(发卡结构)、U、GGG(如图1的f所示)。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：阻断结构(发卡结构)、样本标签、GGG(如图1的g所示)。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：阻断结构(发卡结构)、GGG(如图1的h所示)。According to an embodiment of the present invention, the linker has various forms. According to an embodiment of the present invention, the linker has the following segments from the 5' end to the 3' end in sequence: blocking modification (inter-arm modification), U, sample tag, GGG (as shown in a of Figure 1). According to an embodiment of the present invention, the linker has the following segments from the 5' end to the 3' end in sequence: blocking modification (inter-arm modification), U, sample tag, GGG (as shown in b of Figure 1). As shown). According to an embodiment of the present invention, the linker has the following sections from the 5' end to the 3' end in sequence: blocking modification (spacer modification), sample tag, GGG (as shown in c of Figure 1). According to an embodiment of the present invention, the linker has the following sections from the 5' end to the 3' end in sequence: blocking modification (spacer modification), GGG (as shown in d of Figure 1). According to an embodiment of the present invention, the linker has the following sections from the 5' end to the 3' end in sequence: blocking structure (hairpin structure), U, sample tag, GGG (as shown in e of Figure 1). According to an embodiment of the present invention, the linker has the following sections from the 5' end to the 3' end in sequence: blocking structure (hairpin structure), U, GGG (as shown in f of Figure 1). According to an embodiment of the present invention, the linker has the following sections from the 5' end to the 3' end in sequence: blocking structure (hairpin structure), sample tag, GGG (as shown in g of Figure 1). According to an embodiment of the present invention, the linker has the following sections from the 5' end to the 3' end in sequence: blocking structure (hairpin structure), GGG (as shown in h of Figure 1).

进一步的，所述给平末端双链DNA片段添加接头的方法中还包括模板延伸的步骤。所述模板延伸是添加有接头的双链DNA片段以所述接头为模板进行5’至3’方向的延伸。Furthermore, the method for adding adapters to blunt-ended double-stranded DNA fragments further comprises a template extension step, wherein the template extension is to extend the double-stranded DNA fragments to which the adapters are added in a 5' to 3' direction using the adapters as a template.

根据本发明的实施例，所述模板延伸借助具有DNA聚合活性和/或功能的酶。优选地，所述具有末端转移活性和/或功能的酶与所述具有DNA聚合活性和/或功能的酶是同一种酶。优选地，所述具有末端转移活性和/或功能的酶与所述具有DNA聚合活性和/或功能的酶是不同的酶。According to an embodiment of the present invention, the template extension is performed by an enzyme having DNA polymerization activity and/or function. Preferably, the enzyme having terminal transfer activity and/or function is the same enzyme as the enzyme having DNA polymerization activity and/or function. Preferably, the enzyme having terminal transfer activity and/or function is different from the enzyme having DNA polymerization activity and/or function.

根据本发明的实施例，所述具有DNA聚合活性和/或功能的酶为具有末端转移活性的逆转录酶。优选地，所述具有末端转移活性的逆转录酶为鼠白血病逆转录酶(M-MLV)。优选地，所述具有末端转移活性的逆转录酶为鸟白血病逆转录酶(AMV)。优选地，所述具有末端转移活性的逆转录酶为SuperScriptTMIV逆转录酶、SuperScriptTMI逆转录酶、SuperScriptTMII逆转录酶或SuperScriptTMIII逆转录酶。According to an embodiment of the present invention, the enzyme having DNA polymerization activity and/or function is a reverse transcriptase having terminal transfer activity. Preferably, the reverse transcriptase having terminal transfer activity is murine leukemia reverse transcriptase (M-MLV). Preferably, the reverse transcriptase having terminal transfer activity is avian leukemia reverse transcriptase (AMV). Preferably, the reverse transcriptase having terminal transfer activity is SuperScript ™ IV reverse transcriptase, SuperScript ™ I reverse transcriptase, SuperScript ™ II reverse transcriptase, or SuperScript ™ III reverse transcriptase.

根据本发明的实施例，所述具有DNA聚合活性和/或功能的酶为Klenow片段。根据本发明的实施例，所述具有DNA聚合活性和/或功能的酶为T4 DNA聚合酶。According to an embodiment of the present invention, the enzyme having DNA polymerization activity and/or function is a Klenow fragment. According to an embodiment of the present invention, the enzyme having DNA polymerization activity and/or function is T4 DNA polymerase.

根据本发明的实施例，所述具有末端转移活性和/或功能的酶为具有末端转移活性的逆转录酶。优选地，所述具有末端转移活性的逆转录酶为鼠白血病逆转录酶(M-MLV)。优选地，所述具有末端转移活性的逆转录酶为鸟白血病逆转录酶(AMV)。优选地，所述具有末端转移活性的逆转录酶为SuperScriptTMIV逆转录酶、SuperScriptTMI逆转录酶、SuperScriptTMII逆转录酶或SuperScriptTMIII逆转录酶。According to an embodiment of the present invention, the enzyme having terminal transfer activity and/or function is a reverse transcriptase having terminal transfer activity. Preferably, the reverse transcriptase having terminal transfer activity is murine leukemia reverse transcriptase (M-MLV). Preferably, the reverse transcriptase having terminal transfer activity is avian leukemia reverse transcriptase (AMV). Preferably, the reverse transcriptase having terminal transfer activity is SuperScript ™ IV reverse transcriptase, SuperScript ™ I reverse transcriptase, SuperScript ™ II reverse transcriptase, or SuperScript ™ III reverse transcriptase.

根据本发明的实施例，所述接头可为接头1或接头2。According to an embodiment of the present invention, the connector may be connector 1 or connector 2.

其中，所述接头1为寡聚核苷酸单链衍生物，其结构式如下：接头1是在寡聚核苷酸单链1的5’末端进行阻断修饰得到的；阻断修饰即通过iSp6(Spacer C6，即6个CH2)与3个核苷酸A连接；寡聚核苷酸单链1如结构式中iSp6右侧所示(SEQ ID NO：1)；寡聚核苷酸单链1中，自5’末端第3位为修饰碱基U，自3’末端第1至3位为rG(即核糖核酸G)且第4位为N(N代表A\T\C\G)。Wherein, the linker 1 is a single-stranded oligonucleotide derivative, and its structural formula is as follows: Linker 1 is obtained by performing a blocking modification on the 5' end of single-stranded oligonucleotide 1; the blocking modification is to connect three nucleotide A's via iSp6 (Spacer C6, i.e., six CH2 groups ); single-stranded oligonucleotide 1 is shown to the right of iSp6 in the structural formula (SEQ ID NO: 1); in single-stranded oligonucleotide 1, the third position from the 5' end is a modified base U, and positions 1 to 3 from the 3' end are rG (i.e., ribonucleic acid G), and the fourth position is N (N represents A\T\C\G).

其中，所述接头2为寡聚核苷酸单链衍生物，其结构式如下： 接头2是在寡聚核苷酸单链2的5’末端进行阻断修饰得到的；阻断修饰即通过iSp6(Spacer C6，即6个CH2)与3个核苷酸A连接；寡聚核苷酸单 链2如结构式中iSp6右侧所示(SEQ ID NO：2)；寡聚核苷酸单链2中，自5’末端第1位为修饰碱基U，自3’末端第1至3位为rG(即核糖核酸G)且第4位为N(N代表A\T\C\G)，下划线部分为样本标签(仅为示例性的一种，样本标签用于标记样本，可为任意核苷酸组合)。Wherein, the linker 2 is a single-stranded oligonucleotide derivative, and its structural formula is as follows: The linker 2 is obtained by performing blocking modification on the 5' end of the oligonucleotide single strand 2; the blocking modification is to connect the three nucleotide A's via iSp6 (Spacer C6, i.e., 6 CH2 ); the oligonucleotide single strand Chain 2 is shown to the right of iSp6 in the structural formula (SEQ ID NO: 2). In the oligonucleotide single strand 2, the first position from the 5' end is a modified base U, positions 1 to 3 from the 3' end are rG (i.e., ribonucleic acid G), and the fourth position is N (N represents A\T\C\G). The underlined portion is a sample tag (which is only an exemplary one; the sample tag is used to label the sample and can be any combination of nucleotides).

根据本发明的实施例，所述接头可为接头3或接头4。According to an embodiment of the present invention, the connector may be connector 3 or connector 4.

其中，所述接头3为寡聚核苷酸单链，序列如下： 接头3的5’端具有阻断结构(发卡结构，见波浪下划线标记)，自3’末端第1至3位为rG(即核糖核酸G)且第4位为N(N代表A\T\C\G)。接头3如SEQ ID NO：3所示。Wherein, the linker 3 is a single-stranded oligonucleotide, and the sequence is as follows: The 5' end of linker 3 has a blocking structure (hairpin structure, see wavy underline mark), and the first to third positions from the 3' end are rG (i.e., ribonucleic acid G) and the fourth position is N (N represents A\T\C\G). Linker 3 is shown in SEQ ID NO: 3.

其中，所述接头4为寡聚核苷酸单链，序列如下：接头4的5’端具有阻断结构(发卡结构，见波浪下划线标记)，自3’末端第1至3位为rG(即核糖核酸G)且第4位为N(N代表A\T\C\G)。接头4如SEQ ID NO：4所示。Wherein, the linker 4 is a single-stranded oligonucleotide with the following sequence: The 5' end of linker 4 has a blocking structure (hairpin structure, see wavy underline mark), and the first to third positions from the 3' end are rG (i.e., ribonucleic acid G) and the fourth position is N (N represents A\T\C\G). Linker 4 is shown in SEQ ID NO: 4.

根据本发明的实施例，给平末端双链DNA片段添加接头的反应体系中含有：平末端双链DNA片段、接头和具有末端转移活性和/或功能的酶。根据本发明的实施例，给平末端双链DNA片段添加接头的反应体系中还含有：dNTP和反应缓冲液。根据本发明的实施例，给平末端双链DNA片段添加接头的反应体系中还含有：甜菜碱和MgCl2。根据本发明的实施例，所述接头可为所述接头3和所述接头4。根据本发明的实施例，所述具有末端转移活性和/或功能的酶可为SuperScriptTMIV逆转录酶。根据本发明的实施例，所述反应缓冲液可为5×Superscript IV first-strand buffer。根据本发明的实施例，给平末端双链DNA片段添加接头的反应体系可如表8所示。According to an embodiment of the present invention, the reaction system for adding a linker to a blunt-ended double-stranded DNA fragment contains: a blunt-ended double-stranded DNA fragment, a linker, and an enzyme having terminal transfer activity and/or function. According to an embodiment of the present invention, the reaction system for adding a linker to a blunt-ended double-stranded DNA fragment further contains: dNTP and a reaction buffer. According to an embodiment of the present invention, the reaction system for adding a linker to a blunt-ended double-stranded DNA fragment further contains: betaine and MgCl 2. According to an embodiment of the present invention, the linker may be linker 3 and linker 4. According to an embodiment of the present invention, the enzyme having terminal transfer activity and/or function may be SuperScript TM IV reverse transcriptase. According to an embodiment of the present invention, the reaction buffer may be 5×Superscript IV first-strand buffer. According to an embodiment of the present invention, the reaction system for adding a linker to a blunt-ended double-stranded DNA fragment may be as shown in Table 8.

根据本发明的实施例，所述给平末端双链DNA片段添加接头的反应条件为：40-45℃反应40-80min，然后65-75℃反应5-15min。根据本发明的实施例，所述给平末端双链DNA片段添加接头的反应条件为：42℃反应60min，然后70℃反应10min。根据本发明的实施例，所述给平末端双链DNA片段添加接头的反应条件为：40-45℃反应80-100min。根据本发明的实施例，所述给平末端双链DNA片段添加接头的反应条件为：42℃反应90min。According to an embodiment of the present invention, the reaction conditions for adding adapters to blunt-end double-stranded DNA fragments are: reaction at 40-45°C for 40-80 min, and then reaction at 65-75°C for 5-15 min. According to an embodiment of the present invention, the reaction conditions for adding adapters to blunt-end double-stranded DNA fragments are: reaction at 42°C for 60 min, and then reaction at 70°C for 10 min. According to an embodiment of the present invention, the reaction conditions for adding adapters to blunt-end double-stranded DNA fragments are: reaction at 40-45°C for 80-100 min. According to an embodiment of the present invention, the reaction conditions for adding adapters to blunt-end double-stranded DNA fragments are: reaction at 42°C for 90 min.

根据本发明的实施例，完成反应后，用XP beads进行DNA纯化。根据本发明的实施例，完成反应后，用30μL XP beads进行DNA纯化，纯化后的DNA溶解于20μL TE缓冲液中，即为产物溶液。According to an embodiment of the present invention, after the reaction is complete, DNA is purified using XP beads. According to an embodiment of the present invention, after the reaction is complete, DNA is purified using 30 μL of XP beads, and the purified DNA is dissolved in 20 μL of TE buffer to form the product solution.

在本发明的第二方面，本发明还提供了一种给双链DNA片段末端修复并添加接头的方法，其包括如下过程：In a second aspect of the present invention, the present invention also provides a method for repairing the ends of double-stranded DNA fragments and adding linkers, which comprises the following steps:

①双链DNA片段进行末端修复，得到平末端双链DNA片段；① The double-stranded DNA fragments are repaired at the ends to obtain blunt-ended double-stranded DNA fragments;

②给平末端双链DNA片段添加接头；所述给平末端双链DNA片段添加接头的方法包括如下步骤：将平末端双链DNA片段与具有末端转移活性和/或功能的酶和接头接触；其中，所述酶用于在所述双链DNA片段的每条链的3’末端增加核苷酸片段1；所述核苷酸片段1与所述接头的3’末端的核苷酸片段2反向互补，使得所述接头结合于所述双链DNA片段，获得添加有接头的双链DNA片段。② Adding a linker to a blunt-ended double-stranded DNA fragment; the method for adding a linker to a blunt-ended double-stranded DNA fragment comprises the following steps: contacting the blunt-ended double-stranded DNA fragment with an enzyme having terminal transfer activity and/or function and a linker; wherein the enzyme is used to add a nucleotide fragment 1 to the 3' end of each chain of the double-stranded DNA fragment; the nucleotide fragment 1 is reversely complementary to the nucleotide fragment 2 at the 3' end of the linker, so that the linker binds to the double-stranded DNA fragment, thereby obtaining a double-stranded DNA fragment with an added linker.

优选地，所述给平末端双链DNA片段添加接头的方法如以上“给平末端双链DNA 片段添加接头的方法”中任一所述。Preferably, the method for adding a linker to a blunt-ended double-stranded DNA fragment is as described above in "Adding a linker to a blunt-ended double-stranded DNA fragment" Any of the methods described in "Methods for adding adapters to fragments".

根据本发明的实施例，过程①和②在同一体系中完成。According to an embodiment of the present invention, processes ① and ② are completed in the same system.

根据本发明的实施例，所述双链DNA片段进行末端修复借助具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶。根据本发明的实施例，所述具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶为Klenow片段。根据本发明的实施例，所述具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶为T4 DNA聚合酶。According to an embodiment of the present invention, the double-stranded DNA fragment is end-repaired with the aid of an enzyme having 5'→3' polymerization and 3'→5' exonuclease activity and/or function. According to an embodiment of the present invention, the enzyme having 5'→3' polymerization and 3'→5' exonuclease activity and/or function is a Klenow fragment. According to an embodiment of the present invention, the enzyme having 5'→3' polymerization and 3'→5' exonuclease activity and/or function is T4 DNA polymerase.

在本发明的第三方面，本发明还提供了一种给双链DNA分子片段化、末端修复并添加接头的方法，其包括如下过程：In a third aspect, the present invention further provides a method for fragmenting, end-repairing and adding linkers to double-stranded DNA molecules, which comprises the following steps:

①双链DNA分子进行片段化，得到双链DNA片段；① Fragmentation of double-stranded DNA molecules to obtain double-stranded DNA fragments;

②双链DNA片段进行末端修复，得到平末端双链DNA片段；② The double-stranded DNA fragments are repaired at the ends to obtain blunt-ended double-stranded DNA fragments;

③给平末端双链DNA片段添加接头；所述给平末端双链DNA片段添加接头的方法包括如下步骤：将平末端双链DNA片段与具有末端转移活性和/或功能的酶和接头接触；其中，所述酶用于在所述双链DNA片段的每条链的3’末端增加核苷酸片段1；所述核苷酸片段1与所述接头的3’末端的核苷酸片段2反向互补，使得所述接头结合于所述双链DNA片段，获得添加有接头的双链DNA片段。③ Adding a linker to a blunt-ended double-stranded DNA fragment; the method for adding a linker to a blunt-ended double-stranded DNA fragment comprises the following steps: contacting the blunt-ended double-stranded DNA fragment with an enzyme having terminal transfer activity and/or function and a linker; wherein the enzyme is used to add a nucleotide fragment 1 to the 3' end of each chain of the double-stranded DNA fragment; the nucleotide fragment 1 is reversely complementary to the nucleotide fragment 2 at the 3' end of the linker, so that the linker binds to the double-stranded DNA fragment, thereby obtaining a double-stranded DNA fragment with an added linker.

优选地，所述给平末端双链DNA片段添加接头的方法如以上“给平末端双链DNA片段添加接头的方法”中任一所述。Preferably, the method for adding a linker to a blunt-ended double-stranded DNA fragment is as described in any one of the above “methods for adding a linker to a blunt-ended double-stranded DNA fragment”.

根据本发明的实施例，过程①、②和③在同一体系中完成。According to an embodiment of the present invention, processes ①, ② and ③ are completed in the same system.

根据本发明的实施例，所述双链DNA片段进行末端修复借助具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶。根据本发明的实施例，所述具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶为Klenow片段。根据本发明的实施例，所述具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶为T4 DNA聚合酶。According to an embodiment of the present invention, the double-stranded DNA fragment is end-repaired with the aid of an enzyme having 5'→3' polymerization and 3'→5' exonuclease activity and/or function. According to an embodiment of the present invention, the enzyme having 5'→3' polymerization and 3'→5' exonuclease activity and/or function is a Klenow fragment. According to an embodiment of the present invention, the enzyme having 5'→3' polymerization and 3'→5' exonuclease activity and/or function is T4 DNA polymerase.

根据本发明的实施例，所述双链DNA分子进行片段化的方法选自：物理打断或酶切打断。根据本发明的实施例，所述酶切打断借助具有核酸内切活性和/或功能的酶。根据本发明的实施例，所述具有核酸内切活性和/或功能的酶为Dnase I或Endonuclease V。According to an embodiment of the present invention, the method for fragmenting the double-stranded DNA molecule is selected from: physical fragmentation or enzymatic fragmentation. According to an embodiment of the present invention, the enzymatic fragmentation is performed with the aid of an enzyme having endonuclease activity and/or function. According to an embodiment of the present invention, the enzyme having endonuclease activity and/or function is DNase I or Endonuclease V.

根据本发明的实施例，所述“给双链DNA分子片段化、末端修复并添加接头的方法”的反应体系中含有：双链DNA分子、具有核酸内切活性和/或功能的酶、具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶、具有末端转移活性和/或功能的酶和接头。根据本发明的实施例，所述“给双链DNA分子片段化、末端修复并添加接头的方法”的反应体系中还含有：dNTP和反应缓冲液。根据本发明的实施例，所述“给双链DNA分子片段化、末端修复并添加接头的方法”的反应体系中还含有：甜菜碱和MgCl2。根据本发明的实施例，所述接头可为所述接头1和所述接头2。根据本发明的实施例，所述接头可为所述接头3和所述接头4。根据本发明的实施例，所述具有核酸内切活性和/或功能的酶为Dnase I或Endonuclease V。根据本发明的实施例，所述具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶为Klenow片段。根据本发明的实施例，所述具有末端转移活性和/或功能的酶可为SuperScriptTMIV逆转录酶。根据本发明的实施例，所述反应缓冲液可为5×Superscript IV first-strand buffer。根据本发明的实施例，所述“给双链DNA分子片段化、末端修复并添加接头的方法”的反应体系可如表1所示。根据本发明的实施例，所述“给 双链DNA分子片段化、末端修复并添加接头的方法”的反应体系可如表4所示。According to an embodiment of the present invention, the reaction system of the "method for fragmenting, end-repairing, and adding adapters to double-stranded DNA molecules" contains: double-stranded DNA molecules, an enzyme with endonucleolytic activity and/or function, an enzyme with 5'→3' polymerization and 3'→5' exonucleolytic activity and/or function, an enzyme with terminal transfer activity and/or function, and a adapter. According to an embodiment of the present invention, the reaction system of the "method for fragmenting, end-repairing, and adding adapters to double-stranded DNA molecules" further contains: dNTPs and a reaction buffer. According to an embodiment of the present invention, the reaction system of the "method for fragmenting, end-repairing, and adding adapters to double-stranded DNA molecules" further contains: betaine and MgCl2 . According to an embodiment of the present invention, the adapters may be Adapter 1 and Adapter 2. According to an embodiment of the present invention, the adapters may be Adapter 3 and Adapter 4. According to an embodiment of the present invention, the enzyme with endonucleolytic activity and/or function is DNase I or Endonuclease V. According to an embodiment of the present invention, the enzyme with 5'→3' polymerization and 3'→5' exonucleolytic activity and/or function is a Klenow fragment. According to an embodiment of the present invention, the enzyme having terminal transfer activity and/or function may be SuperScript ™ IV reverse transcriptase. According to an embodiment of the present invention, the reaction buffer may be 5×Superscript IV first-strand buffer. According to an embodiment of the present invention, the reaction system of the “method for fragmenting, end-repairing and adding linkers to double-stranded DNA molecules” may be as shown in Table 1. According to an embodiment of the present invention, the “method for fragmenting, end-repairing and adding linkers to double-stranded DNA molecules” may be as shown in Table 1. The reaction system of the "method for fragmenting double-stranded DNA molecules, repairing ends, and adding adapters" can be shown in Table 4.

根据本发明的实施例，所述“给双链DNA分子片段化、末端修复并添加接头的方法”的反应条件如下所示：在T1℃反应t1分钟，在T2℃反应t2分钟，T3℃反应t3分钟，T4℃反应t4分钟，其中T1：16-40℃，T2：55-65℃，T3：16-55℃，T4：55-75℃，t1：10-60分钟，t2：10-30分钟，t3：10-60分钟，t4：10-30分钟。在一些实施方案中，T1为37℃，T2为60℃，T3为42℃，T4为70℃，t1为10分钟，t2为10分钟，t3为60分钟，t4为10分钟。According to an embodiment of the present invention, the reaction conditions of the "method for fragmenting, end-repairing and adding adapters to double-stranded DNA molecules" are as follows: reaction at T1°C for 1 minute, reaction at T2°C for 2 minutes, reaction at T3°C for 3 minutes, and reaction at T4°C for 4 minutes, wherein T1: 16-40°C, T2: 55-65°C, T3: 16-55°C, T4: 55-75°C, t1 : 10-60 minutes, t2 : 10-30 minutes, t3 : 10-60 minutes, and t4 : 10-30 minutes. In some embodiments, T1 is 37°C, T2 is 60°C, T3 is 42°C, and T4 is 70°C, t1 is 10 minutes, t2 is 10 minutes, t3 is 60 minutes, and t4 is 10 minutes.

在本发明的第四方面，本发明还保护以上任一所述方法在制备核酸文库或高通量测序中的应用。In the fourth aspect of the present invention, the present invention also protects the use of any of the above methods in preparing nucleic acid libraries or high-throughput sequencing.

在本发明的第五方面，本发明还保护一种制备核酸文库的方法，其包括步骤(1)。In the fifth aspect of the present invention, the present invention also protects a method for preparing a nucleic acid library, which comprises step (1).

步骤(1)是为了得到添加有接头的双链DNA片段。Step (1) is to obtain double-stranded DNA fragments with adapters added.

在一些实施方案中，所述步骤(1)为：将平末端双链DNA片段与具有末端转移活性和/或功能的酶和接头接触；其中，所述酶用于在所述双链DNA片段的每条链的3’末端增加核苷酸片段1；其中，所述核苷酸片段1与所述接头的3’末端的核苷酸片段2反向互补，使得所述接头结合于所述双链DNA片段，获得添加有接头的双链DNA片段。In some embodiments, step (1) comprises: contacting a blunt-ended double-stranded DNA fragment with an enzyme having terminal transfer activity and/or function and a linker; wherein the enzyme is used to add a nucleotide fragment 1 to the 3' end of each chain of the double-stranded DNA fragment; wherein the nucleotide fragment 1 is reversely complementary to the nucleotide fragment 2 at the 3' end of the linker, so that the linker binds to the double-stranded DNA fragment, thereby obtaining a double-stranded DNA fragment to which a linker is added.

优选地，所述步骤(1)如以上第一方面“给平末端双链DNA片段添加接头的方法”中任一所述。Preferably, the step (1) is as described in any one of the above first aspects of the "method for adding linkers to blunt-ended double-stranded DNA fragments".

在一些实施方案中，所述步骤(1)包含如下过程：In some embodiments, step (1) comprises the following process:

①双链DNA片段进行末端修复，得到平末端双链DNA片段；① The double-stranded DNA fragments are repaired at the ends to obtain blunt-ended double-stranded DNA fragments;

②给平末端双链DNA片段添加接头；所述给平末端双链DNA片段添加接头的方法包括如下步骤：将平末端双链DNA片段与具有末端转移活性和/或功能的酶和接头接触；其中，所述酶用于在所述双链DNA片段的每条链的3’末端增加核苷酸片段1；所述核苷酸片段1与所述接头的3’末端的核苷酸片段2反向互补，使得所述接头结合于所述双链DNA片段，获得添加有接头的双链DNA片段。② Adding a linker to a blunt-ended double-stranded DNA fragment; the method for adding a linker to a blunt-ended double-stranded DNA fragment comprises the following steps: contacting the blunt-ended double-stranded DNA fragment with an enzyme having terminal transfer activity and/or function and a linker; wherein the enzyme is used to add a nucleotide fragment 1 to the 3' end of each chain of the double-stranded DNA fragment; the nucleotide fragment 1 is reversely complementary to the nucleotide fragment 2 at the 3' end of the linker, so that the linker binds to the double-stranded DNA fragment, thereby obtaining a double-stranded DNA fragment with an added linker.

优选地，过程①和②在同一体系中完成。Preferably, processes ① and ② are completed in the same system.

优选地，所述步骤(1)如以上第二方面“给双链DNA片段末端修复并添加接头的方法”中任一所述。Preferably, the step (1) is as described in any one of the second aspects of the above “method for repairing the ends of double-stranded DNA fragments and adding linkers”.

在一些实施方案中，所述步骤(1)包括如下过程：In some embodiments, step (1) comprises the following process:

①双链DNA分子进行片段化，得到双链DNA片段；① Fragmentation of double-stranded DNA molecules to obtain double-stranded DNA fragments;

②双链DNA片段进行末端修复，得到平末端双链DNA片段；② The double-stranded DNA fragments are repaired at the ends to obtain blunt-ended double-stranded DNA fragments;

③给平末端双链DNA片段添加接头；所述给平末端双链DNA片段添加接头的方法包括如下步骤：将平末端双链DNA片段与具有末端转移活性和/或功能的酶和接头接触；其中，所述酶用于在所述双链DNA片段的每条链的3’末端增加核苷酸片段1；所述核苷酸片段1与所述接头的3’末端的核苷酸片段2反向互补，使得所述接头结合于所述双链DNA片段，获得添加有接头的双链DNA片段。③ Adding a linker to a blunt-ended double-stranded DNA fragment; the method for adding a linker to a blunt-ended double-stranded DNA fragment comprises the following steps: contacting the blunt-ended double-stranded DNA fragment with an enzyme having terminal transfer activity and/or function and a linker; wherein the enzyme is used to add a nucleotide fragment 1 to the 3' end of each chain of the double-stranded DNA fragment; the nucleotide fragment 1 is reversely complementary to the nucleotide fragment 2 at the 3' end of the linker, so that the linker binds to the double-stranded DNA fragment, thereby obtaining a double-stranded DNA fragment with an added linker.

优选地，过程①、②和③在同一体系中完成。Preferably, processes ①, ② and ③ are completed in the same system.

优选地，所述步骤(1)如以上第三方面“给双链DNA分子片段化、末端修复并添加接头的方法”中任一所述。 Preferably, the step (1) is as described in any one of the third aspects above, "a method for fragmenting, end-repairing and adding linkers to double-stranded DNA molecules".

进一步地，在一些实施方案中，所述制备核酸文库的方法还包括步骤(2)：取步骤(1)的产物，进行缺刻补平和阻断切除；所述缺刻补平借助具有DNA连接活性和/或功能的酶；所述阻断切除借助具有特异性切割所述接头上的酶切位点的活性和/或功能的酶。Furthermore, in some embodiments, the method for preparing a nucleic acid library further comprises step (2): taking the product of step (1), performing nick filling and blocking excision; the nick filling is performed with the aid of an enzyme having DNA ligation activity and/or function; the blocking excision is performed with the aid of an enzyme having the activity and/or function of specifically cutting the restriction site on the linker.

优选地，所述步骤(2)在同一体系中完成。Preferably, the step (2) is completed in the same system.

根据本发明的实施例，所述具有DNA连接活性和/或功能的酶为T4 DNA ligase、T3 Taqligase或T7 Taqligase等。According to an embodiment of the present invention, the enzyme having DNA ligation activity and/or function is T4 DNA ligase, T3 Taqligase or T7 Taqligase, etc.

根据本发明的实施例，所述具有特异性切割所述接头上的酶切位点的活性和/或功能的酶为USER Enzyme或UDG酶等。According to an embodiment of the present invention, the enzyme having the activity and/or function of specifically cutting the cleavage site on the connector is USER Enzyme or UDG enzyme, etc.

根据本发明的实施例，所述步骤(2)的反应体系中含有：步骤(1)的产物、具有DNA连接活性和/或功能的酶和具有特异性切割所述接头上的酶切位点的活性和/或功能的酶。根据本发明的实施例，所述步骤(2)的反应体系中还含有：反应缓冲液。根据本发明的实施例，所述具有DNA连接活性和/或功能的酶为T4 DNA ligase。根据本发明的实施例，所述具有特异性切割所述接头上的酶切位点的活性和/或功能的酶为USER Enzyme。根据本发明的实施例，所述反应缓冲液为T4 DNA Ligase Reaction Buffer。根据本发明的实施例，所述步骤(2)的反应体系可如表3所示。According to an embodiment of the present invention, the reaction system of step (2) contains: the product of step (1), an enzyme having DNA ligation activity and/or function, and an enzyme having activity and/or function of specifically cutting the enzyme cleavage site on the connector. According to an embodiment of the present invention, the reaction system of step (2) further contains: a reaction buffer. According to an embodiment of the present invention, the enzyme having DNA ligation activity and/or function is T4 DNA ligase. According to an embodiment of the present invention, the enzyme having activity and/or function of specifically cutting the enzyme cleavage site on the connector is USER Enzyme. According to an embodiment of the present invention, the reaction buffer is T4 DNA Ligase Reaction Buffer. According to an embodiment of the present invention, the reaction system of step (2) may be as shown in Table 3.

根据本发明的实施例，所述步骤(2)的反应条件为30-45℃反应10-30min。根据本发明的实施例，所述步骤(2)的反应条件为37℃反应15min。According to an embodiment of the present invention, the reaction conditions of step (2) are 30-45° C. for 10-30 min. According to an embodiment of the present invention, the reaction conditions of step (2) are 37° C. for 15 min.

根据本发明的实施例，完成所述反应后，用XP beads进行DNA纯化。根据本发明的实施例，完成所述反应后，用40μL XP beads进行DNA纯化，纯化后的DNA溶解于18μL TE缓冲液中，即为核酸文库。According to an embodiment of the present invention, after the reaction is completed, DNA is purified using XP beads. According to an embodiment of the present invention, after the reaction is completed, DNA is purified using 40 μL of XP beads, and the purified DNA is dissolved in 18 μL of TE buffer to form a nucleic acid library.

在一些实施方案中，所述制备核酸文库的方法还包括如下步骤(2)：取步骤(1)的产物，进行缺刻补平和PCR扩增；所述缺刻补平借助具有DNA连接活性和/或功能的酶；所述PCR扩增借助具有DNA聚合活性和/或功能的酶；所述PCR扩增采用的引物对的靶序列对应于所述阻断结构或所述阻断修饰的下游。In some embodiments, the method for preparing a nucleic acid library further comprises the following step (2): taking the product of step (1), performing nick filling and PCR amplification; the nick filling is performed with the aid of an enzyme having DNA ligation activity and/or function; the PCR amplification is performed with the aid of an enzyme having DNA polymerization activity and/or function; the target sequence of the primer pair used in the PCR amplification corresponds to the downstream of the blocking structure or the blocking modification.

优选地，所述步骤(2)在同一体系中完成。Preferably, the step (2) is completed in the same system.

根据本发明的实施例，所述具有DNA连接活性和/或功能的酶为T4 DNA ligase、T3 Taqligase或T7 Taqligase等。According to an embodiment of the present invention, the enzyme having DNA ligation activity and/or function is T4 DNA ligase, T3 Taqligase or T7 Taqligase, etc.

根据本发明的实施例，所述具有DNA聚合活性和/或功能的酶为Tth DNA Polymerase。According to an embodiment of the present invention, the enzyme having DNA polymerization activity and/or function is Tth DNA Polymerase.

优选地，所述PCR扩增采用的引物包含功能区段，所述功能区段中含有样本标签和/或分子标签和/或测序引物。Preferably, the primers used in the PCR amplification comprise a functional segment, and the functional segment contains a sample tag and/or a molecular tag and/or a sequencing primer.

作为一种示例，所述PCR扩增采用的引物对由通用引物1和标签引物1组成。As an example, the primer pair used in the PCR amplification consists of universal primer 1 and index primer 1.

根据本发明的实施例，通用引物1的序列如下：5’(phos)-GAACGACATGGCTACGATCCGACTT-3’。通用引物1的5’末端具有磷酸化修饰，用于匹配后续的测序平台。通用引物1如SEQ ID NO：5所示。According to an embodiment of the present invention, the sequence of universal primer 1 is as follows: 5'(phos)-GAACGACATGGCTACGATCCGACTT-3'. The 5' end of universal primer 1 has a phosphorylation modification for matching the subsequent sequencing platform. Universal primer 1 is shown in SEQ ID NO: 5.

根据本发明的实施例，标签引物1的序列如下： 标签引物1中，下划线部分为样本标签(仅为示例性的一种，样本标签 用于标记样本，可为任意核苷酸组合)。标签引物1如SEQ ID NO：6所示。According to an embodiment of the present invention, the sequence of the tag primer 1 is as follows: In the tag primer 1, the underlined part is the sample tag (only an example, the sample tag Used to label samples, it can be any nucleotide combination). Tag primer 1 is shown in SEQ ID NO: 6.

根据本发明的实施例，所述步骤(2)的反应体系中含有：步骤(1)的产物、具有DNA连接活性和/或功能的酶、具有DNA聚合活性和/或功能的酶和引物。根据本发明的实施例，所述步骤(2)的反应体系中还含有：dNTP和反应缓冲液。根据本发明的实施例，所述步骤(2)的反应体系中还含有：Mg(OAc)2。根据本发明的实施例，所述引物为所述通用引物1和所述标签引物1。根据本发明的实施例，所述具有DNA连接活性和/或功能的酶为T4 DNA ligase。根据本发明的实施例，所述具有DNA聚合活性和/或功能的酶为Tth DNA Polymerase。根据本发明的实施例，所述反应缓冲液为5×Tth RT-PCR Buffer。根据本发明的实施例，所述步骤(2)的反应体系可如表6所示。According to an embodiment of the present invention, the reaction system of step (2) contains: the product of step (1), an enzyme having DNA ligation activity and/or function, an enzyme having DNA polymerization activity and/or function, and a primer. According to an embodiment of the present invention, the reaction system of step (2) further contains: dNTP and reaction buffer. According to an embodiment of the present invention, the reaction system of step (2) further contains: Mg(OAc) 2. According to an embodiment of the present invention, the primers are the universal primer 1 and the tag primer 1. According to an embodiment of the present invention, the enzyme having DNA ligation activity and/or function is T4 DNA ligase. According to an embodiment of the present invention, the enzyme having DNA polymerization activity and/or function is Tth DNA Polymerase. According to an embodiment of the present invention, the reaction buffer is 5×Tth RT-PCR Buffer. According to an embodiment of the present invention, the reaction system of step (2) may be as shown in Table 6.

根据本发明的实施例，所述步骤(2)的反应条件如下所示：第一阶段，在T5℃反应t5分钟；第二阶段，在T6℃反应t6秒钟，T7℃反应t7秒钟，T8℃反应t8秒钟，X个循环；第三阶段，在T9℃反应t9分钟。T5：94-98℃，T6：94-98℃，T7：50-65℃，T8：65-75℃，T9：65-75℃，t5：0.5-2分钟，t6：5-30秒，t7：10-60秒，t8：10-60秒，t9：1-5分钟，X:6-25次。在一些实施方案中，T5为95℃，T6为95℃，T7为62℃，T8为72℃，T9为72℃，t5为2分钟，t6为10秒，t7为30秒，t8为30秒，t9为5分钟。在一些实施方案中，X为10。According to an embodiment of the present invention, the reaction conditions of step (2) are as follows: in the first stage, the reaction is carried out at T5°C for t5 minutes; in the second stage, the reaction is carried out at T6°C for t6 seconds, T7°C for t7 seconds, and T8°C for t8 seconds, for X cycles; in the third stage, the reaction is carried out at T9°C for t9 minutes. T5: 94-98°C, T6: 94-98°C, T7: 50-65°C, T8 : 65-75°C, T9: 65-75°C, t5: 0.5-2 minutes, t6 : 5-30 seconds, t7 : 10-60 seconds, t8 : 10-60 seconds, t9 : 1-5 minutes, and X: 6-25 times. In some embodiments, T5 is 95° C., T6 is 95° C., T7 is 62° C., T8 is 72° C., T9 is 72° C., t5 is 2 minutes, t6 is 10 seconds, t7 is 30 seconds, t8 is 30 seconds, and t9 is 5 minutes. In some embodiments, X is 10.

作为一种示例，完成所述反应后，用XP beads进行DNA纯化。作为一种示例，完成所述反应后，用40μL XP beads进行DNA纯化，纯化后的DNA溶解于30μL TE缓冲液中，即为核酸文库。As an example, after the reaction is complete, DNA is purified using XP beads. As an example, after the reaction is complete, DNA is purified using 40 μL of XP beads and the purified DNA is dissolved in 30 μL of TE buffer to prepare the nucleic acid library.

在本发明的第六方面，本发明还提供了一种试剂盒，其包括如下组件：In a sixth aspect of the present invention, the present invention further provides a kit comprising the following components:

具有末端转移活性和/或功能的酶和接头；Enzymes and linkers having terminal transfer activity and/or function;

所述酶用于在平末端双链DNA片段的每条链的3’末端增加核苷酸片段1；The enzyme is used to add nucleotide fragment 1 to the 3' end of each strand of a blunt-ended double-stranded DNA fragment;

所述核苷酸片段1与所述接头的3’末端的核苷酸片段2反向互补。The nucleotide fragment 1 is reverse complementary to the nucleotide fragment 2 at the 3' end of the linker.

根据本发明的实施例，所述核苷酸片段1由N1个核苷酸组成，所述核苷酸片段2由N2个核苷酸组成；N1和N2分别独立地选自1-10的自然数。优选地，N1和N2分别独立地选自2-5的自然数。优选地，N1为3，N2为3。According to an embodiment of the present invention, the nucleotide fragment 1 consists of N1 nucleotides, and the nucleotide fragment 2 consists of N2 nucleotides; N1 and N2 are each independently selected from a natural number between 1 and 10. Preferably, N1 and N2 are each independently selected from a natural number between 2 and 5. Preferably, N1 is 3 and N2 is 3.

根据本发明的实施例，所述核苷酸片段2中的核苷酸为核糖核苷酸、脱氧核糖核苷酸或经修饰的核苷酸。According to an embodiment of the present invention, the nucleotides in the nucleotide fragment 2 are ribonucleotides, deoxyribonucleotides or modified nucleotides.

根据本发明的实施例，所述核苷酸片段2中的核苷酸为核糖核苷酸。优选地，所述核苷酸片段1为CCC；优选地，所述核苷酸片段2为rGrGrG。需要说明的是，所述rGrGrG代表3个连续的核糖核苷酸G。在一些实施例中，RNA/DNA杂交链的结合性高于RNA/RNA杂交链以及DNA/DNA杂交链。According to an embodiment of the present invention, the nucleotides in nucleotide segment 2 are ribonucleotides. Preferably, nucleotide segment 1 is CCC; preferably, nucleotide segment 2 is rGrGrG. It should be noted that rGrGrG represents three consecutive ribonucleotides G. In some embodiments, the binding affinity of the RNA/DNA hybrid chain is higher than that of the RNA/RNA hybrid chain and the DNA/DNA hybrid chain.

根据本发明的实施例，所述核苷酸片段2中的核苷酸为脱氧核糖核苷酸。优选地，所述核苷酸片段1为CCC；优选地，所述核苷酸片段2为GGG。According to an embodiment of the present invention, the nucleotides in the nucleotide segment 2 are deoxyribonucleotides. Preferably, the nucleotide segment 1 is CCC; preferably, the nucleotide segment 2 is GGG.

根据本发明的实施例，所述核苷酸片段2中的核苷酸为经修饰的核苷酸。优选地，所述经修饰的核苷酸为锁核酸。According to an embodiment of the present invention, the nucleotides in the nucleotide fragment 2 are modified nucleotides. Preferably, the modified nucleotides are locked nucleic acids.

根据本发明的实施例，在所述接头中，与核苷酸片段2的5’端相邻的核苷酸为N。在一些实施例中，所述N代表A或T或C或G。According to an embodiment of the present invention, in the linker, the nucleotide adjacent to the 5' end of nucleotide fragment 2 is N. In some embodiments, the N represents A, T, C, or G.

根据本发明的实施例，所述接头的5’末端具有以阻断核酸延伸为目的的阻断结构或阻断修饰。 According to an embodiment of the present invention, the 5' end of the linker has a blocking structure or blocking modification for the purpose of blocking nucleic acid extension.

根据本发明的实施例，所述阻断结构为发卡结构。According to an embodiment of the present invention, the blocking structure is a hairpin structure.

根据本发明的实施例，所述阻断修饰选自：双脱氧胞苷修饰、反向dt修饰、磷酸基团修饰、硫代基团修饰或间臂修饰。优选地，所述阻断修饰为间臂修饰。优选地，所述间臂修饰为碳骨架修饰或糖骨架修饰。优选地，所述间臂修饰为Spacer C3(即3个CH2)、Spacer C6(即6个CH2)或Spacer C9(即9个CH2)等。According to an embodiment of the present invention, the blocking modification is selected from: dideoxycytidine modification, reverse dt modification, phosphate group modification, thio group modification, or spacer modification. Preferably, the blocking modification is a spacer modification. Preferably, the spacer modification is a carbon backbone modification or a sugar backbone modification. Preferably, the spacer modification is Spacer C3 (i.e., 3 CH 2 ), Spacer C6 (i.e., 6 CH 2 ), or Spacer C9 (i.e., 9 CH 2 ), etc.

根据本发明的实施例，所述接头包含功能区段，所述功能区段位于阻断结构或阻断修饰的下游。优选地，所述功能区段包含样本标签和/或分子标签和/或测序引物。According to an embodiment of the present invention, the linker comprises a functional segment, and the functional segment is located downstream of the blocking structure or blocking modification. Preferably, the functional segment comprises a sample tag and/or a molecular tag and/or a sequencing primer.

根据本发明的实施例，所述接头包含酶切位点，所述酶切位点位于阻断结构或阻断修饰的下游。所述酶切位点用于在后续步骤中通过酶切去除阻断修饰或阻断结构。根据本发明的实施例，所述酶切所用的酶为UDG Enzyme、USER Enzyme或RnaseH等。根据本发明的实施例，所述酶切位点与阻断结构或阻断修饰相邻。根据本发明的实施例，所述酶切位点与阻断结构或阻断修饰间隔N3个核苷酸，N3为自然数。优选地，N3选自1-10的自然数。优选地，N3选自1-5的自然数。优选地，N3为1或2或3。根据本发明的实施例，所述酶切位点选自：dUTP或rNTP。According to an embodiment of the present invention, the linker comprises an enzyme cleavage site, and the enzyme cleavage site is located downstream of the blocking structure or blocking modification. The enzyme cleavage site is used to remove the blocking modification or blocking structure by enzyme cleavage in a subsequent step. According to an embodiment of the present invention, the enzyme used for the enzyme cleavage is UDG Enzyme, USER Enzyme or RnaseH, etc. According to an embodiment of the present invention, the enzyme cleavage site is adjacent to the blocking structure or blocking modification. According to an embodiment of the present invention, the enzyme cleavage site and the blocking structure or blocking modification are separated by N3 nucleotides, and N3 is a natural number. Preferably, N3 is selected from a natural number of 1-10. Preferably, N3 is selected from a natural number of 1-5. Preferably, N3 is 1 or 2 or 3. According to an embodiment of the present invention, the enzyme cleavage site is selected from: dUTP or rNTP.

根据本发明的实施例，所述具有末端转移活性和/或功能的酶具有偏好性。如本文所用，所述偏好性指的是偏好于在链的3’末端增加3个特定的核苷酸。如本文所用，偏好性指的是存在20％以上概率在链的3’末端增加3个特定的核苷酸。根据本发明的实施例，所述3个特定的核苷酸为3个核苷酸C。According to embodiments of the present invention, the enzyme having terminal transfer activity and/or function has a preference. As used herein, the preference refers to a preference for adding three specific nucleotides to the 3' end of a strand. As used herein, the preference refers to a greater than 20% probability of adding three specific nucleotides to the 3' end of a strand. According to embodiments of the present invention, the three specific nucleotides are three C nucleotides.

根据本发明的实施例，接头具有各种形式。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：阻断修饰(间臂修饰)、U、样本标签、GGG(如图1的a所示)。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：自5’末端至3’末端依次具有如下区段：阻断修饰(间臂修饰)、U、GGG(如图1的b所示)。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：阻断修饰(间臂修饰)、样本标签、GGG(如图1的c所示)。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：阻断修饰(间臂修饰)、GGG(如图1的d所示)。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：阻断结构(发卡结构)、U、样本标签、GGG(如图1的e所示)。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：阻断结构(发卡结构)、U、GGG(如图1的f所示)。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：阻断结构(发卡结构)、样本标签、GGG(如图1的g所示)。根据本发明的实施例，所述接头自5’末端至3’末端依次具有如下区段：阻断结构(发卡结构)、GGG(如图1的h所示)。According to an embodiment of the present invention, the connector has various forms. According to an embodiment of the present invention, the connector has the following sections from the 5’ end to the 3’ end in sequence: blocking modification (inter-arm modification), U, sample tag, GGG (as shown in a of Figure 1). According to an embodiment of the present invention, the connector has the following sections from the 5’ end to the 3’ end in sequence: blocking modification (inter-arm modification), U, GGG (as shown in b of Figure 1). According to an embodiment of the present invention, the connector has the following sections from the 5’ end to the 3’ end in sequence: blocking modification (inter-arm modification), sample tag, GGG (as shown in c of Figure 1). According to an embodiment of the present invention, the connector has the following sections from the 5’ end to the 3’ end in sequence: blocking modification (inter-arm modification), GGG (as shown in d of Figure 1). According to an embodiment of the present invention, the connector has the following sections from the 5’ end to the 3’ end in sequence: blocking structure (hairpin structure), U, sample tag, GGG (as shown in e of Figure 1). According to an embodiment of the present invention, the connector has the following sections from the 5' end to the 3' end: blocking structure (hairpin structure), U, GGG (as shown in Figure 1 f). According to an embodiment of the present invention, the connector has the following sections from the 5' end to the 3' end: blocking structure (hairpin structure), sample tag, GGG (as shown in Figure 1 g). According to an embodiment of the present invention, the connector has the following sections from the 5' end to the 3' end: blocking structure (hairpin structure), GGG (as shown in Figure 1 h).

根据本发明的实施例，所述具有末端转移活性和/或功能的酶为具有末端转移活性的逆转录酶。优选地，所述具有末端转移活性的逆转录酶为鼠白血病逆转录酶(M-MLV)。优选地，所述具有末端转移活性的逆转录酶为鸟白血病逆转录酶(AMV)。优选地，所述具有末端转移活性的逆转录酶为SuperScriptTMIV逆转录酶、SuperScriptTMI逆转录酶、SuperScriptTMII逆转录酶或SuperScriptTMIII逆转录酶。According to an embodiment of the present invention, the enzyme having terminal transfer activity and/or function is a reverse transcriptase having terminal transfer activity. Preferably, the reverse transcriptase having terminal transfer activity is murine leukemia reverse transcriptase (M-MLV). Preferably, the reverse transcriptase having terminal transfer activity is avian leukemia reverse transcriptase (AMV). Preferably, the reverse transcriptase having terminal transfer activity is SuperScript ™ IV reverse transcriptase, SuperScript ™ I reverse transcriptase, SuperScript ™ II reverse transcriptase, or SuperScript ™ III reverse transcriptase.

根据本发明的实施例，所述接头可为接头1或接头2。According to an embodiment of the present invention, the connector may be connector 1 or connector 2.

其中，所述接头1为寡聚核苷酸单链衍生物，其结构式如下： 接头1是在寡聚核苷酸单链1的5’末端进行阻断修饰得到的；阻断修饰即通过iSp6(Spacer C6，即6个CH2)与3个核苷酸A连接；寡聚核苷酸单链1如结构式中iSp6右侧所示(SEQ ID NO：1)；寡聚核苷酸单链1中，自5’末端第3位为修饰碱基U，自3’末端第1至3位为rG(即核糖核酸G)且第4位为N(N代表A\T\C\G)。Wherein, the linker 1 is a single-stranded oligonucleotide derivative, and its structural formula is as follows: Linker 1 is obtained by performing a blocking modification on the 5' end of single-stranded oligonucleotide 1; the blocking modification is to connect three nucleotide A's via iSp6 (Spacer C6, i.e., six CH2 groups ); single-stranded oligonucleotide 1 is shown to the right of iSp6 in the structural formula (SEQ ID NO: 1); in single-stranded oligonucleotide 1, the third position from the 5' end is a modified base U, and positions 1 to 3 from the 3' end are rG (i.e., ribonucleic acid G), and the fourth position is N (N represents A\T\C\G).

其中，所述接头2为寡聚核苷酸单链衍生物，其结构式如下： 接头2是在寡聚核苷酸单链2的5’末端进行阻断修饰得到的；阻断修饰即通过iSp6(Spacer C6，即6个CH2)与3个核苷酸A连接；寡聚核苷酸单链2如结构式中iSp6右侧所示(SEQ ID NO：2)；寡聚核苷酸单链2中，自5’末端第1位为修饰碱基U，自3’末端第1至3位为rG(即核糖核酸G)且第4位为N(N代表A\T\C\G)，下划线部分为样本标签(仅为示例性的一种，样本标签用于标记样本，可为任意核苷酸组合)。Wherein, the linker 2 is a single-stranded oligonucleotide derivative, and its structural formula is as follows: Linker 2 is obtained by performing a blocking modification on the 5' end of oligonucleotide single strand 2; the blocking modification is to connect three nucleotides A via iSp6 (Spacer C6, i.e., six CH2 groups ); oligonucleotide single strand 2 is shown to the right of iSp6 in the structural formula (SEQ ID NO: 2); in oligonucleotide single strand 2, the first position from the 5' end is a modified base U, the first to third positions from the 3' end are rG (i.e., ribonucleic acid G), and the fourth position is N (N represents A\T\C\G), and the underlined portion is a sample tag (only an exemplary one, the sample tag is used to label the sample and can be any combination of nucleotides).

根据本发明的实施例，所述接头可为接头3或接头4。According to an embodiment of the present invention, the connector may be connector 3 or connector 4.

其中，所述接头3为寡聚核苷酸单链，序列如下： 接头3的5’端具有阻断结构(发卡结构，见波浪下划线标记)，自3’末端第1至3位为rG(即核糖核酸G)且第4位为N(N代表A\T\C\G)。接头3如SEQ ID NO：3所示。Wherein, the linker 3 is a single-stranded oligonucleotide, and the sequence is as follows: The 5' end of linker 3 has a blocking structure (hairpin structure, see wavy underline mark), and the first to third positions from the 3' end are rG (i.e., ribonucleic acid G) and the fourth position is N (N represents A\T\C\G). Linker 3 is shown in SEQ ID NO: 3.

其中，所述接头4为寡聚核苷酸单链，序列如下：接头4的5’端具有阻断结构(发卡结构，见波浪下划线标记)，自3’末端第1至3位为rG(即核糖核酸G)且第4位为N(N代表A\T\C\G)。接头4如SEQ ID NO：4所示。Wherein, the linker 4 is a single-stranded oligonucleotide with the following sequence: The 5' end of linker 4 has a blocking structure (hairpin structure, see wavy underline mark), and the first to third positions from the 3' end are rG (i.e., ribonucleic acid G) and the fourth position is N (N represents A\T\C\G). Linker 4 is shown in SEQ ID NO: 4.

进一步的，所述试剂盒还包括如下组件：用于模板延伸的组件；Furthermore, the kit further comprises the following components: a component for template extension;

所述模板延伸是添加有接头的双链DNA片段以所述接头为模板进行5’至3’方向的延伸。The template extension is to extend the double-stranded DNA fragment with the adapter added in the 5' to 3' direction using the adapter as a template.

根据本发明的实施例，所述用于模板延伸的组件为具有DNA聚合活性和/或功能的酶。根据本发明的实施例，所述具有DNA聚合活性和/或功能的酶为具有末端转移活性的逆转录酶。优选地，所述具有末端转移活性的逆转录酶为鼠白血病逆转录酶(M-MLV)。优选地，所述具有末端转移活性的逆转录酶为鸟白血病逆转录酶(AMV)。优选地，所述具有末端转移活性的逆转录酶为SuperScriptTMIV逆转录酶、SuperScriptTMI逆转录酶、SuperScriptTMII逆转录酶或SuperScriptTMIII逆转录酶。According to an embodiment of the present invention, the component for template extension is an enzyme having DNA polymerization activity and/or function. According to an embodiment of the present invention, the enzyme having DNA polymerization activity and/or function is a reverse transcriptase having terminal transfer activity. Preferably, the reverse transcriptase having terminal transfer activity is murine leukemia reverse transcriptase (M-MLV). Preferably, the reverse transcriptase having terminal transfer activity is avian leukemia reverse transcriptase (AMV). Preferably, the reverse transcriptase having terminal transfer activity is SuperScript ™ IV reverse transcriptase, SuperScript ™ I reverse transcriptase, SuperScript ™ II reverse transcriptase, or SuperScript ™ III reverse transcriptase.

根据本发明的实施例，所述具有DNA聚合活性和/或功能的酶为Klenow片段。根据本发明的实施例，所述具有DNA聚合活性和/或功能的酶为T4 DNA聚合酶。According to an embodiment of the present invention, the enzyme having DNA polymerization activity and/or function is a Klenow fragment. According to an embodiment of the present invention, the enzyme having DNA polymerization activity and/or function is T4 DNA polymerase.

所述试剂盒用于给平末端双链DNA片段添加接头。The kit is used to add adapters to blunt-ended double-stranded DNA fragments.

进一步的，所述试剂盒还包括如下组件：用于双链DNA片段进行末端修复的组件。Furthermore, the kit also includes the following components: a component for performing end repair on double-stranded DNA fragments.

优选地，所述用于双链DNA片段进行末端修复的组件为具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶。Preferably, the component for end repair of double-stranded DNA fragments is an enzyme having 5'→3' polymerization and 3'→5' exonucleolytic activities and/or functions.

根据本发明的实施例，所述具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶为Klenow片段。根据本发明的实施例，所述具有5'→3'聚合和3'→5'核酸外切活性和/ 或功能的酶为T4 DNA聚合酶。According to an embodiment of the present invention, the enzyme having 5'→3' polymerization and 3'→5' exonuclease activity and/or function is a Klenow fragment. According to an embodiment of the present invention, the enzyme having 5'→3' polymerization and 3'→5' exonuclease activity and/or function is a Klenow fragment. Or the functional enzyme is T4 DNA polymerase.

根据本发明的实施例，所述具有DNA聚合活性和/或功能的酶为Klenow片段。根据本发明的实施例，所述具有DNA聚合活性和/或功能的酶为T4 DNA聚合酶。According to an embodiment of the present invention, the enzyme having DNA polymerization activity and/or function is a Klenow fragment. According to an embodiment of the present invention, the enzyme having DNA polymerization activity and/or function is T4 DNA polymerase.

所述试剂盒用于给双链DNA片段末端修复并添加接头。The kit is used for repairing the ends of double-stranded DNA fragments and adding adapters.

进一步的，所述试剂盒还包括如下组件：用于双链DNA分子进行片段化的组件和用于双链DNA片段进行末端修复的组件。Furthermore, the kit also includes the following components: a component for fragmenting double-stranded DNA molecules and a component for end-repairing double-stranded DNA fragments.

优选地，所述用于双链DNA分子进行片段化的组件为物理打断所需组件或酶切打断所需组件。Preferably, the components for fragmenting double-stranded DNA molecules are components required for physical shearing or components required for enzymatic shearing.

根据本发明的实施例，所述用于双链DNA分子进行片段化的组件为具有核酸内切活性和/或功能的酶。根据本发明的实施例，所述具有核酸内切活性和/或功能的酶为Dnase I或Endonuclease V。According to an embodiment of the present invention, the component for fragmenting double-stranded DNA molecules is an enzyme having endonuclease activity and/or function. According to an embodiment of the present invention, the enzyme having endonuclease activity and/or function is DNase I or Endonuclease V.

根据本发明的实施例，所述用于双链DNA片段进行末端修复的组件为具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶。根据本发明的实施例，所述具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶为Klenow片段。根据本发明的实施例，所述具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶为T4 DNA聚合酶。According to an embodiment of the present invention, the component for end repair of double-stranded DNA fragments is an enzyme having 5'→3' polymerization and 3'→5' exonucleolytic activity and/or function. According to an embodiment of the present invention, the enzyme having 5'→3' polymerization and 3'→5' exonucleolytic activity and/or function is a Klenow fragment. According to an embodiment of the present invention, the enzyme having 5'→3' polymerization and 3'→5' exonucleolytic activity and/or function is T4 DNA polymerase.

所述试剂盒用于给双链DNA分子片段化、末端修复并添加接头。The kit is used for fragmenting, end-repairing and adding linkers to double-stranded DNA molecules.

进一步的，所述试剂盒还包括如下组件：用于缺刻补平的组件和用于阻断切除的组件。Furthermore, the kit also includes the following components: a component for nick filling and a component for blocking excision.

根据本发明的实施例，所述用于缺刻补平的组件为具有DNA连接活性和/或功能的酶。根据本发明的实施例，所述具有DNA连接活性和/或功能的酶为T4 DNA ligase、T3 Taqligase或T7 Taqligase等。According to an embodiment of the present invention, the component for nick filling is an enzyme having DNA ligation activity and/or function. According to an embodiment of the present invention, the enzyme having DNA ligation activity and/or function is T4 DNA ligase, T3 Taqligase, or T7 Taqligase.

根据本发明的实施例，所述用于阻断切除的组件为具有特异性切割所述接头上的酶切位点的活性和/或功能的酶。根据本发明的实施例，所述具有特异性切割所述接头上的酶切位点的活性和/或功能的酶为USER Enzyme或UDG酶等。According to an embodiment of the present invention, the component for blocking excision is an enzyme having the activity and/or function of specifically cleaving the cleavage site on the linker. According to an embodiment of the present invention, the enzyme having the activity and/or function of specifically cleaving the cleavage site on the linker is USER Enzyme or UDG enzyme, etc.

进一步的，所述试剂盒还包括如下组件：用于缺刻补平的组件和用于PCR扩增的组件。Furthermore, the kit also includes the following components: a component for nick filling and a component for PCR amplification.

根据本发明的实施例，所述用于缺刻补平的组件为具有DNA连接活性和/或功能的酶。根据本发明的实施例，所述具有DNA连接活性和/或功能的酶为T4 DNA ligase、T3 Taqligase或T7 Taqligase等。According to an embodiment of the present invention, the component for nick filling is an enzyme having DNA ligation activity and/or function. According to an embodiment of the present invention, the enzyme having DNA ligation activity and/or function is T4 DNA ligase, T3 Taqligase, or T7 Taqligase.

根据本发明的实施例，所述用于PCR扩增的组件包括具有DNA聚合活性和/或功能的酶。根据本发明的实施例，所述具有DNA聚合活性和/或功能的酶为Tth DNA Polymerase。According to an embodiment of the present invention, the component for PCR amplification includes an enzyme having DNA polymerization activity and/or function. According to an embodiment of the present invention, the enzyme having DNA polymerization activity and/or function is Tth DNA Polymerase.

根据本发明的实施例，所述用于PCR扩增的组件还包括PCR扩增采用的引物对。所述PCR扩增采用的引物对的靶序列对应于所述阻断结构或所述阻断修饰的下游。根据本发明的实施例，所述PCR扩增采用的引物包含功能区段，所述功能区段中含有样本标签和/或分子标签和/或测序引物。根据本发明的实施例，所述PCR扩增采用的引物对由通用引物1和标签引物1组成。According to an embodiment of the present invention, the component for PCR amplification further comprises a primer pair for PCR amplification. The target sequence of the primer pair for PCR amplification corresponds to the downstream of the blocking structure or the blocking modification. According to an embodiment of the present invention, the primers for PCR amplification comprise a functional segment containing a sample tag and/or a molecular tag and/or a sequencing primer. According to an embodiment of the present invention, the primer pair for PCR amplification consists of a universal primer 1 and a tag primer 1.

根据本发明的实施例，通用引物1的序列如下：5’(phos)-GAACGACATGGCTACGATCCGACTT-3’。通用引物1的5’末端具有磷酸化 修饰，用于匹配后续的测序平台。通用引物1如SEQ ID NO：5所示。According to an embodiment of the present invention, the sequence of universal primer 1 is as follows: 5'(phos)-GAACGACATGGCTACGATCCGACTT-3'. The 5' end of universal primer 1 has a phosphorylated Modified to match the subsequent sequencing platform. Universal primer 1 is shown in SEQ ID NO: 5.

根据本发明的实施例，标签引物1的序列如下： 标签引物1中，下划线部分为样本标签(仅为示例性的一种，样本标签用于标记样本，可为任意核苷酸组合)。标签引物1如SEQ ID NO：6所示。According to an embodiment of the present invention, the sequence of the tag primer 1 is as follows: In the tag primer 1, the underlined portion is the sample tag (which is only an exemplary one, and the sample tag is used to label the sample and can be any nucleotide combination). The tag primer 1 is shown in SEQ ID NO: 6.

所述试剂盒用于制备核酸文库。The kit is used for preparing a nucleic acid library.

根据本发明的实施例，以上任一所述双链DNA分子指的是基因组DNA、线粒体DNA等。According to an embodiment of the present invention, any of the above double-stranded DNA molecules refers to genomic DNA, mitochondrial DNA, etc.

在本发明的第七方面，本发明还保护一种核酸文库，是通过以上任一所述制备核酸文库的方法制备获得的。In the seventh aspect of the present invention, the present invention also protects a nucleic acid library prepared by any of the above methods for preparing a nucleic acid library.

在本发明的第八方面，本发明还保护所述核酸文库在高通量测序中的应用。In the eighth aspect of the present invention, the present invention also protects the use of the nucleic acid library in high-throughput sequencing.

根据本发明的实施例，所述核酸文库可为DNA文库。According to an embodiment of the present invention, the nucleic acid library may be a DNA library.

本发明公开了利用具有末端转移活性和/功能的酶进行接头连接的方法及其在制备高通量测序文库中的应用。首先，本发明提供了给平末端双链DNA片段添加接头的方法，将平末端双链DNA片段与具有末端转移活性和/或功能的酶和接头接触；所述酶用于在所述双链DNA片段的每条链的3’末端增加核苷酸片段1；所述核苷酸片段1与所述接头的3’末端的核苷酸片段2反向互补，使得所述接头结合于所述双链DNA片段，获得添加有接头的双链DNA片段。进一步，本发明将打断、末端修复和接头添加在一个反应体系中进行，大大降低了文库制备的操作复杂度。本发明解决了高通量测序的文库构建中步骤过多，技术使用门槛高、模板利用率偏低等问题。The present invention discloses a method for connecting adapters using an enzyme having terminal transfer activity and/or function and its application in preparing high-throughput sequencing libraries. First, the present invention provides a method for adding adapters to blunt-end double-stranded DNA fragments, contacting the blunt-end double-stranded DNA fragments with an enzyme having terminal transfer activity and/or function and an adapter; the enzyme is used to add a nucleotide fragment 1 to the 3' end of each chain of the double-stranded DNA fragment; the nucleotide fragment 1 is reversely complementary to the nucleotide fragment 2 at the 3' end of the adapter, so that the adapter is bound to the double-stranded DNA fragment to obtain a double-stranded DNA fragment with an added adapter. Furthermore, the present invention performs shearing, end repair and adapter addition in one reaction system, which greatly reduces the operational complexity of library preparation. The present invention solves the problems of too many steps in the construction of high-throughput sequencing libraries, high threshold for technology use, low template utilization, and the like.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1为接头的各种示例性形式展示。FIG1 shows various exemplary forms of connectors.

图2为实施例1的流程示意图。FIG2 is a schematic flow diagram of Example 1.

图3为实施例2的流程示意图。FIG3 is a schematic flow diagram of Example 2.

图4为实施例3的流程示意图。FIG4 is a schematic flow diagram of Example 3.

图5为实施例1以及实施例2与对比例的流程比较示意图。FIG5 is a schematic diagram showing the process comparison of Example 1 and Example 2 and the comparative example.

具体实施方式DETAILED DESCRIPTION

以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。实施例中的接头为寡聚核苷酸单链或寡聚核苷酸单链衍生物，寡聚核苷酸单链指的是长度为3-100nt的单链寡聚核苷酸。XP beads：Beckman公司的Agencourt AMPure XP磁珠，货号A63881。DNBSEQ-G400：华大智造的DNBSEQ-G400(又称为MGISEQ-2000)高通量基因测序平台。The following examples are for better understanding of the present invention, but they do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged. The linkers in the examples are single-stranded oligonucleotides or single-stranded oligonucleotide derivatives. Single-stranded oligonucleotides refer to single-stranded oligonucleotides with a length of 3-100nt. XP beads: Agencourt AMPure XP magnetic beads from Beckman, catalog number A63881. DNBSEQ-G400: DNBSEQ-G400 (also known as MGISEQ-2000) high-throughput gene sequencing platform manufactured by BGI.

dNTP溶液：即Deoxynucleotide(dNTP)Solution Mix；NEB公司,货号为N0447V。dNTP solution: Deoxynucleotide (dNTP) Solution Mix; NEB, product number N0447V.

模板DNA：NA12878(人类基因组DNA标准品)。Template DNA: NA12878 (human genomic DNA standard).

SuperScriptTMIV逆转录酶(液态试剂；规格为200U/μL)：ThermoFisher公司，货号为18090010。5×Superscript IV first-strand buffer即与SuperScriptTMIV逆转录酶配套出售的5×RT缓冲液。 SuperScript ™ IV reverse transcriptase (liquid reagent; specification: 200 U/μL): ThermoFisher, catalog number: 18090010. 5× Superscript IV first-strand buffer is the 5× RT buffer sold in conjunction with SuperScript ™ IV reverse transcriptase.

Dnase I溶液是将Dnase I用DNase I Reaction Buffer稀释至1000倍体积得到的。Dnase I(液态试剂；规格为2000units/ml)和DNase I Reaction Buffer：NEB公司,货号为M0303S。DNase I solution is prepared by diluting DNase I to 1000 times the volume with DNase I Reaction Buffer. DNase I (liquid reagent; specification: 2000 units/ml) and DNase I Reaction Buffer: NEB, catalog number M0303S.

Klenow片段(液态试剂；规格为10U/μL)：ThermoFisher公司，货号为EP0051。Klenow fragment (liquid reagent; specification: 10 U/μL): ThermoFisher, product number: EP0051.

T4 DNA Ligase(液态试剂；规格为400000units/ml)和T4 DNA Ligase Reaction Buffer(10×)：NEB公司,货号为M0202S。T4 DNA Ligase (liquid reagent; specification: 400,000 units/ml) and T4 DNA Ligase Reaction Buffer (10×): NEB, product number: M0202S.

(液态试剂；规格为1000units/ml)：NEB公司,货号为M5505S。 (Liquid reagent; specification: 1000 units/ml): NEB, product number: M5505S.

Tth DNA Polymerase(液态试剂；规格为5U/μL)和5×Tth RT-PCR Buffer：翊圣公司，货号14607ES72。Tth DNA Polymerase (liquid reagent; specification: 5U/μL) and 5×Tth RT-PCR Buffer: Yishen Company, product number 14607ES72.

实施例1、Example 1

流程示意图见图2。The flow chart is shown in Figure 2.

1、片段化、末端修复、接头连接和模板延伸1. Fragmentation, end repair, adapter ligation, and template extension

按照表1制备反应体系(总体系为20μL)，然后按照表2的程序进行反应。Prepare the reaction system according to Table 1 (total system is 20 μL), and then carry out the reaction according to the procedure in Table 2.

表1
Table 1

接头1为寡聚核苷酸单链衍生物，结构式如下：
Linker 1 is a single-stranded oligonucleotide derivative with the following structural formula:

接头2为寡聚核苷酸单链衍生物，结构式如下：
Linker 2 is a single-stranded oligonucleotide derivative with the following structural formula:

接头1是在寡聚核苷酸单链1的5’末端进行阻断修饰得到的；阻断修饰即通过iSp6(Spacer C6，即6个CH2)与3个核苷酸A连接；寡聚核苷酸单链1如结构式中iSp6右侧所示(SEQ ID NO：1)；寡聚核苷酸单链1中，自5’末端第3位为修饰碱基U，自3’末端第1至3位为rG(即核糖核酸G)且第4位为N(N代表A\T\C\G)。Linker 1 is obtained by performing a blocking modification on the 5' end of single-stranded oligonucleotide 1; the blocking modification is to connect three nucleotide A's via iSp6 (Spacer C6, i.e., six CH2 groups ); single-stranded oligonucleotide 1 is shown to the right of iSp6 in the structural formula (SEQ ID NO: 1); in single-stranded oligonucleotide 1, the third position from the 5' end is a modified base U, and positions 1 to 3 from the 3' end are rG (i.e., ribonucleic acid G), and the fourth position is N (N represents A\T\C\G).

接头2是在寡聚核苷酸单链2的5’末端进行阻断修饰得到的；阻断修饰即通过iSp6(Spacer C6，即6个CH2)与3个核苷酸A连接；寡聚核苷酸单链2如结构式中iSp6右侧所示(SEQ ID NO：2)；寡聚核苷酸单链2中，自5’末端第1位为修饰碱基U，自3’末端第1至3位为rG(即核糖核酸G)且第4位为N(N代表A\T\C\G)，下划线部分为样本标签(仅为示例性的一种，样本标签用于标记样本，可为任意核苷酸组合)。Linker 2 is obtained by performing a blocking modification on the 5' end of oligonucleotide single strand 2; the blocking modification is to connect three nucleotides A via iSp6 (Spacer C6, i.e., six CH2 groups ); oligonucleotide single strand 2 is shown to the right of iSp6 in the structural formula (SEQ ID NO: 2); in oligonucleotide single strand 2, the first position from the 5' end is a modified base U, the first to third positions from the 3' end are rG (i.e., ribonucleic acid G), and the fourth position is N (N represents A\T\C\G), and the underlined portion is a sample tag (only an exemplary one, the sample tag is used to label the sample and can be any combination of nucleotides).

表2

Table 2

本步骤的反应体系中，同时具有Dnase I、Klenow片段和逆转录酶。Dnase I发挥核酸内切功能。逆转录酶发挥末端转移功能和DNA聚合功能。Klenow片段发挥5'→3'聚合功能和3'→5'核酸外切功能和链置换功能。首先，在Dnase I的作用下，模板DNA(双链DNA分子)被随机切割为小片段(双链DNA片段)；然后，在Klenow片段的作用下，双链DNA片段被末端修复为平末端片段(两端均为平末端的双链DNA片段)；然后在逆转录酶的作用下(末端转移功能)，平末端片段的每条链的3’末端增加3个核苷酸C，形成粘末端片段(两端均为3’端突出末端的双链DNA片段)；借助与粘末端的反向互补关系，寡聚核苷酸单链1衍生物和寡聚核苷酸单链2衍生物结合至双链DNA片段(目标物为一端添加寡聚核苷酸单链1衍生物且另一端添加寡聚核苷酸单链2衍生物的双链DNA片段)，然后在逆转录酶的作用下(DNA聚合功能)双链DNA片段的两个3’端突出末端分别以两种接头的寡聚核苷酸单链为模板进行5’至3’方向的延伸。The reaction system in this step contains DNase I, Klenow fragment, and reverse transcriptase. DNase I performs endonucleolytic functions. Reverse transcriptase performs terminal transfer functions and DNA polymerization. Klenow fragment performs 5'→3' polymerization, 3'→5' exonucleolytic functions, and strand displacement functions. First, under the action of DNase I, the template DNA (double-stranded DNA molecule) is randomly cut into small fragments (double-stranded DNA fragments); then, under the action of Klenow fragment, the double-stranded DNA fragments are end-repaired into blunt-end fragments (double-stranded DNA fragments with blunt ends at both ends); then, under the action of reverse transcriptase (terminal transfer function), three nucleotide Cs are added to the 3' end of each chain of the blunt-end fragment to form a sticky-end fragment (double-stranded DNA fragment with 3' protruding ends at both ends); with the help of the reverse complementary relationship with the sticky ends, the oligonucleotide single-stranded 1 derivative and the oligonucleotide single-stranded 2 derivative are bound to the double-stranded DNA fragment (the target is a double-stranded DNA fragment with the oligonucleotide single-stranded 1 derivative added to one end and the oligonucleotide single-stranded 2 derivative added to the other end), and then, under the action of reverse transcriptase (DNA polymerization function), the two 3' protruding ends of the double-stranded DNA fragment are extended in the 5' to 3' direction using the oligonucleotide single strands of the two linkers as templates.

2、缺刻连接和切除阻断修饰2. Nick junction and excision block modification

按照表3制备反应体系(总体系为40μL)，37℃反应15min，然后用40μL XP beads进行DNA纯化，纯化后的DNA溶解于18μL TE缓冲液中，即为文库溶液。Prepare the reaction system according to Table 3 (total system is 40 μL), react at 37°C for 15 min, then purify the DNA with 40 μL XP beads. Dissolve the purified DNA in 18 μL TE buffer to obtain the library solution.

表3
Table 3

本步骤的反应体系中，同时具有T4 DNA ligase和在T4 DNA ligase的作用下，上述步骤的缺刻被修复。在的作用下，U碱基连同其上游的阻断修饰被切除。The reaction system in this step contains both T4 DNA ligase and Under the action of T4 DNA ligase, the nicks in the above steps are repaired. Under the action of , the U base is removed along with its upstream blocking modification.

3、文库上机前准备和上机测序3. Library preparation and sequencing

取步骤2得到的文库溶液，进行DNB制备(采用MGISEQ-2000RS高通量快速测序试剂套装，按说明书操作)并上样于DNBSEQ-G400进行测序(测序类型PE150)。The library solution obtained in step 2 was taken for DNB preparation (using MGISEQ-2000RS High-throughput Rapid Sequencing Reagent Set, according to the instructions) and loaded on DNBSEQ-G400 for sequencing (sequencing type PE150).

实施例2、Example 2

流程示意图见图3。The flow chart is shown in Figure 3.

1、片段化、末端修复、接头连接和模板延伸1. Fragmentation, end repair, adapter ligation, and template extension

按照表4制备反应体系(总体系为20μL)，然后按照表5的程序进行反应，然后用30μLXP beads进行DNA纯化，纯化后的DNA溶解于20μL TE缓冲液中，即为产物溶液。Prepare the reaction system according to Table 4 (total system is 20 μL), then carry out the reaction according to the procedure in Table 5, and then purify the DNA with 30 μL XP beads. Dissolve the purified DNA in 20 μL TE buffer to obtain the product solution.

表4

Table 4

接头3为寡聚核苷酸单链，序列如下：
Linker 3 is a single-stranded oligonucleotide with the following sequence:

接头4为寡聚核苷酸单链，序列如下：
Linker 4 is a single-stranded oligonucleotide with the following sequence:

接头3的5’端具有阻断结构(发卡结构，见波浪下划线标记)，自3’末端第1至3位为rG(即核糖核酸G)且第4位为N(N代表A\T\C\G)。接头3如SEQ ID NO：3所示。The 5' end of adapter 3 has a blocking structure (hairpin structure, see wavy underline mark), and the first to third positions from the 3' end are rG (i.e., ribonucleic acid G) and the fourth position is N (N represents A\T\C\G). Adapter 3 is shown in SEQ ID NO: 3.

接头4的5’端具有阻断结构(发卡结构，见波浪下划线标记)，自3’末端第1至3位为rG(即核糖核酸G)且第4位为N(N代表A\T\C\G)。接头4如SEQ ID NO：4所示。The 5' end of linker 4 has a blocking structure (hairpin structure, see wavy underline mark), and the first to third positions from the 3' end are rG (i.e., ribonucleic acid G) and the fourth position is N (N represents A\T\C\G). Linker 4 is shown in SEQ ID NO: 4.

本步骤的反应体系中，各个酶发挥作用的原理和程序参见实施例1的步骤1中的相关描述。In the reaction system of this step, the principles and procedures of the functions of each enzyme are described in step 1 of Example 1.

表5
Table 5

2、缺刻连接和PCR扩增2. Nick ligation and PCR amplification

按照表6制备反应体系(总体系为50μL)，然后按照表7的程序进行反应，然后用40μLXP beads进行DNA纯化，纯化后的DNA溶解于30μL TE缓冲液中，即为文库溶液。Prepare the reaction system according to Table 6 (total system is 50 μL), then perform the reaction according to the procedure in Table 7, and then purify the DNA with 40 μL XP beads. Dissolve the purified DNA in 30 μL TE buffer to obtain the library solution.

表6
Table 6

通用引物1： Universal Primer 1:

标签引物1：
Index primer 1:

通用引物1的5’末端具有磷酸化修饰，用于匹配后续的测序平台。通用引物1如SEQ ID NO：5所示。Universal Primer 1 has a phosphorylation modification at its 5' end to facilitate compatibility with subsequent sequencing platforms. Universal Primer 1 is shown in SEQ ID NO: 5.

标签引物1中，下划线部分为样本标签(仅为示例性的一种，样本标签用于标记样本，可为任意核苷酸组合)。标签引物1如SEQ ID NO：6所示。In tag primer 1, the underlined portion is the sample tag (this is only an example; the sample tag is used to label the sample and can be any combination of nucleotides). Tag primer 1 is shown in SEQ ID NO: 6.

本步骤的反应体系中，同时具有T4 DNA ligase和Tth DNA Polymerase。在T4 DNA  ligase的作用下，上述步骤的缺刻被修复。在Tth DNA Polymerase的作用下，实现以通用引物1和标签引物1为扩增引物对的PCR扩增。由于PCR扩增产生了大量扩增产物，5’末端具有发卡结构的模板链被极大程度稀释从而被认为去除。The reaction system in this step contains both T4 DNA ligase and Tth DNA Polymerase. The nick created in the previous step was repaired by ligase. PCR amplification was then performed using Universal Primer 1 and Index Primer 1 as the primer pair. Due to the large amount of amplified product generated by PCR, the template strand with a hairpin structure at its 5' end was significantly diluted and thus removed.

表7
Table 7

3、文库上机前准备和上机测序3. Library preparation and sequencing

取步骤2得到的文库溶液，进行DNB制备(采用MGISEQ-2000RS高通量快速测序试剂套装，按说明书操作)并上样于DNBSEQ-G400进行测序(测序类型PE150)。The library solution obtained in step 2 was taken for DNB preparation (using MGISEQ-2000RS High-throughput Rapid Sequencing Reagent Set, according to the instructions) and loaded on DNBSEQ-G400 for sequencing (sequencing type PE150).

实施例3、Example 3

流程示意图见图4。The flow chart is shown in Figure 4.

1、接头连接和模板延伸1. Joint connection and template extension

按照表8制备反应体系(总体系为20μL)，然后按照表9进行反应，然后用30μL XP beads进行DNA纯化，纯化后的DNA溶解于20μL TE缓冲液中，即为产物溶液。Prepare the reaction system according to Table 8 (total system is 20 μL), then carry out the reaction according to Table 9, and then use 30 μL XP beads to purify the DNA. The purified DNA is dissolved in 20 μL TE buffer to obtain the product solution.

表8
Table 8

接头3 Connector 3

接头4 Connector 4

平末端双链DNA片段是将模板DNA利用Dnase I进行片段化，然后利用T4 DNA聚合酶进行末端修复后得到的。Blunt-ended double-stranded DNA fragments are obtained by fragmenting the template DNA using DNase I and then performing end repair using T4 DNA polymerase.

本步骤的反应体系中，具有逆转录酶。逆转录酶发挥末端转移功能和DNA聚合功能。在逆转录酶的作用下(末端转移功能)，平末端双链DNA片段的每条链的3’末端增加3个核苷酸C，形成粘末端片段(两端均为3’端突出末端的双链DNA片段)；借助与粘末端的反向互补关系，寡聚核苷酸单链3和寡聚核苷酸单链4结合至双链DNA片段(目标物为一端添加寡聚核苷酸单链3且另一端添加寡聚核苷酸单链4的双链DNA片段)，然后在逆转录酶的作用下(DNA聚合功能)双链DNA片段的两个3’端突出末端分别以两种 接头的寡聚核苷酸单链为模板进行5’至3’方向的延伸。The reaction system of this step contains reverse transcriptase. Reverse transcriptase performs terminal transfer function and DNA polymerization function. Under the action of reverse transcriptase (terminal transfer function), three nucleotide Cs are added to the 3' end of each chain of the blunt-ended double-stranded DNA fragment to form a sticky-end fragment (a double-stranded DNA fragment with 3' protruding ends at both ends); with the help of the reverse complementary relationship with the sticky end, oligonucleotide single strand 3 and oligonucleotide single strand 4 are bound to the double-stranded DNA fragment (the target is a double-stranded DNA fragment with oligonucleotide single strand 3 added to one end and oligonucleotide single strand 4 added to the other end), and then under the action of reverse transcriptase (DNA polymerization function), the two 3' protruding ends of the double-stranded DNA fragment are respectively converted into two The single-stranded oligonucleotide of the adapter is used as a template for extension from 5' to 3'.

表9
Table 9

2、缺刻连接和PCR扩增2. Nick ligation and PCR amplification

同实施例2的步骤2。Same as step 2 of Example 2.

3、文库上机前准备和上机测序3. Library preparation and sequencing

取步骤2得到的文库溶液，进行DNB制备(采用MGISEQ-2000RS高通量快速测序试剂套装，按说明书操作)并上样于DNBSEQ-G400进行测序(测序类型PE150)。The library solution obtained in step 2 was taken for DNB preparation (using MGISEQ-2000RS High-throughput Rapid Sequencing Reagent Set, according to the instructions) and loaded on DNBSEQ-G400 for sequencing (sequencing type PE150).

对比例、Comparative Example

1、采用对照试剂盒并按说明书操作进行文库制备(模板DNA的加入量为200ng)，得到文库溶液。对照试剂盒：VAHTS Universal DNA Library Prep Kit for MGI；中国诺唯赞公司，货号为NDM607。1. Prepare the library using the control kit according to the manufacturer's instructions (use 200 ng of template DNA) to obtain the library solution. Control kit: VAHTS Universal DNA Library Prep Kit for MGI; Novozymes, China, Cat. No. NDM607.

2、取步骤1得到的文库溶液，进行DNB制备并上样于DNBSEQ-G400进行测序(测序类型PE150)。2. Take the library solution obtained in step 1, prepare DNB and load it on DNBSEQ-G400 for sequencing (sequencing type PE150).

数据分析Data Analysis

数据来源分别为实施例1得到的测序结果、实施例2得到的测序结果、实施例3得到的测序结果和对比例得到的测序结果。按照相同的分析流程进行分析，第一步过滤掉低质量的reads并去除接头序列，根据BWA软件将数据比对到人参考基因组hg19，按照默认参数设置，最后根据Samtools对得到的数据进行统计。The data sources were the sequencing results obtained in Example 1, the sequencing results obtained in Example 2, the sequencing results obtained in Example 3, and the sequencing results obtained in the comparative example. The analysis was performed according to the same analysis process. In the first step, low-quality reads were filtered out and adapter sequences were removed. The data were aligned to the human reference genome hg19 using BWA software with default parameters. Finally, the obtained data were statistically analyzed using Samtools.

结果见表10。发明方案比对照方案有更低的重复率(Dup 0.1-2.1％vs6.3％)。The results are shown in Table 10. The inventive solution had a lower duplication rate than the control solution (Dup 0.1-2.1% vs 6.3%).

表10
Table 10

工业应用Industrial Applications

本发明公开的方法和试剂盒可用于制备文库，从而用于高通量测序，具有操作步骤简单且文库质量高的优势。 The method and kit disclosed in the present invention can be used to prepare a library for high-throughput sequencing, and have the advantages of simple operation steps and high library quality.

Claims (21)

一种给平末端双链DNA片段添加接头的方法，其包括如下步骤：A method for adding a linker to a blunt-ended double-stranded DNA fragment comprises the following steps: 将平末端双链DNA片段与具有末端转移活性和/或功能的酶和接头接触；contacting the blunt-ended double-stranded DNA fragments with an enzyme having terminal transfer activity and/or function and a linker; 其中，所述酶用于在所述双链DNA片段的每条链的3’末端增加核苷酸片段1；wherein the enzyme is used to add nucleotide fragment 1 to the 3' end of each strand of the double-stranded DNA fragment; 所述核苷酸片段1与所述接头的3’末端的核苷酸片段2反向互补，使得所述接头结合于所述双链DNA片段，获得添加有接头的双链DNA片段。The nucleotide fragment 1 is reverse complementary to the nucleotide fragment 2 at the 3' end of the linker, so that the linker is bound to the double-stranded DNA fragment to obtain a double-stranded DNA fragment with the linker added. 根据权利要求1所述的方法，其特征在于：所述核苷酸片段1由N1个核苷酸组成，所述核苷酸片段2由N2个核苷酸组成；N1和N2分别独立地选自1-10的自然数；优选地，N1和N2分别独立地选自2-5的自然数；优选地，N1为3，N2为3；优选地，所述核苷酸片段1为CCC；优选地，所述核苷酸片段2为rGrGrG；优选地，所述接头中，与核苷酸片段2的5’端相邻的核苷酸为N。The method according to claim 1, characterized in that: the nucleotide fragment 1 consists of N1 nucleotides, and the nucleotide fragment 2 consists of N2 nucleotides; N1 and N2 are independently selected from natural numbers of 1-10; preferably, N1 and N2 are independently selected from natural numbers of 2-5; preferably, N1 is 3, and N2 is 3; preferably, the nucleotide fragment 1 is CCC; preferably, the nucleotide fragment 2 is rGrGrG; preferably, in the linker, the nucleotide adjacent to the 5' end of the nucleotide fragment 2 is N. 根据权利要求1所述的方法，其特征在于：所述接头的5’末端具有以阻断核酸延伸为目的的阻断结构或阻断修饰；优选地，所述阻断结构为发卡结构；优选地，所述阻断修饰选自：双脱氧胞苷修饰、反向dt修饰、磷酸基团修饰、硫代基团修饰或间臂修饰；优选地，所述阻断修饰为间臂修饰。The method according to claim 1, characterized in that: the 5' end of the linker has a blocking structure or blocking modification for the purpose of blocking nucleic acid extension; preferably, the blocking structure is a hairpin structure; preferably, the blocking modification is selected from: dideoxycytidine modification, reverse dt modification, phosphate group modification, thio group modification or spacer modification; preferably, the blocking modification is a spacer modification. 根据权利要求1所述的方法，其特征在于：所述接头包含功能区段，所述功能区段位于阻断结构或阻断修饰的下游；优选地，所述功能区段包含样本标签和/或分子标签和/或测序引物。The method according to claim 1, characterized in that: the linker comprises a functional segment, and the functional segment is located downstream of the blocking structure or blocking modification; preferably, the functional segment comprises a sample tag and/or a molecular tag and/or a sequencing primer. 根据权利要求1所述的方法，其特征在于：所述接头包含酶切位点，所述酶切位点位于阻断结构或阻断修饰的下游；优选地，所述酶切位点与阻断结构或阻断修饰相邻；优选地，所述酶切位点与阻断结构或阻断修饰间隔N3个核苷酸，N3选自1-10的自然数；优选地，N3选自1-5的自然数；优选地，N3为0或1或2或3；优选地，所述酶切位点选自：dUTP或rNTP。The method according to claim 1, characterized in that: the linker comprises a restriction enzyme cleavage site, and the restriction enzyme cleavage site is located downstream of the blocking structure or blocking modification; preferably, the restriction enzyme cleavage site is adjacent to the blocking structure or blocking modification; preferably, the restriction enzyme cleavage site and the blocking structure or blocking modification are separated by N3 nucleotides, and N3 is selected from a natural number of 1-10; preferably, N3 is selected from a natural number of 1-5; preferably, N3 is 0 or 1 or 2 or 3; preferably, the restriction enzyme cleavage site is selected from: dUTP or rNTP. 根据权利要求1所述的方法，其特征在于：所述给平末端双链DNA片段添加接头的方法中还包括模板延伸的步骤；所述模板延伸是添加有接头的双链DNA片段以所述接头为模板进行5’至3’方向的延伸；优选地，所述模板延伸借助具有DNA聚合活性和/或功能的酶；优选地，所述具有末端转移活性和/或功能的酶与所述具有DNA聚合活性和/或功能的酶是同一种酶；优选地，所述具有末端转移活性和/或功能的酶与所述具有DNA聚合活性和/或功能的酶是不同的酶；优选地，所述具有DNA聚合活性和/或功能的酶为具有末端转移活性的逆转录酶；优选地，所述具有末端转移活性的逆转录酶为M-MLV或AMV；优选地，所述具有末端转移活性的逆转录酶为SuperScriptTMIV逆转录酶、SuperScriptTMI逆转录酶、SuperScriptTMII逆转录酶或SuperScriptTMIII逆转录酶。The method according to claim 1 is characterized in that: the method of adding adapters to blunt-ended double-stranded DNA fragments further comprises a template extension step; the template extension is to extend the double-stranded DNA fragments to which the adapters are added in the 5' to 3' direction using the adapters as templates; preferably, the template extension is performed with the aid of an enzyme having DNA polymerization activity and/or function; preferably, the enzyme having terminal transfer activity and/or function and the enzyme having DNA polymerization activity and/or function are the same enzyme; preferably, the enzyme having terminal transfer activity and/or function and the enzyme having DNA polymerization activity and/or function are different enzymes; preferably, the enzyme having DNA polymerization activity and/or function is a reverse transcriptase having terminal transfer activity; preferably, the reverse transcriptase having terminal transfer activity is M-MLV or AMV; preferably, the reverse transcriptase having terminal transfer activity is SuperScript ™ IV reverse transcriptase, SuperScript ™ I reverse transcriptase, SuperScript ™ II reverse transcriptase or SuperScript ™ III reverse transcriptase. 一种给双链DNA片段末端修复并添加接头的方法，其包括如下过程：A method for repairing the ends of double-stranded DNA fragments and adding adapters, comprising the following steps: ①双链DNA片段进行末端修复，得到平末端双链DNA片段；① The double-stranded DNA fragments are repaired at the ends to obtain blunt-ended double-stranded DNA fragments; ②给平末端双链DNA片段添加接头；②Add adapters to blunt-ended double-stranded DNA fragments; 所述给平末端双链DNA片段添加接头的方法如权利要求1至6中任一所述；The method for adding a linker to a blunt-ended double-stranded DNA fragment is as described in any one of claims 1 to 6; 优选地，过程①和②在同一体系中完成；Preferably, processes ① and ② are completed in the same system; 优选地，所述双链DNA片段进行末端修复借助具有5'→3'聚合和3'→5'核酸外切 活性和/或功能的酶；优选地，所述具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶为Klenow片段。Preferably, the double-stranded DNA fragments are end-repaired by a 5'→3' polymerization and 3'→5' exonucleolytic Preferably, the enzyme having 5'→3' polymerization and 3'→5' exonucleolytic activity and/or function is a Klenow fragment. 一种给双链DNA分子片段化、末端修复并添加接头的方法，其包括如下过程：A method for fragmenting, end-repairing, and adding adapters to double-stranded DNA molecules, comprising the following steps: ①双链DNA分子进行片段化，得到双链DNA片段；① Fragmentation of double-stranded DNA molecules to obtain double-stranded DNA fragments; ②双链DNA片段进行末端修复，得到平末端双链DNA片段；② The double-stranded DNA fragments are repaired at the ends to obtain blunt-ended double-stranded DNA fragments; ③给平末端双链DNA片段添加接头；③Add adapters to blunt-ended double-stranded DNA fragments; 所述给平末端双链DNA片段添加接头的方法如权利要求1至5中任一所述；The method for adding a linker to a blunt-ended double-stranded DNA fragment is as described in any one of claims 1 to 5; 优选地，过程①、②和③在同一体系中完成；Preferably, processes ①, ② and ③ are completed in the same system; 优选地，所述双链DNA片段进行末端修复借助具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶；优选地，所述具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶为Klenow片段或T4 DNA聚合酶；Preferably, the double-stranded DNA fragment is end-repaired with the aid of an enzyme having 5'→3' polymerization and 3'→5' exonuclease activity and/or function; preferably, the enzyme having 5'→3' polymerization and 3'→5' exonuclease activity and/or function is Klenow fragment or T4 DNA polymerase; 优选地，所述双链DNA分子进行片段化的方法选自：物理打断或酶切打断；Preferably, the method for fragmenting the double-stranded DNA molecule is selected from: physical shearing or enzymatic shearing; 优选地，所述酶切打断借助具有核酸内切活性和/或功能的酶；优选地，所述具有核酸内切活性和/或功能的酶为Dnase I或Endonuclease V。Preferably, the enzymatic cleavage is performed with the aid of an enzyme having endonuclease activity and/or function; preferably, the enzyme having endonuclease activity and/or function is Dnase I or Endonuclease V. 权利要求1至8中任一所述方法在制备核酸文库或高通量测序中的应用。Use of the method according to any one of claims 1 to 8 in preparing a nucleic acid library or high-throughput sequencing. 一种制备核酸文库的方法，其包括如下步骤(1)：采用权利要求1至8中任一所述方法进行操作，得到添加有接头的双链DNA片段。A method for preparing a nucleic acid library, comprising the following step (1): performing an operation according to any one of claims 1 to 8 to obtain double-stranded DNA fragments to which linkers are added. 根据权利要求10所述的方法，其特征在于：所述制备核酸文库的方法还包括如下步骤(2)：取步骤(1)的产物，进行缺刻补平和阻断切除；所述缺刻补平借助具有DNA连接活性和/或功能的酶；所述阻断切除借助具有特异性切割所述接头上的酶切位点的活性和/或功能的酶；优选地，所述步骤(2)在同一体系中完成；优选地，所述具有DNA连接活性和/或功能的酶为T4 DNA ligase、T3 Taqligase或T7 Taqligase；优选地，所述具有特异性切割所述接头上的酶切位点的活性和/或功能的酶为USER Enzyme或UDG酶。The method according to claim 10 is characterized in that: the method for preparing a nucleic acid library further comprises the following step (2): taking the product of step (1), performing nick filling and blocking excision; the nick filling is performed with the aid of an enzyme having DNA ligation activity and/or function; the blocking excision is performed with the aid of an enzyme having the activity and/or function of specifically cutting the enzyme cleavage site on the connector; preferably, the step (2) is completed in the same system; preferably, the enzyme having the activity and/or function of DNA ligation is T4 DNA ligase, T3 Taqligase or T7 Taqligase; preferably, the enzyme having the activity and/or function of specifically cutting the enzyme cleavage site on the connector is USER Enzyme or UDG enzyme. 根据权利要求10所述的方法，其特征在于：所述制备核酸文库的方法还包括如下步骤(2)：取步骤(1)的产物，进行缺刻补平和PCR扩增；所述缺刻补平借助具有DNA连接活性和/或功能的酶；所述PCR扩增借助具有DNA聚合活性和/或功能的酶；所述PCR扩增采用的引物对的靶序列对应于所述阻断结构或所述阻断修饰的下游；优选地，所述PCR扩增采用的引物包含功能区段，所述功能区段中含有样本标签和/或分子标签和/或测序引物；优选地，所述步骤(2)在同一体系中完成；优选地，所述具有DNA连接活性和/或功能的酶为T4 DNA ligase、T3 Taqligase或T7 Taqligase；优选地，用于所述PCR扩增的所述具有DNA聚合活性和/或功能的酶为Tth DNA Polymerase。The method according to claim 10 is characterized in that: the method for preparing a nucleic acid library further comprises the following step (2): taking the product of step (1), performing nick filling and PCR amplification; the nick filling is performed with the aid of an enzyme having DNA ligation activity and/or function; the PCR amplification is performed with the aid of an enzyme having DNA polymerization activity and/or function; the target sequence of the primer pair used for the PCR amplification corresponds to the downstream of the blocking structure or the blocking modification; preferably, the primer used for the PCR amplification comprises a functional segment, and the functional segment contains a sample tag and/or a molecular tag and/or a sequencing primer; preferably, the step (2) is completed in the same system; preferably, the enzyme having DNA ligation activity and/or function is T4 DNA ligase, T3 Taqligase or T7 Taqligase; preferably, the enzyme having DNA polymerization activity and/or function used for the PCR amplification is Tth DNA Polymerase. 一种试剂盒，其包括如下组件：A kit comprising the following components: 具有末端转移活性和/或功能的酶和接头；Enzymes and linkers having terminal transfer activity and/or function; 所述酶用于在平末端双链DNA片段的每条链的3’末端增加核苷酸片段1；The enzyme is used to add nucleotide fragment 1 to the 3' end of each strand of a blunt-ended double-stranded DNA fragment; 所述核苷酸片段1与所述接头的3’末端的核苷酸片段2反向互补。The nucleotide fragment 1 is reverse complementary to the nucleotide fragment 2 at the 3' end of the linker. 根据权利要求13所述的试剂盒，其特征在于：所述核苷酸片段1由N1个核苷酸组成，所述核苷酸片段2由N2个核苷酸组成；N1和N2分别独立地选自1-10的自 然数；优选地，N1和N2分别独立地选自2-5的自然数；优选地，N1为3，N2为3；优选地，所述核苷酸片段1为CCC；优选地，所述核苷酸片段2为rGrGrG；优选地，所述接头中，与核苷酸片段2的5’端相邻的核苷酸为N；The kit according to claim 13, characterized in that: the nucleotide fragment 1 consists of N1 nucleotides, the nucleotide fragment 2 consists of N2 nucleotides; N1 and N2 are independently selected from 1-10 Preferably, N1 and N2 are independently selected from natural numbers of 2-5; preferably, N1 is 3 and N2 is 3; preferably, the nucleotide fragment 1 is CCC; preferably, the nucleotide fragment 2 is rGrGrG; preferably, in the linker, the nucleotide adjacent to the 5' end of the nucleotide fragment 2 is N; 优选地，所述接头的5’末端具有以阻断核酸延伸为目的的阻断结构或阻断修饰；；优选地，所述阻断结构为发卡结构；优选地，所述阻断修饰选自：双脱氧胞苷修饰、反向dt修饰、磷酸基团修饰、硫代基团修饰或间臂修饰；优选地，所述阻断修饰为间臂修饰；Preferably, the 5' end of the linker has a blocking structure or blocking modification for the purpose of blocking nucleic acid extension; preferably, the blocking structure is a hairpin structure; preferably, the blocking modification is selected from: dideoxycytidine modification, reverse dt modification, phosphate group modification, thio group modification or spacer modification; preferably, the blocking modification is spacer modification; 优选地，所述接头包含功能区段，所述功能区段位于阻断结构或阻断修饰的下游；优选地，所述功能区段包含样本标签和/或分子标签和/或测序引物；Preferably, the linker comprises a functional segment, and the functional segment is located downstream of the blocking structure or blocking modification; preferably, the functional segment comprises a sample tag and/or a molecular tag and/or a sequencing primer; 优选地，所述接头包含酶切位点，所述酶切位点位于阻断结构或阻断修饰的下游；优选地，所述酶切位点与阻断结构或阻断修饰相邻；优选地，所述酶切位点与阻断结构或阻断修饰间隔N3个核苷酸，N3选自1-10的自然数；优选地，N3选自1-5的自然数；优选地，N3为0或1或2或3；优选地，所述酶切位点选自：dUTP或rNTP。Preferably, the linker comprises an enzyme cleavage site, which is located downstream of the blocking structure or blocking modification; preferably, the enzyme cleavage site is adjacent to the blocking structure or blocking modification; preferably, the enzyme cleavage site is separated from the blocking structure or blocking modification by N3 nucleotides, and N3 is selected from a natural number of 1-10; preferably, N3 is selected from a natural number of 1-5; preferably, N3 is 0 or 1 or 2 or 3; preferably, the enzyme cleavage site is selected from: dUTP or rNTP. 根据权利要求13所述的试剂盒，其特征在于：The kit according to claim 13, characterized in that: 所述试剂盒还包括如下组件：用于模板延伸的组件；所述模板延伸是添加有接头的双链DNA片段以所述接头为模板进行5’至3’方向的延伸；The kit further comprises the following components: a component for template extension; the template extension is to extend the double-stranded DNA fragment with the adapter added thereto in the 5' to 3' direction using the adapter as a template; 优选地，所述用于模板延伸的组件为具有DNA聚合活性和/或功能的酶；优选地，所述具有末端转移活性和/或功能的酶与所述具有DNA聚合活性和/或功能的酶是同一种酶；优选地，所述具有末端转移活性和/或功能的酶与所述具有DNA聚合活性和/或功能的酶是不同的酶；优选地，所述具有DNA聚合活性和/或功能的酶为具有末端转移活性的逆转录酶；优选地，所述具有末端转移活性的逆转录酶为M-MLV或AMV；优选地，所述具有末端转移活性的逆转录酶为SuperScriptTM IV逆转录酶、SuperScriptTMI逆转录酶、SuperScriptTMII逆转录酶或SuperScriptTM III逆转录酶；优选地，所述具有DNA聚合活性和/或功能的酶为Klenow片段或T4 DNA聚合酶；优选地，所述具有末端转移活性和/或功能的酶为具有末端转移活性的逆转录酶；优选地，所述具有末端转移活性的逆转录酶为M-MLV或AMV；优选地，所述具有末端转移活性的逆转录酶为SuperScriptTM IV逆转录酶、SuperScriptTMI逆转录酶、SuperScriptTMII逆转录酶或SuperScriptTM III逆转录酶。Preferably, the component for template extension is an enzyme having DNA polymerization activity and/or function; preferably, the enzyme having terminal transfer activity and/or function is the same enzyme as the enzyme having DNA polymerization activity and/or function; preferably, the enzyme having terminal transfer activity and/or function is a different enzyme from the enzyme having DNA polymerization activity and/or function; preferably, the enzyme having DNA polymerization activity and/or function is a reverse transcriptase having terminal transfer activity; preferably, the reverse transcriptase having terminal transfer activity is M-MLV or AMV; preferably, the reverse transcriptase having terminal transfer activity is SuperScript ™ IV reverse transcriptase, SuperScript ™ I reverse transcriptase, SuperScript ™ II reverse transcriptase or SuperScript ™ III reverse transcriptase; preferably, the enzyme having DNA polymerization activity and/or function is Klenow fragment or T4 DNA polymerase; preferably, the enzyme having terminal transfer activity and/or function is a reverse transcriptase having terminal transfer activity; preferably, the reverse transcriptase having terminal transfer activity is M-MLV or AMV; preferably, the reverse transcriptase having terminal transfer activity is SuperScript ™ IV reverse transcriptase, SuperScript ™ I reverse transcriptase, SuperScript ™ II reverse transcriptase, or SuperScript ™ III reverse transcriptase. 根据权利要求13所述的试剂盒，其特征在于：The kit according to claim 13, characterized in that: 所述试剂盒还包括如下组件：用于双链DNA片段进行末端修复的组件；The kit also includes the following components: a component for end repair of double-stranded DNA fragments; 优选地，所述用于双链DNA片段进行末端修复的组件为具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶；优选地，所述具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶为Klenow片段或T4 DNA聚合酶。Preferably, the component for end repair of double-stranded DNA fragments is an enzyme having 5'→3' polymerization and 3'→5' exonuclease activity and/or function; preferably, the enzyme having 5'→3' polymerization and 3'→5' exonuclease activity and/or function is Klenow fragment or T4 DNA polymerase. 根据权利要求13所述的试剂盒，其特征在于：The kit according to claim 13, characterized in that: 所述试剂盒还包括如下组件：用于双链DNA分子进行片段化的组件和用于双链DNA片段进行末端修复的组件；The kit further comprises the following components: a component for fragmenting double-stranded DNA molecules and a component for end-repairing double-stranded DNA fragments; 所述用于双链DNA分子进行片段化的组件为物理打断所需组件或酶切打断所需组件；优选地，所述酶切打断所需组件为具有核酸内切活性和/或功能的酶；优选地，所述具有核酸内切活性和/或功能的酶为Dnase I或Endonuclease V； The component for fragmenting double-stranded DNA molecules is a component required for physical shearing or a component required for enzymatic shearing; preferably, the component required for enzymatic shearing is an enzyme having endonuclease activity and/or function; preferably, the enzyme having endonuclease activity and/or function is DNase I or Endonuclease V; 优选地，所述用于双链DNA片段进行末端修复的组件为具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶；优选地，所述具有5'→3'聚合和3'→5'核酸外切活性和/或功能的酶为Klenow片段或T4 DNA聚合酶。Preferably, the component for end repair of double-stranded DNA fragments is an enzyme having 5'→3' polymerization and 3'→5' exonuclease activity and/or function; preferably, the enzyme having 5'→3' polymerization and 3'→5' exonuclease activity and/or function is Klenow fragment or T4 DNA polymerase. 根据权利要求13至17中任一所述的试剂盒，其特征在于：The kit according to any one of claims 13 to 17, characterized in that: 所述试剂盒还包括如下组件：用于缺刻补平的组件和用于阻断切除的组件；The kit further comprises the following components: a component for nick filling and a component for blocking excision; 优选地，所述用于缺刻补平的组件为具有DNA连接活性和/或功能的酶；优选地，所述具有DNA连接活性和/或功能的酶为T4 DNA ligase、T3 Taqligase或T7 Taqligase；Preferably, the component for nick filling is an enzyme having DNA ligation activity and/or function; preferably, the enzyme having DNA ligation activity and/or function is T4 DNA ligase, T3 Taqligase or T7 Taqligase; 优选地，所述用于阻断切除的组件为具有特异性切割所述接头上的酶切位点的活性和/或功能的酶；优选地，所述具有特异性切割所述接头上的酶切位点的活性和/或功能的酶为USER Enzyme或UDG酶。Preferably, the component for blocking excision is an enzyme having the activity and/or function of specifically cutting the cleavage site on the connector; preferably, the enzyme having the activity and/or function of specifically cutting the cleavage site on the connector is USER Enzyme or UDG enzyme. 根据权利要求13至17中任一所述的试剂盒，其特征在于：The kit according to any one of claims 13 to 17, characterized in that: 所述试剂盒还包括如下组件：用于缺刻补平的组件和用于PCR扩增的组件；The kit further comprises the following components: a component for nick filling and a component for PCR amplification; 优选地，所述用于缺刻补平的组件为具有DNA连接活性和/或功能的酶；优选地，所述具有DNA连接活性和/或功能的酶为T4 DNA ligase、T3 Taqligase或T7 Taqligase；Preferably, the component for nick filling is an enzyme having DNA ligation activity and/or function; preferably, the enzyme having DNA ligation activity and/or function is T4 DNA ligase, T3 Taqligase or T7 Taqligase; 优选地，所述用于PCR扩增的组件包括具有DNA聚合活性和/或功能的酶；优选地，所述用于PCR扩增的所述具有DNA聚合活性和/或功能的酶为Tth DNA Polymerase；Preferably, the component for PCR amplification includes an enzyme having DNA polymerization activity and/or function; preferably, the enzyme having DNA polymerization activity and/or function for PCR amplification is Tth DNA Polymerase; 优选地，用于PCR扩增的组件还包括PCR扩增采用的引物对；所述PCR扩增采用的引物对的靶序列对应于所述阻断结构或所述阻断修饰的下游；优选地，所述PCR扩增采用的引物包含功能区段，所述功能区段中含有样本标签和/或分子标签和/或测序引物。Preferably, the components used for PCR amplification also include a primer pair used for PCR amplification; the target sequence of the primer pair used for PCR amplification corresponds to the downstream of the blocking structure or the blocking modification; preferably, the primer used for PCR amplification includes a functional segment, and the functional segment contains a sample tag and/or a molecular tag and/or a sequencing primer. 一种核酸文库，是通过权利要求10至12中任一所述方法制备获得的。A nucleic acid library is prepared by the method according to any one of claims 10 to 12. 权利要求20所述核酸文库在高通量测序中的应用。 Use of the nucleic acid library according to claim 20 in high-throughput sequencing.
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