Method for efficiently and rapidly constructing nucleic acid library

Abstract

The invention belongs to the technical field of library construction, and particularly relates to a method for efficiently and quickly constructing a nucleic acid library. According to the linker composition provided by the invention, the 5 '-end linker and the 3'-end linker can be simultaneously connected to the single-stranded template nucleic acid in the same reaction system through the 5 '-end linker, the first ligase, the 3'-end linker and the second ligase, and the 5 '-end linker and the 3'-end linker do not need to be added step by step; reaction steps are greatly reduced, library building time is greatly shortened, linker connection efficiency is improved, meanwhile, linker connection directionality can be guaranteed, loss of template nucleic acid is reduced, and the utilization rate of original template nucleic acid is increased; the method has the advantages that the method is simple, no extra fixed sequence is added on the template, the sequencing quality is not influenced, the sequencing sequence does not need to be subjected to extra operation of cutting a certain length, and joints are completely not connected with one another.

Description

A method for efficient and rapid nucleic acid library construction Technical Field

[0001] The present invention belongs to the technical field of library construction, and in particular relates to a method for efficiently and quickly constructing a nucleic acid library. Background Art

[0002] High-throughput sequencing has become one of the most important research methods in the life sciences and is widely used in fields such as clinical medicine, forensics, and ancient DNA research. High-throughput sequencing requires the construction of sequencing libraries, the most common of which is DNA sequencing libraries, typically using a sufficient amount of intact genomic DNA as the starting material. Conventional double-stranded DNA library construction methods can be used for such samples. The general steps involve fragmenting the DNA into the desired fragment size, repairing the broken DNA ends, and then ligating adapters to the ends of the DNA template fragments via a simple ligase reaction to generate the sequencing library. However, in addition to common intact DNA samples, there are also specialized samples such as formalin-fixed paraffin-embedded (FFPE) and paraffin-embedded tissue samples, forensic specimens, and paleontological fossils. The DNA extracted from these samples can be severely degraded, resulting in the presence of both double-stranded and single-stranded DNA molecules or single-strand breaks within double-stranded DNA molecules. Furthermore, the amount of DNA extracted can be far lower than that from conventional samples. Furthermore, for specialized applications, such as DNA methylation sequencing, DNA undergoes bisulfite treatment, which breaks the DNA into short fragments, resulting in the loss of most DNA molecules and the conversion to single strands. Furthermore, free-cell DNA in plasma and other body fluid samples is inherently short and present at very low concentrations. Conventional adapter-ligation library construction methods are not suitable for these specialized samples. Conventional methods only utilize intact double-stranded DNA molecules, making it impossible or extremely difficult to attach adapters to all template molecules other than intact double-stranded DNA, leading to significant loss of the original template molecules. Single-stranded library construction technology utilizes these single-stranded DNA template molecules and allows for the addition of adapters. The same method can also be used to attach adapters to double-stranded DNA after denaturation. This minimizes the loss of these irregular double-stranded DNA template molecules, maximizing template utilization and increasing library construction efficiency, ultimately improving both the effective amount and quality of sequencing data. This also allows samples to be better utilized for corresponding application testing, such as liquid biopsy for cell-free extracellular DNA (cfDNA) methylation sequencing, genomic research on ancient biological DNA, forensic identification, and tumor detection in FFPE samples. In addition to DNA samples, RNA sample library construction and sequencing are also very suitable for single-stranded library construction technology. RNA is inherently single-stranded, and the typical library construction method is to reverse transcribe RNA into double-stranded cDNA and then add adapters to the cDNA. Alternatively, single-stranded library construction technology can be used to directly add adapters to the RNA sample, then reverse transcribe it into cDNA to complete the sequencing library construction.

[0003] Existing single-strand library construction technologies include traditional single-strand library construction methods, Swift Adaptase single-strand library construction technology, random primer plus adapter method, terminal transferase plus adapter method and SPALT (Splinted adaptor tagging) technology.

[0004] Traditional single-stranded library construction methods utilize T4 RNA ligase to directly ligate single-stranded adapters to single-stranded DNA template molecules. Typically, T4 RNA ligase 2 (truncated) is used to catalyze the ligation of DNA adapters, pre-adenylated at the 5' end and blocked at the 3' end, to the 3' end of the single-stranded template. After ligation, the 3' end adapter must be digested to prevent subsequent self-ligation. A 5' end adapter and T4 RNA ligase are then added, and the 5' end is ligated back to the nucleic acid fragment to which the 3' end adapter has been attached. After both ends have been attached, PCR amplification is performed. If a truncated adapter was previously ligated, PCR can also be used to complete the remaining adapter.

[0005] Swift's Adaptase single-stranded library construction technology first uses single-stranded ligase to connect the 3' end of the single-stranded DNA template to a universal adapter with random primers, and then uses complementary primers that bind to the 3' end adapter primer sequence to amplify, turning the single-stranded template into a double-stranded chain. Then, through a conventional adapter ligation reaction, the 5' end adapter is added to the 5' end of the newly synthesized chain to complete the library construction.

[0006] The random primer single-strand library construction technology uses a 6-8nt random primer with a linker sequence at the 3' end to bind to the single-stranded DNA template for extension, thereby synthesizing a double strand, and then adding a 5' end linker to the 5' end of the newly synthesized chain to complete the library construction.

[0007] Terminal transferase is similar to the single-strand library construction technology of random primers. First, terminal transferase is used to add continuous identical bases to the 3' end of the single-stranded DNA template. Then the continuous sequence is used as the primer binding sequence to hybridize with the primer and then extended to convert the single-stranded DNA template into a double-stranded one. Finally, a 5' end linker is added to the 5' end of the newly synthesized chain to complete the library construction.

[0008] SPALT (Splinted Adaptor Tagging) technology adds a double-stranded adapter at the 3' end. The double-stranded adapter contains a stretch of random bases for complementary binding to the single-stranded DNA. A double-stranded adapter containing random bases is then added to the 5' end of the single-stranded template DNA. After the adapters are added to both ends of the single-stranded template, PCR amplification is performed to complete the construction of the single-stranded library.

[0009] The traditional single-stranded library construction method involves connecting adapters at both ends separately and performing multiple purifications, resulting in low template utilization and long library construction time.

[0010] Adaptase single-strand library construction technology, random primer plus adapter method, terminal transferase plus adapter method, and SPALT technology all add 3' and 5' adapters respectively. Only after adding adapters at both ends can PCR amplification be performed to amplify the template. These increase the number of experimental steps and lead to the loss of the original template, reducing its utilization rate. In addition, Adaptase single-strand library construction technology and terminal transferase plus adapter method require the addition of a fixed sequence to the template, affecting the sequencing quality. These fixed sequences need to be removed after the sequencing data is obtained, resulting in data loss.

[0011] Summary of the Invention

[0012] The first aspect of the present invention aims to provide a joint composition.

[0013] The second aspect of the present invention aims to provide a nucleic acid library construction kit.

[0014] The third aspect of the present invention is to provide a sequencing reagent kit.

[0015] The fourth aspect of the present invention aims to provide a sequencing system.

[0016] The fifth aspect of the present invention aims to provide a joint connection method.

[0017] The sixth aspect of the present invention aims to provide a method for constructing a nucleic acid library.

[0018] The seventh aspect of the present invention aims to provide a nucleic acid library.

[0019] The eighth aspect of the present invention aims to provide a sequencing method.

[0020] The purpose of the ninth aspect of the present invention is to provide an application of the linker composition of the first aspect, the kit of the second aspect, the sequencing reagent set of the third aspect, or the sequencing system of the fourth aspect.

[0021] In order to achieve the above object, the technical solution adopted by the present invention is:

[0022] The first aspect of the present invention provides a joint composition comprising:

[0023] a 5' end adapter, the 5' end adapter comprising a first oligonucleotide chain, the 3' end of the first oligonucleotide chain of the 5' end adapter being modified with a first modifying group and the 5' end being modified with a first blocking group, wherein the first modifying group comprises a phosphate; and

[0024] a first ligase that can ligate the 3' first modification group end of the oligonucleic acid to the 5' hydroxyl end of the template nucleic acid (phosphate of the 3' first modification group to 5' OH); and

[0025] a 3' end adapter, the 3' end adapter comprising a second oligonucleotide chain, the 3' end of the second oligonucleotide chain of the 3' end adapter being modified with a second blocking group, and the 5' end being modified with a second modifying group, wherein the second modifying group comprises a phosphate; and

[0026] The second ligase can ligate the 5' second modification group end of the oligonucleic acid to the 3' hydroxyl end of the template nucleic acid (phosphate of the 5' second modification group to 3' OH).

[0027] Preferably, the first modification group modification is selected from at least one of the following modifications: phosphorylation modification, 2'3' cyclic phosphate modification, pre-guanosine modification, etc.

[0028] Preferably, the second modification group is selected from at least one of the following modifications: phosphorylation modification and adenylation modification.

[0029] Preferably, the 5' end adapter may further comprise a third oligonucleic acid chain, wherein the third oligonucleic acid chain is complementary to the first oligonucleic acid chain to form a double strand, and the length of the third oligonucleic acid chain is greater than, equal to, or less than the first oligonucleic acid chain.

[0030] Preferably, the difference between the number of bases in the third oligonucleotide chain and the number of bases in the first oligonucleotide chain is greater than or equal to 2.

[0031] Preferably, the 5' end and the 3' end of the third oligonucleic acid chain are modified with a third blocking group and a fourth blocking group, respectively.

[0032] Preferably, the 3' end adapter may further comprise a fourth oligonucleic acid chain, wherein the fourth oligonucleic acid chain is complementary to the second oligonucleic acid chain to form a double strand.

[0033] Preferably, the length of the fourth oligonucleic acid chain is greater than, equal to, or less than that of the second oligonucleic acid chain.

[0034] Preferably, the 5' end and the 3' end of the fourth oligonucleic acid chain are modified with a fifth blocking group and a sixth blocking group, respectively.

[0035] Preferably, the first ligase comprises at least one of: RtcB ligase, an active fragment of RtcB ligase, a derivative of RtcB ligase, and an analog of RtcB ligase.

[0036] Preferably, the linker composition further comprises: a first cofactor, wherein the first cofactor comprises: triphosphate and manganese ions.

[0037] Preferably, the linker composition further comprises: a second cofactor, wherein the second cofactor comprises: at least one of ATP and NAD, and a metal ion.

[0038] Preferably, when the 5' end of the second oligonucleotide chain of the 3' end adapter is phosphorylated, the second ligase comprises at least one of T4 RNA ligase 1, T4 DNA ligase, TS2126 RNA ligase, T3 DNA ligase, Escherichia coli (E. coli) DNA ligase, single-stranded DNA / RNA circular ligase (ssDNA / RNA CircLigase), T4 RNA ligase 2, and Taq DNA ligase.

[0039] Preferably, when the 5' end of the second oligonucleotide chain of the 3' end adapter has an adenylation modification, the second ligase comprises at least one of T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 (truncated K227Q), T4 RNA ligase 2 (truncated KQ), thermostable 5'App DNA / RNA ligase, TS2126 RNA ligase, T3 DNA ligase, Escherichia coli (E. coli) DNA ligase, single-stranded DNA / RNA circular ligase (ssDNA / RNA CircLigase), and Taq DNA ligase.

[0040] Preferably, the first blocking group modification is selected from the following group modifications: amino modification, dideoxy modification, spacer modification, and phosphate modification.

[0041] Preferably, the second blocking group modification is selected from the following group modifications: amino modification, dideoxy modification, spacer modification, and phosphate modification.

[0042] Preferably, the third blocking group modification is selected from the following group modifications: amino modification, dideoxy modification, spacer modification, and phosphate modification.

[0043] Preferably, the fourth blocking group modification is selected from the following group modifications: amino modification, dideoxy modification, spacer modification, and phosphate modification.

[0044] Preferably, the fifth blocking group modification is selected from the following group modifications: amino modification, dideoxy modification, spacer modification, and phosphate modification.

[0045] Preferably, the sixth blocking group modification is selected from the following group modifications: amino modification, dideoxy modification, spacer modification, and phosphate modification.

[0046] Preferably, the linker composition further comprises: phosphatase.

[0047] Preferably, the adaptor composition is used to connect a 5'-end adaptor and a 3'-end adaptor to the 5'-end and 3'-end of a single-stranded template nucleic acid.

[0048] Preferably, the linker composition is used to connect the 5' end linker and the 3' end linker to the 5' end and 3' end of the single-stranded template nucleic acid in the same system; wherein the same system is specifically carried out in the same reaction vessel; further specifically, in the same reaction vessel and at the same time.

[0049] Preferably, the single-stranded template nucleic acid is obtained by the following method: subjecting the nucleic acid to at least one of the following treatments: fragmentation, denaturation, and DNA methylation.

[0050] Preferably, the nucleic acid comprises at least one of DNA and RNA.

[0051] Preferably, the DNA comprises at least one of dsDNA and ssDNA.

[0052] Preferably, the nucleic acid is from the following biological samples: cells, fresh tissues, fresh organs, decayed tissues, formalin-fixed tissues, paraffin-embedded tissues, forensic samples, paleontological fossils, biological materials containing cfDNA or RNA.

[0053] Preferably, the linker composition further comprises: a substance for fragmenting nucleic acids.

[0054] Preferably, the linker composition further comprises: a substance for nucleic acid denaturation.

[0055] Preferably, when the linker composition is used to construct a DNA methylation library, the linker composition further comprises: a DNA methylation modification conversion reagent for converting non-methylated C bases in DNA into U bases.

[0056] Preferably, the first oligonucleotide strand of the 5' end adapter is different from the second oligonucleotide strand of the 3' end adapter.

[0057] Preferably, the first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter are fixed to a solid support.

[0058] Preferably, the non-ligation ends of the first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter are fixed to a solid support.

[0059] Preferably, the solid support is selected from beads, chips, and the like.

[0060] Preferably, the chip can be used in a sequencing platform, such as a sequencing chip.

[0061] Preferably, the beads are selected from agarose gel beads, sepharose beads, magnetic beads, protein A conjugated beads, protein G conjugated beads, protein L conjugated beads, oligo (dT) conjugated beads, silica beads, hydrogel beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof.

[0062] The second aspect of the present invention provides a nucleic acid library construction kit, comprising: the linker composition of the first aspect of the present invention.

[0063] Preferably, the nucleic acid library construction kit further comprises: polynucleotide kinase.

[0064] Preferably, the nucleic acid library construction kit further comprises: PCR primers for performing PCR amplification on the single-stranded template nucleic acid connected to the adapter.

[0065] Preferably, the nucleic acid library construction kit further comprises: a PCR reaction solution.

[0066] Preferably, the nucleic acid library construction kit further comprises: a reverse transcription primer, that is, when the nucleic acid comprises RNA, used to reverse transcribe RNA into cDNA.

[0067] Preferably, the nucleic acid library construction kit further comprises: a reverse transcription reaction solution.

[0068] A third aspect of the present invention provides a sequencing reagent kit comprising: any one of a1) to a2):

[0069] a1) the joint composition according to the first aspect of the present invention;

[0070] a2) The nucleic acid library construction kit according to the second aspect of the present invention.

[0071] Preferably, the sequencing reagent set further comprises: a sequencing reagent kit.

[0072] A fourth aspect of the present invention provides a sequencing system comprising: any one of a1) to a3) and a sequencer:

[0073] a1) the joint composition according to the first aspect of the present invention;

[0074] a2) the nucleic acid library construction kit according to the second aspect of the present invention;

[0075] a3) The sequencing reagent set according to the third aspect of the present invention.

[0076] The fifth aspect of the present invention provides a method for connecting a joint, comprising the step of using the joint composition of the first aspect of the present invention.

[0077] Preferably, the adapter ligation method comprises the following steps: obtaining a single-stranded template nucleic acid, wherein both the 5' end and the 3' end of the single-stranded template nucleic acid have hydroxyl groups; performing an adapter ligation reaction of the 5' end adapter and the 3' end adapter on the single-stranded template nucleic acid using the first ligase, the 5' end adapter, the second ligase and the 3' end adapter in the adapter composition of the first aspect of the present invention.

[0078] Preferably, the linker ligation reactions of the 5' end linker and the 3' end linker are carried out in the same system.

[0079] Preferably, the same system is specifically carried out in the same reaction vessel; further specifically, it is carried out in the same reaction vessel and at the same time.

[0080] Preferably, the connection of the adapter is directional, and the first oligonucleotide chain of the 5'-end adapter and the second oligonucleotide chain of the 3'-end adapter have different nucleic acid sequences.

[0081] Preferably, the method for obtaining the single-stranded template nucleic acid with hydroxyl groups at both the 5' end and the 3' end is dephosphorylation treatment.

[0082] Preferably, the single-stranded template nucleic acid is obtained by the following method: subjecting the nucleic acid to at least one of the following treatments: fragmentation, denaturation, and DNA methylation.

[0083] Preferably, the nucleic acid comprises at least one of DNA and RNA.

[0084] Preferably, the DNA comprises at least one of dsDNA and ssDNA.

[0085] Preferably, the nucleic acid comprises at least one of dsDNA, ssDNA and RNA.

[0086] Preferably, the nucleic acid is from the following biological samples: cells, fresh tissues, fresh organs, decayed tissues, formalin-fixed tissues, paraffin-embedded tissues, forensic samples, paleontological fossils, biological materials containing cfDNA or cfRNA.

[0087] Preferably, the biological material includes but is not limited to: peripheral blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, lymph fluid, alveolar lavage fluid, amniotic fluid, blastocyst cavity fluid, cell culture fluid, embryo culture fluid, microbial culture medium, soil leachate, and bone powder leachate.

[0088] Preferably, when the nucleic acid comprises dsDNA, the process of preparing the single-stranded template nucleic acid from the nucleic acid comprises denaturation treatment.

[0089] Preferably, the denaturation treatment may be performed before or after the phosphorylation treatment.

[0090] Preferably, when the nucleic acid is derived from the following biological samples: cells, fresh tissues, fresh organs, paraffin-embedded tissues, forensic samples, the process of preparing the single-stranded template nucleic acid from the nucleic acid includes a shearing treatment.

[0091] Preferably, the shearing treatment precedes the phosphorylation treatment and the denaturation treatment.

[0092] Preferably, when the nucleic acid is used to construct a DNA methylation library, the process of preparing the single-stranded template nucleic acid from the nucleic acid comprises DNA methylation treatment.

[0093] Preferably, the DNA methylation treatment is performed before the phosphorylation treatment and the denaturation treatment.

[0094] Preferably, the DNA methylation treatment is performed after the shearing treatment.

[0095] The sixth aspect of the present invention provides a method for constructing a nucleic acid library, comprising the steps of the linker ligation method of the fifth aspect of the present invention.

[0096] Preferably, for the PCR-free nucleic acid library construction method, the nucleic acid library construction method further comprises the following steps: dephosphorylating the 3' end of the single-stranded template nucleic acid connected to the adapter, and then performing a circularization reaction to obtain a single-stranded circularized library.

[0097] Preferably, for the method of constructing a nucleic acid library with a PCR process, the method further comprises the following step: performing an amplification reaction to obtain a nucleic acid library.

[0098] Preferably, the amplification reaction is rolling circle amplification or linear amplification.

[0099] Preferably, the amplification reaction uses the PCR primers in the nucleic acid library construction kit of the second aspect of the present invention.

[0100] Preferably, when the nucleic acid comprises RNA, the nucleic acid library construction method may further comprise the following step: a reverse transcription reaction, wherein the reverse transcription reaction may be performed before or after the adapter ligation reaction.

[0101] Preferably, the reverse transcription reaction uses the reverse transcription primers in the nucleic acid library construction kit of the second aspect of the present invention.

[0102] Preferably, the nucleic acid library construction method further comprises the following step: purifying the amplified product.

[0103] Preferably, the purification is performed using magnetic beads.

[0104] Preferably, the linker ligation reaction may be followed by a cyclization reaction.

[0105] Preferably, the nucleic acid library comprises a DNA methylation library.

[0106] The seventh aspect of the present invention provides a nucleic acid library obtained by the nucleic acid library construction method of the sixth aspect of the present invention.

[0107] The eighth aspect of the present invention provides a sequencing method comprising the steps of the nucleic acid library construction method of the sixth aspect of the present invention.

[0108] Preferably, the sequencing method comprises the following steps: obtaining a nucleic acid library; sequencing;

[0109] The method for obtaining a nucleic acid library is the nucleic acid library construction method of the sixth aspect of the present invention.

[0110] Preferably, the sequencing further includes the following steps: library quality inspection.

[0111] The ninth aspect of the present invention provides uses of the linker composition of the first aspect, the kit of the second aspect, the sequencing reagent set of the third aspect, and / or the sequencing system of the fourth aspect.

[0112] Use of the linker composition of the first aspect of the present invention and / or the kit of the second aspect of the present invention in any one of c1) to c6);

[0113] c1) preparing a nucleic acid library;

[0114] c2) preparing products for nucleic acid library construction;

[0115] c3) sequencing;

[0116] c4) preparing products for sequencing;

[0117] c5) DNA methylation detection;

[0118] c6) preparing products for DNA methylation detection.

[0119] Preferably, the nucleic acid library comprises a DNA methylation library.

[0120] Use of the sequencing reagent set of the third aspect of the present invention and / or the sequencing system of the fourth aspect of the present invention in any one of items c3) to c6);

[0121] c3) sequencing;

[0122] c4) preparing products for sequencing;

[0123] c5) DNA methylation detection;

[0124] c6) preparing products for DNA methylation detection.

[0125] Preferably, the product comprises at least one of a reagent, a kit, and a system.

[0126] The beneficial effects of the present invention are:

[0127] The present invention provides a linker composition. The linker composition can connect a 5'-end linker to the 5'-end of a single-stranded template nucleic acid through a first oligonucleotide chain of a 5'-end linker and a first ligase, and can connect a 3'-end linker to the 3'-end of the single-stranded template nucleic acid through a second oligonucleotide chain of a 3'-end linker and a second ligase. Thus, the 5'-end linker and the 3'-end linker can be simultaneously connected to the 5'-end and 3'-end of the single-stranded template nucleic acid in the same reaction system. There is no need to add the 5'-end linker and the 3'-end linker in separate steps, which greatly reduces the reaction steps and library construction time, increases the efficiency of linker connection, and ensures the directionality of linker connection, reduces the loss of template nucleic acid, and improves the utilization rate of the original template nucleic acid. No additional fixed sequence is added to the template, does not affect the sequencing quality, and does not require additional truncation of the sequencing sequence to a certain length. In addition, the linkers are completely non-connected to each other. The linker composition is suitable for constructing nucleic acid (including RNA) libraries (including DNA methylation libraries) of samples with nucleic acid degradation, such as formalin-fixed tissues, paraffin-embedded tissues, forensic samples, decayed tissues, paleontological fossils, and biological samples containing cfDNA or cfRNA.

[0128] Furthermore, the linker composition comprises phosphatase, thereby preventing self-ligation between samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] FIG1 is a schematic diagram showing the principle of the nucleic acid library construction method (with PCR) of the present invention.

[0130] FIG2 is a flow chart of the steps of the nucleic acid library construction method (with PCR) of the present invention.

[0131] FIG3 is a graph showing the percentage of base types detected in each sequencing cycle of the sequencing sequence in Example 1.

[0132] FIG4 is a flow chart showing the principle of the nucleic acid library construction method (without PCR) of the present invention.

[0133] FIG5 is a schematic diagram showing the principle of RtcB enzyme ligation. DETAILED DESCRIPTION

[0134] In the description of the present invention, the terms "first," "second," "third," "fourth," "fifth," and "sixth" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly specifying the number of the technical features indicated. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include at least one of such features. In the description of the present invention, "plurality" means at least two, for example, two, three, etc., unless otherwise specifically defined.

[0135] The first aspect of the present invention provides a joint composition comprising:

[0136] a 5' end adapter, the 5' end adapter comprising a first oligonucleotide chain, the 3' end of the first oligonucleotide chain of the 5' end adapter being modified with a first modifying group and the 5' end being modified with a first blocking group, wherein the first modifying group comprises a phosphate; and

[0137] a first ligase that can ligate the 3' first modification group end of the oligonucleotide to the 5' hydroxyl end of the oligonucleotide (3' first modification group phosphate to 5'OH), i.e., has 3' first modification group phosphate to 5'OH ligase activity; and

[0138] a 3' end adapter, the 3' end adapter comprising a second oligonucleotide chain, the 3' end of the second oligonucleotide chain of the 3' end adapter being modified with a second blocking group, and the 5' end being modified with a second modifying group, wherein the second modifying group comprises a phosphate; and

[0139] The second ligase can connect the 5' second modification group end of the oligonucleotide to the 3' hydroxyl end of the oligonucleotide (5' second modification group phosphate to 3'OH), ie, has 5' second modification group phosphate to 3'OH ligase activity.

[0140] The above-mentioned adapter composition can connect the 5'-end adapter to the 5'-end of the single-stranded template nucleic acid through the first oligonucleotide chain of the 5'-end adapter and the first ligase, and can connect the 3'-end adapter to the 3'-end of the single-stranded template nucleic acid through the second oligonucleotide chain of the 3'-end adapter and the second ligase, so that the 5'-end adapter and the 3'-end adapter can be simultaneously connected to the 5'-end and 3'-end of the single-stranded template nucleic acid in the same reaction system, without the need to add the 5'-end adapter and the 3'-end adapter in steps, which greatly reduces the reaction steps and library construction time, increases the efficiency of adapter connection, and at the same time, can ensure the directionality of adapter connection, reduce the loss of template nucleic acid, and improve the utilization rate of the original template nucleic acid; it will not add additional fixed sequences to the template, will not affect the sequencing quality, and does not require additional truncation of the sequencing sequence to a certain length, and the adapters will not be connected to each other at all.

[0141] Preferably, the phosphate in the first modification group is an artificially added modification.

[0142] Preferably, the phosphate in the second modification group is inherent in the second oligonucleic acid chain.

[0143] Preferably, the first modification group modification is selected from at least one of the following modifications: phosphorylation modification, 2'3' cyclic phosphate modification, pre-guanosine modification, etc.

[0144] Preferably, the second modification group is selected from at least one of the following modifications: phosphorylation modification and adenylation modification.

[0145] Preferably, the 5'-end adapter may further comprise a third oligonucleotide chain, wherein the third oligonucleotide chain is complementary to the first oligonucleotide chain to form a double strand, and the length of the third oligonucleotide chain is greater than, equal to, or less than the first oligonucleotide chain; further preferably, the length of the third oligonucleotide chain is less than the first oligonucleotide chain, so that the 5'-end adapter is a partially double-stranded adapter, and the 3'-end of the partially double-stranded adapter has a protruding structure of multiple bases (the 3'-end of the partially double-stranded adapter having a protruding structure of multiple bases refers to a structure composed of two complementary nucleic acids of unequal lengths, the 3'-end of the short nucleic acid is flush with the 5'-end of the long nucleic acid, and the 3'-end of the long nucleic acid is longer than the 5'-end of the short nucleic acid, that is, the 3'-end of the long nucleic acid has multiple bases in a single-stranded state); that is, the 5'-end adapter may be a single-stranded adapter (excluding the third oligonucleotide chain) or a partially double-stranded adapter (including the third oligonucleotide chain, and the 3'-end of the partially double-stranded adapter has a protruding structure of at least multiple, preferably 2, bases).

[0146] Preferably, the difference between the number of bases in the third oligonucleotide chain and the number of bases in the first oligonucleotide chain is greater than or equal to 2.

[0147] Preferably, the 5' end and the 3' end of the third oligonucleic acid chain are modified with a third blocking group and a fourth blocking group, respectively.

[0148] Preferably, the 3' end adapter may further comprise a fourth oligonucleotide chain, and the fourth oligonucleotide chain is complementary to the second oligonucleotide chain to form a double strand, that is, the 3' end adapter may be a single-stranded adapter (excluding the fourth oligonucleotide chain) or a partially complementary double-stranded adapter (including the fourth oligonucleotide chain).

[0149] Preferably, the length of the fourth oligonucleic acid chain is greater than, equal to, or less than that of the second oligonucleic acid chain.

[0150] Preferably, the 5' end and the 3' end of the fourth oligonucleic acid chain are modified with a fifth blocking group and a sixth blocking group, respectively.

[0151] Preferably, the first ligase is a type of enzyme that can connect the 3' phosphate end of the oligonucleic acid to the 5' hydroxyl end of the oligonucleic acid, and can be selected from: at least one of RtcB ligase, an active fragment of RtcB ligase, a derivative of RtcB ligase, and an analog of RtcB ligase; further comprising: RtcB ligase: wherein the RtcB ligase can be an RtcB ligase from any species; "active fragment of RtcB ligase, derivative of RtcB ligase, analog of RtcB ligase" refers to a polypeptide that substantially maintains the same biological function or activity as the RtcB ligase of the present invention. The derivatives or analogs of the present invention may be (i) polypeptides having one or more (e.g., 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 1-3, 1-2) conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more (e.g., 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 1-3, 1-2) amino acid residues, or (iii) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence or a sequence for purifying the polypeptide or a proprotein sequence, or a fusion protein). According to the definition herein, these active fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0152] In the present invention, an active fragment of RtcB ligase refers to a polypeptide that retains all or part of the function of the full-length RtcB ligase. Typically, the active fragment retains at least 50% of the activity of the full-length RtcB ligase. More preferably, the active fragment retains 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length RtcB ligase.

[0153] In the present invention, RtcB ligase also includes variants having the same function as RtcB ligase. These variants include (but are not limited to): deletion, insertion, and / or substitution of several amino acids (generally 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5), and addition or deletion of one or more amino acids (generally 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5) at the C-terminus and / or N-terminus (particularly the N-terminus). For example, in the art, substitution with amino acids having similar or similar properties generally does not alter the function of the protein. For another example, addition or deletion of one or more amino acids at the C-terminus and / or N-terminus (particularly the N-terminus) generally does not alter the function of the protein.

[0154] Any protein with high homology to the RtcB ligase (e.g., 60% or higher, 70% or higher, 80% or higher homology to the sequence of the RtcB ligase; preferably, 85% or higher homology; more preferably, 90% or higher homology, such as 95%, 98% or 99% homology) and having the same function as the RtcB ligase is also included in the present invention. "Homology" refers to the level of similarity (i.e., sequence similarity or identity) between two or more nucleic acids or polypeptides based on the percentage of identical positions. In this article, variants of the gene can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutagenesis, etc.

[0155] The principle of ligation by RtcB ligase (first ligase) is shown in FIG5 , and is specifically as follows: (1) in the presence of Mn ions, RtcB (first ligase) reacts with triphosphate (e.g., GTP) to form a covalent RtcB–histidine–GMP intermediate and releases pyrophosphate (Ppi); (2) GMP is then transferred to the 3' phosphate or 2'3' cyclic phosphate group at the end of the nucleic acid chain; (3) the nucleic acid chain with 5'-OH then attacks the activated 3'-P nucleic acid chain, forming a 3', 5'-phosphodiester bond and releasing GMP; therefore, any enzyme that can complete nucleic acid ligation according to the above process falls within the scope of protection of the first ligase described in the present invention.

[0156] Preferably, the first ligase comprises: Thermomyces RNA ligase RtcB.

[0157] Preferably, the linker composition further comprises a first auxiliary factor comprising triphosphate and manganese ions, for assisting the first ligase in connecting the 3' first modification group end of the oligonucleic acid to the 5' hydroxyl end of the template nucleic acid.

[0158] The above-mentioned first auxiliary factor is used by the first ligase (e.g., RtcB ligase) to catalyze the ligation reaction between the oligonucleotide with 3' terminal phosphorylation modification (containing 3' phosphate or 2'3' cyclic phosphate group) (3'P oligonucleotide) and the oligonucleotide with 5' terminal hydroxyl modification (5'OH oligonucleotide) through a three-step nucleic acid transfer process: (1) RtcB reacts with triphosphate (e.g., GTP) to form a covalent RtcB-histidine-GMP intermediate and release PPi; (2) GMP is transferred to the 3' phosphate or 2'3' cyclic phosphate group of the oligonucleotide; (3) 5'OH oligonucleotide attacks the activated 3'P oligonucleotide to form a 3', 5'-phosphodiester bond and release GMP.

[0159] Preferably, the triphosphate comprises at least one of NTP and dNTP; and further comprises at least one of ATP, CTP, GTP, TTP, UTP, dATP, dCTP, and dGTP.

[0160] Preferably, the manganese ion is a divalent manganese ion.

[0161] Preferably, the linker composition further comprises a second auxiliary factor comprising at least one of ATP and NAD and metal ions, for assisting the second ligase in connecting the 5' second modified group end of the oligonucleic acid to the 3' hydroxyl end of the template nucleic acid.

[0162] Preferably, the metal ions include at least one of magnesium ions and manganese ions.

[0163] Preferably, the manganese ion is a divalent manganese ion.

[0164] Preferably, when the 5' end of the second oligonucleotide chain of the 3' end adapter is phosphorylated, the second ligase comprises at least one of T4 RNA ligase 1, T4 DNA ligase, TS2126 RNA ligase, T3 DNA ligase, Escherichia coli (E. coli) DNA ligase, single-stranded DNA / RNA circular ligase (ssDNA / RNA CircLigase), T4 RNA ligase 2, and Taq DNA ligase; and further comprises: T4 RNA ligase 1.

[0165] Preferably, when the 5' end of the second oligonucleotide chain of the 3' end adapter has an adenylation modification, the second ligase comprises at least one of T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 (truncated K227Q), T4 RNA ligase 2 (truncated KQ), thermostable 5'App DNA / RNA ligase, TS2126 RNA ligase, T3 DNA ligase, Escherichia coli (E. coli) DNA ligase, single-stranded DNA / RNA circular ligase (ssDNA / RNA CircLigase), and Taq DNA ligase.

[0166] Preferably, the linker composition further comprises: a reaction buffer; which is used for the linker ligation reaction catalyzed by the first ligase and / or the second ligase.

[0167] Preferably, the first blocking group modification is selected from the following groups: amino modification, dideoxy modification, spacer modification, and phosphate modification.

[0168] Preferably, the second blocking group modification is selected from the following groups: amino modification, dideoxy modification, spacer modification, phosphate modification.

[0169] Preferably, the third blocking group modification is selected from the following groups: amino modification, dideoxy modification, spacer modification, and phosphate modification.

[0170] Preferably, the fourth blocking group modification is selected from the following groups: amino modification, dideoxy modification, spacer modification, and phosphate modification.

[0171] Preferably, the fifth blocking group modification is selected from the following group modifications: amino modification, dideoxy modification, spacer modification, and phosphate modification.

[0172] Preferably, the sixth blocking group modification is selected from the following group modifications: amino modification, dideoxy modification, spacer modification, and phosphate modification.

[0173] The first blocking group modification, the second blocking group modification, the third blocking group modification, the fourth blocking group modification, the fifth blocking group modification, and the sixth blocking group modification prevent the linkers from connecting to each other.

[0174] Preferably, the first blocking group modification and the second blocking group modification are amino modifications.

[0175] Preferably, the amino modification includes: at least one of C6 amino modification and C12 amino modification; and further includes: C6 amino modification.

[0176] Preferably, the sequence of the 5' end linker is as shown in SEQ ID NO. 1, wherein the 5' end has a C6 amino modification and the 3' end has a phosphorylation modification.

[0177] Preferably, the sequence of the 3' end linker is as shown in SEQ ID NO. 2, wherein the 5' end thereof has a phosphorylation modification and the 3' end has a C6 amino modification.

[0178] Preferably, the first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter comprise an adapter sequence.

[0179] Preferably, the first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter further comprise a sequencing primer sequence.

[0180] Preferably, the first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter further comprise a sample tag sequence for distinguishing different samples for subsequent multi-sample mixed sequencing, for example, a barcode sequence or an index sequence.

[0181] Preferably, the first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter further comprise a unique molecular identifier (UMI) sequence for counting the copy number of the nucleic acid molecule in the sample.

[0182] Preferably, the first oligonucleotide strand of the 5'-end adapter comprises an adapter sequence.

[0183] Preferably, the first oligonucleotide strand of the 5' end adapter further comprises a sequencing primer sequence.

[0184] Preferably, the first oligonucleotide chain of the 5' end adapter further comprises a sample tag sequence for distinguishing different samples to facilitate subsequent multi-sample mixed sequencing, for example, a barcode sequence or an index sequence.

[0185] Preferably, the first oligonucleotide chain of the 5' end adapter further comprises a unique molecular identifier (UMI) sequence for counting the copy number of the nucleic acid molecule in the sample.

[0186] Preferably, the second oligonucleotide strand of the 3' end adapter comprises an adapter sequence.

[0187] Preferably, the linker sequence of the first oligonucleotide chain of the 5' end linker is the same as or different from the linker sequence of the second oligonucleotide chain of the 3' end linker.

[0188] Adapter Preferably, the second oligonucleotide strand of the 3' end adapter further comprises a sequencing primer sequence.

[0189] Preferably, the sequencing primer sequence of the first oligonucleotide chain of the 5' end adapter is the same as or different from the sequencing primer sequence of the second oligonucleotide chain of the 3' end adapter.

[0190] Preferably, the second oligonucleotide chain of the 3' end adapter further comprises a sample tag sequence for distinguishing different samples to facilitate subsequent multi-sample mixed sequencing, for example, a barcode sequence or an index sequence.

[0191] Preferably, the sample tag sequence of the first oligonucleotide chain of the 5' end adapter is the same as or different from the sample tag sequence of the second oligonucleotide chain of the 3' end adapter.

[0192] Preferably, the second oligonucleotide chain of the 3' end adapter further comprises a unique molecular identifier (UMI) sequence for counting the copy number of the nucleic acid molecule in the sample.

[0193] Preferably, the unique molecular tag sequence of the first oligonucleotide chain of the 5' end adapter is the same as or different from the unique molecular tag sequence of the second oligonucleotide chain of the 3' end adapter.

[0194] Preferably, the length of the sample tag sequence is 5 to 20 bp.

[0195] Preferably, the length of the unique molecular tag is 5 to 20 bp.

[0196] Preferably, the first oligonucleotide strand of the 5' end adapter is different from the second oligonucleotide strand of the 3' end adapter.

[0197] The sequencing primer sequence, sample tag sequence, and unique molecular identifier (UMI) sequence of the first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter can also be introduced through PCR amplification or reverse transcription reaction after the single-stranded template nucleic acid is ligated to the adapter, that is, introduced into the PCR primer or reverse transcription primer.

[0198] Preferably, the linker composition further comprises: a phosphatase, which is used to remove the phosphate groups at the 5' end and / or 3' end of the single-stranded template nucleic acid or the double-stranded nucleic acid containing the single-stranded template nucleic acid, thereby ensuring that the 5' end and 3' end of the single-stranded template nucleic acid or the double-stranded nucleic acid containing the single-stranded template nucleic acid are both hydroxyl groups and will not self-ligate under the action of the ligase.

[0199] Preferably, the phosphatase comprises: at least one of acid phosphatase and alkaline phosphatase; further comprises: alkaline phosphatase; further comprises: at least one of calf intestinal phosphatase, shrimp alkaline phosphatase, Antarctic phosphatase and APEX alkaline phosphatase; further comprises: shrimp alkaline phosphatase.

[0200] Preferably, the adaptor composition is used to connect a 5'-end adaptor and a 3'-end adaptor to the 5'-end and 3'-end of a single-stranded template nucleic acid.

[0201] Preferably, the linker composition is used to connect a 5' end linker and a 3' end linker to the 5' end and 3' end of a single-stranded template nucleic acid in the same system; wherein the same system is specifically carried out in the same reaction vessel; further specifically, in the same reaction vessel and at the same time.

[0202] Preferably, the single-stranded template nucleic acid is obtained by the following method: subjecting the nucleic acid to at least one of the following treatments: fragmentation, denaturation, and DNA methylation.

[0203] Preferably, the nucleic acid comprises at least one of DNA and RNA.

[0204] Preferably, the DNA comprises at least one of dsDNA and ssDNA.

[0205] Preferably, the nucleic acid comprises: dsDNA, ssDNA and RNA.

[0206] Preferably, the nucleic acid is from the following biological samples: cells, fresh tissues, fresh organs, decayed tissues, formalin-fixed tissues, paraffin-embedded tissues, forensic samples, paleontological fossils, biological materials containing cfDNA or RNA.

[0207] Preferably, the biological material includes but is not limited to: peripheral blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, lymph fluid, alveolar lavage fluid, amniotic fluid, blastocyst cavity fluid, cell culture fluid, embryo culture fluid, microbial culture medium, soil leachate, and bone powder leachate.

[0208] More specifically, the nucleic acid can be isolated from a biological sample obtained from an individual (e.g., a test individual). The individual can be any living or non-living organism, including but not limited to humans, non-human animals, plants, bacteria, fungi, protozoa, or pathogens.

[0209] Nucleic acids can be isolated or obtained from any type of suitable biological sample. Nucleic acids can be isolated or obtained from a single cell, a plurality of cells (e.g., cultured cells), a cell culture medium, a conditioned medium, a tissue, an organ, or an organism (e.g., bacteria, yeast, etc.).

[0210] In some cases, nucleic acids can be obtained as part of a forensic analysis. In some embodiments, the kits described herein are applied to forensic samples or specimens. Forensic samples or specimens can include any biological material containing nucleic acids. For example, forensic samples or specimens can include blood, semen, hair, skin, sweat, saliva, decomposed tissue, bone, nail scrapings, licked stamps / envelopes, sluff, contact DNA, razor residue, etc. The specimen can be formalin-fixed tissue and / or paraffin-embedded tissue.

[0211] A biological sample can be any sample isolated or obtained from an individual or part thereof (e.g., a human individual, a pregnant female, a cancer patient, a patient suffering from an infection or infectious disease, a transplant recipient, a fetus, a tumor, an infected organ or tissue, a transplanted organ or tissue, a microbiome). In some embodiments, the biological sample is a cervical swab from an individual. The liquid sample or tissue sample from which nucleic acid is extracted can be acellular (e.g., free of cells). In some embodiments, the biological sample can contain cellular components or cell remnants. In some embodiments, the biological sample can include fetal cells or cancer cells.

[0212] Biological sample can be a liquid sample.Liquid sample can include extracellular nucleic acid (for example, circulating cell-free DNA).The example of liquid sample includes but is not limited to blood or blood products (such as serum, plasma, etc.), urine, cerebrospinal fluid, saliva, sputum, biopsy sample (for example, for detecting liquid biopsy of cancer), above-mentioned liquid sample, analog or its combination.In certain embodiments, biological sample is liquid biopsy, it generally refers to the assessment of the presence, absence, progression or alleviation of the liquid sample from individual about disease (for example, cancer).Liquid biopsy can be used in combination with the biopsy (for example, tumor biopsy) sold or used as its substitute.In some cases, extracellular nucleic acid is analyzed in liquid biopsy.

[0213] The biological sample can be a tumor nucleic acid sample (ie, a nucleic acid sample isolated from a tumor).

[0214] Preferably, when the nucleic acid comprises dsDNA, the process of preparing the single-stranded template nucleic acid from the nucleic acid comprises denaturation treatment.

[0215] Preferably, when the nucleic acid is derived from the following biological samples: cells, fresh tissues, fresh organs, paraffin-embedded tissues, forensic samples, the process of preparing the single-stranded template nucleic acid from the nucleic acid includes a shearing treatment.

[0216] Preferably, when the nucleic acid is used to construct a DNA methylation library, the process of preparing the single-stranded template nucleic acid from the nucleic acid comprises DNA methylation treatment.

[0217] Preferably, the denaturation treatment method comprises: thermal denaturation and / or chemical denaturation.

[0218] More specifically, double-stranded nucleic acids (such as dsDNA) can be chemically denatured using a denaturant (e.g., alkali, glycerol, ethylene glycol, formamide, urea, or a combination thereof) that reduces the melting temperature of the double-stranded nucleic acid. The denaturant can reduce the melting temperature by 5°C-6°C for every 10% (v / v) of denaturant added to the reaction mixture. The denaturant or a combination of denaturants (e.g., 10% glycerol and 6-7% ethylene glycol) can comprise 1%, 5%, 10%, 15%, 20%, or 25% of the reaction mixture (v / v). Salts that reduce hybridization stringency can be included in the reaction buffer at low concentrations to chemically denature the double-stranded nucleic acid at low temperatures. Double-stranded nucleic acids (such as dsDNA) can be heat denatured by, for example, heating the double-stranded nucleic acid (such as dsDNA) at 92-98°C for 3-10 minutes. Immediately after denaturation, place on ice to prevent the melted double-stranded nucleic acid from renaturing.

[0219] Preferably, the interruption treatment method includes at least one of a chemical interruption method and a physical interruption method.

[0220] Preferably, the method of DNA methylation treatment is treatment with a DNA methylation modification conversion reagent.

[0221] Preferably, the DNA methylation modification conversion reagent is selected from at least one of bisulfite, sulfite, bisulfite, and bisulfite.

[0222] Preferably, the linker composition further comprises: a substance for fragmenting nucleic acids.

[0223] Preferably, the substance for fragmenting nucleic acids is a substance selected from one or more of the following fragmentation methods: chemical fragmentation method, physical fragmentation method.

[0224] Preferably, the chemical fragmentation method comprises at least one of a transposase method and a traditional enzyme cleavage method.

[0225] Preferably, the physical breaking method includes at least one of an ultrasonic breaking method and a mechanical breaking method.

[0226] Preferably, the linker composition further comprises: a substance for nucleic acid denaturation.

[0227] Preferably, the substance used for nucleic acid denaturation is a substance selected from one or more of the following denaturation methods: thermal denaturation, chemical denaturation.

[0228] Preferably, when the linker composition is used to construct a DNA methylation library, the linker composition further comprises: a DNA methylation modification conversion reagent for converting non-methylated C bases in DNA into U bases.

[0229] Preferably, the DNA methylation modification conversion reagent is selected from at least one of bisulfite, sulfite, bisulfite, and bisulfite.

[0230] Preferably, the linker composition further comprises: a single-stranded binding protein (SSB) for maintaining the single-stranded state of the single-stranded nucleic acid.

[0231] Single-stranded binding protein refers to any protein that has the function of binding to single-stranded nucleic acids, for example, to prevent premature annealing, protect single-stranded nucleic acids from nuclease digestion, remove secondary structure from nucleic acids, or to facilitate replication of nucleic acids. The term is intended to include, but is not necessarily limited to, proteins that are formally identified as single-stranded binding proteins by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). Exemplary single-stranded binding proteins include, but are not limited to, Escherichia coli (E. coli) SSB, T4 gp32, T7 gene 2.5 SSB, phage π29 SSB, MjA SSB from Methanococcus jannaschii, any homologous protein or protein complex from any phylum, or a functional variant thereof.

[0232] Preferably, the first oligonucleotide chain of the 5' end linker and / or the second oligonucleotide chain of the 3' end linker are fixed to a solid support; further preferably, the non-connected ends of the first oligonucleotide chain of the 5' end linker and / or the second oligonucleotide chain of the 3' end linker (for the first oligonucleotide chain of the 5' end linker, its connecting end is the 3' end of the first oligonucleotide chain of the 5' end linker; for the second oligonucleotide chain of the 3' end linker, its connecting end is the 5' end of the second oligonucleotide chain of the 3' end linker) are fixed to a solid support; the 5' end linker and / or the 3' end linker fixed to the solid support can connect one or more single-stranded template nucleic acids to the solid support through a ligation reaction to generate a solid support with a specific sequence (linker) and a single-stranded template nucleic acid, which is used for single-cell sequencing, spatial omics sequencing, etc.

[0233] Preferably, the solid support is selected from chips, beads and the like.

[0234] Preferably, the chip can be used in a sequencing platform, such as a sequencing chip.

[0235] Preferably, the chip is a high-throughput sequencing chip, such as a high-throughput sequencing chip used in an Illumina, MGI or Thermo Fisher sequencing platform.

[0236] Preferably, the first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter can be immobilized on a solid support (chip) by any suitable method known in the art.

[0237] Preferably, non-limiting examples of the method include nucleic acid hybridization, biotin-streptavidin binding, sulfhydryl binding, photoactivated binding, covalent binding, antibody-antigen, physical confinement via hydrogels or other porous polymers, etc., or any combination thereof.

[0238] Preferably, the chip comprises a material selected from the group consisting of glass, silicon, poly-lysine coating material, nitrocellulose, polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polypropylene, polyethylene, polycarbonate, or any combination thereof.

[0239] Preferably, the beads are selected from agarose gel beads, sepharose beads, magnetic beads, protein A conjugated beads, protein G conjugated beads, protein L conjugated beads, oligo (dT) conjugated beads, silica beads, hydrogel beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof.

[0240] Preferably, the first oligonucleotide strand of the 5' end adapter and / or the second oligonucleotide strand of the 3' end adapter can be fixed to a solid support (bead) by any suitable method known in the art, such as: 1) or 2):

[0241] 1) The solid support (bead) and the nucleic acid (the first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter) are respectively modified with interactive functional units, so that the two react to label the nucleic acid on the solid support;

[0242] 2) The nucleic acid (the first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter) is directly synthesized on a solid support (beads) according to a preset nucleotide sequence.

[0243] More specifically, the first oligonucleotide chain of the 5' end joint and / or the second oligonucleotide chain of the 3' end joint can be connected to the solid support (bead) by "graft to" and / or "graft from". In specific applications, the solid support (bead) can be labeled with nucleic acid (the first oligonucleotide chain of the 5' end joint and / or the second oligonucleotide chain of the 3' end joint) by the "graft to" scheme alone. In specific applications, the solid support (bead) can be labeled with nucleic acid (the first oligonucleotide chain of the 5' end joint and / or the second oligonucleotide chain of the 3' end joint) by the "graft from" scheme alone. In specific applications, the solid support (bead) can be labeled with nucleic acid (the first oligonucleotide chain of the 5' end joint and / or the second oligonucleotide chain of the 3' end joint) by a mixed "graft to" and "graft from" schemes.

[0244] Preferably, the beads comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substances, ceramics, plastics, glass, methylstyrene, acrylic polymers, titanium, latex, agarose gel, cellulose, nylon, silicone, or any combination thereof.

[0245] Preferably, when the "graft to" scheme is adopted, the solid support and the nucleic acid are respectively modified with functional units capable of interacting with each other, and the functional units include but are not limited to one or more of hydroxyl, aldehyde, epoxy, amino, carboxyl and activated forms thereof, phosphate, alkynyl, azide, sulfhydryl, olefin, biotin, avidin, isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, tosyl ester, etc.

[0246] Preferably, when the "graft from" scheme is adopted, the nucleotide sequence can be directly synthesized on the support according to the preset nucleotide sequence.

[0247] The second aspect of the present invention provides a nucleic acid library construction kit, comprising: the linker composition of the first aspect of the present invention.

[0248] Preferably, the nucleic acid library construction kit further comprises: a polynucleotide kinase (preferably T4 polynucleotide kinase) for dephosphorylating the 3' end of the single-stranded template nucleic acid connected to the adapter (preferably the 5' end adapter does not contain the third oligonucleotide chain, the 3' end adapter does not contain the fourth oligonucleotide chain, and the first blocking group modification and the second blocking group modification are phosphate modifications); the single-stranded template nucleic acid connected to the adapter after 3' end dephosphorylation is subsequently subjected to a circularization reaction to obtain a single-stranded circularized library, thereby realizing PCR-free nucleic acid library construction.

[0249] Preferably, the nucleic acid library construction kit further comprises: PCR primers for performing PCR amplification on the single-stranded template nucleic acid connected to the adapter.

[0250] Preferably, the PCR primer comprises a sequence that is partially or entirely identical or complementary to the linker.

[0251] Preferably, the linker comprises a 3' end linker and / or a 5' end linker.

[0252] Preferably, when the linker does not contain a sequencing primer sequence, a sequencing primer sequence can be introduced via a PCR primer, and the PCR primer further contains a sequencing primer sequence.

[0253] Preferably, when the adapter does not contain a sample tag sequence, a sample tag sequence can be introduced through a PCR primer, and the PCR primer also contains a sample tag sequence for distinguishing different samples to facilitate subsequent multi-sample mixed sequencing. For example, it can be a barcode sequence or an index sequence.

[0254] Preferably, when the adapter does not contain a unique molecular index (UMI) sequence, a unique molecular index sequence can be introduced by a PCR primer, wherein the PCR primer also contains a unique molecular index (UMI) sequence for counting the copy number of the nucleic acid molecule in the sample.

[0255] Preferably, when the adapter does not include a sequencing primer sequence, a sample tag sequence, or a unique molecular identifier (UMI) sequence, the PCR primers include a first primer and a second primer;

[0256] Wherein, the first primer comprises a sequence that is partially or entirely identical or complementary to the 5' end of the first oligonucleotide chain of the 5' end adapter, and the second primer comprises a sequencing primer sequence, a sample tag sequence, and a sequence that is partially or entirely identical or complementary to the 3' end of the second oligonucleotide chain of the 3' end adapter; preferably, the second primer further comprises a unique molecular tag sequence; or

[0257] The first primer comprises a sequencing primer sequence, a sample tag sequence, and a sequence that is partially or completely identical or complementary to the 5' end of the first oligonucleotide chain of the 5' end adapter; the second primer comprises a sequence that is partially or completely identical or complementary to the 3' end of the second oligonucleotide chain of the 3' end adapter; preferably, the first primer further comprises a unique molecular tag sequence.

[0258] Preferably, the sequences of the PCR primers are shown as SEQ ID NO. 3 and 4.

[0259] Preferably, the nucleic acid library construction kit further comprises: a PCR reaction solution.

[0260] Preferably, when the nucleic acid comprises RNA, the nucleic acid library construction kit further comprises: a reverse transcription primer for reverse transcribing the RNA to obtain cDNA.

[0261] Preferably, the reverse transcription primer comprises a sequence that is partially or entirely identical to or complementary to the 3' end of the second oligonucleotide chain of the 3' end adapter.

[0262] Preferably, when the adapter does not contain a sequencing primer sequence, a sequencing primer sequence can be introduced via a reverse transcription primer, and the reverse transcription primer further contains a sequencing primer sequence.

[0263] Preferably, when the adapter does not contain a sample tag sequence, a sample tag sequence can be introduced through a reverse transcription primer, which also contains a sample tag sequence for distinguishing different samples to facilitate subsequent multi-sample mixed sequencing. For example, it can be a barcode sequence or an index sequence.

[0264] Preferably, when the adapter does not contain a unique molecular index (UMI) sequence, a unique molecular index sequence can be introduced by a reverse transcription primer, and the reverse transcription primer also contains a unique molecular index (UMI) sequence for counting the copy number of the nucleic acid molecule in the sample.

[0265] Preferably, when the nucleic acid is RNA, the nucleic acid library construction kit further comprises: a reverse transcription reaction solution.

[0266] Preferably, the nucleic acid library construction kit comprises: a nucleic acid extraction reagent, wherein the nucleic acid extraction reagent is: a lysis reagent.

[0267] Preferably, the nucleic acid library construction kit further comprises a nucleic acid extraction reagent combination, specifically a nucleic acid extraction reagent combination for any one of the following methods: alkaline lysis method, phenol chloroform extraction method, chelating resin method, centrifugal column membrane adsorption method and magnetic bead method.

[0268] Preferably, the nucleic acid extraction reagent combination comprises: at least one of: a lysis solution, a washing solution, an eluent, and a nucleic acid adsorbent; further preferably, the nucleic acid extraction reagent combination comprises: a lysis solution, a washing solution, an eluent, and a nucleic acid adsorbent.

[0269] Preferably, the nucleic acid adsorbent comprises at least one of magnetic beads and adsorption membranes.

[0270] Preferably, the nucleic acid library construction kit further comprises: a cyclization reagent combination.

[0271] A third aspect of the present invention provides a sequencing reagent kit comprising: any one of a1) to a2):

[0272] a1) the joint composition according to the first aspect of the present invention;

[0273] a2) The nucleic acid library construction kit according to the second aspect of the present invention.

[0274] Preferably, the sequencing reagent set further comprises: a sequencing reagent kit.

[0275] A fourth aspect of the present invention provides a sequencing system comprising: any one of a1) to a3) and a sequencer:

[0276] a1) the joint composition according to the first aspect of the present invention;

[0277] a2) the nucleic acid library construction kit according to the second aspect of the present invention;

[0278] a3) The sequencing reagent set according to the third aspect of the present invention.

[0279] The fifth aspect of the present invention provides a method for connecting a joint, comprising the step of using the joint composition of the first aspect of the present invention.

[0280] Preferably, the adapter ligation method comprises the following steps: obtaining a single-stranded template nucleic acid, wherein both the 5' end and the 3' end of the single-stranded template nucleic acid have hydroxyl groups; performing an adapter ligation reaction of the 5' end adapter and the 3' end adapter on the single-stranded template nucleic acid using the first ligase, the 5' end adapter, the second ligase and the 3' end adapter in the adapter composition of the first aspect of the present invention.

[0281] Preferably, the linker ligation reaction of the 5' end linker and the 3' end linker is carried out in the same system.

[0282] Preferably, the same system is specifically carried out in the same reaction vessel; further specifically, it is carried out in the same reaction vessel and at the same time.

[0283] Preferably, the reaction temperature of the linker ligation reaction is 15-82°C; further 16-80°C.

[0284] Preferably, the reaction time of the linker ligation reaction is more than 15 minutes; further preferably 30-60 minutes.

[0285] Preferably, the connection of the adapter is directional, and the first oligonucleotide chain of the 5'-end adapter and the second oligonucleotide chain of the 3'-end adapter have different nucleic acid sequences.

[0286] Preferably, the linker ligation reaction system further comprises: the first auxiliary factor in the linker composition of the first aspect of the present invention.

[0287] Preferably, the linker ligation reaction system further comprises: the second auxiliary factor in the linker composition of the first aspect of the present invention.

[0288] Preferably, the linker ligation reaction system further comprises: a reaction buffer in the linker composition of the first aspect of the present invention.

[0289] Preferably, the method for obtaining the single-stranded template nucleic acid having hydroxyl groups at both the 5' and 3' ends is a dephosphorylation treatment, comprising the following steps: treating the single-stranded template nucleic acid or the double-stranded nucleic acid containing the single-stranded template nucleic acid with a phosphatase, thereby removing the phosphate groups at the 5' and / or 3' ends of the single-stranded template nucleic acid or the double-stranded nucleic acid containing the single-stranded template nucleic acid.

[0290] Preferably, the single-stranded template nucleic acid is obtained by the following method: subjecting the nucleic acid to at least one of the following treatments: fragmentation, denaturation, and DNA methylation.

[0291] Preferably, the nucleic acid comprises at least one of DNA and RNA.

[0292] Preferably, the DNA comprises at least one of dsDNA and ssDNA.

[0293] Preferably, the nucleic acid comprises at least one of dsDNA, ssDNA and RNA.

[0294] Preferably, the nucleic acid is from the following biological samples: cells, fresh tissues, fresh organs, decayed tissues, formalin-fixed tissues, paraffin-embedded tissues, forensic samples, paleontological fossils, biological materials containing cfDNA or cfRNA.

[0295] Preferably, the biological material includes but is not limited to: peripheral blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, lymph fluid, alveolar lavage fluid, amniotic fluid, blastocyst cavity fluid, cell culture fluid, embryo culture fluid, microbial culture medium, soil leachate, and bone powder leachate.

[0296] Preferably, when the nucleic acid comprises dsDNA, the process of preparing the single-stranded template nucleic acid from the nucleic acid comprises denaturation treatment.

[0297] Preferably, the denaturation treatment may be performed before or after the phosphorylation treatment.

[0298] Preferably, the denaturation treatment method comprises: thermal denaturation and / or chemical denaturation; further thermal denaturation; and further treatment at 92-98° C. for 3-10 min.

[0299] Preferably, the denaturation treatment may further include a step of preventing the melted double-stranded nucleic acid from renaturing: placing the nucleic acid on ice immediately after the denaturation is completed.

[0300] Preferably, when the nucleic acid is derived from the following biological samples: cells, fresh tissues, fresh organs, paraffin-embedded tissues, forensic samples, the process of preparing the single-stranded template nucleic acid from the nucleic acid includes a shearing treatment.

[0301] Preferably, the shearing treatment precedes the phosphorylation treatment and the denaturation treatment.

[0302] Preferably, the interruption treatment method includes at least one of a chemical interruption method and a physical interruption method.

[0303] Preferably, the chemical fragmentation method comprises at least one of a transposase method and a traditional enzyme cleavage method.

[0304] Preferably, the physical breaking method includes at least one of an ultrasonic breaking method and a mechanical breaking method.

[0305] Preferably, when the nucleic acid is used to construct a DNA methylation library, the process of preparing the single-stranded template nucleic acid from the nucleic acid comprises DNA methylation treatment.

[0306] Preferably, the DNA methylation treatment precedes the phosphorylation treatment and the denaturation treatment.

[0307] Preferably, the DNA methylation treatment is performed after the shearing treatment.

[0308] Preferably, the method of DNA methylation treatment is treatment with a DNA methylation modification conversion reagent.

[0309] Preferably, the DNA methylation modification conversion reagent is selected from at least one of bisulfite, sulfite, bisulfite, and bisulfite.

[0310] The sixth aspect of the present invention provides a method for constructing a nucleic acid library, comprising the steps of the linker ligation method of the fifth aspect of the present invention.

[0311] Preferably, for the method of constructing a nucleic acid library in a PCR-free process, the method further comprises the following steps: dephosphorylating the 3' end of the single-stranded template nucleic acid connected to the adapter, and then performing a circularization reaction to obtain a single-stranded circularized library.

[0312] Preferably, the nucleic acid library construction method, as shown in FIG4 , comprises the following steps:

[0313] Obtaining a single-stranded template nucleic acid connected to an adapter by the adapter connection method of the fifth aspect of the present invention;

[0314] The 3' end of the single-stranded template nucleic acid connected to the adapter is dephosphorylated and then subjected to a circularization reaction to obtain a single-stranded circularized library.

[0315] Preferably, the 5' end adapter in the adapter-ligated single-stranded template nucleic acid does not contain a third oligonucleic acid chain.

[0316] Preferably, the 3' end adapter in the adapter-ligated single-stranded template nucleic acid does not contain a fourth oligonucleic acid chain.

[0317] Preferably, the first blocking group modification and the second blocking group modification in the single-stranded template nucleic acid of the linker are phosphate modifications.

[0318] Preferably, for the method of constructing a nucleic acid library with a PCR process, the method further comprises the following step: performing an amplification reaction on the single-stranded template nucleic acid connected to the adapter to obtain a nucleic acid library.

[0319] Preferably, the nucleic acid library construction method comprises the following steps:

[0320] Obtaining a single-stranded template nucleic acid connected to an adapter by the adapter connection method of the fifth aspect of the present invention;

[0321] The single-stranded template nucleic acid connected to the adapter is amplified to obtain a nucleic acid library.

[0322] Preferably, the amplification reaction is rolling circle amplification or linear amplification.

[0323] Preferably, the amplification reaction uses the PCR primers in the nucleic acid library construction kit of the second aspect of the present invention.

[0324] Preferably, when the nucleic acid comprises RNA, the nucleic acid library construction method may further comprise the following step: a reverse transcription reaction, wherein the reverse transcription reaction may be performed before or after the adapter ligation reaction.

[0325] Preferably, the reverse transcription reaction uses the reverse transcription primers in the nucleic acid library construction kit of the second aspect of the present invention.

[0326] Preferably, the nucleic acid library construction method further comprises the following step: purifying the amplified product.

[0327] Preferably, the purification is performed using magnetic beads.

[0328] Preferably, the linker ligation reaction may be followed by a cyclization reaction.

[0329] Preferably, a method for constructing a nucleic acid library, as schematically shown in Figures 1 and 2, comprises the following steps:

[0330] (1) Obtaining nucleic acid samples: Nucleic acid samples can be: 1) intact genomic DNA or RNA fragmented into certain size fragments; 2) extracted cfDNA or cfRNA; 3) degraded DNA or RNA extracted from various degraded biological samples (e.g., paleontological fossils, FFPE samples); 4) DNA fragments fragmented from intact genomic DNA or DNA purified and recovered after sulfite treatment of extracellular free DNA;

[0331] (2) Denaturing the nucleic acid sample (this step is used for nucleic acid samples containing double-stranded nucleic acids) to obtain a single-stranded template nucleic acid. At the same time, a single-stranded binding protein can be added to maintain the single-stranded state of the nucleic acid sample (single-stranded) or the single-stranded template nucleic acid by utilizing the characteristics of the binding protein. This step can use both double-stranded and single-stranded nucleic acids in the sample;

[0332] (3) Dephosphorylating the single-stranded template nucleic acid by using phosphatase to remove the phosphate groups at the 5' and 3' ends of the single-stranded template nucleic acid so that both ends of all single-stranded template nucleic acids are hydroxyl groups, thereby preventing the template nucleic acids from ligating to each other in the presence of ligase; this step can be performed after step (1) and before step (2);

[0333] (4) Adapter ligation: performing an adapter ligation reaction on the single-stranded template nucleic acid using the first ligase, 5' end adapter, second ligase, and 3' end adapter in the adapter composition of the first aspect of the present invention; preferably, the adapter ligation reaction system further comprises: the first auxiliary factor and the second auxiliary factor in the adapter composition of the first aspect of the present invention; preferably, the adapter ligation reaction system further comprises: the reaction buffer in the adapter composition of the first aspect of the present invention;

[0334] (5) Obtaining a library: performing an amplification reaction on the single-stranded template nucleic acid connected to the adapter to obtain a nucleic acid library; when the nucleic acid is RNA, the amplification reaction further includes a reverse transcription reaction; preferably, the amplification reaction uses the PCR primers in the nucleic acid library construction kit of the second aspect of the present invention; preferably, the reverse transcription reaction uses the reverse transcription primers in the nucleic acid library construction kit of the second aspect of the present invention.

[0335] The seventh aspect of the present invention provides a nucleic acid library obtained by the nucleic acid library construction method of the sixth aspect of the present invention.

[0336] The eighth aspect of the present invention provides a sequencing method comprising the steps of the nucleic acid library construction method of the sixth aspect of the present invention.

[0337] Preferably, the sequencing method comprises the following steps: obtaining a nucleic acid library; sequencing;

[0338] The method for obtaining a nucleic acid library is the nucleic acid library construction method of the sixth aspect of the present invention.

[0339] Preferably, the sequencing further includes the following steps: library quality inspection.

[0340] The ninth aspect of the present invention provides uses of the linker composition of the first aspect, the kit of the second aspect, the sequencing reagent set of the third aspect, and / or the sequencing system of the fourth aspect.

[0341] Use of the linker composition of the first aspect of the present invention and / or the kit of the second aspect of the present invention in any one of c1) to c6);

[0342] c1) preparing a nucleic acid library;

[0343] c2) preparing products for nucleic acid library construction;

[0344] c3) sequencing;

[0345] c4) preparing products for sequencing;

[0346] c5) DNA methylation detection;

[0347] c6) preparing products for DNA methylation detection.

[0348] Preferably, the nucleic acid library comprises a DNA methylation library.

[0349] Use of the sequencing reagent set of the third aspect of the present invention and / or the sequencing system of the fourth aspect of the present invention in any one of items c3) to c6);

[0350] c3) sequencing;

[0351] c4) preparing products for sequencing;

[0352] c5) DNA methylation detection;

[0353] c6) preparing products for DNA methylation detection.

[0354] The present invention is further described in detail below through specific examples.

[0355] It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0356] The experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturers. The materials and reagents used in these examples were commercially available unless otherwise specified.

[0357] Example 1 Construction and sequencing of human peripheral blood free DNA methylation library

[0358] 1. Experimental Materials

[0359] Free DNA extracted from human peripheral blood plasma (sample name: cfDNA-1).

[0360] 2. Experimental steps

[0361] 1. Take 5 ng of human peripheral blood plasma free DNA (cfDNA) and transfer it to a 200 μL PCR tube, with a total volume of 20 μL.

[0362] 2. Use EZ DNA Methylation-Gold Kit (Zymo Research, Cat. No. D5005 / D5006) to treat and purify cfDNA with Bisulfite.

[0363] 3. Sequentially pipette 900μL of NF water, 300μL of M-Dilution Buffer, and 50μL of M-Dissolving Buffer into a tube of CT Conversion Reagent powder (centrifuge briefly before opening the lid). Vortex frequently at room temperature for 10 minutes to prepare the CT Conversion Reagent. Minimize exposure to sunlight and prepare the CT Conversion Reagent immediately before use. The CT Conversion Reagent can be stored at room temperature for up to 1 day, at 4°C for up to 1 week, and at -20°C for up to 1 month. Non-freshly prepared CT Conversion Reagent should be prewarmed to 37°C and vortexed frequently for 10 minutes before use.

[0364] 4. Before opening the bottle cap for the first time, add the correct volume of anhydrous ethanol as indicated on the bottle label and mix thoroughly before use. At room temperature, add the ingredients listed in Table 1 to a new 200 μL PCR tube.

[0365] Table 1

[0366] 5. Place the 200 μL PCR tube in a PCR instrument and perform the reaction according to the Bisulfite treatment conditions shown in Table 2.

[0367] Table 2

[0368] 6. Transfer the reaction product obtained in step 5 to a new 1.5 mL centrifuge tube, add 600 μL of M-Binding Buffer, vortex 6 times, each time for 3 seconds, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0369] 7. Place the Zymo-Spin IC Column into a 2 mL Collection Tube. Transfer the mixture obtained in step 6 onto the Zymo-Spin IC Column. Centrifuge at 13,000 rpm for 30 seconds. Discard the waste liquid and place the Zymo-Spin IC Column back into the Collection Tube.

[0370] 8. Add 100 μL of M-Wash Buffer to the Zymo-Spin IC Column and centrifuge at 13,000 rpm for 30 seconds.

[0371] 9. Add 200 μL of M-Desulphonation Buffer to the Zymo-Spin IC Column, quickly cap the tube, incubate at room temperature for 15-20 minutes, centrifuge at 13,000 rpm for 30 seconds, discard the waste liquid, and return the Zymo-Spin IC Column to the Collection Tube.

[0372] 10. Add 200 μL of M-Wash Buffer to the Zymo-Spin IC Column, centrifuge at 13,000 rpm for 30 seconds, discard the waste liquid, and return the Zymo-Spin IC Column to the Collection Tube.

[0373] 11. Add 200 μL of M-Wash Buffer to the Zymo-Spin IC Column and centrifuge at 13,000 rpm for 30 seconds. Discard the waste liquid and return the Zymo-Spin IC Column to the Collection Tube. Centrifuge at 13,000 rpm for 30 seconds without rotation. Discard the Collection Tube and use a pipette to remove as much liquid as possible from the outer wall of the Zymo-Spin IC Column. Place the column in a new 1.5 mL centrifuge tube.

[0374] 12. Open the Zymo-Spin IC Column tube cap, dry it at room temperature for 2 minutes, and then place the Zymo-Spin IC Column in another new 1.5mL centrifuge tube.

[0375] 13. Slowly add 8 μL of M-Elution Buffer to the center of the Zymo-Spin IC Column filter membrane, let it stand for 1 minute, and centrifuge at 13,000 rpm for 30 seconds. The purified product after Bisulfite treatment is collected in a 1.5 mL centrifuge tube.

[0376] 14. Add 1 μL rSAP (YEASEN, 10322ES72) and 1 μL 10x rSAP buffer (YEASEN, 10322ES72) to the PCR tube, vortex to mix, and centrifuge briefly.

[0377] 15. Place the PCR tube in a PCR instrument and incubate at 25°C for 30 minutes, 65°C for 5 minutes, and 95°C for 3 minutes. Immediately place on ice for 2 minutes after the reaction is complete.

[0378] 16. Add 5 μL of adapter mix (10 μM) to the PCR tube:

[0379] NH 2- 5Ad-uni(5'NH2C6-GAACGACATGGCTACGATCCGACTT(SEQ ID NO.1)-3'P);

[0380] 3Ad-Top-NH2(5'P-AAGTCGGAGGCCAAGCGG (SEQ ID NO. 2)-3'NH2C6).

[0381] 17. Prepare the ligation reaction mixture according to Table 3.

[0382] Table 3

[0383] 18. Add 35 μL of the prepared ligation reaction solution to the PCR tube, vortex to mix, and then centrifuge briefly.

[0384] 19. Place the PCR tube in the PCR instrument and set the program: incubate at 37°C for 30 minutes and store at 4°C.

[0385] 20. After the reaction is complete, centrifuge briefly to collect the liquid in the PCR tube to the bottom of the tube.

[0386] 21. Pipette 50 μL of purified magnetic beads into a 1.5 mL centrifuge tube, transfer the reaction product to the centrifuge tube, and gently pipette at least 10 times to suspend all the magnetic beads. The last time, make sure that all the liquid and magnetic beads in the pipette tip are pumped into the centrifuge tube.

[0387] 22. Incubate at room temperature for 5 minutes, then briefly centrifuge. Place the tube on a magnetic rack and let it sit for 2-5 minutes until the liquid clears. Carefully remove the supernatant with a pipette and discard.

[0388] 23. Keep the centrifuge tube on the magnetic rack and add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. After standing for 30 seconds, carefully aspirate and discard the supernatant.

[0389] 24. Repeat the previous step and try to drain the liquid in the tube. If there is a small amount of liquid remaining on the tube wall, centrifuge the tube instantly. After separation on the magnetic stand, use a small-scale pipette to drain the liquid at the bottom of the tube.

[0390] 25. Place the centrifuge tube on the magnetic rack, open the tube cap, and dry it at room temperature until the surface of the magnetic beads is non-reflective.

[0391] 26. Remove the centrifuge tube from the magnetic stand and add 20 μL TE Buffer (AMBION, AM9858) for elution. Gently pipette at least 10 times to mix thoroughly.

[0392] 27. Incubate at room temperature for 5 minutes.

[0393] 28. Centrifuge the tube briefly, place it on a magnetic rack, and let it sit for 2-5 minutes until the liquid becomes clear. Use a pipette to transfer 19 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0394] 29. Prepare the PCR reaction mixture on ice according to Table 4.

[0395] Table 4

[0396] PCR Barcode Primer Mix (20μM):

[0397] PCR primer1 (10μM): 5'P-GAACGACATGGCTACGA-3', SEQ ID NO.3;

[0398] PCR barcode primer (10 μM): 5′-TGTGAGCCAAGGAGTTGATCGGACCTATTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-3′, SEQ ID NO. 4, the underlined part is the sample tag.

[0399] 30. Use a pipette to draw 31 μL of the prepared PCR reaction solution into the PCR tube containing the purified product. Vortex three times for 3 seconds each time and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0400] 31 Place the PCR tube on the PCR instrument and perform the PCR reaction according to the conditions in Table 5.

[0401] Table 5

[0402] 32. After the reaction is completed, centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0403] 33. Pipette 50 μL of purified magnetic beads into a 1.5 mL centrifuge tube. Transfer the reaction product to the centrifuge tube and gently pipette at least 10 times to resuspend all the magnetic beads. The last time, make sure all the liquid and magnetic beads in the pipette tip are pumped into the centrifuge tube.

[0404] Incubate at room temperature for 5 minutes, then briefly centrifuge. Place the tube on a magnetic rack and let it sit for 2-5 minutes until the liquid clears. Carefully remove the supernatant with a pipette and discard.

[0405] 35. Keep the centrifuge tube on the magnetic rack and add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. After standing for 30 seconds, carefully aspirate and discard the supernatant.

[0406] 36. Repeat the previous step and try to drain the liquid in the tube. If there is a small amount of liquid remaining on the tube wall, centrifuge the tube instantly. After separation on the magnetic rack, use a small-scale pipette to drain the liquid at the bottom of the tube.

[0407] 37. Place the centrifuge tube on the magnetic rack, open the tube cap, and dry it at room temperature until the surface of the magnetic beads is non-reflective.

[0408] 38. Remove the centrifuge tube from the magnetic stand and add 32 μL TE Buffer (AMBION, AM9858) for elution. Gently pipette at least 10 times to mix thoroughly.

[0409] 39. Incubate at room temperature for 5 minutes.

[0410] Centrifuge the tube briefly, place it on a magnetic rack, and let it sit for 2-5 minutes until the liquid becomes clear. Use a pipette to transfer 30 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0411] 41. Use The purified PCR product library was quantified using the dsDNA HS Assay Kit (Theromo Fisher, Q32854) fluorescence quantitative kit according to the operating instructions of the quantitative kit.

[0412] 42. Transfer 280 ng of PCR product to a new 0.2 mL PCR tube and add TE Buffer (AMBION, AM9858) to a total volume of 48 μL.

[0413] 43. Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in Table 6.

[0414] Table 6

[0415] 44. After the reaction is completed, immediately place the PCR tube on ice and let it stand for 2 minutes before adding the single-stranded circularization reaction solution.

[0416] 45. Using the circularization kit from MGI (MGI, 1000005260), prepare the single-stranded circularization reaction solution in advance on ice according to the recipe in Table 7 and the recipe in the table below.

[0417] Table 7

[0418] 46. ​​Use a pipette to draw 12.1 μL of the prepared single-stranded circularization reaction solution into the PCR tube. Vortex three times for 3 seconds each time. Centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0419] 47. Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in Table 8.

[0420] Table 8

[0421] 48. After the reaction is completed, centrifuge the PCR tube briefly and place it on ice before proceeding to the next step.

[0422] 49. Using the circularization kit from MGI (MGI, 1000005260), prepare the enzyme digestion reaction solution in advance on ice according to the recipe in Table 9 and on ice according to the recipe in the table below.

[0423] Table 9

[0424] 50. Use a pipette to draw 4 μL of the prepared enzyme digestion reaction solution into the PCR tube. Vortex 3 times for 3 seconds each time. Centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0425] 51. Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in Table 10.

[0426] Table 10

[0427] 52. After the reaction is completed, centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0428] 53. Pipette 170 μL of purified magnetic beads into a 1.5 mL centrifuge tube. Transfer the reaction product to the centrifuge tube and gently pipette at least 10 times to resuspend all the magnetic beads. The last time, make sure all the liquid and magnetic beads in the pipette tip are pumped into the centrifuge tube.

[0429] Incubate at room temperature for 10 minutes, then briefly centrifuge. Place the tube on a magnetic rack and let it sit for 2-5 minutes until the liquid clears. Carefully remove the supernatant with a pipette and discard.

[0430] 55. Keep the centrifuge tube on the magnetic rack and add 300 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. After standing for 30 seconds, carefully aspirate and discard the supernatant.

[0431] 56. Repeat the previous step and try to drain the liquid in the tube. If there is a small amount of liquid remaining on the tube wall, centrifuge the tube instantly. After separation on the magnetic rack, use a small-scale pipette to drain the liquid at the bottom of the tube.

[0432] 57. Place the centrifuge tube on the magnetic rack, open the tube cap, and dry it at room temperature until the surface of the magnetic beads is non-reflective.

[0433] 58. Remove the centrifuge tube from the magnetic stand and add 22 μL TE Buffer (AMBION, AM9858) for elution. Gently pipette at least 10 times to mix thoroughly.

[0434] 59. Incubate at room temperature for 10 minutes.

[0435] Centrifuge the tube briefly, place it on a magnetic rack, and let it sit for 2-5 minutes until the liquid becomes clear. Use a pipette to transfer 20 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0436] 61. Use ssDNA Assay Kit (Thermo Fisher, Q10212) fluorescence quantitative kit was used to quantify the purified products after enzyme digestion according to the operating instructions of the quantitative kit.

[0437] 62. Sequencing was performed using an MGISEQ-2000 sequencer, and DNB preparation and sequencing were performed using the MGISEQ-2000 High-Throughput Sequencing Reagent Set (PE100) (MGI, 1000012536) according to the instructions.

[0438] 3. Experimental Results

[0439] Using 5 ng of cfDNA as a sample, after sulfite treatment, this method was used to construct a library. With 10 PCR cycles, a 1458.3 ng PCR product library was obtained, indicating a high library yield (Table 11).

[0440] Table 11

[0441] The resulting PCR product library was single-stranded circularized to generate a corresponding single-stranded circle library (36.4 ng). This single-stranded circle library was then sequenced on the DNBSEQ sequencing platform using one lane of an MGISEQ-2000 sequencing chip. The results are shown in Table 12 and Figure 3: The final sequencing data reached 430 Mb, with a Q30 quality of 90.53%. The base distribution diagram (Figure 3) also shows a balanced base distribution in the library, indicating minimal contamination from adapters or other fixed sequences. The reproducibility of the standard analysis results was low (7.0%), demonstrating efficient utilization of the nucleic acid template even with a low sample input.

[0442] Table 12

[0443] Example 2 Construction of human peripheral blood free DNA methylation library

[0444] 1. Experimental Materials

[0445] Free DNA extracted from human peripheral blood plasma (sample name: cfDNA-2).

[0446] 2. Experimental steps

[0447] 1. Take 5 ng of human peripheral blood plasma free DNA (cfDNA-2) and transfer it to a 200 μL PCR tube, with a total volume of 20 μL.

[0448] 2. Use EZ DNA Methylation-Gold Kit (Zymo Research, Cat. No. D5005 / D5006) to treat and purify cfDNA with Bisulfite.

[0449] 3. Sequentially pipette 900μL of NF water, 300μL of M-Dilution Buffer, and 50μL of M-Dissolving Buffer into a tube of CT Conversion Reagent powder (centrifuge briefly before opening the lid). Vortex frequently at room temperature for 10 minutes to prepare the CT Conversion Reagent. Minimize exposure to sunlight and prepare the CT Conversion Reagent immediately before use. The CT Conversion Reagent can be stored at room temperature for up to 1 day, at 4°C for up to 1 week, and at -20°C for up to 1 month. Non-freshly prepared CT Conversion Reagent should be prewarmed to 37°C and vortexed frequently for 10 minutes before use.

[0450] 4. Before opening the bottle cap for the first time, add the correct volume of anhydrous ethanol as indicated on the bottle label and mix thoroughly before use. At room temperature, add the ingredients listed in Table 1 to a new 200 μL PCR tube.

[0451] 5. Place the 200 μL PCR tube in a PCR instrument and perform the reaction according to the Bisulfite treatment conditions shown in Table 2.

[0452] 6. Transfer the reaction product obtained in step 5 to a new 1.5 mL centrifuge tube, add 600 μL of M-Binding Buffer, vortex 6 times, each time for 3 seconds, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0453] 7. Place the Zymo-Spin IC Column into a 2 mL Collection Tube. Transfer the mixture obtained in step 6 onto the Zymo-Spin IC Column. Centrifuge at 13,000 rpm for 30 seconds. Discard the waste liquid and place the Zymo-Spin IC Column back into the Collection Tube.

[0454] 8. Add 100 μL of M-Wash Buffer to the Zymo-Spin IC Column and centrifuge at 13,000 rpm for 30 seconds.

[0455] 9. Add 200 μL of M-Desulphonation Buffer to the Zymo-Spin IC Column, quickly cap the tube, incubate at room temperature for 15-20 minutes, centrifuge at 13,000 rpm for 30 seconds, discard the waste liquid, and return the Zymo-Spin IC Column to the Collection Tube.

[0456] 10. Add 200 μL of M-Wash Buffer to the Zymo-Spin IC Column, centrifuge at 13,000 rpm for 30 seconds, discard the waste liquid, and return the Zymo-Spin IC Column to the Collection Tube.

[0457] 11. Add 200 μL of M-Wash Buffer to the Zymo-Spin IC Column and centrifuge at 13,000 rpm for 30 seconds. Discard the waste liquid and return the Zymo-Spin IC Column to the Collection Tube. Centrifuge at 13,000 rpm for 30 seconds without rotation. Discard the Collection Tube and use a pipette to remove as much liquid as possible from the outer wall of the Zymo-Spin IC Column. Place the column in a new 1.5 mL centrifuge tube.

[0458] 12. Open the Zymo-Spin IC Column tube cap, dry it at room temperature for 2 minutes, and then place the Zymo-Spin IC Column in another new 1.5mL centrifuge tube.

[0459] 13. Slowly add 8 μL of M-Elution Buffer to the center of the Zymo-Spin IC Column filter membrane, let it stand for 1 minute, and centrifuge at 13,000 rpm for 30 seconds. The purified product after Bisulfite treatment is collected in a 1.5 mL centrifuge tube.

[0460] 14. Add 1 μL rSAP (YEASEN, 10322ES72) and 1 μL 10x rSAP buffer (YEASEN, 10322ES72) to the PCR tube, vortex to mix, and centrifuge briefly.

[0461] 15. Place the PCR tube in a PCR instrument and incubate at 25°C for 30 minutes, 65°C for 5 minutes, and 95°C for 3 minutes. Immediately place on ice for 2 minutes after the reaction is complete.

[0462] 16. Add 1 μL of adapter mix (50 μM) to the PCR tube:

[0463] NH 2- 5Ad-uni(5'NH2C6-GAACGACATGGCTACGATCCGACTT(SEQ ID NO.1)-3'P);

[0464] 3Ad-Top-NH2(5'P-AAGTCGGAGGCCAAGCGG (SEQ ID NO. 2)-3'NH2C6).

[0465] 17. Prepare the ligation reaction mixture according to Table 13.

[0466] Table 13

[0467] 18. Add 19 μL of the prepared ligation reaction solution to the PCR tube, vortex to mix, and then centrifuge briefly.

[0468] 19. Place the PCR tube in the PCR instrument and set the program: incubate at 16°C for 60 minutes and store at 4°C.

[0469] 20. After the reaction is complete, centrifuge briefly to collect the liquid in the PCR tube to the bottom of the tube.

[0470] 21. Pipette 30 μL of purified magnetic beads into a 1.5 mL centrifuge tube, transfer the reaction product to the centrifuge tube, and gently pipette at least 10 times to suspend all the magnetic beads. The last time, make sure that all the liquid and magnetic beads in the pipette tip are pumped into the centrifuge tube.

[0471] 22. Incubate at room temperature for 5 minutes, then briefly centrifuge. Place the tube on a magnetic rack and let it sit for 2-5 minutes until the liquid clears. Carefully remove the supernatant with a pipette and discard.

[0472] 23. Keep the centrifuge tube on the magnetic rack and add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. After standing for 30 seconds, carefully aspirate and discard the supernatant.

[0473] 24. Repeat the previous step and try to drain the liquid in the tube. If there is a small amount of liquid remaining on the tube wall, centrifuge the tube instantly. After separation on the magnetic stand, use a small-scale pipette to drain the liquid at the bottom of the tube.

[0474] 25. Place the centrifuge tube on the magnetic rack, open the tube cap, and dry it at room temperature until the surface of the magnetic beads is non-reflective.

[0475] 26. Remove the centrifuge tube from the magnetic stand and add 20 μL TE Buffer (AMBION, AM9858) for elution. Gently pipette at least 10 times to mix thoroughly.

[0476] 27. Incubate at room temperature for 5 minutes.

[0477] 28. Centrifuge the tube briefly, place it on a magnetic rack, and let it sit for 2-5 minutes until the liquid becomes clear. Use a pipette to transfer 19 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0478] 29. Prepare the PCR reaction mixture on ice according to Table 4, where the PCR Barcode Primer Mix is ​​the same as that in Example 1.

[0479] 30. Use a pipette to draw 31 μL of the prepared PCR reaction solution into the PCR tube containing the purified product. Vortex three times for 3 seconds each time and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0480] 31 Place the PCR tube on the PCR instrument and perform the PCR reaction according to the conditions in Table 5.

[0481] 32. After the reaction is completed, centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0482] 33. Pipette 50 μL of purified magnetic beads into a 1.5 mL centrifuge tube. Transfer the reaction product to the centrifuge tube and gently pipette at least 10 times to resuspend all the magnetic beads. The last time, make sure all the liquid and magnetic beads in the pipette tip are pumped into the centrifuge tube.

[0483] Incubate at room temperature for 5 minutes, then briefly centrifuge. Place the tube on a magnetic rack and let it sit for 2-5 minutes until the liquid clears. Carefully remove the supernatant with a pipette and discard.

[0484] 35. Keep the centrifuge tube on the magnetic rack and add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. After standing for 30 seconds, carefully aspirate and discard the supernatant.

[0485] 36. Repeat the previous step and try to drain the liquid in the tube. If there is a small amount of liquid remaining on the tube wall, centrifuge the tube instantly. After separation on the magnetic rack, use a small-scale pipette to drain the liquid at the bottom of the tube.

[0486] 37. Place the centrifuge tube on the magnetic rack, open the tube cap, and dry it at room temperature until the surface of the magnetic beads is non-reflective.

[0487] 38. Remove the centrifuge tube from the magnetic stand and add 32 μL TE Buffer (AMBION, AM9858) for elution. Gently pipette at least 10 times to mix thoroughly.

[0488] 39. Incubate at room temperature for 5 minutes.

[0489] Centrifuge the tube briefly, place it on a magnetic rack, and let it sit for 2-5 minutes until the liquid becomes clear. Use a pipette to transfer 30 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0490] 41. Use The purified library was quantified using the dsDNA HS Assay Kit (Theromo Fisher, Q32854) according to the kit's instructions. The resulting PCR library concentration for the cfDNA-2 sample was 39.5 ng / μL. Therefore, the library yield using this method was 1185 ng, indicating high library yield and high utilization of the cfDNA template.

[0491] Example 3 Construction of human peripheral blood free DNA methylation library

[0492] 1. Experimental Materials

[0493] Cell-free DNA extracted from human peripheral blood plasma (sample name: cfDNA-3).

[0494] 2. Experimental steps

[0495] 1. Take 5 ng of human peripheral blood plasma free DNA (cfDNA-3) and transfer it to a 200 μL PCR tube, with a total volume of 20 μL.

[0496] 2. Use EZ DNA Methylation-Gold Kit (Zymo Research, Cat. No. D5005 / D5006) to treat and purify cfDNA with Bisulfite.

[0497] 3. Sequentially pipette 900μL of NF water, 300μL of M-Dilution Buffer, and 50μL of M-Dissolving Buffer into a tube of CT Conversion Reagent powder (centrifuge briefly before opening the lid). Vortex frequently at room temperature for 10 minutes to prepare the CT Conversion Reagent. Minimize exposure to light and use the CT Conversion Reagent immediately after preparation. The CT Conversion Reagent can be stored at room temperature for up to 1 day, at 4°C for up to 1 week, and at -20°C for up to 1 month. Non-freshly prepared CT Conversion Reagent should be prewarmed to 37°C and vortexed frequently for 10 minutes at room temperature before use.

[0498] 4. Before opening the bottle cap for the first time, add the correct volume of anhydrous ethanol as indicated on the bottle label and mix thoroughly before use. At room temperature, add the ingredients listed in Table 1 to a new 200 μL PCR tube.

[0499] 5. Place the 200 μL PCR tube in a PCR instrument and perform the reaction according to the Bisulfite treatment conditions shown in Table 2.

[0500] 6. Transfer the reaction product obtained in step 5 to a new 1.5 mL centrifuge tube, add 600 μL of M-Binding Buffer, vortex 6 times, each time for 3 seconds, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0501] 7. Place the Zymo-Spin IC Column into a 2 mL Collection Tube. Transfer the mixture obtained in step 6 onto the Zymo-Spin IC Column. Centrifuge at 13,000 rpm for 30 seconds. Discard the waste liquid and place the Zymo-Spin IC Column back into the Collection Tube.

[0502] 8. Add 100 μL of M-Wash Buffer to the Zymo-Spin IC Column and centrifuge at 13,000 rpm for 30 seconds.

[0503] 9. Add 200 μL of M-Desulphonation Buffer to the Zymo-Spin IC Column, quickly cap the tube, incubate at room temperature for 15-20 minutes, centrifuge at 13,000 rpm for 30 seconds, discard the waste liquid, and return the Zymo-Spin IC Column to the Collection Tube.

[0504] 10. Add 200 μL of M-Wash Buffer to the Zymo-Spin IC Column, centrifuge at 13,000 rpm for 30 seconds, discard the waste liquid, and return the Zymo-Spin IC Column to the Collection Tube.

[0505] 11. Add 200 μL of M-Wash Buffer to the Zymo-Spin IC Column and centrifuge at 13,000 rpm for 30 seconds. Discard the waste liquid and return the Zymo-Spin IC Column to the Collection Tube. Centrifuge at 13,000 rpm for 30 seconds without rotation. Discard the Collection Tube and use a pipette to remove as much liquid as possible from the outer wall of the Zymo-Spin IC Column. Place the column in a new 1.5 mL centrifuge tube.

[0506] 12. Open the Zymo-Spin IC Column tube cap, dry it at room temperature for 2 minutes, and then place the Zymo-Spin IC Column in another new 1.5mL centrifuge tube.

[0507] 13. Slowly add 8 μL of M-Elution Buffer to the center of the Zymo-Spin IC Column filter membrane, let it stand for 1 minute, and centrifuge at 13,000 rpm for 30 seconds. The purified product after Bisulfite treatment is collected in a 1.5 mL centrifuge tube.

[0508] 14. Add 1 μL rSAP (YEASEN, 10322ES72) and 1 μL 10x rSAP buffer (YEASEN, 10322ES72) to the PCR tube, vortex to mix, and centrifuge briefly.

[0509] 15. Place the PCR tube in a PCR instrument and incubate at 25°C for 30 minutes, 65°C for 5 minutes, and 95°C for 3 minutes. Immediately place on ice for 2 minutes after the reaction is complete.

[0510] 16. Add 5 μL of adapter mix (10 μM) to the PCR tube:

[0511] NH 2- 5Ad-uni(5'NH2C6-GAACGACATGGCTACGATCCGACTT(SEQ ID NO.1)-3'P);

[0512] 3Ad-Top-NH2(5'P-AAGTCGGAGGCCAAGCGG (SEQ ID NO. 2)-3'NH2C6).

[0513] 17. Prepare the ligation reaction mixture according to Table 14.

[0514] Table 14 Note: Pho RtcB ligase is the purified expression product of the RtcB gene of the extremely thermophilic archaeon Pyrococcus horikoshii (UniProt accession number A0A832T3E6).

[0515] 18. Add 35 μL of the prepared ligation reaction solution to the PCR tube, vortex to mix, and then centrifuge briefly.

[0516] 19. Place the PCR tube in the PCR instrument and set the program: incubate at 65°C for 30 minutes, incubate at 80°C for 5 minutes, and store at 4°C.

[0517] 20. After the reaction is complete, centrifuge briefly to collect the liquid in the PCR tube to the bottom of the tube.

[0518] 21. Pipette 50 μL of purified magnetic beads into a 1.5 mL centrifuge tube, transfer the reaction product to the centrifuge tube, and gently pipette at least 10 times to suspend all the magnetic beads. The last time, make sure that all the liquid and magnetic beads in the pipette tip are pumped into the centrifuge tube.

[0519] 22. Incubate at room temperature for 5 minutes, then briefly centrifuge. Place the tube on a magnetic rack and let it sit for 2-5 minutes until the liquid clears. Carefully remove the supernatant with a pipette and discard.

[0520] 23. Keep the centrifuge tube on the magnetic rack and add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. After standing for 30 seconds, carefully aspirate and discard the supernatant.

[0521] 24. Repeat the previous step and try to drain the liquid in the tube. If there is a small amount of liquid remaining on the tube wall, centrifuge the tube instantly. After separation on the magnetic stand, use a small-scale pipette to drain the liquid at the bottom of the tube.

[0522] 25. Place the centrifuge tube on the magnetic rack, open the tube cap, and dry it at room temperature until the surface of the magnetic beads is non-reflective.

[0523] 26. Remove the centrifuge tube from the magnetic stand and add 20 μL TE Buffer (AMBION, AM9858) for elution. Gently pipette at least 10 times to mix thoroughly.

[0524] 27. Incubate at room temperature for 5 minutes.

[0525] 28. Centrifuge the tube briefly, place it on a magnetic rack, and let it sit for 2-5 minutes until the liquid becomes clear. Use a pipette to transfer 19 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0526] 29. Prepare the PCR reaction mixture on ice according to Table 4, where the PCR Barcode Primer Mix is ​​the same as that in Example 1.

[0527] 30. Use a pipette to draw 31 μL of the prepared PCR reaction solution into the PCR tube containing the purified product. Vortex three times for 3 seconds each time and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0528] 31 Place the PCR tube on the PCR instrument and perform the PCR reaction according to the conditions in Table 5.

[0529] 32. After the reaction is completed, centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0530] 33. Pipette 50 μL of purified magnetic beads into a 1.5 mL centrifuge tube. Transfer the reaction product to the centrifuge tube and gently pipette at least 10 times to resuspend all the magnetic beads. The last time, make sure all the liquid and magnetic beads in the pipette tip are pumped into the centrifuge tube.

[0531] Incubate at room temperature for 5 minutes, then briefly centrifuge. Place the tube on a magnetic rack and let it sit for 2-5 minutes until the liquid clears. Carefully remove the supernatant with a pipette and discard.

[0532] 35. Keep the centrifuge tube on the magnetic rack and add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. After standing for 30 seconds, carefully aspirate and discard the supernatant.

[0533] 36. Repeat the previous step and try to drain the liquid in the tube. If there is a small amount of liquid remaining on the tube wall, centrifuge the tube instantly. After separation on the magnetic rack, use a small-scale pipette to drain the liquid at the bottom of the tube.

[0534] 37. Place the centrifuge tube on the magnetic rack, open the tube cap, and dry it at room temperature until the surface of the magnetic beads is non-reflective.

[0535] 38. Remove the centrifuge tube from the magnetic stand and add 32 μL TE Buffer (AMBION, AM9858) for elution. Gently pipette at least 10 times to mix thoroughly.

[0536] 39. Incubate at room temperature for 5 minutes.

[0537] Centrifuge the tube briefly, place it on a magnetic rack, and let it sit for 2-5 minutes until the liquid becomes clear. Use a pipette to transfer 30 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0538] 41. Use The purified library was quantified using the dsDNA HS Assay Kit (Theromo Fisher, Q32854) according to the kit's instructions. The resulting PCR library concentration for the cfDNA-2 sample was 43 ng / μL. Therefore, the library yield using this method was 1290 ng, indicating high library yield and high utilization of the cfDNA template.

[0539] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A joint composition comprising: A 5' end adapter, the 5' end adapter comprising a first oligonucleic acid chain, the 3' end of the first oligonucleic acid chain of the 5' end adapter is modified with a first modifying group, the 5' end is modified with a first blocking group, and the first modifying group comprises phosphate; and a first ligase, which can ligate the 3' first modification group end of the oligonucleic acid to the 5' hydroxyl end of the template nucleic acid; and A 3' end adapter, wherein the 3' end adapter comprises a second oligonucleic acid chain, wherein the 3' end of the second oligonucleic acid chain of the 3' end adapter is modified with a second blocking group, and the 5' end is modified with a second modifying group, wherein the second modifying group comprises phosphate; and The second ligase can ligate the 5' second modification group end of the oligonucleic acid to the 3' hydroxyl end of the template nucleic acid.

2. The joint composition according to claim 1, characterized in that: The first modification group modification is selected from at least one of the following modifications: phosphorylation modification, 2'3' cyclic phosphate modification, and pre-guanosine modification.

3. The joint composition according to claim 1, characterized in that: The second modification group modification is selected from at least one of the following modifications: phosphorylation modification and adenylation modification.

4. The joint composition according to claim 1, characterized in that: The 5' end adapter may further comprise a third oligonucleotide chain, wherein the third oligonucleotide chain is complementary to the first oligonucleotide chain to form a double strand, and the length of the third oligonucleotide chain is greater than, equal to or less than the first oligonucleotide chain; Preferably, the difference between the number of bases in the third oligonucleotide chain and the number of bases in the first oligonucleotide chain is greater than or equal to 2; Preferably, the 5' end and the 3' end of the third oligonucleic acid chain are modified with a third blocking group and a fourth blocking group, respectively.

5. The joint composition according to claim 1 or 4, characterized in that: The 3' end adapter may further comprise a fourth oligonucleic acid strand, wherein the fourth oligonucleic acid strand is complementary to the second oligonucleic acid strand to form a double strand; Preferably, the length of the fourth oligonucleotide strand is greater than, equal to or less than the length of the second oligonucleotide strand; Preferably, the 5' end and the 3' end of the fourth oligonucleic acid strand are modified with a fifth blocking group and a sixth blocking group, respectively.

6. The joint composition according to claim 1, characterized in that: The linker composition further comprises: a phosphatase.

7. The joint composition according to claim 1, characterized in that: The first ligase is selected from: RtcB ligase, an active fragment of RtcB ligase, a derivative of RtcB ligase, At least one analog of RtcB ligase.

8. The joint composition according to claim 3, characterized in that: When the 5' end of the second oligonucleotide chain of the 3' end adapter is phosphorylated, the second ligase comprises at least one of T4 RNA ligase 1, T4 DNA ligase, TS2126 RNA ligase, T3 DNA ligase, Escherichia coli DNA ligase, single-stranded DNA / RNA circular ligase, T4 RNA ligase 2, and Taq DNA ligase; or When the 5' end of the second oligonucleotide chain of the 3' end connector has an adenylation modification, the second ligase comprises at least one of T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 (truncated K227Q), T4 RNA ligase 2 (truncated KQ), thermostable 5'App DNA / RNA ligase, TS2126 RNA ligase, T3 DNA ligase, Escherichia coli DNA ligase, single-stranded DNA / RNA circular ligase, and Taq DNA ligase.

9. The joint composition according to claim 1, 4 or 5, characterized in that: The first blocking group modification is selected from the following group modifications: amino modification, dideoxy modification, spacer modification, phosphate modification; preferably, the second blocking group modification is selected from the following group modifications: amino modification, dideoxy modification, spacer modification, phosphate modification; Preferably, the third blocking group modification is selected from the following group modifications: amino modification, dideoxy modification, spacer modification, phosphate modification; Preferably, the fourth blocking group modification is selected from the following group modifications: amino modification, dideoxy modification, spacer modification, phosphate modification; Preferably, the fifth blocking group modification is selected from the following group modifications: amino modification, dideoxy modification, spacer modification, phosphate modification; Preferably, the sixth blocking group modification is selected from the following group modifications: amino modification, dideoxy modification, spacer modification, phosphate modification.

10. The joint composition according to claim 1, characterized in that: The linker composition further comprises: a first cofactor, wherein the first cofactor comprises: triphosphate and manganese ions; Preferably, the linker composition further comprises: a second cofactor, wherein the second cofactor comprises: at least one of ATP and NAD and a metal ion; Preferably, the linker composition further comprises: a substance for breaking nucleic acid; Preferably, the linker composition further comprises: a substance for nucleic acid denaturation; Preferably, when the linker composition is used to construct a DNA methylation library, the linker composition further comprises: a DNA methylation modification conversion reagent; Preferably, the linker composition further comprises: a single-chain binding protein.

11. The joint composition according to claim 1, characterized in that: The first oligonucleotide strand of the 5' end adapter and / or the second oligonucleotide strand of the 3' end adapter comprise a sequencing primer sequence; Preferably, the first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter further comprises a sample tag sequence; Preferably, the first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter further comprise a unique molecular tag sequence.

12. The joint composition according to claim 1, characterized in that: The first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter are fixed to a solid support; Preferably, the non-ligated ends of the first oligonucleotide strand of the 5' end adapter and / or the second oligonucleotide strand of the 3' end adapter are fixed to a solid support; Preferably, the solid support is selected from beads, chips and the like.

13. The joint composition according to claim 12, characterized in that: The chip can be used in a sequencing platform, such as a sequencing chip; Preferably, the beads are selected from agarose gel beads, agarose beads, magnetic beads, protein A conjugated beads, protein G conjugated beads, protein L conjugated beads, oligo (dT) conjugated beads, silica beads, hydrogel beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. 14 . A nucleic acid library construction kit comprising the linker composition according to claim 1 .

15. The nucleic acid library construction kit according to claim 14, characterized in that: The nucleic acid library construction kit further comprises: polynucleotide kinase; Preferably, the nucleic acid library construction kit further comprises: PCR primers; Preferably, the nucleic acid library construction kit further comprises: a cyclization reagent combination; Preferably, when the nucleic acid is RNA, the nucleic acid library construction kit further comprises: a reverse transcription primer.

16. A method for connecting a joint, comprising the following steps: Obtaining a single-stranded template nucleic acid, wherein both the 5' end and the 3' end of the single-stranded template nucleic acid have hydroxyl groups; performing a 5' end adapter and a 3' end adapter adapter ligation reaction on the single-stranded template nucleic acid using a first ligase, a 5' end adapter, a second ligase and a 3' end adapter; The 5' end adapter comprises a first oligonucleotide chain, the 3' end of the first oligonucleotide chain of the 5' end adapter is modified with a first modification group, the 5' end is modified with a first blocking group, and the first modification group comprises phosphate; The first ligase can connect the 3' first modification group end of the oligonucleic acid to the 5' hydroxyl end of the template nucleic acid; The 3' end adapter comprises a second oligonucleic acid chain, the 3' end of the second oligonucleic acid chain of the 3' end adapter is modified with a second blocking group, and the 5' end is modified with a second modifying group, and the second modifying group comprises phosphate; The second ligase can ligate the 5' second modification group end of the oligonucleic acid to the 3' hydroxyl end of the template nucleic acid.

17. The joint connection method according to claim 16, characterized in that: in, The adapter ligation reaction of the 5' end adapter and the 3' end adapter is carried out in the same system.

18. The joint connection method according to claim 16, characterized in that: The connection of the adapter is directional, and the first oligonucleotide chain of the 5'-end adapter and the second oligonucleotide chain of the 3'-end adapter have different nucleic acid sequences.

19. The joint connection method according to claim 16, characterized in that: The method for obtaining the single-stranded template nucleic acid with hydroxyl groups at both the 5' end and the 3' end is dephosphorylation treatment.

20. The joint connection method according to claim 16, characterized in that: The first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter are fixed to a solid support and are contacted with a single-stranded template nucleic acid to generate a solid support with a specific sequence and a single-stranded template nucleic acid.

21. The joint connection method according to claim 16, characterized in that: The single-stranded template nucleic acid is obtained by the following method: subjecting the nucleic acid to at least one of the following treatments: shearing, denaturation, and DNA methylation; Preferably, the nucleic acid comprises: at least one of DNA and RNA; Preferably, the DNA comprises: at least one of dsDNA and ssDNA; Preferably, the nucleic acid is from the following biological samples: cells, fresh tissues, fresh organs, decayed tissues, formalin-fixed tissues, paraffin-embedded tissues, forensic samples, paleontological fossils, biological materials containing cfDNA or cfRNA; Preferably, the biological material includes but is not limited to: peripheral blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, lymph fluid, alveolar lavage fluid, amniotic fluid, blastocyst cavity fluid, cell culture fluid, embryo culture fluid, microbial culture medium, soil extract, bone powder extract.

22. The joint connection method according to claim 21, characterized in that: When the nucleic acid comprises dsDNA, the process of preparing the single-stranded template nucleic acid by the nucleic acid comprises denaturation treatment; Preferably, the denaturation treatment may be performed before or after the phosphorylation treatment.

23. The joint connection method according to claim 22, characterized in that: When the nucleic acid is used to construct a DNA methylation library, the process of preparing the single-stranded template nucleic acid from the nucleic acid includes DNA methylation treatment; Preferably, the methylation treatment is before the phosphorylation treatment and the denaturation treatment; Preferably, the methylation treatment is performed after the disruption treatment.

24. A method for constructing a nucleic acid library, comprising the following steps: obtain a single-stranded template nucleic acid, wherein both the 5' end and the 3' end of the single-stranded template nucleic acid are hydroxyl groups; use a first ligase, The 5' end adapter, the second ligase and the 3' end adapter are used to perform adapter ligation reaction on the single-stranded template nucleic acid to obtain the single-stranded template nucleic acid ligated with an adapter; and Amplification reaction to obtain a nucleic acid library; The 5' end adapter comprises a first oligonucleotide chain, the 3' end of the first oligonucleotide chain of the 5' end adapter is modified with a first modification group, the 5' end is modified with a first blocking group, and the first modification group comprises phosphate; The first ligase can connect the 3' first modification group end of the oligonucleic acid to the 5' hydroxyl end of the template nucleic acid; The 3' end adapter comprises a second oligonucleic acid chain, the 3' end of the second oligonucleic acid chain of the 3' end adapter is modified with a second blocking group, and the 5' end is modified with a second modifying group, and the second modifying group comprises phosphate; The second ligase can ligate the 5' second modification group end of the oligonucleic acid to the 3' hydroxyl end of the template nucleic acid.

25. The method according to claim 24, characterized in that: The first oligonucleotide chain of the 5' end adapter and / or the second oligonucleotide chain of the 3' end adapter are fixed to a solid support, and a ligation reaction occurs by contacting with a single-stranded template nucleic acid to generate a solid support with a specific sequence and a single-stranded template nucleic acid; Preferably, when the nucleic acid is RNA, the nucleic acid library construction method may further comprise the following steps: reverse transcription reaction; Preferably, the linker ligation reaction is followed by a cyclization reaction; Preferably, the nucleic acid library comprises a DNA methylation library.

26. The method according to claim 24, characterized in that: The amplification reaction is rolling circle amplification or linear amplification.

27. A nucleic acid library, obtained by the following steps: Obtaining a single-stranded template nucleic acid, wherein both the 5' end and the 3' end of the single-stranded template nucleic acid have hydroxyl groups; performing a 5' end adapter and a 3' end adapter adapter ligation reaction on the single-stranded template nucleic acid using a first ligase, a 5' end adapter, a second ligase, and a 3' end adapter to obtain an adapter-attached single-stranded template nucleic acid; and Amplification reaction to obtain a nucleic acid library; The 5' end adapter comprises a first oligonucleotide chain, the 3' end of the first oligonucleotide chain of the 5' end adapter is modified with a first modification group, the 5' end is modified with a first blocking group, and the first modification group comprises phosphate; The first ligase can connect the 3' first modification group end of the oligonucleic acid to the 5' hydroxyl end of the template nucleic acid; The 3' end adapter comprises a second oligonucleic acid chain, the 3' end of the second oligonucleic acid chain of the 3' end adapter is modified with a second blocking group, and the 5' end is modified with a second modifying group, and the second modifying group comprises phosphate; The second ligase can ligate the 5' second modification group end of the oligonucleic acid to the 3' hydroxyl end of the template nucleic acid.

Images