Single-stranded nucleic acid molecule sequencing library construction method

Abstract

The invention provides a single-stranded nucleic acid molecule sequencing library construction method which comprises the following steps: (1) connecting the 3'end of a single-stranded nucleic acid molecule with a 3 '-end joint so as to obtain a connection product; (2) carrying out extension and amplification reaction on a first primer, DNA polymerase, reverse transcriptase and the connection product so as to obtain amplification products of which the 3'end and the 5 'end are respectively connected with joints, and forming the sequencing library by the amplification products; wherein the reverse transcriptase has terminal transferase activity. The sequencing library obtained by using the sequencing library construction method simultaneously contains a 3'end joint and a 5 'end joint with known sequences, the joint connection efficiency is high, the original template loss is less, the utilization rate is high, the reaction time is short, the cost is low, and the sequencing library construction method is suitable for wide application.

Description

Method for constructing single-stranded nucleic acid molecule sequencing library Technical Field

[0001] The present invention relates to the biological field, and in particular to a method for constructing a single-stranded nucleic acid molecule sequencing library. Background Art

[0002] Currently, the most common method for DNA sequencing library construction is to use double-stranded DNA to construct libraries. The starting material generally consists of sufficient, intact, undegraded genomic DNA, or DNA with minimal degradation and damage. The general steps for double-stranded library construction are to first fragment the DNA into fragments of appropriate length, then perform end-repair, and then connect double-stranded adapters to the ends of the DNA fragments using a simple ligase reaction. PCR is then performed to generate the library required for sequencing.

[0003] However, double-stranded DNA library construction has certain limitations for samples such as formalin-fixed and paraffin-embedded tissue samples, forensic specimens, and paleontological fossils. The DNA extracted from these samples is severely degraded and suffers DNA strand damage, 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 very low. Furthermore, in methylation sequencing, DNA is typically subjected to bisulfite conversion, a process in which prolonged exposure to acidic and high temperatures severely damages the DNA, leading to the loss of most DNA molecules and fragmentation into short, single-stranded fragments. Furthermore, free extracellular DNA in plasma and other body fluid samples is inherently short and present in very low concentrations. Conventional double-stranded adapter ligation library construction methods are unsuitable for these specialized samples. This is because conventional library construction methods only utilize intact double-stranded DNA molecules. Adaptors are rarely or impossible to attach to template molecules other than intact double-stranded DNA, resulting in the loss of a significant amount of the original template molecules. In addition to double-stranded library construction methods, there are also library construction methods for single-stranded nucleic acids. These single-stranded DNA template molecules can be used for library construction, minimizing the loss of these incomplete double-stranded DNA template molecules, maximizing template utilization, increasing library construction efficiency, and improving both the effective amount of sequencing data and data quality. Single-stranded library construction technology can also better utilize samples and can be used in a variety of applications, such as liquid biopsies for extracellular free DNA (cfDNA) methylation sequencing, genomic research on paleontological DNA, forensic identification, and tumor detection in FFPE samples.

[0004] 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 and terminal transferase plus adapter method.

[0005] Traditional single-stranded library construction methods use T4 RNA ligase to directly ligate single-stranded sequencing adapters onto single-stranded DNA template molecules. Typically, T4 RNA ligase 2 (truncated) is used to catalyze the ligation of pre-adenylated 5'-end DNA adapters to the 3' end of the single-stranded template. The 3' end of the adapter is blocked to minimize the formation of adapter dimers. After purification, a 5'-end adapter and T4 RNA ligase are added, and the 5' end is ligated to the template to which the 3'-end adapter has been attached. After both ends of the adapter have been added, PCR amplification is performed. If a truncated adapter was previously ligated, PCR can also be used to complete the remaining adapter. To create a single-stranded circular library, single-stranded denaturation is required, followed by circular ligation using DNA ligase and oligonucleotide chains that are partially complementary to the adapters at both ends.

[0006] 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 sequencing adapter with random primers. Amplification is then performed using primers complementary to the 3'-end adapter primer binding sequence, converting the single-stranded template into a double-stranded sequence. A conventional adapter ligation reaction then adds the 5'-end sequencing adapter to the 5' end of the newly synthesized chain to complete library construction. If a single-stranded circular library is to be created, single-strand denaturation is required, followed by circular ligation using DNA ligase and oligonucleotide chains complementary to the adapters at both ends.

[0007] The random primer single-strand library construction technique uses 6-8 nt random primers to bind to a single-stranded DNA template and extend it to synthesize a double-stranded DNA. A sequencing adapter is then added to the 5' end of the newly synthesized chain to complete the library construction. If a single-stranded circular library is to be created, the single strand must be denatured and then circularized using DNA ligase and oligonucleotide chains with complementary adapters at both ends.

[0008] Terminal transferase (TdT) is similar to random-primed single-strand library construction. First, a terminal transferase is used to add consecutive bases to the 3' end of the single-stranded DNA template. The consecutive sequence is then used as a primer binding site for extension, converting the single-stranded DNA template into a double-stranded DNA template. A 5' sequencing adapter is then added to the 5' end of the newly synthesized strand to complete the library construction. To create a single-stranded circular library, single-strand denaturation is required, followed by circular ligation using DNA ligase and oligonucleotide chains with complementary adapters at both ends.

[0009] However, traditional single-stranded library construction methods use T4 RNA ligase 2 (truncated form) and require DNA adapters with 5'-end pre-adenylation modifications. These 5'-end pre-adenylation modifications are expensive and difficult to synthesize, with only a very small number of manufacturers able to produce them, resulting in high costs. Furthermore, the adapters are ligated separately at both ends and require multiple purifications, resulting in low template utilization and lengthy library construction times.

[0010] Adaptase single-strand library construction technology, random primer plus adapter method, and terminal transferase plus adapter method 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, the random primer method, due to the use of random primers, can also generate unpredictable products, resulting in a decrease in the proportion of valid libraries and thus reducing the effective sequencing data.

[0011] Therefore, the method of constructing single-stranded DNA libraries still needs to be studied.

[0012] Summary of the Invention

[0013] The present invention aims to, at least to some extent, address at least one of the technical problems existing in the prior art. The present invention provides a method for constructing a single-stranded nucleic acid molecule sequencing library, a sequencing library, a sequencing method, an adapter and a kit for constructing a single-stranded nucleic acid molecule sequencing library, and uses thereof. The sequencing library obtained using the sequencing library construction method of the present invention contains both 3'-end adapters and 5'-end adapters of known sequences, and exhibits high adapter ligation efficiency, minimal loss of original template, high utilization rate, short reaction time, and low cost, making it suitable for a wide range of applications.

[0014] In one aspect of the present invention, the present invention provides a method for constructing a single-stranded nucleic acid molecule sequencing library. According to an embodiment of the present invention, the method comprises: (1) connecting the 3' end of the single-stranded nucleic acid molecule to a 3' end adapter to obtain a connection product; (2) extending and amplifying the connection product with a first primer, a DNA polymerase, and a reverse transcriptase to obtain an amplification product with adapters connected to the 3' end and the 5' end, respectively, and the amplification product constitutes the sequencing library; wherein the 3' end adapter comprises: a main chain, the main chain is used to connect to the 3' end of the single-stranded nucleic acid molecule; an auxiliary chain, the 3' end of the auxiliary chain contains multiple N bases, the multiple N bases are used to hybridize with the 3' end of the single-stranded nucleic acid molecule; the 5' end sequence of the auxiliary chain is reverse complementary to at least a portion of the sequence of the 5' end of the main chain; the reverse transcriptase has terminal transferase activity, and the 3' end of the resulting chain obtained by the extension reaction has multiple identical base sequences; the first primer comprises a 5' end adapter sequence and a complementary sequence, and the complementary sequence is reverse complementary to the multiple identical base sequences at the 3' end of the resulting chain.

[0015] In the method according to an embodiment of the present invention, the 3' end adapter is first connected to the single-stranded nucleic acid molecule to obtain a connection product. Then, the hydroxyl group at the 3' end of the auxiliary chain is used as the starting point and extended under the action of reverse transcriptase to obtain a double-stranded nucleic acid, wherein the 3' end of the generated chain in the double-stranded nucleic acid has multiple identical base sequences. Since the auxiliary chain and the main chain are at least partially reverse complementary in sequence, the first primer includes a 5' end adapter sequence and a complementary sequence that is reverse complementary to the multiple identical base sequences at the 3' end of the generated chain. Therefore, the double-stranded nucleic acid is amplified using the first primer and the 3' end adapter under the action of DNA polymerase to obtain a sequencing library.

[0016] In addition, the present invention designs the 3' end adapter to be a local double-stranded structure, so that it can be stably connected to the 3' end of the single-stranded nucleic acid molecule, thereby improving the adapter connection efficiency and avoiding the occurrence of adapter self-ligation.

[0017] As a result, the sequencing library obtained using the method of the present invention contains both 3'-end adapters and 5'-end adapters of known sequence. The extension and amplification reactions are performed in a single tube, requiring only a single step. This increases adapter ligation efficiency and reduces original template loss, ultimately improving original template utilization and shortening reaction time. Furthermore, the sequencing adapters do not require special modifications or expensive special ligases, thereby reducing costs and making them suitable for widespread application.

[0018] In another aspect, the present invention provides a sequencing method. According to an embodiment of the present invention, the sequencing method comprises: constructing a sequencing library according to the method described above; and sequencing the sequencing library.

[0019] In yet another aspect, the present invention provides an adapter for constructing a single-stranded nucleic acid molecule sequencing library. According to an embodiment of the present invention, the adapter comprises: a backbone for attaching to the 3' end of a single-stranded nucleic acid molecule; an auxiliary chain, the 3' end of the auxiliary chain comprising a plurality of N-bases for hybridizing with the 3' end of the single-stranded nucleic acid molecule; and a 5' end sequence of the auxiliary chain that is reverse complementary to at least a portion of the 5' end sequence of the backbone.

[0020] In another aspect of the present invention, a kit for constructing a single-stranded nucleic acid molecule sequencing library is provided. According to an embodiment of the present invention, the kit includes the aforementioned adapter.

[0021] In another aspect of the present invention, the present invention provides the use of the aforementioned linker or kit in constructing a single-stranded nucleic acid molecule sequencing library or sequencing.

[0022] 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

[0023] 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:

[0024] 1 to 3 respectively show a schematic flow chart of a method for constructing a single-stranded nucleic acid molecule sequencing library according to an embodiment of the present invention. DETAILED DESCRIPTION

[0025] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0026] The present invention provides a method for constructing a single-stranded nucleic acid molecule sequencing library, a sequencing library, a sequencing method, a linker and a kit for constructing a single-stranded nucleic acid molecule sequencing library, and uses thereof, which are described in detail below.

[0027] Method for constructing single-stranded nucleic acid molecule sequencing library

[0028] In one aspect, the present invention provides a method for constructing a single-stranded nucleic acid molecule sequencing library. According to an embodiment of the present invention, the method includes: S100 linker ligation, S200 extension and amplification reaction, each of which will be described in detail below.

[0029] S100 Connector

[0030] In this step, the 3' end of the single-stranded nucleic acid molecule is connected to the 3' end adapter to obtain a ligation product, so that the final sequencing library contains the 3' end adapter.

[0031] According to an embodiment of the present invention, the 3' end connector includes: a main chain, which is used to connect to the 3' end of the single-stranded nucleic acid molecule; an auxiliary chain, the 3' end of the auxiliary chain contains multiple N bases, and the multiple N bases are used to hybridize with the 3' end of the single-stranded nucleic acid molecule; the 5' end sequence of the auxiliary chain is reverse complementary to at least part of the sequence of the 5' end of the main chain.

[0032] If an adapter is directly attached to a single-stranded nucleic acid molecule, the ligation efficiency is low and unstable, and the adapter is prone to self-ligation. Furthermore, the inventors of the present invention designed the 3'-end adapter to be a partially double-stranded structure. Specifically, the main chain is the 3'-end adapter of the final amplification product; the 3' end of the auxiliary chain contains multiple N bases, which can hybridize with the 3' end of the template chain, and the 5' end sequence of the auxiliary chain is reverse complementary to at least part of the 5' end sequence of the main chain. As a result, the design of the auxiliary chain can stably bind the entire main chain to the template chain, improve the adapter ligation efficiency, and avoid adapter self-ligation.

[0033] Traditional single-stranded library construction methods typically use T4 RNA ligase 2 (truncated form) to catalyze the ligation of 5'-end pre-adenylated DNA adapters to the 3' end of single-stranded templates. The 3' end of the adapter is blocked to reduce the formation of adapter dimers. T4 RNA ligase 2 (truncated form) can only catalyze reactions with adenylated DNA. This method utilizes the backbone and auxiliary strands to form a partial double-strand with the template DNA, allowing direct use of DNA ligase (such as T4 DNA ligase), without the need for additional special modification of the adapters.

[0034] According to an embodiment of the present invention, the 3' end of the main chain is pre-blocked to reduce the generation of linker dimers. Specifically, the blocking treatment includes phosphorylation, C3-spacer modification, C5-spacer modification, or amino modification.

[0035] According to an embodiment of the present invention, the main chain and the auxiliary chain are provided in an independent form. Before performing step (1), the main chain and the auxiliary chain are annealed so as to partially hybridize the main chain and the auxiliary chain to form a locally double-stranded 3' end joint.

[0036] According to an embodiment of the present invention, the number of bases N is selected from an integer of 6 to 12. N represents a random base. When the number is 6 to 12, the auxiliary chain can stably bind to the template chain, thereby improving the efficiency of the linker connection and avoiding linker self-ligation.

[0037] According to an embodiment of the present invention, the single-stranded nucleic acid molecule is selected from an RNA molecule or a single-stranded DNA molecule.

[0038] According to an embodiment of the present invention, the single-stranded DNA molecules are derived from interrupted genomic DNA molecules, extracellular free DNA molecules, degraded DNA molecules and / or sulfite-treated DNA molecules.

[0039] According to an embodiment of the present invention, for a sample containing both double-stranded nucleic acid molecules and single-stranded nucleic acid molecules, the sample is denatured before performing step (1) to obtain a sample consisting entirely of single-stranded nucleic acid molecules.

[0040] According to an embodiment of the present invention, in step S100, T4 DNA ligase is used to connect the 3' end of the single-stranded nucleic acid molecule to the 3' end adapter. Traditional single-stranded library construction methods are based on T4 RNA ligase to directly connect single-stranded sequencing adapters to single-stranded DNA template molecules. Specifically, T4 RNA ligase 2 (truncated) is generally used to catalyze the connection of the 5' end pre-adenylated DNA adapter to the 3' end of the single-stranded template, and the 3' end of the adapter is blocked to reduce the production of adapter dimers. T4 RNA ligase 2 (truncated) can only catalyze the reaction of DNA with adenylation. T4 RNA ligase 2 (truncated) is expensive.

[0041] This method uses the main chain and auxiliary chain to form a local double strand with the template DNA, and can be directly used with T4 DNA ligase. T4 DNA ligase is the most commonly used ligase and is low in cost.

[0042] S200 extension and amplification

[0043] In this embodiment, a first primer, a DNA polymerase, and a reverse transcriptase are subjected to an extension and amplification reaction with the ligation product to obtain an amplification product having adapters connected to the 3' and 5' ends, respectively, and the amplification product constitutes the sequencing library, wherein the reverse transcriptase has terminal transferase activity, and the 3' end of the generated chain obtained by the extension reaction has multiple identical base sequences; the first primer includes a 5' end adapter sequence and a complementary sequence, and the complementary sequence is reverse complementary to the multiple identical base sequences at the 3' end of the generated chain.

[0044] According to an embodiment of the present invention, the 5' end sequence of the auxiliary chain is reverse complementary to the 3' end sequence of the main chain, and the 3' end of the auxiliary chain is pre-blocked by modification with a reversible blocking group. Under this condition, step S200 includes: removing the reversible blocking group on the 3' end of the auxiliary chain so that the 3' end of the auxiliary chain is a hydroxyl group; starting with the 3' end hydroxyl group of the auxiliary chain, extending under the action of a reverse transcriptase to obtain a double-stranded nucleic acid, wherein the 3' end of the generated chain in the double-stranded nucleic acid has multiple identical base sequences; using the first primer and the auxiliary chain under the action of a DNA polymerase to amplify the double-stranded nucleic acid to obtain an amplified product, and the amplified product constitutes the sequencing library.

[0045] It should be noted that the "double-stranded nucleic acid" described in the present invention can refer to both double-stranded DNA and RNA-DNA hybrids. Specifically, when the single-stranded nucleic acid molecule is DNA, it is extended starting from the 3'-terminal hydroxyl group of the auxiliary strand under the action of reverse transcriptase to obtain double-stranded DNA; when the single-stranded nucleic acid molecule is RNA, it is extended starting from the 3'-terminal hydroxyl group of the auxiliary strand under the action of reverse transcriptase to obtain RNA-DNA hybrids.

[0046] According to a specific embodiment of the present invention, when the single-stranded nucleic acid molecule to be tested is RNA, the 5' end sequence of the auxiliary chain is reversely complementary to the 3' end sequence of the main chain, and the 3' end of the auxiliary chain is pre-blocked by modification with a reversible blocking group. Under this condition, step S200 includes: removing the reversible blocking group on the 3' end of the auxiliary chain so that the 3' end of the auxiliary chain is a hydroxyl group; starting with the 3' end hydroxyl group of the auxiliary chain, extending under the action of reverse transcriptase to obtain an RNA-DNA hybrid chain, wherein the 3' end of the DNA generated chain in the RNA-DNA hybrid chain has multiple identical base sequences; using the first primer and the 3' end adapter to extend the RNA-DNA hybrid chain under the action of DNA polymerase, replacing the RNA chain to obtain a double-stranded DNA amplification product, and the amplification product constitutes the sequencing library.

[0047] The specially designed 3'-end adapter structure of the present invention not only stably binds to the template strand, improving adapter ligation efficiency and preventing adapter self-ligation, but also allows direct extension from the 3' end of the auxiliary strand to generate double-stranded DNA. Because the first primer includes a 5'-end adapter sequence and a complementary sequence that is reverse complementary to multiple identical base sequences at the 3' end of the generated strand, the auxiliary strand of the 3'-end adapter is at least partially complementary to the main strand. Therefore, the first primer and 3'-end adapter can be used to amplify double-stranded DNA to generate a sequencing library.

[0048] According to an embodiment of the present invention, the blocking treatment includes phosphorylation, 3'-O-propylene modification, or 3'-O-azidomethyl modification. Thus, the blocking treatment can prevent the linker from self-ligating and reversibly remove the blocking group, thereby allowing auxiliary chain extension.

[0049] According to an embodiment of the present invention, the 5' end sequence of the auxiliary chain is partially reverse complementary to the 3' end sequence of the main chain; the step (2) further comprises: extending and amplifying the connection product with a first primer, a second primer, a DNA polymerase, and a reverse transcriptase to obtain an amplification product with a connector connected to the 3' end and the 5' end respectively, and the amplification product constitutes the sequencing library; wherein the sequence of the second primer is at least reverse complementary to the 3' end sequence of the main chain.

[0050] Under these conditions, step S200 includes: hybridizing the second primer with the ligation product and performing an extension reaction under the action of a reverse transcriptase to obtain a generated chain, wherein the 3' end of the generated chain has multiple identical base sequences; using the generated chain as a template chain, hybridizing the first primer with the multiple identical base sequences at the 3' end of the generated chain and performing amplification under the action of a DNA polymerase to obtain a double-stranded nucleic acid; and amplifying the double-stranded nucleic acid using the second primer and the first primer under the action of a DNA polymerase to obtain an amplified product, wherein the amplified product constitutes the sequencing library.

[0051] Using the ligation product obtained in step S100 as a template, the sequence of the second primer is at least reverse complementary to the 3' end sequence of the main chain, so the second primer will hybridize with the template chain and extend toward the 5' end of the template chain under the action of the reverse transcriptase. When it reaches the 5' end, due to the terminal transferase activity of the reverse transcriptase, multiple identical base sequences can be formed at the 3' end of the generated chain.

[0052] Because the first primer includes a 5'-end adapter sequence and a complementary sequence that is reverse complementary to the multiple identical base sequences at the 3' end of the generated strand, it can hybridize with the generated strand and be extended by DNA polymerase to produce a double-stranded nucleic acid. The first and second primers are then used to amplify the double-stranded nucleic acid under the action of DNA polymerase to produce a sequencing library.

[0053] According to an embodiment of the present invention, the 3' end of the auxiliary chain is pre-blocked; the blocking treatment includes phosphorylation, 3'-O-propylene modification, or 3'-O-azidomethyl modification, thereby preventing the linker from self-ligating.

[0054] According to an embodiment of the present invention, the number of the multiple identical bases is 1 to 3.

[0055] According to an embodiment of the present invention, the bases in the plurality of identical bases are C bases.

[0056] According to an embodiment of the present invention, the backbone further includes at least one of the following: a binding sequence of a sequencing primer, a tag sequence, and a binding sequence of a universal amplification primer.

[0057] According to an embodiment of the present invention, the tag sequence length is 5 to 12 bp.

[0058] According to an embodiment of the present invention, the reverse transcriptase is selected from MMLV reverse transcriptase. In some embodiments, the reverse transcriptase is selected from SuperScript II reverse transcriptase or Alpha reverse transcriptase. These reverse transcriptases all have terminal transferase activity and can form multiple identical base sequences at the 5' end of the generated strand, facilitating subsequent complementary hybridization with the first primer sequence.

[0059] According to an embodiment of the present invention, the DNA polymerase is selected from Q5 hot start high-fidelity DNA polymerase, KAPA hot start high-fidelity DNA polymerase, Platinum hot start high-fidelity DNA polymerase or Pfu DNA polymerase, thereby achieving efficient amplification.

[0060] According to an embodiment of the present invention, the extension reaction and the amplification reaction are performed in one step in the same reaction system, thereby shortening the reaction time and improving the library construction efficiency.

[0061] According to an embodiment of the present invention, step S200 further includes: denaturing the amplified product to obtain single-stranded DNA; and 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, wherein the circularized product constitutes a single-stranded circular sequencing library. Specifically, the single-stranded circularization reagent is selected from DNA ligase.

[0062] According to an embodiment of the present invention, referring to Fig. 2 and Fig. 3, the single-stranded nucleic acid molecule sequencing library construction method includes the step of connection and amplification of 3' end joints. Specifically, the joint connection step is to utilize T4 DNA ligase to connect the joint of the local double chain to the 3' end of the single-stranded DNA template. Wherein the joint includes a main chain and an auxiliary chain, and the 3' end of the auxiliary chain has a random base sequence of 6 N to 12 N, which can be hybridized and complementary with the 3' end of the single-stranded DNA template, and its 5' end is reversely complementary to the 5' end partial sequence of the main chain. Like this, a local double chain is formed between the auxiliary chain and the main chain and the single-stranded template. The 3' end of the main chain can utilize phosphorylation, C3-spacer, C5-spacer to modify or amino modification to be blocked, and the 3' end of the auxiliary chain also can be blocked accordingly.

[0063] After the adapter is attached, amplification is performed. This involves first extending the single-stranded template using a sequence complementary to the main strand (the 3' adapter) as primer 1 and reverse transcriptase. After extension to the 5' end of the single-stranded template, the terminal transferase activity of the reverse transcriptase adds three cytosine bases. Primer 2 (containing the sequence of the 5' adapter and a 3' end with three Gs) is then used to hybridize with the resulting strand from the previous step, and extension is continued using primer 2 as a template. PCR amplification of the double-stranded nucleic acid from the previous step is then performed using DNA polymerase and primers 1 and 2.

[0064] This allows the addition of 5'-end adapters and the simultaneous PCR amplification reaction in a single-step reaction, yielding a library with 2-end sequencing adapters. For the Illumina platform, after amplification, purification is performed to obtain the library. For the DNBSEQ platform, the PCR amplification products are denatured and quenched at high temperature to become single-stranded. The single-stranded DNA is then circularized, and the uncircularized molecules are digested with a linear digestion enzyme to obtain a circular library suitable for single-stranded DNA.

[0065] The specific steps are as follows:

[0066] 1. After intact genomic DNA is sheared, the fragments are screened to 100-500 bp in size, or free extracellular DNA is extracted, or degraded DNA is extracted from various degraded biological samples (such as paleontological fossils, FFPE samples). DNA fragments after intact genomic DNA shearing or free extracellular DNA are purified and recovered after sulfite treatment.

[0067] 2. Denature the DNA sample to convert the double-stranded DNA in the sample into single-stranded DNA. This is usually done at high temperature (usually 92-98°C for 3-10 minutes). Immediately place the sample on ice to prevent the melted double-stranded DNA from renaturing.

[0068] 3. After denaturation, the DNA sample is ligated to the 3' end with an adapter. First, the auxiliary strand is annealed to the backbone to form a partially double-stranded adapter. T4 DNA ligase is then used to ligate the single-stranded DNA template to the adapter. This results in a 3' end adapter attached to the single-stranded DNA template. The adapter sequence can be compatible with the 5' and 3' sequencing adapters of the Illumina or DNBSEQ platforms.

[0069] 4. After the first step is completed, the adapter-added DNA is purified. Using PCR primer 1 complementary to the 3'-end adapter sequence and a 5'-end sequencing adapter sequence with three Gs as primer 2, reverse transcriptase, and DNA polymerase, the single-stranded DNA obtained in the previous step is used as a template for simultaneous extension and PCR amplification reactions. The 3-12 nucleotides at the 3' end of primer 1 are modified with deoxyribonucleotides or locked nucleotides, and the three Gs at the 3' end of primer 2 are also modified with deoxyribonucleotides or locked nucleotides. The 5' end of primer 2 is phosphorylated.

[0070] 5. After completing the PCR reaction in the previous step, purify the PCR product. The single-stranded circular DNA library of the DNBSEQ platform requires high-temperature denaturation of the purified product (generally 92-98°C for 3-10 minutes). Immediately place on ice after denaturation to prevent the melted double-stranded DNA from renaturing.

[0071] 6. Use ligase to directly circularize the denatured product into a single-stranded molecule. The circularized single-stranded DNA ring contains the template and the sequencing adapter required for sequencing.

[0072] 7. Use linear digestion enzyme to digest the circularized product to obtain the final single-stranded DNA circular library.

[0073] Sequencing methods

[0074] In another aspect, the present invention provides a sequencing method. According to an embodiment of the present invention, the sequencing method comprises: constructing a sequencing library according to the aforementioned method; and sequencing the sequencing library. This method can effectively obtain more accurate nucleic acid sequence data, providing more favorable support for subsequent analysis. Furthermore, the method is simple to operate, time-efficient, and low-cost, making it suitable for widespread application.

[0075] It should be noted that the features and advantages described above for the method for constructing a single-stranded nucleic acid molecule sequencing library are also applicable to this sequencing method and will not be repeated here.

[0076] Adapters for constructing single-stranded nucleic acid molecule sequencing libraries

[0077] In yet another aspect, the present invention provides an adapter for constructing a single-stranded nucleic acid molecule sequencing library. According to an embodiment of the present invention, the adapter comprises: a backbone, which is configured to connect to the 3' end of a single-stranded nucleic acid molecule; an auxiliary chain, wherein the 3' end of the auxiliary chain comprises a plurality of N-bases, which are configured to hybridize with the 3' end of the single-stranded nucleic acid molecule; and a 5' end sequence of the auxiliary chain that is reverse complementary to at least a portion of the 5' end sequence of the backbone chain.

[0078] The linker according to an embodiment of the present invention can form a local double-stranded structure, where the main chain is the 3' end linker of the final amplified product, the 3' end of the auxiliary chain contains multiple N bases, which can hybridize with the 3' end of the template chain, and the 5' end sequence of the auxiliary chain is reverse complementary to at least part of the 5' end sequence of the main chain. Therefore, the design of the auxiliary chain can make the entire main chain stably bound to the template chain, improve the linker connection efficiency, and avoid the occurrence of linker self-ligation.

[0079] According to an embodiment of the present invention, the number of bases N is selected from an integer of 6 to 12. N represents a random base. When the number is 6 to 12, the auxiliary chain can stably bind to the template chain, thereby improving the efficiency of the linker connection and avoiding linker self-ligation.

[0080] According to an embodiment of the present invention, the 3' ends of the main chain and the auxiliary chain have blocking group modifications. Specifically, the 3' end of the main chain has phosphorylation modification, C3-spacer modification, C5-spacer modification or amino modification, thereby, it is possible to reduce the generation of joint dimers. The 3' end of the auxiliary chain has phosphorylation modification, 3'-O-propylene modification or 3'-O-azidomethyl modification, thereby, it is possible to reduce the generation of joint dimers. After connecting the joint, the above-mentioned modifying group can be removed so that the 3' end of the auxiliary chain is hydroxyl, thereby it is possible to extend with the 3' end of the auxiliary chain as a starting point to generate a double-stranded nucleic acid.

[0081] According to an embodiment of the present invention, the backbone further includes at least one of the following: a binding sequence of a sequencing primer, a tag sequence, and a binding sequence of a universal amplification primer.

[0082] According to an embodiment of the present invention, the tag sequence length is 5 to 12 bp.

[0083] Kit for constructing single-stranded nucleic acid molecule sequencing library

[0084] In yet another aspect, the present invention provides a kit for constructing a single-stranded nucleic acid molecule sequencing library. According to an embodiment of the present invention, the kit includes the aforementioned adapter. Thus, the kit can be used to construct a single-stranded nucleic acid molecule library.

[0085] According to an embodiment of the present invention, the main chain and the auxiliary chain are provided in separate packages. Thus, when the linker is used, the main chain and the auxiliary chain can form a local double-stranded structure through annealing.

[0086] According to an embodiment of the present invention, the kit further comprises: a reverse transcriptase, a DNA polymerase, a DNA ligase, a second primer and a first primer; wherein the second primer sequence is reverse complementary to at least a portion of the sequence of the main chain; and the first primer comprises a 5' end linker sequence and a sequence containing multiple identical bases.

[0087] DNA ligase can ligate an adapter to the 3' end of a single-stranded nucleic acid molecule, producing a ligation product. Using this ligation product as the template strand, the second primer, whose sequence is reverse complementary to at least a portion of the adapter sequence, hybridizes to the template strand and is then extended toward the 5' end of the template strand by the action of reverse transcriptase. Once extended to the 5' end, the terminal transferase activity of reverse transcriptase allows the formation of multiple identical base sequences at the 3' end of the resulting strand.

[0088] Because the first primer includes a 5' adapter sequence and a complementary sequence that is the reverse complement of the multiple identical base sequences, it can hybridize with the generated strand and be extended by DNA polymerase to produce a double-stranded nucleic acid. The second primer and the first primer are then amplified by DNA polymerase to produce a sequencing library.

[0089] Thus, the nucleic acid molecules (amplification products) in the sequencing library obtained by using the kit of the present invention to construct the library contain both 3'-end adapters and 5'-end adapters, and the extension and amplification reactions are carried out in one tube, requiring only a one-step reaction, thereby increasing the efficiency of adding adapters and reducing the loss of the original template. Overall, the utilization rate of the original template is improved and the reaction time is shortened.

[0090] According to an embodiment of the present invention, the number of the multiple identical bases is 1 to 3.

[0091] According to an embodiment of the present invention, the bases in the plurality of identical bases are C bases.

[0092] According to an embodiment of the present invention, the reverse transcriptase is selected from MMLV reverse transcriptase. In some embodiments, the reverse transcriptase is selected from SuperScript II reverse transcriptase or Alpha reverse transcriptase. These reverse transcriptases all have terminal transferase activity and can form multiple identical base sequences at the 5' end of the generated strand, facilitating complementary hybridization with the first primer sequence.

[0093] According to an embodiment of the present invention, the DNA polymerase is selected from Q5 hot start high-fidelity DNA polymerase, KAPA hot start high-fidelity DNA polymerase, Platinum hot start high-fidelity DNA polymerase or Pfu DNA polymerase, thereby achieving efficient amplification.

[0094] According to an embodiment of the present invention, the DNA ligase is selected from T4 DNA ligase, thereby facilitating the connection of a linker to the 3' end of a single-stranded nucleic acid molecule with high ligation efficiency and low cost.

[0095] It should be noted that the features and advantages described above for the connector are also applicable to this kit and will not be repeated here.

[0096] use

[0097] In yet another aspect, the present invention provides the use of the aforementioned adapters or kits for constructing single-stranded nucleic acid sequencing libraries or sequencing. Sequencing libraries constructed using the adapters or kits of the present invention contain both 3'-end adapters and 5'-end adapters of known sequence, resulting in high adapter ligation efficiency, minimal loss of original template, high utilization rate, short reaction times, and low cost. Furthermore, sequencing using these sequencing libraries can effectively obtain more authentic nucleic acid sequencing data, providing more favorable support for subsequent analysis.

[0098] It should be noted that the features and advantages described above for the connector and the kit are also applicable to this use and will not be repeated here. Beneficial effects

[0099] The present invention only requires adding a 3' end adapter to the single-stranded DNA template, and then using reverse transcriptase to complete the 5' end adapter ligation and PCR amplification in a one-step reaction. This eliminates the need to add different sequencing adapters to both ends of the DNA, and only requires a single-step ligation reaction. Simultaneously, the addition of the 5' end adapter and the PCR amplification are performed in a single tube, increasing the efficiency of the adapter ligation and reducing the loss of the original template. Overall, it improves the utilization rate of the original template and reduces the reaction time. In addition, the sequencing adapter does not require the use of special modifications or special high-cost ligases, thereby reducing costs.

[0100] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0101] Example 1 Construction and sequencing of human peripheral blood free DNA methylation library

[0102] 1. Experimental Materials

[0103] Free DNA extracted from human peripheral blood plasma

[0104] 2. Experimental steps

[0105] 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.

[0106] 2. Use EZ DNA Methylation-Gold Kit (Zymo Research, Cat. No. D5005 / D5006) to treat and purify the adapter ligation product with Bisulfite.

[0107] 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 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 before use.

[0108] 4. Before opening the M-Wash Buffer for the first time, add the correct volume of anhydrous ethanol as indicated on the bottle label and mix thoroughly before use. Add the following ingredients to a new 200 μL PCR tube at room temperature.

[0109] Component Volume CT Conversion Reagent 130 μL cfDNA 20 μL Total 150 μL

[0110] 5. Place the 200 μL PCR tube on the PCR instrument and react according to the Bisulfite treatment conditions.

[0111] Temperature Time Heat Cover On 98℃ 10min 64℃ 2.5h 4℃ Hold

[0112] 6. Transfer the reaction product to a new 1.5 mL centrifuge tube, add 600 μL M-Binding Buffer, vortex 6 times, 3 seconds each time, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0113] 7. Place the Zymo-Spin IC Column into a 2 mL Collection Tube, transfer the mixed solution 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.

[0114] 8. Add 100 μL M-Wash Buffer to the Zymo-Spin IC Column and centrifuge at 13,000 rpm for 30 seconds.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 13. Slowly add 10 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.

[0120] 14. Place all Bisulfite-treated and purified products in a new 200 μL PCR tube and add molecular-grade water to make up the total volume to 20 μL.

[0121] 15. Prepare adapters by annealing and hybridizing 20 μM sequencing adapter 1 (main strand) and 20 μM sequencing adapter 2 (auxiliary strand) at a 1:1 ratio to form a partially double-stranded adapter mix. Reaction conditions: 95°C for 3 minutes, then cool to 4°C. Set the PCR instrument to a cooling rate of 0.1°C / s.

[0122] Sequence of sequencing adapter 1:

[0123] 5'Pho-AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAATAGGTCCGATCAACTCCTTGGCTCACA-3'Pho (SEQ ID NO: 1)

[0124] Sequence of sequencing adapter 2:

[0125] 5'-ACATGGCTACGATCCGACTTNNNNNNNNNNNN-3'Pho (SEQ ID NO: 2)

[0126] 16. Use T4 DNA ligase and ligation buffer from the MGI DNA library preparation kit (1000005254, MGI) to perform adapter ligation. Prepare the ligation reaction solution on ice according to the table below.

[0127] Component volume: T4 DNA ligase 1.6 μL, ligation buffer 23.4 μL, Adapter Mix 10 μL

[0128] 17. Add 35 μL of the prepared ligation reaction solution to the PCR tube containing the denatured DNA, mix thoroughly using a shaker, and then centrifuge briefly to centrifuge the liquid to the bottom of the tube.

[0129] 18. Place the centrifuged PCR tube on a PCR instrument and incubate at 30°C for 30 minutes for ligation reaction.

[0130] 19. After the ligation reaction is complete, transfer the reaction product to a 1.5 mL centrifuge tube. Then add 96 μL of purified magnetic beads, pipette 10 times to mix thoroughly, and incubate at room temperature for 5 minutes.

[0131] 20. After instant centrifugation, place the 1.5 mL centrifuge tube on a magnetic rack and let it stand for 5 minutes until the liquid becomes clear. Use a pipette to aspirate and discard the supernatant.

[0132] 21. Add 200 μL of 75% ethanol to a 1.5 mL centrifuge tube and let it stand for 30 seconds to wash the magnetic beads.

[0133] 22. Repeat the previous step.

[0134] 23. Try to drain the liquid in the tube. If there is a small amount of liquid remaining on the wall of the tube, centrifuge the 1.5mL centrifuge tube instantly. After separation on the magnetic stand, use a small-scale pipette to drain the liquid at the bottom of the tube.

[0135] 24. Keep the 1.5 mL centrifuge tube fixed on the magnetic rack, open the cap of the 1.5 mL centrifuge tube, and dry it at room temperature until the surface of the magnetic beads is non-reflective and free of cracks.

[0136] 25. Remove the 1.5 mL centrifuge tube from the magnetic stand, add 16 μL of molecular grade water to elute the DNA, and gently pipette 10 times to mix thoroughly.

[0137] 26. Incubate at room temperature for 5 minutes.

[0138] 27. Centrifuge briefly, place the 1.5 mL centrifuge tube on a magnetic rack, let it stand for 5 minutes until the liquid becomes clear, and transfer 15 μL of the supernatant to a new 200 μL PCR tube.

[0139] 28. Prepare the amplification reaction solution on an ice box according to the table below.

[0140]

[0141]

[0142] 29. Add 35 μL of the prepared amplification reaction solution to the PCR tube containing the purified DNA from step 27. Mix thoroughly using a shaker and centrifuge briefly to remove the liquid from the tube.

[0143] Primer1: 5'-TGTGAGCCAAGGAG / iXNA_T / / iXNA_T / / iXNA_G / -3' (SEQ ID NO: 3)

[0144] Pirmer2:

[0145] 5'Pho-GAACGACATGGCTACGATCCGACTT / iXNA_G / / iXNA_G / / iXNA_G / -3' (SEQ ID NO: 4) (iXNA_ represents a locked nucleotide modification)

[0146] 30. Place the PCR tube on the PCR plate and perform the reaction according to the procedure in the table below.

[0147]

[0148] 31. After the reaction is completed, centrifuge briefly to collect the liquid in the PCR tube to the bottom of the tube.

[0149] 32. Add 80ul of XP purified magnetic beads to the centrifuge tube, pipette 10 times to mix thoroughly, and incubate at room temperature for 10 minutes.

[0150] 33. After instant centrifugation, place the 1.5 mL centrifuge tube on a magnetic stand and let it stand for 5 minutes until the liquid becomes clear. Use a pipette to aspirate and discard the supernatant.

[0151] 34. Add 200 μl of 75% ethanol to a 1.5 mL centrifuge tube and let it stand for 30 seconds to wash the magnetic beads.

[0152] 35. Repeat the previous step. Aspirate as much liquid as possible from the tube. If a small amount remains on the tube wall, centrifuge the 1.5 mL tube briefly. After separation on a magnetic stand, use a small-range pipette to aspirate the liquid at the bottom of the tube.

[0153] 36. Keep the 1.5 mL centrifuge tube fixed on the magnetic rack, open the 1.5 mL centrifuge tube cap, and dry it at room temperature until the surface of the magnetic beads is non-reflective and free of cracks.

[0154] 37. Remove the 1.5 mL centrifuge tube from the magnetic stand, add 32 μL of molecular grade water to elute the DNA, and gently pipette 10 times to mix thoroughly.

[0155] 38. Incubate at room temperature for 5 minutes.

[0156] 39. Centrifuge briefly, place the 1.5 mL centrifuge tube on a magnetic rack, let it stand for 5 minutes until the liquid becomes clear, and transfer 30 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0157] 40. 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.

[0158] 41. Transfer 360 ng of PCR product to a new 0.2 mL PCR tube and add TE Buffer to a total volume of 48 μL.

[0159] 42. Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in the table below.

[0160] Temperature time 105℃ hot cover On 95℃ 3min

[0161] 43. After the reaction is completed, immediately place the PCR tube on ice and let it stand for 2 minutes.

[0162] 44. Use the cyclization kit from MGI (MGI, 1000005260) to prepare the cyclization reaction solution on ice according to the recipe in the table below in advance.

[0163] Components Single Reaction Volume (μL) Splint Buffer 11.6 DNA Rapid Ligase 0.5 Total Volume 12.1

[0164] 45. Use a pipette to draw 12.1 μL of the prepared single-stranded circularization reaction solution into the PCR tube in step 43. Vortex 3 times for 3 seconds each time, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0165] 46. ​​Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in the table below.

[0166] Temperature Time 75℃ Hot Cover On 37℃ 30min 4℃ Hold

[0167] 47. After the reaction is completed, centrifuge the PCR tube briefly and place it on ice, and immediately proceed to step 49.

[0168] 48. Use the circularization kit from MGI (MGI, 1000005260) to prepare the enzyme digestion reaction solution on ice in advance according to the recipe in the table below.

[0169] Components Single Reaction Volume (μL) Digestion Buffer 1.4 Digestion Enzyme 2.6 Total Volume 4

[0170] 49. Use a pipette to draw 4 μL of the prepared enzyme digestion reaction solution into the PCR tube in step 47. Vortex 3 times for 3 seconds each time, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0171] 50. Place the PCR tube on the PCR instrument and perform the reaction according to the conditions in the table below.

[0172] Temperature Time 75℃ Hot Cover On 37℃ 30min 4℃ Hold

[0173] 51. After the reaction is completed, centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0174] 52. Immediately add 7.5 μL Digestion Stop Buffer (from MGI circularization kit, MGI, 1000005260) to the PCR tube and vortex three times for 3 seconds each time. Centrifuge briefly to collect the reaction solution at the bottom of the tube. Pipette all the reaction solution into a new 1.5 mL centrifuge tube.

[0175] 53. Remove the DNA Clean Beads in advance and equilibrate them at room temperature for at least 30 minutes. Vortex thoroughly before use. Pipette 170 μL of DNA Clean Beads into the digested product. Gently pipette at least 10 times to resuspend all the beads. Be sure to pipette all the liquid and beads into the centrifuge tube with the last stroke.

[0176] 54. Incubate at room temperature for 10 minutes.

[0177] 55. Centrifuge the centrifuge tube briefly, place it on a magnetic rack, and let it stand for 2 to 5 minutes until the liquid becomes clear. Use a pipette to carefully aspirate the supernatant and discard it.

[0178] 56. Keep the centrifuge tube on the magnetic stand, add 500 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. After standing for 30 seconds, carefully aspirate the supernatant and discard it.

[0179] 57. 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.

[0180] 58. Keep the centrifuge tube on the magnetic rack, open the tube cap, and dry at room temperature until the magnetic beads are no longer reflective. Remove the tube from the magnetic rack and add 22 μL of TE Buffer to elute the DNA. Gently pipette at least 10 times to resuspend all the magnetic beads.

[0181] 59. Incubate at room temperature for 10 minutes.

[0182] 60. Centrifuge the centrifuge tube briefly, place it on a magnetic rack, and let it stand for 2 to 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.

[0183] 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.

[0184] 62. Use MGISEQ-2000 sequencer for sequencing, use MGISEQ-2000 High-throughput Sequencing Reagent Set (PE100) (MGI, catalog number 1000012536), and perform DNB preparation and sequencing according to the instructions.

[0185] 3. Experimental Results

[0186] 1. Database construction results

[0187]

[0188] 2. Sequencing results

[0189] Index results: Number of original reads 1339286228 Number of reads after filtering 1133839420 Read length (bp) 100 Alignment rate (%) 91.62 Duplication rate (%) 25.26 Unique alignment rate (%) 86.85 Paired-end alignment rate (%) 79.31 Single alignment rate (%) 12.31 Coverage (%) 97.95 Average depth 24.28 BS conversion rate (%) 97.6 Number of detected CG sites (1x) 51988782

[0190] The experimental results are shown in the table above. After adding 5 ng of cfDNA, the resulting double-stranded DNA library had a concentration of 38.5 ng / μL. The concentration of the single-stranded circular DNA library constructed from the double-stranded DNA library was 3.4 ng / μL. 8 ng of the single-stranded circular library was used to prepare DNA nanoballs (DNBs), a unique feature of the DNBSEQ platform. The resulting DNB concentration was 28.5 ng / μL, meeting expectations. After sequencing, data analysis revealed an alignment rate of 91.62%, a duplication rate of 25.26%, and a coverage of 97.95%, all meeting expectations. This demonstrates that this method for library construction effectively utilizes template DNA and produces high-quality sequencing data.

[0191] 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.

[0192] 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 method for constructing a single-stranded nucleic acid molecule sequencing library, characterized in that: include: (1) connecting the 3' end of the single-stranded nucleic acid molecule to the 3' end adapter to obtain a ligation product; (2) performing an extension and amplification reaction on the ligation product with a first primer, a DNA polymerase, and a reverse transcriptase to obtain an amplification product with a linker connected to the 3' end and the 5' end, respectively, wherein the amplification product constitutes the sequencing library; in, The 3' end linker comprises: A backbone, the backbone being used to connect to the 3' end of the single-stranded nucleic acid molecule; An auxiliary chain, wherein the 3' end of the auxiliary chain contains a plurality of N bases, and the plurality of N bases are used to hybridize with the 3' end of the single-stranded nucleic acid molecule; the 5' end sequence of the auxiliary chain is reverse complementary to at least a portion of the 5' end sequence of the main chain; The reverse transcriptase has terminal transferase activity, and the 3' end of the generated chain obtained by the extension reaction has multiple identical base sequences; The first primer includes a 5' end adapter sequence and a complementary sequence, and the complementary sequence is reverse complementary to the multiple identical base sequences at the 3' end of the generated chain.

2. The method according to claim 1, characterized in that The 5' end sequence of the auxiliary chain is reverse complementary to the 3' end sequence of the main chain, and the 3' end of the auxiliary chain is pre-blocked by modification with a reversible blocking group.

3. The method according to claim 2, characterized in that Step (2) includes: removing the reversible blocking group on the 3' end of the auxiliary chain, so that the 3' end of the auxiliary chain is a hydroxyl group; Starting from the 3'-terminal hydroxyl group of the auxiliary chain, extending under the action of reverse transcriptase to obtain a double-stranded nucleic acid, wherein the 3'-end of the generated chain in the double-stranded nucleic acid has multiple identical base sequences; The double-stranded nucleic acid is amplified using the first primer and the auxiliary chain under the action of DNA polymerase to obtain an amplified product, which constitutes the sequencing library.

4. The method according to claim 2 or 3, characterized in that The blocking treatment includes: phosphorylation modification, 3'-O-propylene modification or 3'-O-azidomethyl modification.

5. The method according to claim 1, wherein The 5' end sequence of the auxiliary chain is partially reverse complementary to the 5' end sequence of the main chain; The step (2) further comprises: performing an extension and amplification reaction on the ligation product with the first primer, the second primer, a DNA polymerase, and a reverse transcriptase to obtain an amplification product with a linker connected to the 3' end and the 5' end, respectively, wherein the amplification product constitutes the sequencing library; The sequence of the second primer is at least reverse complementary to the 3' end sequence of the main chain.

6. The method according to claim 5, characterized in that Step (2) includes: Using the ligation product as a template chain, the second primer hybridizes with the 3' linker of the ligation product, and performs an extension reaction under the action of a reverse transcriptase to obtain a generated chain, wherein the 3' end of the generated chain has multiple identical base sequences; The first primer hybridizes with the multiple identical base sequences at the 3' end of the generated chain and is extended under the action of DNA polymerase to obtain a double-stranded nucleic acid molecule; The double-stranded nucleic acid molecule is amplified using the first primer and the second primer under the action of DNA polymerase to obtain an amplification product, which constitutes the sequencing library.

7. The method according to claim 5 or 6, characterized in that The 3' end of the auxiliary chain is pre-blocked; The blocking treatment includes: phosphorylation modification, 3'-O-propylene modification or 3'-O-azidomethyl modification.

8. The method according to any one of claims 1 to 7, characterized in that The 3' end of the main chain is pre-blocked; The 3' end of the main chain has phosphorylation modification, C3-spacer modification, C5-spacer modification or amino modification.

9. The method according to any one of claims 1 to 8, characterized in that The main chain and the auxiliary chain are provided in separate forms. Before performing the step (1), the main chain and the auxiliary chain are annealed so as to partially hybridize the main chain and the auxiliary chain to form a partially double-stranded 3' end linker.

10. The method according to any one of claims 1 to 9, characterized in that The number of the N bases is selected from integers ranging from 6 to 12.

11. The method according to any one of claims 1 to 10, characterized in that The single-stranded nucleic acid molecule is selected from an RNA molecule or a single-stranded DNA molecule.

12. The method according to claim 11, characterized in that The single-stranded DNA molecules are derived from interrupted genomic DNA molecules, extracellular free DNA molecules, degraded DNA molecules and / or sulfite-treated DNA molecules.

13. The method according to any one of claims 1 to 12, characterized in that For a sample containing both double-stranded nucleic acid molecules and single-stranded nucleic acid molecules, the sample is denatured before performing step (1) to obtain a sample consisting entirely of single-stranded nucleic acid molecules.

14. The method according to any one of claims 1 to 13, characterized in that In step (1), the 3' end of the single-stranded nucleic acid molecule is connected to the 3' end adapter using T4 DNA ligase.

15. The method according to any one of claims 1 to 14, characterized in that The number of the plurality of identical bases is 1 to 3, and the bases in the plurality of identical bases are C bases.

16. The method according to any one of claims 1 to 15, characterized in that The backbone further comprises at least one of the following: a binding sequence of a sequencing primer, a tag sequence, and a binding sequence of a universal amplification primer.

17. The method according to claim 16, characterized in that The tag sequence length is 5 to 12 bp.

18. The method according to any one of claims 1 to 17, characterized in that The reverse transcriptase is selected from MMLV reverse transcriptase.

19. The method according to any one of claims 1 to 18, characterized in that The reverse transcriptase is selected from SuperScript II reverse transcriptase or Alpha reverse transcriptase.

20. The method according to any one of claims 1 to 19, characterized in that The extension reaction and amplification reaction are performed in one step in the same reaction system.

21. The method according to any one of claims 1 to 20, characterized in that Step (2) further comprises: Denaturing the amplified product to obtain single-stranded DNA; In the presence of a single-stranded circularization reagent, the single-stranded DNA is subjected to a single-stranded circularization reaction to obtain a circularization product, which constitutes a single-stranded circular sequencing library.

22. A sequencing method, characterized in that: include: constructing a sequencing library according to the method according to any one of claims 1 to 21; and The sequencing library is sequenced.

23. A linker for constructing a single-stranded nucleic acid molecule sequencing library, characterized in that: The connector comprises: A backbone, the backbone being used to connect to the 3' end of the single-stranded nucleic acid molecule; The auxiliary chain has a plurality of N bases at its 3' end, and the plurality of N bases are used to hybridize with the 3' end of the single-stranded nucleic acid molecule; the 5' end sequence of the auxiliary chain is reverse complementary to at least part of the 5' end sequence of the main chain.

24. The connector according to claim 23, wherein The number of the N bases is selected from integers ranging from 6 to 12.

25. The joint according to claim 23 or 24, characterized in that The 3' ends of the main chain and the auxiliary chain are modified with blocking groups.

26. The connector according to claim 25, wherein The 3' end of the main chain has phosphorylation modification, C3-spacer modification, C5-spacer modification or amino modification; The 3' end of the auxiliary chain has phosphorylation modification, 3'-O-propylene modification or 3'-O-azidomethyl modification, phosphorylation modification, C3-spacer modification, C5-spacer modification or amino modification.

27. The joint according to any one of claims 23 to 26, characterized in that: The backbone further comprises at least one of the following: a binding sequence of a sequencing primer, a tag sequence, and a binding sequence of a universal amplification primer.

28. The connector according to claim 27, wherein The tag sequence length is 5 to 12 bp.

29. A kit for constructing a single-stranded nucleic acid molecule sequencing library, characterized in that: The joint comprises the joint according to any one of claims 23 to 28.

30. The kit according to claim 29, characterized in that The main chain and auxiliary chain are provided in separate packages.

31. The kit according to claim 29 or 30, characterized in that Further including: Reverse transcriptase, DNA polymerase, DNA ligase, a first primer, and a second primer; Wherein, the first primer comprises a 5' end linker sequence and a sequence containing multiple identical bases, The second primer sequence is reverse complementary to at least a portion of the backbone sequence.

32. The kit according to claim 31, characterized in that The reverse transcriptase is selected from MMLV reverse transcriptase.

33. The kit according to claim 31 or 32, characterized in that The reverse transcriptase is selected from SuperScript II reverse transcriptase or Alpha reverse transcriptase.

34. The kit according to claim 31, wherein The DNA polymerase is selected from Q5 hot start super-fidelity DNA polymerase, KAPA hot start super-fidelity DNA polymerase, Platinum hot start super-fidelity DNA polymerase or Pfu DNA polymerase.

35. The kit according to claim 31, wherein The number of the plurality of identical bases is 1 to 3, and the bases in the plurality of identical bases are C bases.

36. The kit according to claim 31, wherein The DNA ligase is selected from T4 DNA ligase.

37. Use of the linker according to any one of claims 23 to 28 or the kit according to any one of claims 29 to 36 in constructing a single-stranded nucleic acid molecule sequencing library or sequencing.

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