WO2023240610A1 - 单链核酸分子测序文库构建方法 - Google Patents

单链核酸分子测序文库构建方法 Download PDF

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WO2023240610A1
WO2023240610A1 PCT/CN2022/099505 CN2022099505W WO2023240610A1 WO 2023240610 A1 WO2023240610 A1 WO 2023240610A1 CN 2022099505 W CN2022099505 W CN 2022099505W WO 2023240610 A1 WO2023240610 A1 WO 2023240610A1
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sequence
nucleic acid
stranded nucleic
primer
dna
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PCT/CN2022/099505
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English (en)
French (fr)
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夏军
孔格致
杨林
张艳艳
宋喆
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深圳华大智造科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the present invention relates to the biological field. Specifically, the present invention relates to a method for constructing a single-stranded nucleic acid molecule sequencing library.
  • the common method for DNA sequencing library construction is to use double-stranded DNA to construct libraries.
  • the general objects are complete genomic DNA with sufficient starting amount and no degradation, or DNA with slight degradation and damage.
  • the general steps of the double-stranded library construction method are to first break the DNA into fragments of appropriate length, then repair the ends, connect the double-stranded adapters to both ends of the DNA fragment through a simple ligase reaction, and then perform PCR.
  • the library required for sequencing is obtained.
  • double-stranded library construction has certain limitations for samples, such as formalin-fixed and paraffin-embedded biological tissue samples, forensic samples, and paleontological fossils.
  • the DNA extracted from these samples is severely degraded, and the DNA strands are Damage results in the presence of both double-stranded and single-stranded molecules in the DNA molecules, or single-strand breaks in the double-stranded DNA molecules, and the amount of DNA extracted will also be very low.
  • DNA usually undergoes bisulfite conversion treatment. During this process, due to long-term acidic and high-temperature environments, DNA will also be severely damaged, resulting in the loss of most DNA molecules. And the DNA will break into short fragments and remain single-stranded.
  • Single-stranded library construction technology can also make samples better utilized and can be used in a variety of applications, such as liquid biopsy for extracellular cell-free DNA (cfDNA) methylation sequencing, genomic research on paleontological DNA, forensic identification, and FFPE samples. tumor detection, etc.
  • cfDNA extracellular cell-free DNA
  • Existing single-stranded library construction technologies include traditional single-stranded library construction methods, Swift's Adaptase single-stranded library construction technology, random primer plus adapter method, and terminal transferase plus adapter method.
  • T4RNA ligase 2 (truncated type) is used to catalyze the 5' end pre-adenylation of the DNA adapter. Connected to the 3' end of the single-stranded template, and the 3' end of the linker is blocked, which can reduce the generation of linker dimers. After purification, add the 5' end adapter and T4RNA ligase, and then connect the 5' end to the template with the 3' end adapter added. After both ends of the adapters are added, PCR amplification is performed.
  • PCR also has the function of completing the remaining adapters. If you want to make a single-stranded circular library, you need to perform single-strand denaturation, and then use DNA ligase and oligonucleotide chains with complementary connectors at both ends to perform circular ligation.
  • Swift's Adaptase single-stranded library construction technology first uses a single-stranded ligase to connect the 3' end of a single-stranded DNA template to a universal sequencing adapter with a random primer, and then uses a complementary primer that binds to the 3' end adapter primer sequence. Amplify, convert the single-stranded template into double-stranded, and then add a 5'-end sequencing adapter to the 5' end of the newly synthesized strand through a conventional adapter ligation reaction to complete the construction of the library. If you want to make a single-stranded circular library, you need to perform single-strand denaturation, and then use DNA ligase and oligonucleotide chains with complementary connectors at both ends to perform circular ligation.
  • the single-stranded library construction technology of random primers uses 6 to 8nt random primers to combine with a single-stranded DNA template for extension, thereby synthesizing a double strand, and then adding a 5'-end sequencing adapter to the 5' end of the newly synthesized strand to complete the process.
  • Library construction If you want to make a single-stranded circular library, you also need to perform single-strand denaturation, and then use DNA ligase and oligonucleotide chains with complementary connectors at both ends to perform circular ligation.
  • Terminal transferase is similar to the single-stranded library construction technology of random primers. First, terminal transferase is used to add continuous bases to the 3' end of the single-stranded DNA template, and then the continuous sequence is used as the primer binding position for extension. The single-stranded DNA template is converted into a double-stranded DNA, and a 5'-end sequencing adapter is added to the 5' end of the newly synthesized strand to complete the construction of the library. If you want to make a single-stranded circular library, you need to perform single-strand denaturation, and then use DNA ligase and oligonucleotide chains with complementary connectors at both ends to perform circular ligation.
  • the traditional single-stranded library construction method uses T4 RNA ligase 2 (truncated type), which requires a 5'-end pre-adenylation modified DNA adapter.
  • the 5'-end pre-adenylation modification is expensive and difficult to synthesize. Only a few manufacturers can produce it, resulting in high costs.
  • the adapters at both ends are connected separately, and multiple purifications are required, resulting in low template utilization and long library construction time.
  • Adaptase single-stranded library construction technology, random primer plus adapter method, and terminal transferase plus adapter method all add 3’ and 5’ adapters respectively. Only after adding both end adapters can PCR amplification be performed to amplify the template. These increase the number of experimental steps, lead to the loss of the original template, and reduce the utilization rate. Moreover, the random primer method will also generate unpredictable products due to the use of random primers, resulting in a reduction in the proportion of effective libraries and thus reduction in effective sequencing data.
  • the present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.
  • the present invention proposes a single-stranded nucleic acid molecule sequencing library construction method, a sequencing library, a sequencing method, a connector and a kit for constructing a single-stranded nucleic acid molecule sequencing library, and their uses.
  • the sequencing library obtained by utilizing the sequencing library construction method of the present invention also contains
  • the 3' end adapter and 5' end adapter of known sequence have high adapter connection efficiency, less loss of original template, high utilization rate, short reaction time, low cost, and are suitable for wide application.
  • the present invention proposes a method for constructing a single-stranded nucleic acid molecule sequencing library.
  • the method includes: (1) connecting the 3' end of the single-stranded nucleic acid molecule to the 3' end adapter to obtain a ligation product; (2) combining the first primer, DNA polymerase, reverse transcription The enzyme performs extension and amplification reactions with the ligation product to obtain an amplification product with adapters connected to the 3' end and 5' end respectively, and the amplification product constitutes the sequencing library; wherein, the 3' end adapter It 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 a plurality of N bases, and the plurality of N bases are hybridizes with the 3' end of the single-stranded nucleic acid molecule; the 5' end sequence of the auxiliary strand is reverse complementary
  • the 3' end adapter is first connected to the single-stranded nucleic acid molecule to obtain the connection product. Then, starting from the hydroxyl group at the 3' end of the auxiliary strand, it is extended under the action of reverse transcriptase to obtain a double-stranded nucleic acid, in which the 3' end of the generated chain in the double-stranded nucleic acid has multiple identical base sequences. Since the auxiliary chain is reverse complementary to at least part of the sequence of the main chain, the first primer includes a 5' end linker sequence and a complementary sequence that is reverse complementary to multiple identical base sequences at the 3' end of the generated chain. Therefore, using this first The primers and 3'-end adapter amplify the double-stranded nucleic acid under the action of DNA polymerase to obtain a sequencing library.
  • the present invention designs the 3' end linker into a partial double-stranded structure, so that it can be stably connected to the 3' end of a single-stranded nucleic acid molecule, thereby improving the linker connection efficiency and avoiding linker self-ligation.
  • the sequencing library obtained by the method of the present invention contains both the 3' end adapter and the 5' end adapter of the known sequence, and the extension and amplification reactions are performed in one tube, requiring only one step, which increases the number of adapters to be connected. efficiency, but also reduces the loss of the original template, overall improves the utilization of the original template, and shortens the reaction time.
  • sequencing adapters do not require special modifications, nor do they require special high-cost ligases, thus reducing costs and making them suitable for widespread use.
  • the invention proposes a sequencing method.
  • the sequencing method includes: constructing a sequencing library according to the method described above; and sequencing the sequencing library.
  • the present invention provides an adapter for constructing a single-stranded nucleic acid molecule sequencing library.
  • the linker includes: a main chain, the main chain is used to connect to the 3' end of a single-stranded nucleic acid molecule; an auxiliary strand, the 3' end of the auxiliary strand 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 strand is reverse complementary to at least part of the sequence of the 5' end of the main chain.
  • the present invention provides a kit for constructing a single-stranded nucleic acid molecule sequencing library.
  • the kit includes the aforementioned connector.
  • the present invention proposes the use of the aforementioned adapter or kit in constructing a single-stranded nucleic acid molecule sequencing library or sequencing.
  • Figures 1 to 3 respectively show a schematic flow chart of a method for constructing a single-stranded nucleic acid molecule sequencing library according to one embodiment of the present invention.
  • the present invention proposes 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 their uses, which will be described in detail below.
  • the present invention proposes a method for constructing a single-stranded nucleic acid molecule sequencing library.
  • the method includes: S100 ligation adapter, S200 extension and amplification reaction. Each step will be described in detail below.
  • the 3' end of the single-stranded nucleic acid molecule is connected to the 3' end adapter to obtain a ligation product.
  • the final sequencing library contains the 3’ end adapter.
  • the 3' end linker includes: a main chain, the main chain is used to connect to the 3' end of the single-stranded nucleic acid molecule; an auxiliary strand, the 3' end of the auxiliary strand contains polypeptide N bases, 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 strand is at least partially reversed to the 5' end sequence of the main chain. complementary.
  • the connection efficiency is low and unstable, and the adapter is prone to self-ligation.
  • the inventor of the present invention designed the 3'-end linker into a partial double-stranded structure.
  • the main chain is the 3'-end linker of the finally obtained amplification product;
  • the 3' end of the auxiliary chain contains multiple N bases.
  • the design of the auxiliary strand can make the entire main chain stably bind to the template strand. to improve the joint connection efficiency and avoid joint self-connection.
  • the traditional single-stranded library construction method generally uses T4 RNA ligase 2 (truncated type) 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, which can reduce the production of Linker dimer.
  • T4 RNA ligase 2 (truncated type) can only catalyze reactions with adenylated DNA.
  • This method uses the main chain and auxiliary strand to form partial double strands with the template DNA, and can directly use DNA ligase (such as T4 DNA ligase) without additional special modifications to the linker.
  • the 3' end of the main chain has been blocked in advance.
  • the generation of linker dimers can be reduced.
  • the blocking treatment includes phosphorylation, C3-spacer modification, C5-spacer modification or amino modification.
  • the main chain and the auxiliary chain are provided in an independent form.
  • the main chain and the auxiliary chain are annealed, so that the main chain and the auxiliary chain Partially hybridizes to form a partially double-stranded 3' end linker.
  • the number of N bases is selected from an integer of 6 to 12.
  • N represents a random base.
  • the auxiliary strand can be stably combined with the template strand, improving the efficiency of linker connection and avoiding self-ligation of the linker.
  • the single-stranded nucleic acid molecule is selected from RNA molecules or single-stranded DNA molecules.
  • 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.
  • the sample is denatured to obtain a sample containing all single-stranded nucleic acid molecules.
  • T4 DNA ligase is used to connect the 3' end of the single-stranded nucleic acid molecule to the 3' end adapter.
  • the traditional single-stranded library construction method is based on T4 RNA ligase to directly connect the single-stranded sequencing adapter to the single-stranded DNA template molecule.
  • T4 RNA ligase 2 (truncated type) is generally used to catalyze the 5'-end pre-densation The glycosylated DNA linker is connected to the 3' end of the single-stranded template, and the 3' end of the linker is blocked, which can reduce the generation of linker dimers.
  • T4 RNA ligase 2 (truncated type) can only catalyze reactions with adenylated DNA.
  • T4 RNA ligase 2 (truncated form) is expensive.
  • T4 DNA ligase is the most commonly used ligase with low cost.
  • the first primer, DNA polymerase, reverse transcriptase and the ligation product are subjected to extension and amplification reactions to obtain an amplification product with adapters connected to the 3' end and the 5' end respectively.
  • the amplification product constitutes the sequencing library, wherein the reverse transcriptase has terminal transferase activity, and the 3' end of the generated chain generated by the extension reaction has multiple identical base sequences;
  • the first primer includes a 5' end A linker sequence and a complementary sequence, the complementary sequence being reverse complementary to multiple identical base sequences at the 3' end of the generated chain.
  • 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 from the 3' end hydroxyl group of the auxiliary chain.
  • extension is performed under the action of reverse transcriptase to obtain a double-stranded nucleic acid, in which the 3' end of the generated strand has multiple identical base sequences; the first primer and the auxiliary strand are used in the DNA
  • the double-stranded nucleic acid is amplified under the action of polymerase to obtain an amplification product, and the amplification product constitutes the sequencing library.
  • double-stranded nucleic acid can refer to either double-stranded DNA or RNA-DNA hybrid strand.
  • the single-stranded nucleic acid molecule is DNA
  • the 3'-terminal hydroxyl group of the auxiliary strand is used as the starting point and extended under the action of reverse transcriptase to obtain double-stranded DNA
  • the single-stranded nucleic acid molecule is RNA
  • Starting from the 3'-terminal hydroxyl group of the auxiliary strand it is extended under the action of reverse transcriptase to obtain the RNA-DNA hybrid strand.
  • 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 from the 3' end hydroxyl group of the auxiliary chain. Start by extending under the action of reverse transcriptase to obtain an RNA-DNA hybrid chain.
  • the 3' end of the DNA generating 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.
  • the specially designed 3’-end joint structure of the present invention can not only be stably combined with the template chain, but also improve the joint connection efficiency and avoid joint self-connection. Moreover, it can be directly extended from the 3' end of the auxiliary strand to generate double-stranded DNA. Since the first primer includes a 5' end linker sequence and a complementary sequence that is reverse complementary to multiple identical base sequences at the 3' end of the generated chain, the auxiliary chain of the 3' end linker is at least partially complementary to the main chain, so the above-mentioned The first primer and the 3' end adapter amplify the double-stranded DNA to obtain a sequencing library.
  • the blocking treatment includes: phosphorylation modification, 3'-O-propenyl modification or 3'-O-azidomethyl modification. Therefore, the above-mentioned blocking treatment method can not only prevent self-ligation of the linker, but also reversibly remove the blocking group, thereby extending the auxiliary chain.
  • the 5' end sequence of the auxiliary strand is partially reverse complementary to the 3' end sequence of the main chain; the step (2) further includes: combining the first primer, the second primer, and DNA Polymerase and reverse transcriptase perform extension and amplification reactions with the ligation product to obtain an amplification product with adapters connected to the 3' end and 5' end respectively, and the amplification product constitutes the sequencing library; wherein, the The sequence of the second primer is at least reverse complementary to the 3' end sequence of the main chain.
  • step S200 includes: the second primer hybridizes to the ligation product, and an extension reaction is performed under the action of reverse transcriptase to obtain a generated chain, the 3' end of the generated chain has multiple identical bases base sequence; using the generated strand as a template strand, the first primer hybridizes with multiple identical base sequences at the 3' end of the generated strand, and amplifies under the action of DNA polymerase to obtain double-stranded nucleic acid; The second primer and the first primer are used to amplify the double-stranded nucleic acid under the action of DNA polymerase to obtain an amplification product, and 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, so the second primer will hybridize with the template strand and transfer to the template under the action of reverse transcriptase.
  • the chain extends in the 5' end direction. When extended to the 5' end, due to the terminal transferase activity of reverse transcriptase, multiple identical base sequences can be formed at the 3' end of the generated chain.
  • the first primer includes a 5' end linker sequence and a complementary sequence that is reverse complementary to multiple identical base sequences at the 3' end of the generated strand, it can hybridize with the generated strand and extend under the action of DNA polymerase to obtain a double strand. nucleic acids.
  • the first primer and the second primer are then used to amplify the double-stranded nucleic acid under the action of DNA polymerase to obtain a sequencing library.
  • the 3' end of the auxiliary chain has been blocked in advance; the blocking treatment includes: phosphorylation modification, 3'-O-propenyl modification or 3'-O-azidomethyl modification. In this way, self-connection of the joints can be avoided.
  • the number of the plurality of identical bases is 1 to 3.
  • the base among the plurality of identical bases is a C base.
  • the main chain 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.
  • the length of the tag sequence is 5 to 12 bp.
  • the reverse transcriptase is selected from MMLV reverse transcriptase.
  • the reverse transcriptase is selected from SuperScript II reverse transcriptase or Alpha reverse transcriptase.
  • the above-mentioned reverse transcriptase enzymes all have terminal transferase activity and can form multiple identical base sequences on the 5' end of the generated chain, which is helpful for subsequent complementary hybridization with the first primer sequence.
  • the DNA polymerase is selected from the group consisting of Q5 hot-start ultra-fidelity DNA polymerase, KAPA hot-start ultra-fidelity DNA polymerase, Platinum hot-start ultra-fidelity DNA polymerase or Pfu DNA polymerase. This enables efficient amplification.
  • the extension reaction and amplification reaction are performed in one step in the same reaction system.
  • the reaction time is shortened and the efficiency of library construction is improved.
  • step S200 further includes: denaturing the amplification product to obtain single-stranded DNA; performing a single-stranded cyclization reaction on the single-stranded DNA in the presence of a single-stranded cyclization reagent,
  • the circularization product constitutes a single-stranded circular sequencing library.
  • the single-strand cyclizing agent is selected from DNA ligases.
  • the single-stranded nucleic acid molecule sequencing library construction method includes the steps of ligation and amplification of 3' end adapters.
  • the adapter ligation step uses T4 DNA ligase to connect the partially double-stranded adapter to the 3' end of the single-stranded DNA template.
  • the linker includes a main chain and an auxiliary strand.
  • the 3' end of the auxiliary strand has a random base sequence of 6 N to 12 N, which can be hybridized and complementary to the 3' end of the single-stranded DNA template, and its 5' end is complementary to the main chain.
  • the 5' end partial sequence was reverse complementary hybridized.
  • the amplification includes first using the sequence complementary to the main chain (3' end adapter) as primer 1 and reverse transcriptase for extension. After extending to the 5' end of the single-stranded template, reverse transcriptase is used. With terminal transferase activity plus 3 cytosine bases. Then use primer 2 (containing the sequence of the 5' end adapter and the 3' end with three G's) to hybridize with the generated chain obtained in the previous step, and then use primer 2 as a template to continue extending. Then use DNA polymerase and primer 1 and primer 2 to perform PCR amplification of the double-stranded nucleic acid obtained in the previous step.
  • the PCR amplification reaction can be completed while adding the 5'-end adapter in a one-step reaction, and a library with a 2-end sequencing adapter can be obtained.
  • the library can be obtained by purification.
  • the PCR amplification products need to be denatured and quenched at high temperature to become single-stranded, then the single-stranded DNA is circularized, and then linear digestion enzymes are used to digest the non-circularized molecules to obtain a suitable single-stranded DNA circular library. .
  • DNA fragments after fragmentation are purified and recovered after sulfite treatment.
  • the 3’-end adapter is connected.
  • the auxiliary chain and the main chain are annealed to form a partial double-stranded linker.
  • T4 DNA ligase is then used to ligate the single-stranded DNA template to the adapter.
  • the adapter sequence can be a sequence that is adapted to the 5’ end and 3’ end sequencing adapter of the Illumina or DNBSEQ platform.
  • PCR primer 1 complementary to the 3'-end adapter sequence and the 5'-end sequencing adapter sequence with 3 Gs as primer 2, as well as reverse transcriptase and DNA polymerase, and use the single-stranded DNA obtained in the above step as a template to simultaneously extend and PCR amplification reaction.
  • the 3 to 12 nucleotides at the 3' end of primer 1 are modified with deoxyribonucleotides or locked nucleotides, and the 3 G's at the 3' end of primer 2 are also modified with deoxyribonucleotides or locked nucleotides.
  • the 5' end of primer 2 is phosphorylated.
  • the single-stranded circular DNA library of the DNBSEQ platform requires high-temperature denaturation of the purified product (generally placed at 92 to 98°C for 3 to 10 minutes). Immediately after the denaturation is completed, place it on ice to prevent the melted double-stranded DNA from denaturing. Restoration.
  • the circularized single-stranded DNA ring contains the template and sequencing adapters required for sequencing.
  • the invention proposes a sequencing method.
  • the sequencing method includes: constructing a sequencing library according to the method described above; and sequencing the sequencing library.
  • the present invention provides an adapter for constructing a single-stranded nucleic acid molecule sequencing library.
  • the linker includes: a main chain, the main chain is used to connect to the 3' end of a single-stranded nucleic acid molecule; an auxiliary strand, the 3' end of the auxiliary strand 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 strand is reverse complementary to at least part of the sequence of the 5' end of the main chain.
  • the linker according to the embodiment of the present invention can form a partial double-stranded structure.
  • the main chain is the 3' end linker of the finally obtained amplification product.
  • the 3' end of the auxiliary strand contains multiple N bases and can be connected with the 3' end of the template strand.
  • the 5' end sequence of the auxiliary strand is reverse complementary to at least part of the 5' end sequence of the main chain. Therefore, the design of the auxiliary strand can make the entire main chain stably combine with the template strand, improve the efficiency of adapter connection, and avoid the occurrence of The connectors are self-connecting.
  • the number of N bases is selected from an integer of 6 to 12.
  • N represents a random base.
  • the auxiliary strand can be stably combined with the template strand, improving the efficiency of linker connection and avoiding self-ligation of the linker.
  • the 3' ends of the main chain and the auxiliary chain are modified with blocking groups.
  • the 3' end of the main chain has phosphorylation modification, C3-spacer modification, C5-spacer modification or amino modification, thereby reducing the generation of linker dimers.
  • the 3' end of the auxiliary chain has phosphorylation modification, 3'-O-propenyl modification or 3'-O-azidomethyl modification, thereby reducing the generation of linker dimers.
  • the above-mentioned modifying group can be removed so that the 3' end of the auxiliary chain is a hydroxyl group, so that the 3' end of the auxiliary chain can be used as a starting point for extension to generate a double-stranded nucleic acid.
  • the main chain 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.
  • the length of the tag sequence is 5 to 12 bp.
  • the present invention provides a kit for constructing a single-stranded nucleic acid molecule sequencing library.
  • the kit includes the aforementioned connector. Therefore, this kit can be used to build a library of single-stranded nucleic acid molecules.
  • the main chain and the auxiliary chain are provided in independent packages. Therefore, when using this linker, the main chain and the auxiliary chain can form a partial double-stranded structure through annealing.
  • the kit further includes: reverse transcriptase, DNA polymerase, DNA ligase, a second primer and a first primer; wherein the second primer sequence is identical to at least a part of the main chain The sequence is reverse complementary; the first primer includes a 5' end linker sequence and a sequence containing multiple identical bases.
  • DNA ligase can ligate adapters at the 3' end of single-stranded nucleic acid molecules to obtain ligation products.
  • the second primer since the sequence of the second primer can be reverse complementary to at least part of the sequence of the adapter, the second primer will hybridize with the template strand and move toward the 5' end of the template strand under the action of reverse transcriptase.
  • reverse transcriptase When extending to the 5' end, because reverse transcriptase has terminal transferase activity, multiple identical base sequences can be formed at the 3' end of the generated chain.
  • the first primer includes a 5' end linker sequence and a complementary sequence that is reverse complementary to the above-mentioned multiple identical base sequences, it can hybridize with the generated strand and extend under the action of DNA polymerase to obtain a double-stranded nucleic acid.
  • the second primer and the first primer are then used to amplify the double-stranded nucleic acid under the action of DNA polymerase to obtain a sequencing library.
  • the nucleic acid molecules (amplification products) in the sequencing library obtained by using the kit of the present invention to build 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 one step.
  • the reaction increases the efficiency of adding joints, while also reducing the loss of the original template, overall improving the utilization of the original template and shortening the reaction time.
  • the number of the plurality of identical bases is 1 to 3.
  • the base among the plurality of identical bases is a C base.
  • the reverse transcriptase is selected from MMLV reverse transcriptase.
  • the reverse transcriptase is selected from SuperScript II reverse transcriptase or Alpha reverse transcriptase.
  • the above-mentioned reverse transcriptase enzymes all have terminal transferase activity and can form multiple identical base sequences on the 5' end of the generated chain, which is helpful for complementary hybridization with the first primer sequence.
  • the DNA polymerase is selected from the group consisting of Q5 hot-start ultra-fidelity DNA polymerase, KAPA hot-start ultra-fidelity DNA polymerase, Platinum hot-start ultra-fidelity DNA polymerase or Pfu DNA polymerase. This enables efficient amplification.
  • the DNA ligase is selected from T4 DNA ligase. This makes it easy to connect the adapter to the 3' end of the single-stranded nucleic acid molecule, with high connection efficiency and low cost.
  • the present invention proposes the use of the aforementioned adapter or kit in constructing a single-stranded nucleic acid molecule sequencing library or sequencing.
  • the sequencing library constructed using the adapter or kit of the present invention contains both the 3'-end adapter and the 5'-end adapter of known sequences.
  • the adapter connection efficiency is high, the loss of the original template is small, the utilization rate is high, the reaction time is short, and the cost is low.
  • using this sequencing library for sequencing can effectively obtain more realistic nucleic acid molecule sequencing data, which can provide more favorable support for subsequent analysis.
  • the present invention only needs to first add a 3'-end adapter to the single-stranded DNA template, and then use reverse transcriptase to realize the connection of the 5'-end adapter and PCR amplification in one step, eliminating the need to add different ends of the DNA separately.
  • the sequencing adapter requires only one step of ligation reaction. At the same time, the addition of the 5' end adapter and the PCR amplification are performed in one tube, which increases the efficiency of the adapter and reduces the loss of the original template. Overall, the original template is improved. The utilization rate is reduced and the response time is reduced. In addition, sequencing adapters do not require special modifications or special high-cost ligases, thus reducing costs.
  • cfDNA human peripheral blood plasma cell-free DNA
  • CT Conversion Reagent should reduce exposure, and it is best to prepare it for immediate use.
  • 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-prepared CT Conversion Reagent should be preheated to 37°C before use. Vortex frequently for 10 minutes at room temperature before use.
  • T4 DNA ligase and ligation buffer from the MGI digested DNA library preparation kit (1000005254, MGI) to perform adapter ligation. Prepare the ligation reaction solution on an ice box according to the table below.
  • step 29 Add 35 ⁇ L of the prepared amplification reaction solution into the PCR tube containing the purified DNA in step 27. Use a oscillation mixer to mix thoroughly and then centrifuge briefly to centrifuge the liquid to the bottom of the tube.
  • Primer1 5’-TGTGAGCCAAGGAG/iXNA_T//iXNA_T//iXNA_G/-3’(SEQ ID NO: 3)
  • Centrifuge the centrifuge tube momentarily, place it on a magnetic stand, and let it sit for 2 to 5 minutes until the liquid is clear. Use a pipette to carefully absorb the supernatant and discard it.
  • Centrifuge the centrifuge tube momentarily, place it on a magnetic stand, and let it stand for 2 to 5 minutes until the liquid is clear. Use a pipette to absorb 20 ⁇ L of the supernatant and transfer it to a new 1.5 mL centrifuge tube.
  • MGISEQ-2000 sequencer for sequencing, use the MGISEQ-2000 high-throughput sequencing reagent set (PE100) (MGI, product number 1000012536), and follow the instructions to prepare and sequence DNB.
  • PE100 high-throughput sequencing reagent set
  • the experimental results are shown in the table above. It can be seen that after adding 5ng cfDNA, the concentration of the double-stranded DNA library obtained after the reaction is 38.5ng/ ⁇ L. The concentration of the single-stranded circular DNA library constructed from the double-stranded DNA library is 3.4ng/ ⁇ L. 8ng of the single-stranded circular library was taken to prepare DNA nanospheres (DNB) unique to the DNBSEQ platform. The obtained DNB concentration was 28.5ng/ ⁇ L, and these indicators met expectations. After sequencing, the data was analyzed and the comparison rate reached 91.62%, the repetition rate was 25.26%, and the coverage was 97.95%. These indicators all met expectations. It shows that using this method to build a library has a high utilization rate of template DNA and obtains high-quality sequencing data.
  • references to the terms “one embodiment,” “some embodiments,” “an example,” “specific examples,” or “some examples” or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

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Abstract

提供了单链核酸分子测序文库构建方法，所述方法包括：(1)使单链核酸分子的3'端与3'端接头相连，以便得到连接产物；(2)将第一引物、DNA聚合酶、逆转录酶与所述连接产物进行延伸和扩增反应，以便得到3'端和5'端分别连有接头的扩增产物，所述扩增产物构成所述测序文库；其中，所述逆转录酶具有末端转移酶活性。利用所述的测序文库构建方法获得的测序文库同时含有已知序列的3'端接头和5'端接头，接头连接效率高，原始模板损失少、利用率高，反应时间短，成本低，适于广泛应用。

Description

单链核酸分子测序文库构建方法 技术领域
本发明涉及生物领域。具体地，本发明涉及单链核酸分子测序文库构建方法。
背景技术
目前，DNA测序建库的常见建库方法是利用DNA双链构建文库，一般的对象都是起始量足够且没有发生降解的完整的基因组DNA，或者有轻微降解和损伤的DNA。双链建库方法的一般步骤是先将DNA打断成长度合适的片段，之后再对其进行末端修复，通过简单的连接酶反应将双链接头连接到DNA片段的两端，再进行PCR，从而得到测序需要的文库。
但是，双链建库对于样本有一定的局限性，比如福尔马林固定和石蜡包埋的生物组织样本、法医样本和古生物化石等，这些样本提取出的DNA存在严重的降解，DNA链存在损伤，导致DNA分子中会同时存在双链和单链的分子，或者双链DNA分子中存在单链断裂的现象，同时提取的DNA量也会非常低。此外，在甲基化测序中，DNA通常要经过亚硫酸氢盐转化处理，在这个过程中，由于长时间的酸性和高温环境，DNA也会受到严重损伤，从而丢失绝大多数的DNA分子，并且DNA会断裂为短片段，一直保持单链的状态。还有血浆或其他体液样本中的胞外游离的DNA，其本身就是片段短，且含量也非常低。对于这些特殊的样本，不适合采用常见的双链接头连接的建库方法，因为常规的建库方法只能利用完整的双链DNA分子，除完整双链DNA分子以外的所有模板分子无法或极少能加上接头，从而造成大量原始模板分子的损失。除了双链建库的方法以外，还有针对单链核酸的建库方法，可以将这些单链的DNA模板分子利用起来进行建库，最大限度地避免这些非完整双链的DNA模板分子的损失，最大程度上的提高模板利用率，增加了建库的效率，使测序的有效数据量和数据质量都得到了提升。单链建库技术也可以让样本更好被利用，可以被用到多种应用中，比如胞外游离DNA(cfDNA)甲基化测序的液体活检、古生物DNA的基因组研究、法医鉴定、FFPE样本的肿瘤检测等。
现有的单链建库的技术包括传统的单链建库方法、Swift的Adaptase单链建库技术、随机引物加接头法和末端转移酶加接头法。
传统的单链建库方法：基于T4RNA连接酶将单链测序接头直接连接在单链DNA模板分子上，一般是使用T4RNA连接酶2(截短型)催化5’端预腺苷酸化的DNA接头与单链模板3’端连接，且接头的3’端封闭，可以减少产生接头二聚体。纯化后，再加入5’端接头和T4RNA 连接酶，将5’端再连到已经加上3’端接头的模板上。两端接头都加上之后，再进行PCR放大，如果之前连接的是截短的接头，PCR还有将剩余部分接头补全的功能。如果要做成单链环环文库，则还需要进行单链变性，再利用DNA连接酶和两端接头部分互补的寡核苷酸链进行环化连接。
Swift的Adaptase单链建库技术是先通过单链连接酶将单链的DNA模板3’端连接上一段带有随机引物的通用测序接头，在利用与3’端接头引物结合序列的互补引物进行扩增，将单链模板在变成双链，再通过常规的接头连接反应在新合成的链的5’端加上5’端的测序接头，完成文库的构建。如果要做成单链环环文库，则还需要进行单链变性，再利用DNA连接酶和两端接头部分互补的寡核苷酸链进行环化连接。
随机引物的单链建库技术是采用6～8nt的随机引物与单链的DNA模板结合进行延伸，从而合成双链，再给新合成的链的5’端加上5’端的测序接头，完成文库的构建。如果要做成单链环状文库，则还需要进行单链变性，再利用DNA连接酶和两端接头部分互补的寡核苷酸链进行环化连接。
末端端转移酶与随机引物的单链建库技术类似，先是利用末端转移酶给单链DNA模板的3’端加上连续的碱基，再利用连续的序列作为引物结合位置，进行延伸，将单链DNA模板变成双链，再给新合成的链的5’端加上5’端的测序接头，完成文库的构建。如果要做成单链环环文库，则还需要进行单链变性，再利用DNA连接酶和两端接头部分互补的寡核苷酸链进行环化连接。
然而，传统的单链建库方法使用T4RNA连接酶2(截短型)，需要5’端预腺苷酸化修饰的DNA接头，其中5’端预腺苷酸化修饰的价格贵，且合成困难，只有极少数厂家可以生产，导致成本高。而且两端接头分别连接，还要进行多次纯化，导致模板利用率低，建库时间长。
Adaptase单链建库技术、随机引物加接头法、末端转移酶加接头法也是都是分别加3’和5’的接头，只有在加上两端接头之后才能进行PCR扩增来放大模板。这些都使得实验步骤增加，也导致了原始的模板的损失，使利用率降低。并且随机引物的方法由于使用了随机引物，也会生成无法预测的产物，导致有效的文库比例降低，从而降低了有效的测序数据。
因此，目前针对单链DNA建库方法仍有待研究。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。本发明提出 了单链核酸分子测序文库构建方法、测序文库、测序方法、用于构建单链核酸分子测序文库的接头和试剂盒及用途，利用本发明的测序文库构建方法获得的测序文库同时含有已知序列的3’端接头和5’端接头，接头连接效率高，原始模板损失少、利用率高，反应时间短，成本低，适于广泛应用。
在本发明的一个方面，本发明提出了一种单链核酸分子测序文库构建方法。根据本发明的实施例，所述方法包括：(1)使单链核酸分子的3’端与3’端接头相连，以便得到连接产物；(2)将第一引物、DNA聚合酶、逆转录酶与所述连接产物进行延伸和扩增反应，以便得到3’端和5’端分别连有接头的扩增产物，所述扩增产物构成所述测序文库；其中，所述3’端接头包括：主链，所述主链用于与所述单链核酸分子的3’端相连；辅助链，所述辅助链的3’端含有多个N碱基，所述多个N碱基用于与所述单链核酸分子的3’端杂交；所述辅助链的5’端序列与所述主链的5’端至少部分序列反向互补；所述逆转录酶具有末端转移酶活性，所述延伸反应所得生成链的3’末端具有多个相同碱基序列；所述第一引物包括5’端接头序列和互补序列，所述互补序列与所述生成链的3’末端多个相同碱基序列反向互补。
根据本发明实施例的方法中，首先将3’端接头与单链核酸分子相连，得到连接产物。接着以辅助链3’端的羟基为起始在逆转录酶作用下进行延伸，得到双链核酸，其中双链核酸中生成链3’末端具有多个相同碱基序列。由于辅助链与主链至少部分序列反向互补，第一引物包括5’端接头序列和与上述生成链的3’末端多个相同碱基序列反向互补的互补序列，因此，利用该第一引物和3’端接头在DNA聚合酶的作用下对双链核酸进行扩增，获得测序文库。
另外，本发明将3’端接头设计为局部双链结构，使其可以稳定地连接在单链核酸分子的3’端上，提高接头连接效率，避免出现接头自连。
由此，采用本发明的方法获得的测序文库同时含有已知序列的3’端接头和5’端接头，并且延伸和扩增反应在一管中进行、仅需要一步反应，增加了连接接头的效率，同时也降低了原始模板的损失，整体上提升了原始模板的利用率，缩短了反应时间。同时测序接头不需要使用特殊的修饰，也不需要特殊的高成本连接酶，从而降低了成本，适于广泛应用。
在本发明的另一方面，本发明提出了一种测序方法。根据本发明的实施例，所述测序方法包括：根据前面所述的方法构建测序文库；和对所述测序文库进行测序。
在本发明的又一方面，本发明提出了一种用于构建单链核酸分子测序文库的接头。根据本发明的实施例，所述接头包括：主链，所述主链用于与单链核酸分子的3’端相连；辅助链，所述辅助链的3’端含有多个N碱基，所述多个N碱基用于与单链核酸分子的3’ 端杂交；所述辅助链的5’端序列与所述主链的5’端至少部分序列反向互补。
在本发明的又一方面，本发明提出了一种用于构建单链核酸分子测序文库的试剂盒。根据本发明的实施例，所述试剂盒包括前面所述接头。
在本发明的又一方面，本发明提出了前面所述接头或试剂盒在构建单链核酸分子测序文库或测序中的用途。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1-图3分别显示了根据本发明一个实施例的单链核酸分子测序文库构建方法流程示意图。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
本发明提出了单链核酸分子测序文库构建方法、测序文库、测序方法、用于构建单链核酸分子测序文库的接头和试剂盒及用途，下面将分别对其进行详细描述。
单链核酸分子测序文库构建方法
在本发明的一个方面，本发明提出了一种单链核酸分子测序文库构建方法。根据本发明的实施例，该方法包括：S100连接接头、S200延伸和扩增反应，下面将分别对各步骤进行详细描述。
S100连接接头
在该步骤中，使单链核酸分子的3’端与3’端接头相连，以便得到连接产物。由此，以便最终获得的测序文库中含有3’端接头。
根据本发明的实施例，所述3’端接头包括：主链，所述主链用于与所述单链核酸分子的3’端相连；辅助链，所述辅助链的3’端含有多个N碱基，所述多个N碱基用于与所述单链核酸分子的3’端杂交；所述辅助链的5’端序列与所述主链的5’端至少部分序列 反向互补。
若直接在单链核酸分子上连接接头，则连接效率低且不稳定，接头容易发生自连。进而，本发明的发明人通过将3’端接头设计为局部双链结构，具体地，主链为最终获得的扩增产物的3’端接头；辅助链的3’端含有多个N碱基，可以与模板链的3’端杂交，辅助链的5’端序列与主链的5’端至少部分序列反向互补，由此，辅助链的设计可以使得整个主链稳定地结合在模板链上，提高接头连接效率，避免出现接头自连。
传统的单链建库方法一般是使用T4RNA连接酶2(截短型)催化5’端预腺苷酸化的DNA接头与单链模板3’端连接，且接头的3’端封闭，可以减少产生接头二聚体。T4RNA连接酶2(截短型)只能催化带腺苷酸化的DNA进行反应。本方法利用主链和辅助链与模板DNA形成局部双链，直接使用DNA连接酶(如T4 DNA连接酶)即可，无需对接头进行额外的特殊修饰。
根据本发明的实施例，所述主链的3’端预先经过封闭处理。由此，可以减少接头二聚体产生。具体地，所述封闭处理包括磷酸化、C3-spacer修饰、C5-spacer修饰或氨基修饰。
根据本发明的实施例，所述主链和辅助链是以独立形式提供的，进行所述步骤(1)之前，将所述主链和辅助链进行退火，以便使所述主链和辅助链部分杂交，形成局部双链的3’端接头。
根据本发明的实施例，所述N碱基的个数选自6～12的整数。N表示随机碱基，当其个数为6～12时，辅助链可以稳定地与模板链结合，提高接头连接效率，避免出现接头自连。
根据本发明的实施例，所述单链核酸分子选自RNA分子或单链DNA分子。
根据本发明的实施例，所述单链DNA分子来源于打断的基因组DNA分子、胞外游离DNA分子、降解的DNA分子和/或亚硫酸处理的DNA分子。
根据本发明的实施例，针对同时含有双链核酸分子和单链核酸分子的样本，进行所述步骤(1)之前，将所述样本进行变性，以便得到全部为单链核酸分子的样本。
根据本发明的实施例，步骤S100中，利用T4 DNA连接酶使单链核酸分子的3’端与3’端接头相连。传统的单链建库方法是基于T4 RNA连接酶将单链测序接头直接连接在单链DNA模板分子上，具体地，一般是使用T4 RNA连接酶2(截短型)催化5’端预腺苷酸化的DNA接头与单链模板3’端连接，且接头的3’端封闭，可以减少产生接头二聚体。T4 RNA连接酶2(截短型)只能催化带腺苷酸化的DNA进行反应。T4 RNA连接酶2(截短型)成本高。
本方法利用主链和辅助链与模板DNA形成局部双链，直接使用T4 DNA连接酶即可。T4 DNA连接酶是最常用连接酶，成本低。
S200延伸和扩增
在该实施例中，将第一引物、DNA聚合酶、逆转录酶与所述连接产物进行延伸和扩增反应，以便得到3’端和5’端分别连有接头的扩增产物，所述扩增产物构成所述测序文库，其中，所述逆转录酶具有末端转移酶活性，所述延伸反应所得生成链的3’末端具有多个相同碱基序列；所述第一引物包括5’端接头序列和互补序列，所述互补序列与所述生成链的3’末端多个相同碱基序列反向互补。
根据本发明的实施例，所述辅助链的5’端序列与所述主链的3’端序列反向互补，所述辅助链的3’端预先采用可逆阻断基团修饰的方式进行封闭处理。在此条件下，步骤S200包括：除去所述辅助链的3’端上的可逆阻断基团，使得所述辅助链的3’端为羟基；以所述辅助链的3’端羟基为起始，在逆转录酶的作用下进行延伸，以便得到双链核酸，所述双链核酸中的生成链的3’末端具有多个相同碱基序列；利用所述第一引物和辅助链在DNA聚合酶的作用下对所述双链核酸进行扩增，以便得到扩增产物，所述扩增产物构成所述测序文库。
需要说明的是，本发明所描述的“双链核酸”既可以指双链DNA，也可以指RNA-DNA杂交链。具体地，当单链核酸分子为DNA时，经以辅助链的3’端羟基为起始，在逆转录酶的作用下进行延伸，以便得到双链DNA；当单链核酸分子为RNA时，经以辅助链的3’端羟基为起始，在逆转录酶的作用下进行延伸，以便得到RNA-DNA杂交链。
根据本发明的具体实施例，待测核酸单链分子为RNA时，所述辅助链的5’端序列与所述主链的3’端序列反向互补，所述辅助链的3’端预先采用可逆阻断基团修饰的方式进行封闭处理。在此条件下，步骤S200包括：除去所述辅助链的3’端上的可逆阻断基团，使得所述辅助链的3’端为羟基；以所述辅助链的3’端羟基为起始，在逆转录酶的作用下进行延伸，以便得到RNA-DNA杂交链，所述RNA-DNA杂交链中的DNA生成链的3’末端具有多个相同碱基序列；利用所述第一引物和3’端接头在DNA聚合酶的作用下对所述RNA-DNA杂交链进行延伸，替换掉RNA链得到双链DNA扩增产物，所述扩增产物构成所述测序文库。
本发明特殊设计的3’端接头结构不仅能够稳定地结合在模板链上，提高接头连接效率，避免出现接头自连。而且，可以以辅助链的3’端为起点直接延伸，生成双链DNA。由于第一引物包括5’端接头序列和与上述生成链的3’末端多个相同碱基序列反向互补的互补序列，3’端接头的辅助链与主链至少部分互补，因此可以利用上述第一引物和3’端接头对双链DNA进行扩增，获得测序文库。
根据本发明的实施例，所述封闭处理包括：磷酸化修饰、3’-O-丙烯基修饰或3’-O- 叠氮甲基修饰。由此，采用上述封闭处理方式既可以防止接头发生自连，也可以可逆地除去阻断基团，从而实施辅助链的延伸。
根据本发明的实施例，所述辅助链的5’端序列与所述主链的3’端序列部分反向互补；所述步骤(2)进一步包括：将第一引物、第二引物、DNA聚合酶、逆转录酶与所述连接产物进行延伸和扩增反应，以便得到3’端和5’端分别连有接头的扩增产物，所述扩增产物构成所述测序文库；其中，所述第二引物的序列至少与所述主链的3’端序列反向互补。
在此条件下，步骤S200包括：所述第二引物与所述连接产物杂交，在逆转录酶的作用下进行延伸反应，以便得到生成链，所述生成链的3’末端具有多个相同碱基序列；以所述生成链为模板链，所述第一引物与所述生成链3’末端的多个相同碱基序列杂交，在DNA聚合酶作用下进行扩增，以便得到双链核酸；利用所述第二引物和第一引物在DNA聚合酶的作用下对所述双链核酸进行扩增，以便得到扩增产物，所述扩增产物构成所述测序文库。
以步骤S100所得连接产物作为模板，所述第二引物的序列至少与所述主链的3’端序列反向互补，所以第二引物将与模板链杂交，在逆转录酶的作用下向模板链的5’端方向延伸，待延伸至5’末端时，由于逆转录酶具有末端转移酶活性，可以在生成链的3’末端形成多个相同碱基序列。
由于第一引物包括5’端接头序列和与上述生成链的3’末端多个相同碱基序列反向互补的互补序列，因此可以与生成链杂交，在DNA聚合酶作用下延伸，得到双链核酸。再利用第一引物和第二引物在DNA聚合酶的作用下对双链核酸进行扩增，获得测序文库。
根据本发明的实施例，所述辅助链的3’端预先经过封闭处理；所述封闭处理包括：磷酸化修饰、3’-O-丙烯基修饰或3’-O-叠氮甲基修饰。由此，可以避免出现接头自连。
根据本发明的实施例，所述多个相同碱基的个数为1～3个。
根据本发明的实施例，所述多个相同碱基中的碱基为C碱基。
根据本发明的实施例，所述主链进一步包括下列至少之一：测序引物的结合序列、标签序列和通用扩增引物的结合序列。
根据本发明的实施例，所述标签序列长度为5～12bp。
根据本发明的实施例，所述逆转录酶选自MMLV逆转录酶。在一些实施例中，所述逆转录酶选自SuperScript II逆转录酶或Alpha逆转录酶。上述逆转录酶均具有末端转移酶活性，可以在生成链的5’末端上形成多个相同碱基序列，有助于后续与第一引物序列互补杂交。
根据本发明的实施例，所述DNA聚合酶选自Q5热启动超保真DNA聚合酶、KAPA热启动超保真DNA聚合酶、Platinum热启动超保真DNA聚合酶或Pfu DNA聚合酶。由此， 以便实现高效扩增。
根据本发明的实施例，所述延伸反应和扩增反应是在同一反应体系中进行一步反应。由此，缩短了反应时间，提高了建库效率。
根据本发明的实施例，步骤S200进一步包括：将所述扩增产物进行变性，以便得到单链DNA；在单链环化试剂的存在下，将所述单链DNA进行单链环化反应，以便得到环化产物，所述环化产物构成单链环状测序文库。具体地，单链环化试剂选自DNA连接酶。
根据本发明的实施例，参见图2和图3，所述单链核酸分子测序文库构建方法包括3’端接头的连接和扩增的步骤。具体地，接头连接步骤是利用T4 DNA连接酶将局部双链的接头连接到单链DNA模板的3’端。其中接头包括主链和辅助链，辅助链的3’端有6个N到12个N的随机碱基序列，可以与单链DNA模板的3’端进行杂交互补，其5’端与主链的5’端部分序列反向互补杂交。这样辅助链和主链与单链模板之间形成局部双链。主链的3’端会利用磷酸化、C3-spacer、C5-spacer修饰或氨基修饰进行封闭，辅助链的3’端也会进行相应的封闭。
连接上接头之后进行扩增，扩增包括先利用与主链(3’端接头)互补的序列作为引物1和逆转录酶进行延伸，延伸到单链模板的5’末端后会利用逆转录酶带有的末端转移酶的活性加上3个胞嘧啶碱基。再利用引物2(含有5’端接头的序列以及带有3个G的3’端)与上步所得生成链进行杂交，然后以引物2为模板继续延伸。之后再利用DNA聚合酶和引物1和引物2对上步所得双链核酸进行PCR扩增。
如此，可以在一步反应中加上5’端接头的同时完成PCR扩增反应，得到带有2端测序接头的文库。对于illumina平台，扩增完成后，纯化即可得到文库。对于DNBSEQ平台，需要对PCR扩增产物进行高温变性淬火后都变成单链，再对单链DNA进行环化，再用线性消化酶消化没有环化的分子，得到适合单链DNA环状文库。
具体步骤如下：
1、完整的基因组DNA进行打断后片段筛选到100～500bp的大小DNA片段，或提取得到的胞外游离DNA，或者从各种降解的生物样本中提取的降解的DNA(例如古生物化石，FFPE样本)。完整的基因组DNA打断后的DNA片段或胞外游离DNA进行亚硫酸盐处理后纯化回收的DNA。
2、对DNA样本进行变性，让样本中存在的双链DNA变成单链DNA。一般进行高温变性(一般为92～98℃放置3～10分钟)，变性结束后立即放到冰上，使解链的双链DNA不至于复性。
3、DNA样本经过变性后，进行3’端的接头连接。先将辅助链与主链进行退火，形成 局部双链的接头。然后使用T4 DNA连接酶将单链DNA模板与接头进行连接。使得单链DNA模板加上3’端接头。其中接头序列可以是适配illumina或者DNBSEQ平台的5’端和3’端测序接头的序列。
4、一步完成之后，对加上接头的DNA进行纯化后。利用与3’端接头序列互补的PCR引物1和带有3个G的5’端测序接头序列作为引物2，以及逆转录酶和DNA聚合酶，以上步所得单链DNA为模板同时进行延伸和PCR扩增反应。其中引物1的3’端的3～12个核苷酸为脱氧核糖核苷酸或锁核苷酸修饰，引物2的3’端的3个G也为脱氧核糖核苷酸或锁核苷酸修饰，且引物2的5’端进行磷酸化修饰。
5、完成上一步的PCR反应后，对PCR产物进行纯化。DNBSEQ平台的单链环状DNA文库需要对纯化后的产物进行高温变性(一般为92～98℃放置3～10分钟)，变性结束后立即放到冰上，使解链的双链DNA不至于复性。
6、使用连接酶对变性后的产物直接进行单链分子内的环化，环化的后的单链DNA环包含模板和测序需要的测序接头。
7、使用线性消化酶对环化后的产物进行消化后得到最终的单链DNA环状文库。
测序方法
在本发明的另一方面，本发明提出了一种测序方法。根据本发明的实施例，所述测序方法包括：根据前面所述的方法构建测序文库；和对所述测序文库进行测序。由此，可以有效地获得更真实的核酸分子测序数据，可以为后续分析提供更有利的支持。并且，该方法操作简便、用时短、成本低，适于广泛应用。
需要说明的是，前面针对单链核酸分子测序文库构建方法所描述的特征和优点，同样适用于该测序方法，在此不再赘述。
用于构建单链核酸分子测序文库的接头
在本发明的又一方面，本发明提出了一种用于构建单链核酸分子测序文库的接头。根据本发明的实施例，所述接头包括：主链，所述主链用于与单链核酸分子的3’端相连；辅助链，所述辅助链的3’端含有多个N碱基，所述多个N碱基用于与单链核酸分子的3’端杂交；所述辅助链的5’端序列与所述主链的5’端至少部分序列反向互补。
根据本发明实施例的接头可以形成局部双链结构，主链为最终获得的扩增产物的3’端接头，辅助链的3’端含有多个N碱基，可以与模板链的3’端杂交，辅助链的5’端序列与主链的5’端至少部分序列反向互补，由此，辅助链的设计可以使得整个主链稳定地结 合在模板链上，提高接头连接效率，避免出现接头自连。
根据本发明的实施例，所述N碱基的个数选自6～12的整数。N表示随机碱基，当其个数为6～12时，辅助链可以稳定地与模板链结合，提高接头连接效率，避免出现接头自连。
根据本发明的实施例，所述主链和辅助链的3’端具有封闭基团修饰。具体地，所述主链的3’端具有磷酸化修饰、C3-spacer修饰、C5-spacer修饰或氨基修饰，由此，可以减少接头二聚体产生。所述辅助链的3’端具有磷酸化修饰、3’-O-丙烯基修饰或3’-O-叠氮甲基修饰，由此，可以减少接头二聚体产生。在连接接头之后可以除去上述修饰基团以使得辅助链3’端为羟基，从而可以以辅助链3’端作为起点进行延伸，生成双链核酸。
根据本发明的实施例，所述主链进一步包括下列至少之一：测序引物的结合序列、标签序列和通用扩增引物的结合序列。
根据本发明的实施例，所述标签序列长度为5～12bp。
用于构建单链核酸分子测序文库的试剂盒
在本发明的又一方面，本发明提出了一种用于构建单链核酸分子测序文库的试剂盒。根据本发明的实施例，所述试剂盒包括前面所述接头。由此，利用该试剂盒可以实现单链核酸分子建库。
根据本发明的实施例，所述主链和辅助链分别以独立包装形式提供。由此，在使用该接头时，通过退火可以使得主链和辅助链形成局部双链结构。
根据本发明的实施例，所述试剂盒进一步包括：逆转录酶、DNA聚合酶、DNA连接酶、第二引物和第一引物；其中，所述第二引物序列与所述主链的至少一部分序列反向互补；所述第一引物与包括5’端接头序列和含有多个相同碱基的序列。
DNA连接酶可以在单链核酸分子的3’端连接接头，获得连接产物。以该连接产物作为模板链，由于第二引物的序列可以与接头的至少一部分序列反向互补，所以第二引物将与模板链杂交，在逆转录酶的作用下向模板链的5’端方向延伸，待延伸至5’末端时，由于逆转录酶具有末端转移酶活性，可以在生成链的3’末端形成多个相同碱基序列。
由于第一引物包括5’端接头序列和与上述多个相同碱基序列反向互补的互补序列，因此可以与生成链杂交，在DNA聚合酶作用下延伸，得到双链核酸。再利用第二引物和第一引物在DNA聚合酶的作用下对双链核酸进行扩增，获得测序文库。
由此，采用本发明的试剂盒建库获得的测序文库中核酸分子(扩增产物)同时含有3’端接头和5’端接头，并且延伸和扩增反应在一管中进行、仅需要一步反应，增加了增加接头的效率，同时也降低了原始模板的损失，整体上提升了原始模板的利用率，缩短了反 应时间。
根据本发明的实施例，所述多个相同碱基的个数为1～3个。
根据本发明的实施例，所述多个相同碱基中的碱基为C碱基。
根据本发明的实施例，所述逆转录酶选自MMLV逆转录酶。在一些实施例中，所述逆转录酶选自SuperScript II逆转录酶或Alpha逆转录酶。上述逆转录酶均具有末端转移酶活性，可以在生成链的5’末端上形成多个相同碱基序列，有助于与第一引物序列互补杂交。
根据本发明的实施例，所述DNA聚合酶选自Q5热启动超保真DNA聚合酶、KAPA热启动超保真DNA聚合酶、Platinum热启动超保真DNA聚合酶或Pfu DNA聚合酶。由此，以便实现高效扩增。
根据本发明的实施例，所述DNA连接酶选自T4 DNA连接酶。由此，以便于在单链核酸分子的3’端连接接头，连接效率高，成本低。
需要说明的是，前面针对接头所描述的特征和优点，同样适用于该试剂盒，在此不再赘述。
用途
在本发明的又一方面，本发明提出了前面所述接头或试剂盒在构建单链核酸分子测序文库或测序中的用途。利用本发明的接头或试剂盒构建的测序文库同时含有已知序列的3’端接头和5’端接头，接头连接效率高，原始模板损失少、利用率高，反应时间短，成本低。进一步地，利用该测序文库进行测序可以有效地获得更真实的核酸分子测序数据，可以为后续分析提供更有利的支持。
需要说明的是，前面针对接头和试剂盒所描述的特征和优点，同样适用于该用途，在此不再赘述。
有益效果
本发明只需要先让单链DNA模板加上3’端接头，之后利用逆转录酶实现5’端接头的连接和PCR扩增在一步反应中完成，让原来需要分别在DNA两端加上不同的测序接头，只需要一步连接反应，同时5’端接头的添加和PCR的扩增在一管中进行，增加了连接接头的效率，同时也降低了原始模板的损失，整体上提升了原始模板的利用率，减少了反应时间。另外，测序接头也不需要使用特殊的修饰，也不需要特殊的高成本的连接酶，从而降低了成本。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1 人外周血游离DNA甲基化文库构建与测序
一、实验材料
人外周血血浆提取的游离DNA
二、实验步骤
1、取5ng人外周血血浆游离DNA(cfDNA)到200μL PCR管中，总体积在20μL。
2、使用EZ DNA Methylation-Gold Kit(Zymo Research,Cat.No.D5005/D5006)对接头连接产物进行Bisulfite处理和纯化。
3、依次吸取900μL NF water，300μL M-Dilution Buffer和50μL M-Dissolving Buffer到一管CT Conversion Reagent粉末(开盖前需瞬时离心)，在室温下频繁涡旋震荡10min，完成CT Conversion Reagent的配制。CT Conversion Reagent应减少曝光，最好现配现用。CT Conversion Reagent在室温下可最多保存1天，在4℃下可最多保存1周，在-20℃下可最多保存1个月，非现配的CT Conversion Reagent使用前应预热到37℃，在室温下频繁涡旋震荡10min方可使用。
4、M-Wash Buffer第一次开盖使用前需按照瓶子标签所示加入正确体积的无水乙醇，混匀方可使用在室温下，将以下成分加入新的200μL PCR管中。
组分 体积
CT Conversion Reagent 130μL
cfDNA 20μL
Total 150μL
5、将200μL PCR管置于PCR仪上，按照Bisulfite处理条件进行反应
温度 时间
热盖 On
98℃ 10min
64℃ 2.5h
4℃ Hold
6、将反应产物转移到新的1.5mL离心管中，加入600μL M-Binding Buffer，涡旋震荡6次，每次3s，瞬时离心将反应液收集至管底。
7、把Zymo-Spin IC Column放入2mL Collection Tube，将混合液转移至Zymo-Spin IC Column上，13000rpm离心30s，弃废液，将Zymo-Spin IC Column放回Collection Tube。
8、向Zymo-Spin IC Column加入100μL M-Wash Buffer，13000rpm离心30s。
9、向Zymo-Spin IC Column加入200μL M-Desulphonation Buffer，迅速盖紧管盖后，室温孵育15～20min，13000rpm离心30s，弃废液，将Zymo-Spin IC Column放回Collection Tube。
10、向Zymo-Spin IC Column加入200μL M-Wash Buffer，13000rpm离心30s，弃废液，将Zymo-Spin IC Column放回Collection Tube。
11、向Zymo-Spin IC Column加入200μL M-Wash Buffer，13000rpm离心30s，弃废液，将Zymo-Spin IC Column放回Collection Tube。13000rpm空转离心30s，弃Collection Tube，用移液器将Zymo-Spin IC Column外壁液体尽量吸干，放在新的1.5mL离心管中。
12、打开Zymo-Spin IC Column管盖，室温干燥2min，然后将Zymo-Spin IC Column放在另一新的1.5mL离心管中。
13、缓慢在Zymo-Spin IC Column滤膜中央加入10μL M-Elution Buffer，静置1min，13000rpm离心30s，纯化后的Bisulfite处理后的纯化产物即被收集在1.5mL离心管中。
14、取全部Bisulfite处理和纯化后产物于新200μL PCR管中，加入分子级水将总体积补至20μL。
15、准备接头，将20μM的测序接头1(主链)和20μM的测序接头2(辅助链)按照1:1的比例进行退火杂交，形成局部双链的接头Adapter mix。反应条件为：95℃3min,之后降温到4℃，设置PCR仪的降温速率为0.1℃/s。
测序接头1的序列：
5’Pho-AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAATAGGTCCGATCAACTCCTTGGCTCACA-3’Pho(SEQ ID NO：1)
测序接头2的序列：
5’-ACATGGCTACGATCCGACTTNNNNNNNNNNNN-3’Pho(SEQ ID NO：2)
16、使用来自MGI的酶切DNA文库制备试剂盒(1000005254，MGI)中的T4 DNA连接酶和连接buffer进行接头连接，按照下表在冰盒上配制连接反应液。
组分 体积
T4 DNA ligase 1.6μL
Ligation buffer 23.4μL
Adapter Mix 10μL
17、将配制完成的连接反应液35μL加入到装有变性后的DNA的PCR管中，使用振荡混匀仪进行充分混匀，再瞬时离心，将液体离心到管底。
18、将离心好的PCR管放置到PCR仪上30℃反应30min进行连接反应。
19、连接反应结束后，将反应产物转移到1.5mL离心管中。之后加入96μL的纯化磁珠，用移液器吹打10次到充分混匀后，常温孵育5min。
20、瞬时离心后，将1.5mL离心管放置于磁力架上，静置5min至液体澄清，用移液器吸取并丢弃上清液。
21、向1.5mL离心管中加入200μL 75％乙醇，静置30s，洗涤磁珠。
22、重复上一步。
23、尽量吸干管内液体，有少量残留在管壁时可将1.5mL离心管瞬时离心，在磁力架上分离后，用小量程的移液器将管底液体吸干。
24、保持1.5mL离心管固定于磁力架上，打开1.5mL离心管管盖，室温干燥，直至磁珠表面无反光、无开裂。
25、将1.5mL离心管从磁力架上取下，加入16μL分子级水进行DNA洗脱，用移液器轻轻吹打10次至完全混匀。
26、室温下孵育5min。
27、瞬时离心，将1.5mL离心管置于磁力架上，静置5min至液体澄清，将15μL上清液转移到新的200ul PCR管中。
28、按照下表在冰盒上配制扩增反应液
Figure PCTCN2022099505-appb-000001
Figure PCTCN2022099505-appb-000002
29、将配制完成的扩增反应液35μL加入到装有步骤27纯化后的DNA的PCR管中。使用振荡混匀仪进行充分混匀后瞬时离心，将液体离心到管底。
Primer1：5’-TGTGAGCCAAGGAG/iXNA_T//iXNA_T//iXNA_G/-3’(SEQ ID NO：3)
Pirmer2：
5’Pho-GAACGACATGGCTACGATCCGACTT/iXNA_G//iXNA_G//iXNA_G/-3’(SEQ ID NO：4)(iXNA_.代表锁核苷酸修饰)
30、将PCR管置于PCR以上，按照下表程序进行反应。
Figure PCTCN2022099505-appb-000003
31、反应完成后，瞬时离心收集PCR管中的液体到管底。
32、向离心管中加入80ul XP纯化磁珠，用移液器吹打10次到充分混匀后，常温孵育10min。
33、瞬时离心后，将1.5mL离心管放置与磁力架上，静置5min至液体澄清，用移液器吸取并丢弃上清液。
34、向1.5mL离心管中加入200ul 75％乙醇，静置30s，洗涤磁珠。
35、重复上一步。尽量吸干管内液体，有少量残留在管壁时可将1.5mL离心管瞬时离心，在磁力架上分离后，用小量程的移液器将管底液体吸干。
36、保持1.5mL离心管固定于磁力架上，打开1.5mL离心管管盖，室温干燥，直至磁珠表面无反光、无开裂。
37、将1.5mL离心管从磁力架上取下，加入32μL分子级水进行DNA洗脱，用移液器轻轻吹打10次至完全混匀。
38、室温下孵育5min。
39、瞬时离心，将1.5mL离心管置于磁力架上，静置5min至液体澄清，将30μL上清液转移到新的1.5mL离心管中。
40、使用
Figure PCTCN2022099505-appb-000004
dsDNA HS Assay Kit(Theromo Fisher，Q32854)荧光定量试剂盒，按照定量试剂盒的操作说明对酶切消化纯化后产物进行定量。
41、取360ng PCR产物至新的0.2mL PCR管中，用TE Buffer补充至总体积48μL。
42、将PCR管置于PCR仪上，按照下表的条件进行反应。
温度 时间
105℃热盖 On
95℃ 3min
43、反应结束后，立即将PCR管置于冰上，静置2min。
44、使用来自MGI的环化试剂盒(MGI，1000005260)，提前按照下表的配方在冰上按照下表的配方在冰上配制环化反应液。
组分 单个反应体积(μL)
Splint Buffer 11.6
DNA Rapid Ligase 0.5
总体积 12.1
45、用移液器吸取12.1μL配制好的单链环化反应液加入步骤43的PCR管中，涡旋振荡3次，每次3s，瞬时离心将反应液收集至管底。
46、将PCR管置于PCR仪上，按照下表的条件进行反应。
温度 时间
75℃热盖 On
37℃ 30min
4℃ Hold
47、反应结束后，将PCR管瞬时离心并置于冰上，立即进入步骤49反应。
48、使用来自MGI的环化试剂盒(MGI，1000005260)，提前按照下表的配方在冰上配制酶切消化反应液。
组分 单个反应体积(μL)
Digestion Buffer 1.4
Digestion Enzyme 2.6
总体积 4
49、用移液器吸取4μL配制好的酶切消化反应液加入步骤47的PCR管中，涡旋振荡3次，每次3s，瞬时离心将反应液收集至管底。
50、将PCR管置于PCR仪上，按照下表的条件进行反应。
温度 时间
75℃热盖 On
37℃ 30min
4℃ Hold
51、反应结束后，瞬时离心将反应液收集至管底。
52、立即向PCR管中加入7.5μL Digestion Stop Buffer(来自MGI的环化试剂盒MGI，1000005260)涡旋振荡3次，每次3s，瞬时离心将反应液收集至管底，吸取全部反应液转移到新的1.5mL离心管中。
53、提前取出DNA Clean Beads，置于室温平衡至少30min，使用前充分震荡混匀。吸取170μL DNA Clean Beads至酶切消化产物中，用移液器轻轻吹打至少10次至所有磁珠悬浮，最后一次应确保将吸头中所有液体及磁珠都打入离心管中。
54、室温孵育10min。
55、将离心管瞬时离心，置于磁力架上，静置2～5min至液体澄清，用移液器小心吸取上清并丢弃。
56、保持离心管置于磁力架上，加入500μL新鲜配制的80％乙醇漂洗磁珠及管壁，静置30s后小心吸取上清并丢弃。
57、重复步骤上一步，尽量吸干管内液体，有少量残留在管壁时可将离心管瞬时离心，在磁力架上分离后，用小量程的移液器将管底液体吸干。
58、保持离心管置于磁力架上，打开离心管管盖，室温干燥，直至磁珠表面无反光。将离心管从磁力架上取下，加入22μL TE Buffer进行DNA洗脱，用移液器轻轻吹打至少10次至所有磁珠悬浮。
59、室温下孵育10min。
60、将离心管瞬时离心，置于磁力架上，静置2～5min至液体澄清，用移液器吸取20μL上清液转移到新的1.5mL离心管中。
61、使用
Figure PCTCN2022099505-appb-000005
ssDNA Assay Kit(Thermo Fisher，Q10212)荧光定量试剂盒，按照定量试剂盒的操作说明对酶切消化纯化后产物进行定量。
62、使用MGISEQ-2000测序仪进行测序，使用MGISEQ-2000高通量测序试剂套装(PE100)(MGI,货号1000012536)，按照说明书指导进行DNB制作和测序。
三、实验结果
1、建库结果
Figure PCTCN2022099505-appb-000006
2、测序结果
指标 结果
原始reads数目 1339286228
过滤后reads数目 1133839420
Reads长度(bp) 100
比对率(％) 91.62
重复率(％) 25.26
唯一比对率(％) 86.85
双端比对率(％) 79.31
单独比对率(％) 12.31
覆盖度(％) 97.95
平均深度 24.28
BS转化率(％) 97.6
检测到CG位点数目(1x) 51988782
实验结果如上表所示，可以看出，投入5ng cfDNA后，经过反应得到的双链DNA文库浓度为38.5ng/μL。以双链DNA文库构建的单链环DNA文库的浓度为3.4ng/μL。取8ng的单链环文库进行DNBSEQ平台特有的DNA纳米球(DNB)的制备，得到的DNB浓度是28.5ng/μL，这些指标都达到预期。测序之后，对数据进行分析，比对率能达到91.62％，重复率在25.26％，覆盖度能到97.95％，这些指标都达到了预期。说明使用本方法进行建库，对模板DNA的利用率高，得到测序数据质量高。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须 针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (37)

  1. 一种单链核酸分子测序文库构建方法，其特征在于，包括：
    (1)使单链核酸分子的3’端与3’端接头相连，以便得到连接产物；
    (2)将第一引物、DNA聚合酶、逆转录酶与所述连接产物进行延伸和扩增反应，以便得到3’端和5’端分别连有接头的扩增产物，所述扩增产物构成所述测序文库；
    其中，
    所述3’端接头包括：
    主链，所述主链用于与所述单链核酸分子的3’端相连；
    辅助链，所述辅助链的3’端含有多个N碱基，所述多个N碱基用于与所述单链核酸分子的3’端杂交；所述辅助链的5’端序列与所述主链的5’端至少部分序列反向互补；
    所述逆转录酶具有末端转移酶活性，所述延伸反应所得生成链的3’末端具有多个相同碱基序列；
    所述第一引物包括5’端接头序列和互补序列，所述互补序列与所述生成链的3’末端多个相同碱基序列反向互补。
  2. 根据权利要求1所述的方法，其特征在于，所述辅助链的5’端序列与所述主链的3’端序列反向互补，所述辅助链的3’端预先采用可逆阻断基团修饰的方式进行封闭处理。
  3. 根据权利要求2所述的方法，其特征在于，步骤(2)包括：
    除去所述辅助链的3’端上的可逆阻断基团，使得所述辅助链的3’端为羟基；
    以所述辅助链的3’端羟基为起始，在逆转录酶的作用下进行延伸，以便得到双链核酸，所述双链核酸中的生成链的3’末端具有多个相同碱基序列；
    利用所述第一引物和辅助链在DNA聚合酶的作用下对所述双链核酸进行扩增，以便得到扩增产物，所述扩增产物构成所述测序文库。
  4. 根据权利要求2或3所述的方法，其特征在于，所述封闭处理包括：磷酸化修饰、3’-O-丙烯基修饰或3’-O-叠氮甲基修饰。
  5. 根据权利要求1所述的方法，其特征在于，所述辅助链的5’端序列与所述主链的5’端序列部分反向互补；
    所述步骤(2)进一步包括：将第一引物、第二引物、DNA聚合酶、逆转录酶与所述连接产物进行延伸和扩增反应，以便得到3’端和5’端分别连有接头的扩增产物，所述扩增产物构成所述测序文库；
    其中，所述第二引物的序列至少与所述主链的3’端序列反向互补。
  6. 根据权利要求5所述的方法，其特征在于，步骤(2)包括：
    以所述连接产物为模板链，所述第二引物与所述连接产物3’接头杂交，在逆转录酶的作用下进行延伸反应，以便得到生成链，所述生成链的3’末端具有多个相同碱基序列；
    所述第一引物与所述生成链3’末端的多个相同碱基序列杂交，在DNA聚合酶作用下进行延伸，以便得到双链核酸分子；
    利用所述第一引物和第二引物在DNA聚合酶的作用下对所述双链核酸分子进行扩增，以便得到扩增产物，所述扩增产物构成所述测序文库。
  7. 根据权利要求5或6所述的方法，其特征在于，所述辅助链的3’端预先经过封闭处理；
    所述封闭处理包括：磷酸化修饰、3’-O-丙烯基修饰或3’-O-叠氮甲基修饰。
  8. 根据权利要求1-7任一项所述的方法，其特征在于，所述主链的3’端预先经过封闭处理；
    所述主链的3’端具有磷酸化修饰、C3-spacer修饰、C5-spacer修饰或氨基修饰。
  9. 根据权利要求1-8任一项所述的方法，其特征在于，所述主链和辅助链是以独立形式提供的，进行所述步骤(1)之前，将所述主链和辅助链进行退火，以便使所述主链和辅助链部分杂交，形成局部双链的3’端接头。
  10. 根据权利要求1-9任一项所述的方法，其特征在于，所述N碱基的个数选自6～12的整数。
  11. 根据权利要求1-10任一项所述的方法，其特征在于，所述单链核酸分子选自RNA分子或单链DNA分子。
  12. 根据权利要求11所述的方法，其特征在于，所述单链DNA分子来源于打断的基因组DNA分子、胞外游离DNA分子、降解的DNA分子和/或亚硫酸处理的DNA分子。
  13. 根据权利要求1-12任一项所述的方法，其特征在于，针对同时含有双链核酸分子和单链核酸分子的样本，进行所述步骤(1)之前，将所述样本进行变性，以便得到全部为单链核酸分子的样本。
  14. 根据权利要求1-13任一项所述的方法，其特征在于，步骤(1)中，利用T4 DNA连接酶使单链核酸分子的3’端与3’端接头相连。
  15. 根据权利要求1-14任一项所述的方法，其特征在于，所述多个相同碱基的个数为1～3个，所述多个相同碱基中的碱基为C碱基。
  16. 根据权利要求1-15任一项所述的方法，其特征在于，所述主链进一步包括下列至少之一：测序引物的结合序列、标签序列和通用扩增引物的结合序列。
  17. 根据权利要求16所述的方法，其特征在于，所述标签序列长度为5～12bp。
  18. 根据权利要求1-17任一项所述的方法，其特征在于，所述逆转录酶选自MMLV逆转录酶。
  19. 根据权利要求1-18任一项所述的方法，其特征在于，所述逆转录酶选自SuperScript II逆转录酶或Alpha逆转录酶。
  20. 根据权利要求1-19任一项所述的方法，其特征在于，所述延伸反应和扩增反应是在同一反应体系中进行一步反应。
  21. 根据权利要求1-20任一项所述的方法，其特征在于，步骤(2)进一步包括：
    将所述扩增产物进行变性，以便得到单链DNA；
    在单链环化试剂的存在下，将所述单链DNA进行单链环化反应，以便得到环化产物，所述环化产物构成单链环状测序文库。
  22. 一种测序方法，其特征在于，包括：
    根据权利要求1～21任一项所述的方法构建测序文库；和
    对所述测序文库进行测序。
  23. 一种用于构建单链核酸分子测序文库的接头，其特征在于，所述接头包括：
    主链，所述主链用于与单链核酸分子的3’端相连；
    辅助链，所述辅助链的3’端含有多个N碱基，所述多个N碱基用于与单链核酸分子的3’端杂交；所述辅助链的5’端序列与所述主链的5’端至少部分序列反向互补。
  24. 根据权利要求23所述的接头，其特征在于，所述N碱基的个数选自6～12的整数。
  25. 根据权利要求23或24所述的接头，其特征在于，所述主链和辅助链的3’端具有封闭基团修饰。
  26. 根据权利要求25所述的接头，其特征在于，所述主链的3’端具有磷酸化修饰、C3-spacer修饰、C5-spacer修饰或氨基修饰；
    所述辅助链的3’端具有磷酸化修饰、3’-O-丙烯基修饰或3’-O-叠氮甲基修饰磷酸化修饰、C3-spacer修饰、C5-spacer修饰或氨基修饰。
  27. 根据权利要求23～26任一项所述的接头，其特征在于，所述主链进一步包括下列至少之一：测序引物的结合序列、标签序列和通用扩增引物的结合序列。
  28. 根据权利要求27所述的接头，其特征在于，所述标签序列长度为5～12bp。
  29. 一种用于构建单链核酸分子测序文库的试剂盒，其特征在于，包括权利要求23～28任一项所述的接头。
  30. 根据权利要求29所述的试剂盒，其特征在于，所述主链和辅助链分别以独立包装 形式提供。
  31. 根据权利要求29或30所述的试剂盒，其特征在于，进一步包括：逆转录酶、DNA聚合酶、DNA连接酶、第一引物和第二引物；
    其中，所述第一引物与包括5’端接头序列和含有多个相同碱基的序列，
    所述第二引物序列与所述主链的至少一部分序列反向互补。
  32. 根据权利要求31所述的试剂盒，其特征在于，所述逆转录酶选自MMLV逆转录酶。
  33. 根据权利要求31或32所述的试剂盒，其特征在于，所述逆转录酶选自SuperScript II逆转录酶或Alpha逆转录酶。
  34. 根据权利要求31所述的试剂盒，其特征在于，所述DNA聚合酶选自Q5热启动超保真DNA聚合酶、KAPA热启动超保真DNA聚合酶、Platinum热启动超保真DNA聚合酶或Pfu DNA聚合酶。
  35. 根据权利要求31所述的试剂盒，其特征在于，所述多个相同碱基的个数为1～3个，所述多个相同碱基中的碱基为C碱基。
  36. 根据权利要求31所述的试剂盒，其特征在于，所述DNA连接酶选自T4 DNA连接酶。
  37. 权利要求23～28任一项所述接头或权利要求29～36任一项所述试剂盒在构建单链核酸分子测序文库或测序中的用途。
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