Method for carrying out 3'end phosphorylation modification and library establishment on nucleic acid

Abstract

The invention provides a method for carrying out 3 '-end phosphorylation modification and library establishment on nucleic acid. The library building method comprises the following steps: carrying out phosphorylation modification on the 3'end of a nucleic acid fragment; carrying out connection treatment on the nucleic acid fragment with phosphorylation modification at the 3 '-end and a linker under the action of RtcB ligase so as to connect the 3'-end of the nucleic acid fragment with the 5 '-end of the linker; and carrying out amplification treatment on the connection treatment product so as to obtain the sequencing library.

Description

Method for 3' end phosphorylation modification and library construction of nucleic acids Technical Field

[0001] The present invention relates to the field of gene detection, and in particular to a method for phosphorylating and modifying the 3' end of a nucleic acid, isolating the nucleic acid, constructing a sequencing library, and sequencing. 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 are DNA sequencing libraries, typically constructed from a sufficient amount of intact genomic DNA. For such samples, conventional double-stranded DNA library construction methods can be used. 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 through a simple ligase reaction to generate the library required for sequencing.

[0003] However, for specialized samples, such as formalin-fixed (FFPE) and paraffin-embedded tissue samples, forensic specimens, and paleontological fossils, the DNA extracted from these samples can be severely degraded, resulting in the coexistence of 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 requires sulfite treatment, which breaks the DNA into short fragments, resulting in the loss of most DNA molecules and the conversion to single strands. Furthermore, extracellular free DNA in plasma and other body fluid samples is inherently short and present in very low concentrations. Conventional adapter-ligation library construction methods are unsuitable for these specialized samples. Conventional methods only utilize intact double-stranded DNA molecules, making it impossible or extremely difficult to add adapters to all template molecules other than the intact double-stranded DNA, leading to the loss of a significant amount of the original template molecules.

[0004] Single-stranded library construction technology utilizes these single-stranded DNA template molecules by adding adapters to them. Double-stranded DNA can also be denatured to single strands using the same method. This minimizes the loss of these irregular double-stranded DNA template molecules, maximizes template utilization, increases library construction efficiency, and improves both the effective amount and quality of sequencing data. This also allows for better sample utilization in specific applications, such as methylation sequencing of cell-free extracellular DNA (cfDNA) for liquid biopsies, genomic research using paleontological DNA, forensic analysis, and tumor detection in FFPE samples. In addition to DNA samples, RNA samples are also well-suited for library construction using single-stranded library construction technology. RNA is inherently single-stranded, and typical library construction methods involve reverse transcription of RNA into double-stranded cDNA, followed by adapter addition. Alternatively, single-stranded library construction technology can be used to directly add adapters to RNA samples, reverse transcribe them into cDNA, and complete the sequencing library.

[0005] Existing single-stranded library construction technologies include traditional single-stranded library construction methods, Swift's Adaptase single-stranded library construction technology, random primers and adapters, terminal transferase and adapters, and SPALT (Splinted Adapter Tagging) technology. Because traditional single-stranded library construction methods use T4 RNA ligase 2 (truncated form), they require separate ligation of adapters at both ends and require multiple purifications, resulting in low template utilization and prolonged library construction time. Furthermore, the template itself can undergo self-ligation, causing template loss and the formation of chimeric templates, which can affect the detection results of certain structural variants. Adaptase single-stranded library construction technology, random primers and adapters, terminal transferase and adapters, and SPALT all add 3' and 5' adapters separately. PCR amplification of the template is only possible after adding adapters at both ends. These methods increase the number of experimental steps, lead to loss of the original template, and reduce utilization. Furthermore, the template itself can undergo self-ligation, causing template loss and the formation of chimeric templates, which can affect the detection results of certain structural variants.

[0006] Therefore, there is an urgent need in this field to develop a method for efficiently constructing sequencing libraries.

[0007] Summary of the Invention

[0008] The present invention aims to solve one of the technical problems in the related art at least to a certain extent.

[0009] The existing library construction methods have the following problems: PCR amplification is required to amplify the template, multiple purifications are time-consuming and labor-intensive, the template utilization rate is low, and template self-ligation and chimeric templates are generated.

[0010] In order to solve the problem of template self-ligation, the present application uses a terminal transferase enzyme or a ligase to block the 3' end of the nucleic acid template with phosphate under reaction conditions in the presence of deoxyribonucleotides (such as deoxyadenosine 5'-triphosphate, 3'-phosphate, deoxyadenosine 3', 5'-diphosphate, deoxycytidine 5'-triphosphate, 3'-phosphate, deoxyguanosine 5'-triphosphate, 3'-phosphate or deoxythymidine 5'-triphosphate, 3'-phosphate, etc.) and ribonucleotides (such as adenosine 5'-triphosphate, 3'-phosphate, adenosine 5'-phosphate, 2', 3'-cyclic-phosphate, adenosine 5'-triphosphate, 2', 3'-cyclic-phosphate or adenosine 3', 5'-diphosphate, etc.) to prevent the nucleic acid template from self-ligating.

[0011] To improve template utilization, this application further utilizes T4 polynucleotide kinase (T4PNK) to phosphorylate the 5' end of the nucleic acid template, providing a phosphate group for the ligation adapter, improving ligation efficiency and preventing false-positive PCR results. This application uses nucleic acid templates with phosphate groups at both ends, along with T4 RNA ligase and RtcB enzyme, to build libraries, increasing adapter ligation efficiency and significantly improving template utilization.

[0012] Specifically, in its first aspect, the present invention provides a method for phosphorylating the 3' end of a nucleic acid. According to an embodiment of the present invention, the method comprises: ligating the 3' end of the nucleic acid to a deoxyribonucleotide or a ribonucleotide, wherein the 3-carbon atom of the deoxyribonucleotide or ribonucleotide has a phosphate group attached thereto.

[0013] The inventors discovered that the above method can be used to phosphorylate the 3' end of nucleic acids (such as DNA and RNA), thereby modifying the 3' end of the nucleic acid template with a phosphate group and preventing self-ligation of the nucleic acid template. Furthermore, this method has broad application value in library construction (improving adapter ligation efficiency and preventing false positive results in PCR) and probe synthesis.

[0014] According to an embodiment of the present invention, the phosphorylation modification method may further include at least one of the following technical features:

[0015] According to an embodiment of the present invention, the nucleic acid includes at least one selected from single-stranded DNA, double-stranded DNA or RNA.

[0016] It should be noted that DNA single stranding can be achieved by denaturing double-stranded DNA or by using single-stranded binding protein or DNA gyrase to coil DNA into a supercoil structure to induce single stranding.

[0017] According to an embodiment of the present invention, the deoxyribonucleotide is at least one of deoxyadenosine 5'-triphosphate, 3'-phosphate, deoxyadenosine 3', 5'-diphosphate, deoxycytidine 5'-triphosphate, 3'-phosphate, deoxycytidine 3', 5'-diphosphate, deoxyguanosine 5'-triphosphate, 3'-phosphate, deoxyguanosine 3', 5'-diphosphate, deoxythymidine 5'-triphosphate, 3'-phosphate, deoxythymidine 3', 5'-diphosphate, deoxyuridine 5'-triphosphate, 3'-phosphate, and deoxyuridine 3', 5'-diphosphate.

[0018] It should be noted that the deoxyribonucleotides described in this application include but are not limited to deoxyadenosine 5'-triphosphate, 3'-phosphate, deoxyadenosine 3', 5'-diphosphate, deoxycytidine 5'-triphosphate, 3'-phosphate, deoxycytidine 3', 5'-diphosphate, deoxyguanosine 5'-triphosphate, 3'-phosphate, deoxyguanosine 3', 5'-diphosphate, deoxythymidine 5'-triphosphate, 3'-phosphate, deoxythymidine 3', 5'-diphosphate, deoxyuridine 5'-triphosphate, 3'-phosphate, deoxyuridine 3', 5'-diphosphate, for example, may also include deoxyadenosine 3', 5'-phosphate, etc.

[0019] According to an embodiment of the present invention, the ribonucleotides are adenosine 5'-triphosphate, 3'-phosphate, adenosine 5'-phosphate, 2', 3'-cyclic-phosphate, adenosine 5'-phosphate, 2', 3'-cyclic-phosphate, adenosine 3', 5'-diphosphate, cytidine 5'-triphosphate, 3'-phosphate, cytidine 5'-phosphate, 2', 3'-cyclic-phosphate, cytidine 5'-phosphate, 2', 3'-cyclic-phosphate, cytidine 3', 5'-diphosphate, guanidine At least one of guanosine 5'-triphosphate, 3'-phosphate, guanosine 5'-phosphate, 2',3'-cyclic-phosphate, guanosine 5'-phosphate, 2',3'-cyclic-phosphate, guanosine 3',5'-diphosphate, guanosine 5'-triphosphate, 3'-phosphate, uridine 5'-triphosphate, 3'-phosphate, uridine 5'-phosphate, 2',3'-cyclic-phosphate, uridine 5'-phosphate, 2',3'-cyclic-phosphate, and uridine 3',5'-diphosphate.

[0020] It should be noted that the ribonucleotides described in this application include but are not limited to adenosine 5'-triphosphate, 3'-phosphate, adenosine 5'-phosphate, 2', 3'-cyclic-phosphate, adenosine 5'-phosphate, 2', 3'-cyclic-phosphate, adenosine 3', 5'-diphosphate, cytidine 5'-triphosphate, 3'-phosphate, cytidine 5'-phosphate, 2', 3'-cyclic-phosphate, cytidine 5'-phosphate, 2', 3'-cyclic-phosphate, cytidine 3', 5'-diphosphate, guanosine 5'-triphosphate, 3'-phosphate, Guanosine 5'-phosphate, 2', 3'-cyclic-phosphate, guanosine 5'-phosphate, 2', 3'-cyclic-phosphate, guanosine 3', 5'-diphosphate, guanosine 5'-triphosphate, 3'-phosphate, uridine 5'-triphosphate, 3'-phosphate, uridine 5'-phosphate, 2', 3'-cyclic-phosphate, uridine 5'-phosphate, 2', 3'-cyclic-phosphate, uridine 3', 5'-diphosphate, for example, may also include adenosine 5'-diphosphate, 3'-phosphate or adenosine 5'-phosphate, 3'-phosphate, etc.

[0021] According to an embodiment of the present invention, the ligation process is performed under the catalysis of terminal transferase, poly(A) polymerase, poly(G) polymerase or T4 RNA ligase 1.

[0022] According to an embodiment of the present invention, the nucleic acid is a single-stranded DNA, and the ligation process is performed under the catalysis of terminal transferase. Terminal transferase is an enzyme that can add unpaired nucleotides to the 3' end of DNA. The inventors utilized the property of terminal transferase to add unpaired nucleotides to the end of a DNA chain and used terminal transferase to catalyze the DNA molecule, thereby adding a phosphorylated deoxyadenosine or adenosine residue or several other nucleoside residues at the 3' end of the DNA.

[0023] According to an embodiment of the present invention, the nucleic acid is RNA, and the ligation process is performed under the catalysis of poly(A) polymerase or poly(G) polymerase. The poly(A) polymerase can catalyze the addition of an adenosine residue to the 3' terminus of the RNA, thereby adding an adenosine residue that is phosphorylated at the C-3 position to the 3' terminus of the RNA. The poly(G) polymerase can catalyze the addition of a guanosine residue to the 3' terminus of the RNA, thereby adding a guanosine residue that is phosphorylated at the C-3 position to the 3' terminus of the RNA.

[0024] According to an embodiment of the present invention, the nucleic acid is a mixture of single-stranded DNA and RNA, and the ligation process is performed under the catalysis of terminal transferase and poly(A) polymerase or poly(G) polymerase. According to an embodiment of the present invention, the terminal transferase is used to connect a deoxyribonucleotide or ribonucleotide modified by phosphorylation at the C-3 position to the 3' end of the DNA, and the poly(A) polymerase or poly(G) polymerase is used to connect an adenosine residue or guanosine residue modified by phosphorylation at the C-3 position to the 3' end of the RNA.

[0025] According to an embodiment of the present invention, the nucleic acid is a single-stranded DNA, RNA, or a mixture of single-stranded DNA and RNA, and the ligation process is performed under the catalysis of T4 RNA ligase 1. According to an embodiment of the present invention, the T4 RNA ligase is used to connect deoxyribonucleotides or ribonucleotides with phosphorylation modification at the C-3 position to the 3' end of DNA and RNA.

[0026] According to an embodiment of the present invention, the 5' end of the nucleic acid is connected to a phosphate group. The inventors phosphorylated the 3' end of the nucleic acid under the condition that the 5' end of the nucleic acid is connected to a phosphate group to ensure that the nucleic acid does not self-ligate.

[0027] According to an embodiment of the present invention, the nucleic acid having a phosphate group attached to the 5' end is obtained by subjecting the nucleic acid to the catalytic action of T4 phosphokinase. According to an embodiment of the present invention, the T4 phosphokinase does not have 3' end phosphatase activity, thereby ensuring that only the 5' end of the nucleic acid is phosphorylated.

[0028] According to an embodiment of the present invention, the 5' end of the nucleic acid is -OH.

[0029] According to an embodiment of the present invention, the nucleic acid with -OH at the 5' end is obtained by subjecting the nucleic acid to phosphatase catalysis.

[0030] For example, as shown in FIG6 , the nucleic acid with -OH at the 5' end can be a sample itself carrying a hydroxyl group at the 5' end, or can be obtained by subjecting the nucleic acid to phosphatase catalysis.

[0031] In the second aspect of the present invention, the present invention proposes a method for library construction. According to an embodiment of the present invention, the method comprises: performing phosphorylation modification on the 3' end of the nucleic acid fragment using the method described in the first aspect of the present invention; ligating the nucleic acid fragment with phosphorylation modification at the 3' end to the adapter under the action of RtcB ligase so that the 3' end of the nucleic acid fragment is connected to the 5' end of the adapter; and amplifying the ligation product to obtain a sequencing library. The above method is used to construct a sequencing library. On the one hand, it prevents the self-ligation problem of the nucleic acid template; on the other hand, the use of RtcB enzyme for library construction increases the efficiency of adapter ligation and improves template utilization.

[0032] According to an embodiment of the present invention, the above-mentioned library construction method may further include at least one of the following technical features:

[0033] According to an embodiment of the present invention, the 5' end of the nucleic acid fragment is phosphorylated, and the ligation process further includes: ligating the nucleic acid fragment with the phosphorylated 5' end and the adapter under the action of T4 RNA ligase 1, so that the 5' end of the nucleic acid fragment is connected to the 3' end of the adapter. Here, both the 5' end and the 3' end of the nucleic acid fragment to be connected to the adapter are phosphorylated.

[0034] According to an embodiment of the present invention, the phosphorylation modification of the 5' end of the nucleic acid fragment is obtained by subjecting the nucleic acid fragment to the action of T4 phosphokinase.

[0035] It should be noted that the T4 phosphokinase only has 5' end phosphokinase activity.

[0036] According to an embodiment of the present invention, the phosphorylation modification of the 5' end and the phosphorylation modification of the 3' end of the nucleic acid fragment are performed simultaneously or stepwise. Based on the difference in the activity of the enzymes used, either a one-step method or a stepwise method can be used to phosphorylate the 5' end and the 3' end of the nucleic acid fragment, ensuring that both ends of all nucleic acid fragments in the reaction system are phosphorylated.

[0037] According to an embodiment of the present invention, the nucleic acid fragment is a DNA fragment, and the method includes: subjecting the nucleic acid fragment to 5'-end phosphorylation treatment under the action of T4 phosphokinase; subjecting the 5'-end phosphorylation treatment product to 3'-end phosphorylation treatment under the action of terminal transferase; ligating the 3'-end phosphorylation treatment product to a linker under the action of T4 RNA ligase 1 and RtcB ligase; and performing PCR amplification on the ligation treatment product to obtain a sequencing library.

[0038] It should be noted that when the nucleic acid fragment is a DNA fragment, the nucleic acid fragment is subjected to 5'-end phosphorylation treatment (3'-end is a hydroxyl group) under the action of T4 phosphokinase; the 5'-end phosphorylation treatment product is subjected to 3'-end phosphorylation treatment under the action of terminal transferase (refer to the 3'-end phosphorylation modification method described in the first aspect); the 3'-end phosphorylated product is further ligated to a sequencing adapter under the action of T4 RNA ligase 1 and RtcB ligase; and the ligation product is amplified by PCR to obtain a sequencing library.

[0039] According to an embodiment of the present invention, the nucleic acid fragment is an RNA fragment, and the method includes: subjecting the nucleic acid fragment to 5'-end phosphorylation and 3'-end phosphorylation under the catalysis of T4 phosphokinase and poly(A) polymerase or poly(G) polymerase (refer to the 3'-end phosphorylation modification method described in the first aspect); ligating the phosphorylated product to a linker under the action of T4 RNA ligase 1 and RtcB ligase; and reverse transcribing and PCR amplifying the ligated product to obtain a sequencing library.

[0040] It should be noted that when the nucleic acid fragment is RNA, 5'-end phosphorylation treatment and 3'-end phosphorylation treatment (one-step method or step-by-step method) are performed under the catalysis of T4 phosphokinase and poly (A) polymerase (poly (G) polymerase) (refer to the 3'-end phosphorylation modification method described in the first aspect); the phosphorylated product is ligated to the sequencing adapter under the action of T4 RNA ligase 1 and RtcB ligase; the ligated product is reverse transcribed and PCR amplified to obtain a sequencing library.

[0041] It should be noted that the RtcB ligase used in this application is an enzyme that has self-cleaving enzyme activity and can transfer the 5' end phosphate of the nucleic acid molecule to the 3' end of the nucleic acid molecule, and can also form a phosphodiester bond between the 3' end phosphate and the 5' end hydroxyl of the nucleic acid molecule.

[0042] According to an embodiment of the present invention, the RtcB ligase catalyzed reaction is carried out in a reaction system containing triphosphate and divalent manganese ions.

[0043] According to an embodiment of the present invention, the triphosphate is selected from at least one of GTP, dGTP or ATP.

[0044] According to an embodiment of the present invention, the RtcB ligase catalyzed reaction is carried out at 15-85° C. for 15-120 min.

[0045] According to an embodiment of the present invention, the nucleic acid fragment is a mixture of DNA fragments and RNA fragments, and the method includes: subjecting the nucleic acid fragment to 5'-end phosphorylation and 3'-end phosphorylation under the catalysis of T4 phosphokinase, terminal transferase, and poly(A) polymerase or poly(G) polymerase (refer to the 3'-end phosphorylation modification method described in the first aspect); ligating the phosphorylated product to a linker under the action of T4 RNA ligase 1 and RtcB ligase; and reverse transcription and PCR amplification of the ligated product to obtain a sequencing library.

[0046] It should be noted that the nucleic acid fragment is a mixture of DNA fragments and RNA fragments, and the nucleic acid fragment is subjected to 5'-end phosphorylation and 3'-end phosphorylation (one-step method or step-by-step method) under the catalysis of T4 phosphokinase, terminal transferase, and poly(A) polymerase or poly(G) polymerase (refer to the 3'-end phosphorylation modification method described in the first aspect); the phosphorylated product is ligated to the sequencing adapter under the action of T4 RNA ligase 1 and RtcB ligase; the ligated product is reverse transcribed (this operation is performed when the nucleic acid fragment contains RNA) and PCR amplified to obtain a sequencing library.

[0047] According to an embodiment of the present invention, the reverse transcription is performed under the catalysis of reverse transcriptase.

[0048] According to an embodiment of the present invention, the reverse transcriptase is selected from at least one of SuperScript II reverse transcriptase or Alpha reverse transcriptase.

[0049] According to an embodiment of the present invention, the nucleic acid fragment is a DNA fragment or an RNA fragment or a mixture of DNA and RNA fragments, and the method includes: dephosphorylating the nucleic acid under the action of a phosphatase; phosphorylating the dephosphorylated product at the 3' end by T4 RNA ligase 1; phosphorylating the 3' end phosphorylated product at the 5' end by T4 phosphokinase; ligating the 5' end phosphorylated product to a linker under the action of T4 RNA ligase 1 and RtcB ligase; and reverse transcribing and PCR amplifying the ligated product to obtain a sequencing library.

[0050] It should be noted that the present invention also provides another library construction method when the nucleic acid fragment is a DNA fragment or an RNA fragment or a mixture of DNA and RNA fragments. The method comprises: dephosphorylating the nucleic acid under the action of a phosphatase (both ends of the nucleic acid are hydroxyl groups); phosphorylating the dephosphorylated product at the 3' end (replacing the 3' end hydroxyl group with a phosphate group) by T4 RNA ligase 1; phosphorylating the 3' end phosphorylated product at the 5' end (replacing the 5' end hydroxyl group with a phosphate group) by T4 phosphokinase; ligating the 5' end phosphorylated product to a sequencing adapter under the action of T4 RNA ligase 1 and RtcB ligase; and reverse transcribing the ligated product (this operation is performed when the nucleic acid fragment contains RNA) and PCR amplifying it to obtain a sequencing library.

[0051] According to an embodiment of the present invention, the reverse transcription is performed under the catalysis of reverse transcriptase.

[0052] According to an embodiment of the present invention, the reverse transcriptase is selected from at least one of SuperScript II reverse transcriptase or Alpha reverse transcriptase.

[0053] According to an embodiment of the present invention, the RtcB ligase catalyzed reaction is carried out in a reaction system containing triphosphate and divalent manganese ions.

[0054] According to an embodiment of the present invention, the triphosphate is selected from at least one of GTP, dGTP or ATP.

[0055] According to an embodiment of the present invention, the RtcB ligase catalyzed reaction is carried out at 15-85° C. for 15-120 min.

[0056] According to an embodiment of the present invention, the DNA fragments include interrupted genomic DNA molecules, extracellular free DNA molecules, degraded DNA molecules and / or sulfite-treated DNA molecules.

[0057] It should be noted that the aforementioned disrupted genomic DNA molecules, extracellular free DNA molecules, degraded DNA molecules, and / or sulfite-treated DNA molecules are all single-stranded DNA molecules. Furthermore, the conversion of double-stranded DNA molecules to single-stranded DNA molecules includes, but is not limited to, the aforementioned methods and may also include high-temperature denaturation, ligation of single-stranded binding proteins, and the like.

[0058] According to an embodiment of the present invention, after the ligation process and before the amplification process, the method further comprises subjecting the single-stranded DNA to a single-stranded circularization reaction in the presence of a single-stranded circularization reagent to obtain a circularized product.

[0059] In a third aspect, the present invention provides an isolated nucleic acid. According to an embodiment of the present invention, the isolated nucleic acid is obtained by the method described in the first aspect of the present invention. Nucleic acids with 3'-terminal phosphorylation modifications can prevent self-ligation when used as nucleic acid templates. Using such nucleic acids for library construction can increase linker ligation efficiency and improve template utilization.

[0060] In a fourth aspect, the present invention provides a sequencing library. According to an embodiment of the present invention, the sequencing library is obtained using the method described in the second aspect of the present invention. Using the method described in the second aspect, 3'-end phosphorylation-modified nucleic acid templates can be obtained for library construction, thereby increasing adapter ligation efficiency and improving template utilization.

[0061] In the fifth aspect of the present invention, the present invention proposes a kit for constructing a 3'-end phosphorylation-modified nucleic acid. According to an embodiment of the present invention, the kit comprises a deoxyribonucleotide or a ribonucleotide with a phosphate group connected to the 3-carbon atom. The kit has high stability and can conveniently and efficiently construct a 3'-end phosphorylation-modified nucleic acid. According to an embodiment of the present invention, the kit for constructing a 3'-end phosphorylation-modified nucleic acid further comprises at least one selected from reverse transcriptase, terminal transferase, poly(A) polymerase, poly(G) polymerase, T4 RNA ligase 1, T4 phosphokinase or phosphatase. The enzyme included in the kit of the present invention can perform phosphate blocking on the 3' end of the nucleic acid template, so that both ends of the nucleic acid template are modified with phosphate groups to prevent the nucleic acid template from self-ligating.

[0062] In a sixth aspect, the present invention provides a use of the kit of the fifth aspect in constructing a nucleic acid with a phosphorylation modification at the 3' end. According to an embodiment of the present invention, the kit of the present invention can be used to construct a nucleic acid with a phosphorylation modification at the 3' end.

[0063] In a seventh aspect, the present invention provides a kit for constructing a sequencing library. According to an embodiment of the present invention, the kit comprises a deoxyribonucleotide or ribonucleotide having a phosphate group attached to the 3-carbon atom and RtcB ligase. According to an embodiment of the present invention, the kit can improve library construction efficiency and shorten library construction time, and can be widely used in scientific research.

[0064] According to an embodiment of the present invention, the kit for constructing a sequencing library further comprises at least one selected from the group consisting of a reverse transcriptase, a terminal transferase, a polyadenylate polymerase, a polyguanylate polymerase, a T4 RNA ligase 1, a T4 phosphokinase, and a phosphatase. The enzyme included in the kit of the present invention can phosphate-block the 3' end of the nucleic acid template, thereby modifying both ends of the nucleic acid template with phosphate groups and preventing self-ligation of the nucleic acid template.

[0065] In an eighth aspect of the present invention, the present invention provides a use of the kit described in the seventh aspect in constructing a sequencing library. According to an embodiment of the present invention, the kit can be used to efficiently construct a sequencing library.

[0066] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0068] FIG1 is a schematic diagram of a DNA template library construction process according to an embodiment of the present invention;

[0069] FIG2 is a schematic diagram of the RNA template library construction process according to an embodiment of the present invention;

[0070] FIG3 is a schematic diagram of a process for co-constructing a library of DNA and RNA templates according to an embodiment of the present invention;

[0071] FIG4 shows the molecular formulas of adenylate and four deoxynucleotides modified with a 3' phosphate group according to an embodiment of the present invention;

[0072] FIG5 is a molecular formula of adenosine 5'-triphosphate, 2',3'-cyclic-phosphate according to an embodiment of the present invention;

[0073] FIG6 is a schematic diagram of a process for co-building a library of DNA and RNA templates according to an embodiment of the present invention;

[0074] FIG7 is a molecular formula of adenosine 3',5'-diphosphate and adenosine 5'-phosphate, 2',3'-cyclic-phosphate according to an embodiment of the present invention;

[0075] FIG8 is a schematic diagram of the process of co-constructing a library of DNA and RNA templates according to an embodiment of the present invention. DETAILED DESCRIPTION

[0076] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0077] Definition and Description

[0078] As used herein, unless otherwise indicated, the singular forms "a," "an," and the like include plural referents (more than one); "a set" or "a plurality" refers to two or more.

[0079] In this document, unless otherwise specified, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated; features specified as "first", "second", etc. may explicitly or implicitly include one or more of the said features.

[0080] As used herein, unless otherwise indicated, nucleotides refer to the four natural nucleotides (e.g., dATP, dCTP, dGTP, and dTTP, or ATP, CTP, GTP, and UTP) or their derivatives, and are sometimes also directly represented by the bases they contain (A, T / U, C, and G). A person of ordinary skill in the art will be able to identify the reference to a nucleotide or base in a specific embodiment based on the context.

[0081] In this article, the so-called "sequencing" is sequence determination, which is the same as "nucleic acid sequencing" or "gene sequencing", referring to the determination of the base order in the nucleic acid sequence; it includes synthesis sequencing and / or ligation sequencing; it includes DNA sequencing and / or RNA sequencing; it includes long fragment sequencing and / or short fragment sequencing, and the so-called long fragments and short fragments are relative, such as nucleic acid molecules longer than 1Kb, 2Kb, 5Kb or 10Kb can be called long fragments, and those shorter than 1Kb or 800bp can be called short fragments; it includes double-end sequencing, single-end sequencing and / or paired-end sequencing, etc., and the so-called double-end sequencing or paired-end sequencing can refer to the reading of any two segments or two parts of the same nucleic acid molecule that do not completely overlap.

[0082] Nucleic acid 3' end phosphorylation modification method

[0083] In one aspect of the present invention, a method for phosphorylation modification of the 3' end of a nucleic acid is provided.

[0084] According to an embodiment of the present invention, when the nucleic acid is a single-stranded DNA, the single-stranded DNA is catalytically treated with deoxyribonucleotides (such as deoxyadenosine 5'-triphosphate, 3'-phosphate, deoxyadenosine 3', 5'-diphosphate, deoxycytidine 5'-triphosphate, 3'-phosphate, deoxyguanosine 5'-triphosphate, 3'-phosphate or deoxythymidine 5'-triphosphate, 3'-phosphate, etc.) under the action of terminal transferase to obtain a single-stranded DNA with a phosphorylation modification at the 3' end.

[0085] In addition, the 5' end of the DNA single strand can also be connected to a phosphate group to prevent the nucleic acid fragment from self-ligating. If the 5' end of the DNA single strand does not have a phosphate group, T4 phosphokinase can be added to the reaction system to catalyze the reaction. T4 phosphokinase does not have 3' end phosphatase activity.

[0086] It should be noted that when the nucleic acid is double-stranded DNA, the above operation can also be performed to phosphorylate the 3' end of the double-stranded DNA, which will not be described in detail here.

[0087] According to an embodiment of the present invention, when the nucleic acid is RNA, the RNA is treated with ribonucleotides (such as adenosine 5'-triphosphate, 3'-phosphate, adenosine 5'-phosphate, 2', 3'-cyclic-phosphate, adenosine 5'-triphosphate, 2', 3'-cyclic-phosphate or adenosine 3', 5'-diphosphate, etc.) under conditions catalyzed by poly(A) polymerase or poly(Gu) polymerase, so as to obtain a single-stranded RNA with a phosphorylation modification at the 3' end.

[0088] In addition, the 5' end of the RNA single strand can also be linked to a phosphate group to prevent nucleic acid self-ligation. If the 5' end of the RNA strand lacks a phosphate group, T4 phosphokinase can be added to the reaction system to catalyze the reaction. T4 phosphokinase has no 3' end phosphatase activity.

[0089] According to an embodiment of the present invention, when the nucleic acid is a mixture of single-stranded DNA and RNA, deoxyribonucleotides (such as deoxyadenosine 5'-triphosphate, 3'-phosphate, deoxyadenosine 3', 5'-diphosphate, deoxycytidine 5'-triphosphate, 3'-phosphate, deoxyguanosine 5'-triphosphate, 3'-phosphate or deoxythymidine 5'-triphosphate, 3'-phosphate, etc.) and ribonucleotides (such as adenosine 5'-triphosphate, 3'-phosphate, adenosine 5'-phosphate, 2', 3'-cyclic-phosphate, adenosine 5'-triphosphate, 2', 3'-cyclic-phosphate or adenosine 3', 5'-diphosphate, etc.) are catalytically treated with the single-stranded nucleic acid mixture under the conditions of T4 phosphokinase (without 3'-end phosphatase activity), terminal transferase and poly(A) polymerase or poly(G) polymerase, so as to obtain single-stranded DNA and single-stranded RNA with phosphorylation modification at the 3' end.

[0090] In addition, the 5' ends of the RNA and DNA single strands can also be linked to phosphate groups to prevent the nucleic acids from self-ligating. If the 5' ends of the single-stranded nucleic acids do not have phosphate groups, T4 phosphokinase should be added to the reaction system to catalyze the reaction. T4 phosphokinase does not have 3' end phosphatase activity.

[0091] Database construction method

[0092] In another aspect of the present invention, the present invention proposes a library construction method. According to an embodiment of the present invention, the library construction method includes: using any of the aforementioned single-stranded nucleic acid 3' end phosphorylation modification methods to perform phosphorylation modification on the 3' end of the nucleic acid fragment; the nucleic acid fragment with phosphorylation modification at the 3' end and the connector are connected under the action of RtcB ligase or T4 RNA ligase 1 and phosphokinase, so that the 3' end of the nucleic acid fragment is connected to the 5' end of the connector; the connection treatment product is amplified to obtain a sequencing library. The use of the above method for sequencing library construction, on the one hand, prevents the self-ligation problem of the nucleic acid template; on the other hand, the use of T4 RNA ligase and RtcB enzyme for library construction increases the efficiency of connector connection and improves template utilization.

[0093] The specific steps are as follows:

[0094] 1) Obtain nucleic acid samples: Use the following methods to obtain nucleic acid samples according to experimental requirements.

[0095] The nucleic acid sample (DNA, RNA or a mixture of the two) is sheared into fragments of a certain size, or the extracellular free DNA or RNA is extracted, or the degraded DNA or RNA is extracted from various degraded biological samples (such as paleontological fossils, FFPE samples) or sulfite-treated DNA molecules.

[0096] 2) Sample Processing: Denature the nucleic acid sample to convert the double-stranded nucleic acid in the nucleic acid template into a single-stranded nucleic acid. Perform high-temperature denaturation (92-98°C for 3-10 minutes). Immediately after denaturation, place on ice. Selectively add a single-stranded binding protein to maintain the single-stranded state of the nucleic acid sample using the properties of the binding protein.

[0097] 3) When the single-stranded nucleic acid is a DNA fragment or an RNA fragment or a mixture of DNA fragments and RNA fragments, different library construction methods are selected for library construction.

[0098] The following describes each method in detail. The specific steps are as follows:

[0099] As shown in Figure 1, when the nucleic acid fragment is a DNA fragment, the 5' end of the single-stranded DNA fragment is first phosphorylated using T4 phosphokinase. Then, under the catalysis of terminal transferase, a deoxyribonucleotide (such as deoxyadenosine 5'-triphosphate, 3'-phosphate, deoxyadenosine 3', 5'-diphosphate, deoxycytidine 5'-triphosphate, 3'-phosphate, deoxyguanosine 5'-triphosphate, 3'-phosphate, or deoxythymidine 5'-triphosphate, 3'-phosphate) is added to phosphorylate the 3' end of the single-stranded DNA fragment. The 3'-phosphorylated DNA single strand is mixed with a sequencing adapter sequence and ligated with sequencing adapters at both ends under the catalysis of T4 RNA ligase 1 and RtcB ligase. The ligation product is then amplified by PCR to generate a sequencing library.

[0100] It should be noted that the 5' end phosphorylation treatment and the 3' end phosphorylation treatment can be performed simultaneously or in steps, and the reverse transcription and PCR amplification treatment can be performed simultaneously or in steps.

[0101] As shown in Figure 2, when the nucleic acid fragment is RNA, the 5' end of the RNA fragment is phosphorylated using T4 phosphokinase. Under the catalysis of poly(A) polymerase or poly(G) polymerase, ribonucleotides (such as adenosine 5'-triphosphate, 3'-phosphate, adenosine 5'-phosphate, 2',3'-cyclic-phosphate, adenosine 5'-triphosphate, 2',3'-cyclic-phosphate, or adenosine 3',5'-diphosphate) are added to phosphorylate the 3' end of the RNA to obtain a single-stranded RNA with phosphorylation at both ends. The phosphorylated RNA strands are then mixed with sequencing adapter sequences and ligated with sequencing adapters at both ends under the catalysis of T4 RNA ligase 1 and RtcB ligase. The ligated products are then reverse transcribed and amplified by PCR to obtain a sequencing library.

[0102] It should be noted that the 5' end phosphorylation treatment and the 3' end phosphorylation treatment can be performed simultaneously or in steps, and the reverse transcription and PCR amplification treatment can be performed simultaneously or in steps.

[0103] As shown in Figure 3, when the nucleic acid fragment is a mixture of DNA and RNA, the 5' ends of the single-stranded DNA and RNA fragments are phosphorylated using T4 phosphokinase. Then, under the catalysis of terminal transferase, deoxyribonucleotides (such as deoxyadenosine 5'-triphosphate, 3'-phosphate, deoxyadenosine 3', 5'-diphosphate, deoxycytidine 5'-triphosphate, 3'-phosphate, deoxyguanosine 5'-triphosphate, 3'-phosphate, or deoxythymidine 5'-triphosphate, 3'-phosphate) are added to phosphorylate the 3' ends of the single-stranded DNA fragments. At the same time, under the catalysis of poly(A) polymerase or poly(G) polymerase, ribonucleotides (such as adenosine 5'-triphosphate, 3'-phosphate, adenosine 5'-phosphate, 2',3'-cyclic-phosphate, adenosine 5'-triphosphate, 2',3'-cyclic-phosphate, or adenosine 3',5'-diphosphate) are added to phosphorylate the 3' end of the RNA to obtain a single-stranded RNA with phosphorylation at both ends. Finally, the phosphorylated DNA and RNA single strands are mixed with sequencing adapter sequences and ligated with sequencing adapters at both ends under the catalysis of T4 RNA ligase 1 and RtcB ligase. The ligation products are reverse transcribed and PCR amplified to obtain a sequencing library.

[0104] It should be noted that the 5'-end phosphorylation treatment and the 3'-end phosphorylation treatment can be performed simultaneously or in steps, and the reverse transcription and PCR amplification treatment can be performed simultaneously or in steps. The molecular formulas of adenosine 5'-triphosphate, 3'-phosphate, deoxyadenosine 5'-triphosphate, 3'-phosphate, deoxycytidine 5'-triphosphate, 3'-phosphate, deoxyguanosine 5'-triphosphate, 3'-phosphate, or deoxythymidine 5'-triphosphate, 3'-phosphate are shown in Figure 4. The molecular formula of adenosine 5'-triphosphate, 2', 3'-cyclic-phosphate is shown in Figure 5. The molecular formulas of adenosine 3', 5'-diphosphate and adenosine 5'-phosphate, 2', 3'-cyclic-phosphate are shown in Figure 7.

[0105] As shown in FIG6 , when the nucleic acid fragment is a mixture of DNA and RNA, the nucleic acid fragment mixture is subjected to dephosphorylation treatment of the single-stranded nucleic acid under phosphatase catalysis conditions, and then T4 RNA ligase 1 phosphorylates the 3' ends of DNA and RNA fragments in the presence of deoxyribonucleotides (such as deoxyadenosine 5'-triphosphate, 3'-phosphate, deoxyadenosine 3', 5'-diphosphate, deoxycytidine 5'-triphosphate, 3'-phosphate, deoxyguanosine 5'-triphosphate, 3'-phosphate or deoxythymidine 5'-triphosphate, 3'-phosphate, etc.) and ribonucleotides (such as adenosine 5'-triphosphate, 3'-phosphate, adenosine 5'-phosphate, 2', 3'-cyclic-phosphate, adenosine 5'-triphosphate, 2', 3'-cyclic-phosphate or adenosine 3', 5'-diphosphate, etc.), and further phosphorylates the 5' ends of DNA and RNA fragments using T4 phosphokinase (without 3' end phosphatase activity) to obtain nucleic acid products with phosphorylated modifications at both ends. Finally, the nucleic acid products with phosphorylated modifications at both ends are mixed with sequencing adapter sequences on T4 The sequencing adapters at both ends are connected under the catalysis of RNA ligase 1 and RtcB ligase, and the ligation products are reverse transcribed and PCR amplified to obtain the sequencing library.

[0106] It should be noted that the 5' end phosphorylation treatment and the 3' end phosphorylation treatment can be performed simultaneously or in steps, and the reverse transcription and PCR amplification treatment can be performed simultaneously or in steps.

[0107] As shown in FIG8 , when the nucleic acid fragment is a mixture of DNA and RNA, the nucleic acid fragment mixture, deoxyribonucleotides (such as deoxyadenosine 5'-triphosphate, 3'-phosphate, deoxyadenosine 3', 5'-diphosphate, deoxycytidine 5'-triphosphate, 3'-phosphate, deoxyguanosine 5'-triphosphate, 3'-phosphate or deoxythymidine 5'-triphosphate, 3'-phosphate, etc.) and ribonucleotides (such as adenosine 5'-triphosphate, 3'-phosphate, adenosine 5'-phosphate, 2', 3'-cyclic-phosphate, adenosine 5'-triphosphate, 2', 3'-cyclic-phosphate or adenosine 3', 5'-diphosphate, etc.) are phosphorylated to modify single-stranded DNA and RNA under the catalytic conditions of T4 phosphokinase, terminal transferase and poly(A) polymerase or poly(G) polymerase, and the phosphorylated products at both ends are further subjected to T4 The 5' end adapter is ligated under the catalysis of RNA ligase 1, and then the 3' end adapter is ligated under the catalysis of T4 phosphokinase (which converts the phosphate group at the 3' end into hydroxyl -OH), T4 RNA ligase 1 or T4 RNA ligase 2 truncated form. Finally, the ligation product is reverse transcribed and PCR amplified to obtain the sequencing library.

[0108] It should be noted that the reverse transcription processes involved in this application are all carried out under the catalytic conditions of reverse transcriptase, and the reverse transcriptase includes but is not limited to SuperScript II reverse transcriptase or Alpha reverse transcriptase. Suitable reverse transcriptases can be selected for experiments as needed.

[0109] It should be noted that the T4 phosphokinase involved in the present application can be replaced by an enzyme with the same function, such as alkaline phosphatase.

[0110] Isolated nucleic acids

[0111] In another aspect, the present invention provides an isolated nucleic acid. According to an embodiment of the present invention, the isolated nucleic acid has a 3' end phosphorylation modification, and the isolated nucleic acid is constructed based on the aforementioned nucleic acid 3' end phosphorylation modification method.

[0112] Sequencing library

[0113] In another aspect of the present invention, a sequencing library is provided. According to an embodiment of the present invention, the sequencing library is obtained by any of the aforementioned library construction methods.

[0114] Kit for constructing 3' end phosphorylation modified nucleic acids

[0115] In another aspect of the present invention, a kit is provided for constructing a nucleic acid with a phosphorylation modification at the 3' end.

[0116] Kits for constructing sequencing libraries

[0117] In yet another aspect of the present invention, the present invention provides a kit for constructing a sequencing library. Beneficial effects

[0118] The present invention phosphorylates both the 5' and 3' ends of the nucleic acid template, preventing self-ligation under the action of a ligase. Subsequently, a modified single-stranded adapter sequence is ligated to both ends of the phosphorylated nucleic acid sample in the same reaction system, ensuring the directionality of the adapter ligation and eliminating the need for adding adapters at the 5' and 3' ends in separate steps. This approach solves the problem of nucleic acid template self-ligation and reduces template loss; it prevents adapters from ligating to each other, reducing adapter loss and increasing adapter ligation efficiency; and it simultaneously connects the adapters at both ends in a single step, reducing reaction steps, shortening library construction time, and improving the utilization rate of the original nucleic acid template.

[0119] The embodiments of the present invention will be described in more detail below, examples of which are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0120] Example 1: Construction and sequencing of human peripheral blood free DNA methylation library

[0121] A: Experimental materials

[0122] Cell-free DNA extracted from human peripheral blood plasma.

[0123] B: Experimental steps

[0124] 1. Take 5 ng of human peripheral blood plasma free DNA (cfDNA) and transfer it to a 200 μL PCR tube, label the sample S1, and the total volume is 20 μL;

[0125] 2. Bisulfite treatment and purification of the adapter ligation products using the EZ DNA Methylation-Gold Kit (Zymo Research, Cat. No. D5005 / D5006);

[0126] 3. Sequentially pipette 900 μL of NF water, 300 μL of M-Dilution Buffer, and 50 μL 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 complete the preparation of the CT Conversion Reagent (CT Conversion Reagent should be kept out of the reach of the eyes and should ideally be prepared and used immediately). 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 preheated to 37°C and vortexed frequently for 10 minutes at room temperature before use.

[0127] 4. Before opening the bottle cap for the first use, add the correct volume of anhydrous ethanol as indicated on the bottle label and mix thoroughly before use. At room temperature, add the ingredients in Table 1 to a new 200 μL PCR tube.

[0128] Table 1:

[0129] 5. Place the 200 μL PCR tube in a PCR instrument and perform the reaction according to the Bisulfite treatment conditions;

[0130] Table 2:

[0131] 6. Transfer the reaction product to a new 1.5 mL centrifuge tube, add 600 μL of M-Binding Buffer, vortex 6 times for 3 seconds each time, and centrifuge briefly to collect the reaction solution at the bottom of the tube;

[0132] 7. Place the Zymo-Spin IC Column into a 2 mL Collection Tube, transfer the mixture 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.

[0133] 8. Add 100 μL M-Wash Buffer to the Zymo-Spin IC Column and centrifuge at 13,000 rpm for 30 seconds.

[0134] 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;

[0135] 10. Add 200 μL 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;

[0136] 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.

[0137] 12. Open the Zymo-Spin IC Column tube cap and dry it at room temperature for 2 minutes. Then place the Zymo-Spin IC Column in another new 1.5 mL centrifuge tube.

[0138] 13. Slowly add 10 μL of M-Elution Buffer to the center of the Zymo-Spin IC Column filter, 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 200 μL PCR tube;

[0139] 14. Prepare the phosphorylation reaction mixture according to Table 3;

[0140] Table 3:

[0141] 15. Add 10 μL of the prepared phosphorylation reaction mixture to the PCR tube from the previous step, vortex to mix, and centrifuge briefly;

[0142] 16. Place the PCR tube in a PCR instrument and incubate at 37°C for 15 minutes, then at 75°C for 10 minutes, and then maintain at 4°C. After the reaction is complete, remove the PCR tube and centrifuge briefly.

[0143] 17. Prepare the terminal modification reaction mixture according to Table 4;

[0144] Table 4:

[0145] 18. Add 30 μL of the prepared end-modification reaction mixture to the PCR tube from the previous step, vortex to mix, and centrifuge briefly;

[0146] 19. Place the PCR tube in a PCR instrument and incubate at 37°C for 15 minutes, then maintain at 4°C. After the reaction is complete, remove the PCR tube and centrifuge briefly.

[0147] 20. Pipette 75 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;

[0148] 21. 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 becomes clear. Carefully remove the supernatant with a pipette and discard.

[0149] 22. Keep the centrifuge tube on the magnetic stand and add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. Let it stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0150] 23. 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.

[0151] 24. Place the centrifuge tube on the magnetic rack, open the tube cap, and dry at room temperature until the surface of the magnetic beads is non-reflective.

[0152] 25. Remove the centrifuge tube from the magnetic stand and add 21 μL TE Buffer (AMBION, AM9858) for elution. Gently pipette at least 10 times to mix thoroughly.

[0153] 26. Incubate at room temperature for 5 minutes;

[0154] 27. 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 200 μL PCR tube.

[0155] 28. Add 5 μL of adapter mix (10 μM) to the PCR tube;

[0156] NH2-5Ad-uni-1(5'NH2 C6-GAACGACATGGCTACGATCCGACTT-3'OH)

[0157] 3Ad-Top-NH2-1(5'OH-AAGTCGGAGGCCAAGCGG-3'NH2 C6)

[0158] 29. Prepare the ligation reaction mixture according to Table 5;

[0159] Table 5:

[0160] 30. Add 25 μL of the prepared ligation reaction solution to the PCR tube, vortex to mix, and centrifuge briefly;

[0161] 31. Place the PCR tube in a PCR instrument and set the program: incubate at 37°C for 30 minutes and store at 4°C. After the reaction is complete, centrifuge the tube to collect the liquid at the bottom of the tube.

[0162] 32. Pipette 75 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 all the liquid and magnetic beads in the pipette tip are pumped into the centrifuge tube;

[0163] 33. 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.

[0164] 34. 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. Let it stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0165] 35. 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.

[0166] 36. Place the centrifuge tube on the magnetic rack, open the tube cap, and dry at room temperature until the surface of the magnetic beads is non-reflective.

[0167] 37. 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.

[0168] 38. Incubate at room temperature for 5 minutes;

[0169] 39. Centrifuge the tube briefly, place it on a magnetic rack, and let it stand 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.

[0170] 40. Prepare PCR reaction solution on ice according to Table 6;

[0171] Table 6:

[0172] PCR Barcode Primer Mix:

[0173] PCR primer 1(5'P-GAACGACATGGCTACGA-3')

[0174] PCR Barcode primer (5'-TGTGAGCCAAGGAGTTGATCGGACCTATTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-3') The bold part is the sample label

[0175] 41. 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.

[0176] 42. Place the PCR tube on a PCR instrument and perform the PCR reaction according to the conditions in Table 7;

[0177] Table 7:

[0178] 43. After the reaction is completed, centrifuge briefly to collect the reaction solution at the bottom of the tube;

[0179] 44. 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 all the liquid and magnetic beads in the pipette tip are pumped into the centrifuge tube;

[0180] 45. 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 clarifies. Carefully remove the supernatant with a pipette and discard.

[0181] 46. ​​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. Let it stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0182] 47. 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.

[0183] 48. Place the centrifuge tube on the magnetic rack, open the tube cap, and dry at room temperature until the surface of the magnetic beads is non-reflective.

[0184] 49. 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.

[0185] 50. Incubate at room temperature for 5 minutes;

[0186] 51. Centrifuge the tube briefly, place it on a magnetic rack, and let it stand 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.

[0187] 52. Use dsDNA HS Assay Kit (Theromo Fisher, Q32854) fluorescence quantitative kit was used to quantify the purified products after enzyme digestion according to the operating instructions of the quantitative kit;

[0188] 53. 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.

[0189] 54. Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in Table 8;

[0190] Table 8:

[0191] 55. 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;

[0192] 56. 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 9;

[0193] Table 9:

[0194] 57. Use a pipette to draw 12.1 μL of the prepared single-stranded circularization 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.

[0195] 58. Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in Table 10;

[0196] Table 10:

[0197] 59. After the reaction is completed, centrifuge the PCR tube and place it on ice before proceeding to the next step.

[0198] 60. Prepare the enzyme digestion reaction solution on ice in advance using the circularization kit from MGI (MGI, 1000005260) according to the recipe in Table 11;

[0199] Table 11:

[0200] 61. Use a pipette to draw 4 μL of the prepared enzyme digestion reaction solution into the PCR tube, vortex and shake three times for 3 seconds each time, and centrifuge briefly to collect the reaction solution at the bottom of the tube;

[0201] 62. Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in Table 12;

[0202] Table 12:

[0203] 63. After the reaction is completed, centrifuge briefly to collect the reaction solution at the bottom of the tube;

[0204] 64. 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 suspend 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.

[0205] 65. 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 clarifies. Carefully remove the supernatant with a pipette and discard.

[0206] 66. 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. Let it stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0207] 67. 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.

[0208] 68. Place the centrifuge tube on the magnetic rack, open the tube cap, and dry at room temperature until the surface of the magnetic beads is non-reflective.

[0209] 69. 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.

[0210] 70. Incubate at room temperature for 10 minutes;

[0211] 71. Centrifuge the tube briefly, place it on a magnetic rack, and let it stand 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.

[0212] 72. 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;

[0213] 73. Sequencing was performed using the MGISEQ-2000 sequencer, using the MGISEQ-2000 High-Throughput Sequencing Reagent Set (PE100) (MGI, 1000012536). DNB production and sequencing were performed according to the instructions, and the sequencing type was Custom PE101.

[0214] 74. For data analysis, first truncate the reads and remove the first base of all reads to reduce the impact of the added base on the data. Then, analyze and compile the data according to the standard process.

[0215] C: Result Analysis

[0216] Using 5 ng of cfDNA as the sample (S1), after sulfite treatment, the library was constructed using the aforementioned steps. With 10 PCR cycles, a 1182.5 ng PCR product library was finally obtained, with a high library yield.

[0217] Table 13:

[0218] The resulting PCR product library was further circularized to generate a corresponding single-stranded circle library, which was then sequenced on the DNBSEQ sequencing platform. Using a single lane of an MGISEQ-2000 sequencing chip, the resulting sequencing data reached 403M, with a Q30 quality score exceeding 89% (Table 14), indicating minimal contamination from adapters or other fixed sequences. Furthermore, the reproducibility of standard analysis results was less than 10%, demonstrating efficient utilization of the nucleic acid template even with low sample input.

[0219] Table 14:

[0220] Example 2: Construction and sequencing of a universal human reference RNA (UHRR) sample library

[0221] A: Experimental materials

[0222] Universal Human Reference RNA (UHRR) sample (Agilent, 750500)

[0223] B: Experimental steps

[0224] 1. Take 200 ng of universal human reference RNA (UHRR) sample (Agilent, 750500) into a 1.5 mL non-stick tube and make up the volume to 50 μL with NF Water;

[0225] 2. Use the mRNA Capture Beads kit (Novozymes, N401-01) to enrich mRNA according to the kit instructions to obtain a final volume of 10 μL of mRNA;

[0226] 3. Transfer 10 μL of mRNA to a PCR tube and add 2 μL of E.coli poly A polymerase reaction buffer (10X) (NEB, M0276L). Pipette 10 times to mix, centrifuge briefly, and place in a PCR instrument. Incubate at 94°C for 8 minutes. After the reaction is complete, place on ice for 2 minutes and centrifuge briefly for 10 seconds.

[0227] 4. Prepare the terminal modification reaction mixture according to Table 15;

[0228] Table 15:

[0229] 5. Add 8 μL of the prepared end-modification reaction mixture to the PCR tube in the previous step, vortex to mix, and then centrifuge briefly;

[0230] 6. Place the PCR tube in a PCR instrument and incubate at 37°C for 30 minutes, then maintain at 4°C. After the reaction is complete, remove the PCR tube and centrifuge briefly.

[0231] 7. Add 5 μL of adapter mix (10 μM) to the PCR tube;

[0232] NH2-5Ad-uni-1(5'NH2 C6-GAACGACATGGCTACGATCCGACTT-3'OH)

[0233] 3Ad-Top-NH2-1(5'OH-AAGTCGGAGGCCAAGCGG-3'NH2 C6)

[0234] 8. Prepare the ligation reaction mixture according to Table 16;

[0235] Table 16:

[0236] 9. Add 25 μL of the prepared ligation reaction solution to the PCR tube, vortex to mix, and then centrifuge briefly;

[0237] 10. Place the PCR tube in a PCR instrument and set the program: incubate at 37°C for 30 minutes and store at 4°C. After the reaction is complete, centrifuge the tube to collect the liquid at the bottom of the tube.

[0238] 11. Pipette 75 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;

[0239] 12. 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 becomes clear. Carefully remove the supernatant with a pipette and discard.

[0240] 13. Keep the centrifuge tube on the magnetic stand and add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. Let it stand for 30 seconds, then carefully aspirate and discard the supernatant;

[0241] 14. Repeat the previous step and try to dry 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 dry the liquid at the bottom of the tube.

[0242] 15. Keep the centrifuge tube on the magnetic rack, open the centrifuge tube cover, and dry it at room temperature until the surface of the magnetic beads is non-reflective;

[0243] 16. 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.

[0244] 17. Incubate at room temperature for 5 minutes;

[0245] 18. Centrifuge the tube briefly, place it on a magnetic rack, and let it stand for 2-5 minutes until the liquid becomes clear. Use a pipette to transfer 16 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0246] 19. Prepare reverse transcription and PCR reaction mixtures according to Table 17;

[0247] Table 17:

[0248] PCR Barcode Primer Mix:

[0249] PCR primer 1(5'P-GAACGACATGGCTACGA-3')(15μM)

[0250] PCR Barcode primer (5'-TGTGAGCCAAGGAGTTGATCGGACCTATTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-3') (15μM) The bold part is the sample label

[0251] 20. Use a pipette to draw 34 μL of the prepared reverse transcription and PCR reaction mixture 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.

[0252] 21. Place the PCR tube on a PCR instrument and perform the PCR reaction according to the conditions in Table 18;

[0253] Table 18:

[0254] 22. After the reaction is completed, centrifuge briefly to collect the reaction solution at the bottom of the tube;

[0255] 23. 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;

[0256] 24. 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 becomes clear. Carefully remove the supernatant with a pipette and discard.

[0257] 25. 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. Let it stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0258] 26. Repeat the previous step and try to dry 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 dry the liquid at the bottom of the tube.

[0259] 27. Keep 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;

[0260] 28. 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.

[0261] 29. Incubate at room temperature for 5 minutes;

[0262] 30. Centrifuge the tube briefly, place it on a magnetic rack, and let it stand 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.

[0263] 31. Use dsDNA HS Assay Kit (Theromo Fisher, Q32854) fluorescence quantitative kit was used to quantify the purified products after enzyme digestion according to the operating instructions of the quantitative kit;

[0264] 32. 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.

[0265] 33. Place the PCR tube on a PCR instrument and perform the reaction according to the conditions in Table 19;

[0266] Table 19:

[0267] 34. 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;

[0268] 35. 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 20;

[0269] Table 20:

[0270] 36. 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, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0271] 37. Place the PCR tube on a PCR instrument and perform the reaction according to the conditions in Table 21;

[0272] Table 21:

[0273] 38. After the reaction is complete, centrifuge the PCR tube and place it on ice before proceeding to the next step.

[0274] 39. Prepare the enzyme digestion reaction solution on ice in advance using the circularization kit from MGI (MGI, 1000005260) according to the recipe in Table 22;

[0275] Table 22:

[0276] 40. 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, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0277] 41. Place the PCR tube on a PCR instrument and perform the reaction according to the conditions in Table 23;

[0278] Table 23:

[0279] 42. After the reaction is completed, centrifuge briefly to collect the reaction solution at the bottom of the tube;

[0280] 43. 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 suspend 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;

[0281] 44. 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 becomes clear. Carefully remove the supernatant with a pipette and discard.

[0282] 45. 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. Let it stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0283] 46. ​​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.

[0284] 47. Place the centrifuge tube on the magnetic rack, open the tube cap, and dry at room temperature until the surface of the magnetic beads is non-reflective.

[0285] 48. 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.

[0286] 49. Incubate at room temperature for 10 minutes;

[0287] 50. Centrifuge the tube briefly, place it on a magnetic rack, and let it stand 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.

[0288] 51. 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;

[0289] 52. Sequencing was performed using the MGISEQ-2000 sequencer, using the MGISEQ-2000 High-Throughput Sequencing Reagent Set (PE100) (MGI, 1000012536). DNB production and sequencing were performed according to the instructions, and the sequencing type was Custom PE101.

[0290] 53. For off-machine data analysis, first truncate the reads and remove the first base of all reads to reduce the impact of the added base on the data. Then, analyze and compile the data according to standard procedures.

[0291] C: Result Analysis

[0292] Using 200 ng of total RNA as the sample, after removing rRNA, the mRNA library was constructed. After mRNA fragmentation, end modification and adapter ligation, as well as reverse transcription and PCR, a 1450 ng PCR product library was finally obtained, with high library construction yield.

[0293] The resulting PCR product library was further circularized to generate a corresponding single-stranded circle library. This single-stranded circle library was then sequenced on the DNBSEQ sequencing platform, using one lane of an MGISEQ-2000 sequencing chip. The resulting sequencing data reached 403.07M, with a Q30 quality of 91.44, indicating minimal contamination from adapters or other fixed sequences. Analysis of the same data set (50M reads) revealed genome and gene alignment rates of 89.6% and 71.7%, respectively. The analyzed number of genes was 20,409, and the number of transcripts was 32,432, demonstrating excellent performance.

[0294] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0295] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0296] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A library construction method, characterized in that: include: Phosphorylation modification of the 3' end of the nucleic acid fragment; The nucleic acid fragment with phosphorylation modification at the 3' end is connected to the adapter under the action of RtcB ligase, so that the 3' end of the nucleic acid fragment is connected to the 5' end of the adapter; The ligation products are amplified to obtain a sequencing library.

2. The method according to claim 1, characterized in that The phosphorylation modification of the 3' end of the nucleic acid fragment is carried out in the following manner: The 3' end of the nucleic acid is ligated with a deoxyribonucleotide or a ribonucleotide, wherein the 3-carbon atom of the deoxyribonucleotide or the ribonucleotide is linked with a phosphate group.

3. The method according to claim 1, characterized in that The nucleic acid comprises at least one selected from single-stranded DNA, double-stranded DNA or RNA.

4. The method according to claim 2, characterized in that: The deoxyribonucleotide is at least one of deoxyadenosine 5'-triphosphate, 3'-phosphate, deoxyadenosine 3', 5'-diphosphate, deoxycytidine 5'-triphosphate, 3'-phosphate, deoxycytidine 3', 5'-diphosphate, deoxyguanosine 5'-triphosphate, 3'-phosphate, deoxyguanosine 3', 5'-diphosphate, deoxythymidine 5'-triphosphate, 3'-phosphate, deoxythymidine 3', 5'-diphosphate, deoxyuridine 5'-triphosphate, 3'-phosphate, and deoxyuridine 3', 5'-diphosphate.

5. The method according to claim 2, characterized in that: The ribonucleotides are adenosine 5'-triphosphate, 3'-phosphate, adenosine 5'-phosphate, 2',3'-cyclic-phosphate, adenosine 5'-phosphate, 2',3'-cyclic-phosphate, adenosine 3',5'-diphosphate, cytidine 5'-triphosphate, 3'-phosphate, cytidine 5'-phosphate, 2',3'-cyclic-phosphate, cytidine 5'-phosphate, 2',3'-cyclic-phosphate, cytidine 3',5'-diphosphate, guanosine 5'-triphosphate, 3' ... phosphate, 3'-phosphate, guanosine 5'-phosphate, 2',3'-cyclic-phosphate, guanosine 5'-phosphate, 2',3'-cyclic-phosphate, guanosine 3',5'-diphosphate, guanosine 5'-triphosphate, 3'-phosphate, uridine 5'-triphosphate, 3'-phosphate, uridine 5'-phosphate, 2',3'-cyclic-phosphate, uridine 5'-phosphate, 2',3'-cyclic-phosphate, uridine 3',5'-diphosphate.

6. The method according to claim 2, characterized in that The ligation process is carried out under the catalysis of terminal transferase, polyadenylic acid polymerase, polyguanylic acid polymerase or T4 RNA ligase 1.

7. The method according to claim 2, characterized in that The nucleic acid is a single-stranded DNA, and the connection process is performed under the catalysis of terminal transferase.

8. The method according to claim 2, characterized in that: The nucleic acid is RNA, and the ligation process is performed under the catalysis of polyadenylic acid polymerase or polyguanylic acid polymerase.

9. The method according to claim 2, characterized in that: The nucleic acid is a mixture of single-stranded DNA and RNA, and the connection process is performed under the catalysis of terminal transferase and polyadenylic acid polymerase or polyguanylic acid polymerase.

10. The method according to claim 1, characterized in that The nucleic acid is a single-stranded DNA, RNA or a mixture of single-stranded DNA and RNA, and the ligation process is performed under the catalysis of T4 RNA ligase 1.

11. The method according to claim 1, characterized in that: The 5' end of the nucleic acid fragment is phosphorylated, and the ligation process further comprises: The nucleic acid fragment with phosphorylation modification at the 3' end is ligated with a linker under the action of T4 RNA ligase 1, so that the 5' end of the nucleic acid fragment is connected to the 3' end of the linker.

12. The method according to claim 11, characterized in that The phosphorylation modification of the 5' end of the nucleic acid fragment is obtained by subjecting the nucleic acid fragment to the action of T4 phosphokinase.

13. The method according to claim 12, characterized in that The phosphorylation modification of the 5' end and the phosphorylation modification of the 3' end of the nucleic acid fragment are performed simultaneously or step by step.

14. The method according to claim 13, characterized in that The nucleic acid fragment is a DNA fragment, and the method comprises: The nucleic acid fragments are phosphorylated at the 5' end under the action of T4 phosphokinase; The 5'-end phosphorylation product is subjected to 3'-end phosphorylation treatment under the action of terminal transferase; The 3'-end phosphorylation product and the adapter are ligated under the action of T4 RNA ligase 1 and RtcB ligase; The ligation products are amplified by PCR to obtain a sequencing library.

15. The method according to claim 13, characterized in that The nucleic acid fragment is an RNA fragment, and the method comprises: The nucleic acid fragments are subjected to 5'-end phosphorylation treatment and 3'-end phosphorylation treatment under the catalysis of T4 phosphokinase and polyadenylic acid polymerase or polyguanylic acid polymerase; The phosphorylation product and the adapter are connected under the action of T4 RNA ligase 1 and RtcB ligase; The ligation products were reverse transcribed and PCR amplified to obtain a sequencing library.

16. The method according to claim 13, characterized in that The nucleic acid fragments are a mixture of DNA fragments and RNA fragments, and the method comprises: The nucleic acid fragment is subjected to 5'-end phosphorylation treatment and 3'-end phosphorylation treatment under the catalysis of T4 phosphokinase and terminal transferase and polyadenylic acid polymerase or polyguanylic acid polymerase; The phosphorylation product and the adapter are connected under the action of T4 RNA ligase 1 and RtcB ligase; The ligation products were reverse transcribed and PCR amplified to obtain a sequencing library.

17. The method according to claim 13, characterized in that The nucleic acid fragment is a DNA fragment or an RNA fragment or a mixture of DNA and RNA fragments, and the method comprises: Dephosphorylating the nucleic acid under the action of phosphatase; The dephosphorylated product was phosphorylated at the 3' end by T4 RNA ligase 1; The 3'-end phosphorylation product is phosphorylated at the 5' end under the action of T4 phosphokinase; The 5'-end phosphorylation product and the adapter are ligated under the action of T4 RNA ligase 1 and RtcB ligase; The ligation products were reverse transcribed and PCR amplified to obtain a sequencing library.

18. The method according to claim 13, characterized in that The nucleic acid fragment is a mixture of DNA fragments and RNA fragments, and the method comprises: The nucleic acid fragment is subjected to 5'-end phosphorylation treatment and 3'-end phosphorylation treatment under the catalysis of T4 phosphokinase and terminal transferase and polyadenylic acid polymerase or polyguanylic acid polymerase; The phosphorylated product and the adapter are connected to the 5-end adapter under the action of T4 RNA ligase 1; The ligated product is subjected to ligation treatment under the action of T4 phosphokinase and T4 RNA ligase 1 or a truncated version of T4 RNA ligase 2; The ligation products were reverse transcribed and PCR amplified to obtain a sequencing library.

19. The method according to any one of claims 15 to 18, characterized in that: The reverse transcription is carried out under the catalysis of reverse transcriptase.

20. The method according to claim 19, characterized in that The reverse transcriptase is selected from at least one of SuperScript II reverse transcriptase and Alpha reverse transcriptase.

21. The method according to claim 14, characterized in that The RtcB ligase catalyzed reaction is carried out in a reaction system containing triphosphate and divalent manganese ions.

22. The method according to claim 21, characterized in that The triphosphate is selected from at least one of GTP, dGTP or ATP.

23. The method according to claim 21, characterized in that The RtcB ligase catalyzed reaction is carried out at 15-85° C. for 15-120 min.

24. The method according to any one of claims 14 or 16 to 18, characterized in that: The DNA fragments include interrupted genomic DNA molecules, extracellular free DNA molecules, degraded DNA molecules and / or sulfite-treated DNA molecules.

25. The method according to claim 14, characterized in that After the ligation treatment and before the amplification treatment, the method further comprises subjecting the ligation treatment product to a single-stranded circularization reaction in the presence of a single-stranded circularization agent to obtain a circularized product.

26. A method for phosphorylation modification of the 3' end of a nucleic acid, characterized in that: include: The 3' end of the nucleic acid is ligated with a deoxyribonucleotide or a ribonucleotide, wherein the 3-carbon atom of the deoxyribonucleotide or the ribonucleotide is linked with a phosphate group.

27. The method according to claim 26, characterized in that The nucleic acid is a single-stranded DNA, and the connection process is performed under the catalysis of terminal transferase.

28. The method according to claim 26, characterized in that The nucleic acid is RNA, and the ligation process is performed under the catalysis of polyadenylic acid polymerase or polyguanylic acid polymerase.

29. The method according to claim 26, characterized in that The nucleic acid is a mixture of single-stranded DNA and RNA, and the connection process is performed under the catalysis of terminal transferase and polyadenylic acid polymerase or polyguanylic acid polymerase.

30. The method according to claim 26, characterized in that The nucleic acid is a single-stranded DNA, RNA or a mixture of single-stranded DNA and RNA, and the ligation process is performed under the catalysis of T4 RNA ligase 1.

31. The method according to any one of claims 27 to 29, characterized in that: The 5' end of the nucleic acid is linked to a phosphate group.

32. The method according to claim 31, characterized in that The nucleic acid with a phosphate group connected to the 5' end is obtained by catalyzing the nucleic acid with T4 phosphokinase.

33. The method according to claim 30, characterized in that The 5' end of the nucleic acid is -OH.

34. The method according to claim 33, characterized in that The nucleic acid with -OH at the 5' end is obtained by subjecting the nucleic acid to the catalysis of phosphatase.

35. A kit for constructing a 3'-terminal phosphorylation-modified nucleic acid, characterized in that: The kit comprises a deoxyribonucleotide or a ribonucleotide with a phosphate group connected to the 3-carbon atom.

36. The kit according to claim 35, characterized in that The kit further comprises at least one selected from reverse transcriptase, terminal transferase, polyadenylic acid polymerase, polyguanylic acid polymerase, T4 RNA ligase 1, T4 phosphokinase or phosphatase.

37. Use of the kit according to claim 35 or 36 in constructing a nucleic acid with phosphorylation modification at the 3' end.

38. A kit for constructing a sequencing library, characterized in that: The kit comprises a deoxyribonucleotide or a ribonucleotide with a phosphate group connected to the 3-carbon atom and an RtcB ligase.

39. The kit according to claim 38, characterized in that The kit further comprises at least one selected from reverse transcriptase, terminal transferase, polyadenylic acid polymerase, polyguanylic acid polymerase, T4 RNA ligase 1, T4 phosphokinase or phosphatase.

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