WO2024259564A1 - 一种一步法构建靶向文库的方法及其应用 - Google Patents

一种一步法构建靶向文库的方法及其应用 Download PDF

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Publication number
WO2024259564A1
WO2024259564A1 PCT/CN2023/101079 CN2023101079W WO2024259564A1 WO 2024259564 A1 WO2024259564 A1 WO 2024259564A1 CN 2023101079 W CN2023101079 W CN 2023101079W WO 2024259564 A1 WO2024259564 A1 WO 2024259564A1
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sequence
universal
specific
oligonucleotide
specific oligonucleotide
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English (en)
French (fr)
Inventor
杨林
张艳艳
刘锋
普珺
杨贵芳
夏军
陈芳
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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Priority to CN202380096087.4A priority Critical patent/CN120882880A/zh
Priority to PCT/CN2023/101079 priority patent/WO2024259564A1/zh
Publication of WO2024259564A1 publication Critical patent/WO2024259564A1/zh
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the invention belongs to the technical field of library construction, and particularly relates to a one-step method for constructing a targeted library and an application thereof.
  • Target region capture technology can be roughly divided into two types: one is hybridization-based capture sequencing technology, and the other is multiplex PCR-based capture technology.
  • the former has a cumbersome experimental process and a high probe cost, which limits its clinical application.
  • the latter has simple experimental operation, strong flexibility, low cost and wider adaptability.
  • the targeted sequencing library of multiplex PCR is a library preparation method that selects target genes or regions for amplification through specific PCR technology, and adds sequencing adapters and sample tag sequences.
  • the complete sequencing adapter and sample label sequence that need to be added in the second step PCR can be integrated into the 5' end of the specific primer in the first step PCR.
  • the corresponding adapter and sample label sequence are added to the 5' end of the library.
  • RNA targeted library preparation can not only perform targeted amplification of DNA, but also prepare targeted libraries for RNA.
  • RNA targeted library preparation requires an additional reverse transcription process. Usually, all RNAs are reverse transcribed using the N6 primer to obtain a single-strand cDNA product, and then the cDNA is amplified and enriched using specific primers. The overall operation is relatively complicated.
  • the Ampliseq method of Thermo and the Clean plex method of Paragon genomics are currently commonly used library construction methods. However, they both involve multiple steps and the library construction operation is cumbersome. Therefore, it is necessary to develop a simpler library construction method.
  • the first aspect of the present invention aims to provide an oligonucleotide combination.
  • the second aspect of the present invention aims to provide a kit.
  • the third aspect of the present invention aims to provide a multiplex PCR amplification method.
  • the fourth aspect of the present invention aims to provide a method for constructing a targeted library.
  • the first aspect of the present invention provides an oligonucleotide combination, comprising: a specific oligonucleotide and a universal primer;
  • the universal primers include: a first universal primer and a second universal primer;
  • the specific oligonucleotides include: an upstream specific oligonucleotide and a downstream specific oligonucleotide: wherein,
  • the upstream specific oligonucleotide comprises, from the 5' end to the 3' end, an upstream specific sequence and a first universal sequence
  • the downstream specific oligonucleotide comprises, from the 5' end to the 3' end, a downstream specific sequence and a second universal sequence;
  • the 3' end sequence of the first universal primer interacts with a part or all of the first universal sequence of the upstream specific oligonucleotide;
  • the 3' end sequence of the second universal primer is complementary to a part or all of the second universal sequence of the downstream specific oligonucleotide;
  • the 3' end sequence of the second universal primer is complementary to a part or all of the first universal sequence of the upstream specific oligonucleotide, and the 3' end sequence of the first universal primer is complementary to a part or all of the second universal sequence of the downstream specific oligonucleotide;
  • the sequence of the upstream specific oligonucleotide contains one or more cleavage sites that can be cleaved by a specific cleavage agent;
  • the sequence of the downstream specific oligonucleotide comprises one or more cleavage sites that can be cleaved by a specific cleavage agent.
  • the specific cleavage agent comprises an enzymatic cleavage agent.
  • the enzymatic cleavage agent comprises at least one of a ribonuclease (e.g., ribonuclease H), a glycosylase (e.g., uracil-DNA glycosylase (UDG), a USER enzyme (e.g., a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII), a 3-methyladenine DNA glycosylase, a 3-methyladenine DNA glycosylase II, a pyrimidine hydrate-DNA glycosylase, a FaPy-DNA glycosylase, and a thymine mismatch-DNA glycosylase (e.g., a hypoxanthine-DNA glycosylase, a uracil DNA glycosylase (UDG), a 5-hydroxymethyluracil DNA glycosylase (HmUDG), a 5-hydroxymethylcytosine DNA glycosylase, and a 1,
  • the enzymatic cleavage agent is selected from at least one of RNase H and UDG; and is further selected from RNase H.
  • the ribonuclease H comprises at least one of ribonuclease HI and ribonuclease HII.
  • the specific cleavage agents in the upstream specific oligonucleotide and the downstream specific oligonucleotide may be the same or different.
  • the multiple specific cleavage agents in the upstream specific oligonucleotide may be the same or different.
  • the multiple specific cleavage agents in the downstream specific oligonucleotide may be the same or different.
  • the cleavage site comprises at least one of c1) to c2):
  • rNTP Ribonucleotides
  • the cleavage site comprises c1).
  • the cleavage sites in the upstream specific oligonucleotide and the downstream specific oligonucleotide may be the same or different.
  • the multiple cleavage sites in the upstream specific oligonucleotide may be the same or different.
  • the multiple cleavage sites in the downstream specific oligonucleotide may be the same or different.
  • the first universal primer comprises a functional nucleic acid sequence.
  • the first universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.
  • the first universal primer comprises one or more sequencing primer sequences or sequencing primer complementary sequences; further preferably, the first universal primer comprises two or more sequencing primer sequences or sequencing primer complementary sequences.
  • the first universal primer further comprises a first tag sequence.
  • the first tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence; further preferably, the first tag sequence is located in the middle of two segments of the sequencing primer sequence or the sequencing primer complementary sequence.
  • the second universal primer comprises a functional nucleic acid sequence.
  • the second universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.
  • the second universal primer comprises one or more sequencing primer sequences or sequencing primer complementary sequences; further preferably, the second universal primer comprises two or more sequencing primer sequences or sequencing primer complementary sequences.
  • the second universal primer further comprises a second tag sequence.
  • the second tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence; further preferably, the second tag sequence is located in the middle of two segments of the sequencing primer sequence or the sequencing primer complementary sequence.
  • the first tag sequence and the second tag sequence are the same or different.
  • the first universal sequence and the second universal sequence are each independently selected from a partial sequencing adapter sequence, a complete sequencing adapter sequence, a sequencing primer binding sequence or any fixed sequence (such as comprising a restriction site).
  • the first universal sequence is the same as or different from the second universal sequence; further preferably, the first universal sequence is different from the second universal sequence.
  • the 3' end of the upstream specific oligonucleotide is blocked; and/or
  • the 3' end of the downstream specific oligonucleotide is subjected to blocking modification.
  • the blocking modification comprises: at least one of: phosphorylation modification, spacer modification, and amino modification; further preferably, the blocking modification comprises: phosphorylation modification.
  • the blocking modifications of the upstream-specific oligonucleotide and the downstream-specific oligonucleotide are the same or different.
  • the upstream specific sequence and the downstream specific sequence in the specific oligonucleotide are respectively identical to or complementary to the two segments of the target region to be detected.
  • the 5' end of the first universal primer contains a phosphate group
  • the 5' end of the second universal primer contains a phosphate group.
  • the number of cleavage sites that can be cleaved by a specific cleavage agent is an integer.
  • the upstream specific sequence of the upstream specific oligonucleotide has 15 to 45 nucleotides.
  • the first universal sequence of the upstream specific oligonucleotide has 15-45 nucleotides.
  • the upstream specific oligonucleotide has 30 to 90 nucleotides.
  • the number of nucleotides in the downstream specific sequence of the downstream specific oligonucleotide is 15-45.
  • the second universal sequence of the downstream specific oligonucleotide has 15-45 nucleotides.
  • the number of nucleotides in the downstream specific oligonucleotide is 30-90.
  • the first universal primer has 15-90 nucleotides.
  • the second universal primer has 15-90 nucleotides.
  • the plurality of cleavage sites that can be cleaved by a specific cleavage agent are randomly distributed in the upstream specific oligonucleotide.
  • the plurality of cleavage sites that can be cleaved by a specific cleavage agent are randomly distributed in the downstream specific oligonucleotide.
  • the specific oligonucleotides comprise multiple pairs of specific oligonucleotides; and further comprise multiple pairs of specific oligonucleotides targeting different target regions to be detected.
  • the universal primers comprise a plurality of pairs of universal primers.
  • the second aspect of the present invention provides a kit comprising: the oligonucleotide combination of the first aspect of the present invention.
  • the kit further comprises: at least one of a reverse transcriptase and a DNA polymerase; and further comprises a reverse transcriptase and a DNA polymerase.
  • the kit further comprises: the specific cleavage agent in the first aspect of the present invention.
  • the kit further comprises: a reverse transcription primer.
  • the kit comprises: a nucleic acid extraction reagent combination.
  • the nucleic acid extraction reagent combination is a nucleic acid extraction reagent combination for any one of the following methods: alkaline lysis method, phenol chloroform extraction method, chelating resin method, centrifugal column membrane adsorption method and magnetic bead method.
  • the nucleic acid extraction reagent combination comprises: at least one of: a lysis solution, a washing solution, an eluent, and a nucleic acid adsorbent; further preferably, the nucleic acid extraction reagent combination comprises: a lysis solution, a washing solution, an eluent, and a nucleic acid adsorbent.
  • the nucleic acid adsorbent comprises at least one of magnetic beads and adsorption membranes.
  • the third aspect of the present invention provides a multiplex PCR amplification method, comprising the step of using the oligonucleotide combination of the first aspect of the present invention.
  • the multiplex PCR amplification method comprises the following steps: performing a multiplex PCR reaction on nucleic acid using the oligonucleotide combination of the first aspect of the present invention.
  • reaction steps include:
  • Amplification reaction was performed using PCR reaction primers.
  • the extension reaction and amplification reaction are carried out in the same system.
  • the multiplex PCR reaction further comprises: a reverse transcription reaction.
  • the reverse transcription reaction and the multiplex PCR reaction are performed in the same system or in different systems; further preferably, the reverse transcription reaction and the multiplex PCR reaction are performed in the same system.
  • the same system is specifically carried out in the same reaction system.
  • the fourth aspect of the present invention provides a method for constructing a targeted library, comprising the steps of the multiplex PCR amplification method of the third aspect of the present invention to obtain a sequencing library.
  • a circularization reaction may be further included before obtaining the sequencing library, that is, circularizing the linear library obtained by amplification.
  • the method further comprises the step of: purifying the sequencing library.
  • the purification is performed using magnetic beads.
  • the present invention provides an oligonucleotide combination, comprising: a specific oligonucleotide and a universal primer; wherein the 5' end to the 3' end of the specific oligonucleotide sequentially comprises: a specific sequence and a universal sequence, the specific oligonucleotide comprises one or more cleavage sites that can be cleaved by a specific cleavage agent; the 3' end of the universal primer comprises a sequence complementary to the universal sequence of the specific oligonucleotide; thus, a long-chain primer comprising a universal primer sequence and a sequence complementary to the specific oligonucleotide can be generated by the specific oligonucleotide and the universal primer (short-chain oligonucleotide), the long-chain primer realizes the enrichment of the target region and the addition of the universal primer sequence (such as a sequencing adapter sequence and a sample tag sequence) to the product during the PCR process, thereby realizing the one-
  • RNA targeted libraries 1) in the RT reaction, the 3' end sequence diversity of specific oligonucleotides and universal primers is low, the possibility of primer-template binding is low, and even non-specific products generated by binding to the template will not accumulate in the subsequent PCR amplification process; 2) during the RT process, the 3' end sequence diversity of specific oligonucleotides and universal primers is low, the possibility of forming dimers with each other is small, and the probability of generating dimers is small; long-chain primers containing universal primer sequences and sequences complementary to specific oligonucleotides are generated by short-chain oligonucleotides (specific oligonucleotides and universal primers) and are compatible with the RNA reverse transcription process, realizing the reverse transcription step, long-chain primer generation, enrichment of PCR target areas, and addition of universal primer sequences (such as sequencing adapter sequences and sample label sequences); the process of reverse transcription, amplification, and library preparation is realized in one tube
  • FIG1 is a schematic diagram of the reaction structure of specific oligonucleotides and universal primers.
  • FIG. 2 is a flow chart of the method for constructing a targeted library in Example 1.
  • FIG. 3 is a flow chart of the method for constructing a targeted library in Example 2.
  • FIG4 is a flowchart showing the advantages of the method for constructing a targeted library in Example 2 compared to Comparative Example 2.
  • first”, “second”, and “third” are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features.
  • a feature defined as “first”, “second”, and “third” may explicitly or implicitly include at least one of the features.
  • the meaning of “plurality” is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
  • the first aspect of the present invention provides an oligonucleotide combination, comprising: a specific oligonucleotide and a universal primer;
  • the universal primers include: a first universal primer and a second universal primer;
  • the specific oligonucleotides include: an upstream specific oligonucleotide and a downstream specific oligonucleotide: wherein,
  • the upstream specific oligonucleotide comprises, from the 5' end to the 3' end, an upstream specific sequence and a first universal sequence
  • the downstream specific oligonucleotide comprises, from the 5' end to the 3' end, a downstream specific sequence and a second universal sequence;
  • the 3' end sequence of the first universal primer is complementary to a part or all of the first universal sequence of the upstream specific oligonucleotide
  • the 3' end sequence of the second universal primer is complementary to a part or all of the second universal sequence of the downstream specific oligonucleotide
  • the 3' end sequence of the second universal primer is complementary to a part or all of the first universal sequence of the upstream specific oligonucleotide, and the 3' end sequence of the first universal primer is complementary to a part or all of the second universal sequence of the downstream specific oligonucleotide;
  • the sequence of the upstream specific oligonucleotide contains one or more cleavage sites that can be cleaved by a specific cleavage agent;
  • the sequence of the downstream specific oligonucleotide comprises one or more cleavage sites that can be cleaved by a specific cleavage agent.
  • the 3' end sequence of the first universal primer is complementary to a portion or all of the first universal sequence of the upstream specific oligonucleotide
  • the 3' end sequence of the second universal primer is complementary to a portion or all of the second universal sequence of the downstream specific oligonucleotide.
  • the 3' end sequence of the first universal primer is complementary to the entire sequence of the first universal sequence of the upstream specific oligonucleotide
  • the 3' end sequence of the second universal primer is complementary to the entire sequence of the second universal sequence of the downstream specific oligonucleotide.
  • the method based on two-step PCR can be: 1) firstly amplify the target gene by PCR in the first step, obtain the product and then perform the second step PCR to add universal primer sequences such as sequencing adapter sequence and sample tag sequence (multiple steps are required, the operation is relatively complicated and time-consuming); or 2) introduce the universal primer sequences such as sequencing adapter sequence and sample tag sequence required to be added in the second step PCR into the 5' end of the specific primer of the first step PCR (the primer is long, the accuracy is reduced, and the cost is increased);
  • the present application designs specific oligonucleotides and universal primers, wherein the 5' end to the 3' end of the specific oligonucleotide sequentially comprises: a specific sequence and a universal sequence, the specific oligonucleotide comprises one or more cleavage sites that can be cleaved by a specific cleavage agent; the 3' end of the universal primer comprises a sequence complementary to the universal sequence of the specific oligonucleotide; thereby, the specific oligonucleotide and the universal primer form a double-stranded DNA containing the universal primer sequence and the specific oligonucleotide sequence under the action of a reverse transcriptase and/or a polymerase ( Figure 1, when the 3' end of the specific oligonucleotide is preferably modified with a blocking group, it cannot be extended, and therefore, the double-stranded DNA is an incompletely complementary double-stranded DNA, wherein one chain (herein referred to as the short chain)
  • the long chain can be complementary to the target region to be detected, and the target region is amplified under the action of a polymerase to obtain a product containing a universal primer sequence and a target sequence to be detected, i.e., a targeted library, thereby achieving one-step reaction to complete the capture of the target region and the addition of a universal primer sequence (such as a sequencing adapter sequence and a sample label sequence), i.e., a long-chain primer containing a universal primer sequence and a target sequence to be detected is generated by a short-chain oligonucleotide (specific oligonucleotide and universal primer), thereby achieving enrichment of the target region and addition of a universal primer sequence (such as a sequencing adapter sequence and a sample label sequence), and realizing one-step construction of a targeted library, which has the advantages of simple operation, short time, short designed primers/specific oligonucleotides, high accuracy, and low cost.
  • a universal primer sequence such as a sequencing adapt
  • RT-mPCR reverse transcript multiplex PCR
  • RT reaction and mPCR The reaction is two independent reactions, which usually require reverse transcription with random primers first, and then add multiple PCR primers to perform PCR amplification of the target region.
  • the steps are relatively complicated. If the one-step method is used for RT-mPCR, specific primers need to be added at the beginning of the reaction, which has the following disadvantages: the 3' end sequence diversity of multiple specific primers is relatively strong.
  • the low-temperature RT process 1) they will produce non-specific binding with the template to produce non-specific products, and accumulate exponentially in the subsequent PCR process; 2) the specific primer sequence diversity is relatively high, and it is easy to complementarily bind and produce dimers under the action of reverse transcriptase, which accumulate exponentially in the subsequent PCR process, resulting in reaction termination.
  • the present application invents a method in which specific primers are compatible with reverse transcription reactions without producing non-specificity and dimers.
  • the 3' end sequences of the specific oligonucleotides and universal primers in the application method are universal and have low diversity.
  • the 3' ends of the specific oligonucleotides and universal primers are single sequences with low diversity and template The possibility of mutual binding is low, and even non-specific products generated by binding to the template will not accumulate in the subsequent PCR amplification process; 2)
  • the 3' ends of specific oligonucleotides and universal primers are both single sequences with low diversity, and the possibility of forming dimers between each other is small, and the probability of generating dimers is small.
  • the process of generating long-chain primers from specific short-chain oligonucleotides can be compatible with the reverse transcription process; first, N6 reverse transcribes RNA to generate cDNA, and at the same time, short-chain oligonucleotides (specific oligonucleotides and universal primers) generate long-chain primers containing universal primer sequences and target sequences to be tested, and then the cDNA target region is amplified under the action of polymerase to obtain a product containing the universal primer sequence and the target sequence to be tested, i.e., a targeted library, to achieve the reverse transcription step, long-chain primer generation and enrichment of the target region and the addition of universal primer sequences (such as sequencing adapter sequences and sample label sequences), so as to complete RT and mPCR reactions in one step, i.e., to achieve one-step method for constructing a targeted library for RNA.
  • the specific cleavage agent comprises an enzymatic cleavage agent.
  • the enzymatic cleavage agent comprises a ribonuclease (e.g., ribonuclease H), a glycosylase (e.g., uracil-DNA glycosylase (UDG), a USER enzyme (e.g., a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII), 3-methyladenine DNA glycosylase, 3-methyladenine DNA glycosylase II, pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase, thymine mismatch-DNA glycosylase (e.g., hypoxanthine-DNA At least one of glycosylase, uracil DNA glycosylase (UDG), 5-hydroxymethyluracil DNA glycosylase (HmUDG), 5-hydroxymethylcytosine DNA glycosylase, 1,N6-vinylidene adenine DNA glycosylase); further comprising at least one of
  • the enzymatic cleavage agent is selected from at least one of RNase H and UDG; and is further selected from RNase H.
  • the ribonuclease H comprises at least one of ribonuclease HI and ribonuclease HII.
  • the specific cleavage agents in the upstream specific oligonucleotide and the downstream specific oligonucleotide may be the same or different.
  • the multiple specific cleavage agents in the upstream specific oligonucleotide may be the same or different.
  • the multiple specific cleavage agents in the downstream specific oligonucleotide may be the same or different.
  • the cleavage site comprises at least one of c1) to c2); further comprises c1):
  • rNTPs ribonucleotides
  • rATP ribonucleotide
  • rGTP ribonuclease
  • rUTP ribonuclease
  • its corresponding specific cleavage agent may also be uracil-DNA glycosylase (UDG) or USER enzyme (such as a mixture of uracil-DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII);
  • dUTP uracil deoxyribonucleotide
  • UDG uracil-DNA glycosylase
  • USER enzyme for example: a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-cleavage enzyme Endo VIII.
  • the cleavage sites in the upstream specific oligonucleotide and the downstream specific oligonucleotide may be the same or different.
  • the multiple cleavage sites in the upstream specific oligonucleotide may be the same or different.
  • the multiple cleavage sites in the downstream specific oligonucleotide may be the same or different.
  • the first universal primer comprises a functional nucleic acid sequence.
  • the 5' end of the first universal primer contains a functional nucleic acid sequence.
  • the first universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.
  • the 5' end of the first universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.
  • the first universal primer comprises one or more sequencing primer sequences or sequencing primer complementary sequences; further preferably, the first universal primer comprises two or more sequencing primer sequences or sequencing primer complementary sequences.
  • the 5' end of the first universal primer contains one or more sequencing primer sequences or sequencing primer complementary sequences; further preferably, the 5' end of the first universal primer contains two or more sequencing primer sequences or sequencing primer complementary sequences.
  • the first universal primer further comprises a first tag sequence for distinguishing different samples to facilitate subsequent mixed sequencing of multiple samples, such as a barcode sequence or an index sequence.
  • a first tag sequence for distinguishing different samples to facilitate subsequent mixed sequencing of multiple samples, such as a barcode sequence or an index sequence.
  • the 5' end of the first universal primer also contains a first tag sequence for distinguishing different samples for subsequent mixed sequencing of multiple samples, for example, a barcode sequence or an index sequence.
  • the first tag sequence may be a unique molecular identifier (UMI) for counting the copy number of nucleic acid molecules in a sample.
  • UMI unique molecular identifier
  • the length of the first tag sequence is 5 to 20 bp.
  • the first tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence; further preferably, the first tag sequence is located in the middle of two segments of the sequencing primer sequence or the sequencing primer complementary sequence.
  • the second universal primer comprises a functional nucleic acid sequence.
  • the 5' end of the second universal primer contains a functional nucleic acid sequence.
  • the second universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.
  • the 5' end of the second universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.
  • the second universal primer comprises one or more sequencing primer sequences or sequencing primer complementary sequences; further preferably, the second universal primer comprises two or more sequencing primer sequences or sequencing primer complementary sequences.
  • the 5' end of the second universal primer contains one or more sequencing primer sequences or sequencing primer complementary sequences; further preferably, the 5' end of the second universal primer contains two or more sequencing primer sequences or sequencing primer complementary sequences.
  • the second universal primer further comprises a second tag sequence for distinguishing different samples to facilitate subsequent mixed sequencing of multiple samples, such as a barcode sequence or an index sequence.
  • a second tag sequence for distinguishing different samples to facilitate subsequent mixed sequencing of multiple samples, such as a barcode sequence or an index sequence.
  • the 5' end of the second universal primer also contains a second tag sequence for distinguishing different samples for subsequent mixed sequencing of multiple samples, for example, a barcode sequence or an index sequence.
  • the second tag sequence may be a unique molecular identifier (UMI) for counting the copy number of nucleic acid molecules in a sample.
  • UMI unique molecular identifier
  • the length of the second tag sequence is 5 to 20 bp.
  • the second tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence; further preferably, the second tag sequence is located in the middle of two segments of the sequencing primer sequence or the sequencing primer complementary sequence.
  • the first tag sequence and the second tag sequence are the same or different.
  • the first tag sequence and the second tag sequence are different.
  • the first universal sequence and the second universal sequence are each independently selected from a partial sequencing adapter sequence, a complete sequencing adapter sequence, a sequencing primer binding sequence or any fixed sequence (such as comprising a restriction site).
  • the sequencing adapter sequence can be any sequencing adapter of any sequencing platform.
  • the first universal sequence is the same as or different from the second universal sequence; further preferably, the first universal sequence is different from the second universal sequence.
  • the 3' end of the upstream specific oligonucleotide is blocked; and/or
  • the 3' end of the downstream specific oligonucleotide is subjected to blocking modification.
  • the 3' end of the upstream specific oligonucleotide is subjected to blocking modification; and/or
  • the 3' end of the downstream specific oligonucleotide is subjected to blocking modification.
  • the blocking modification comprises: at least one of phosphorylation modification, spacer modification, and amino modification; further preferably, the Blocking modifications include: phosphorylation modifications.
  • the blocking modifications of the upstream-specific oligonucleotide and the downstream-specific oligonucleotide are the same or different.
  • the upstream specific sequence and the downstream specific sequence in the specific oligonucleotide are respectively identical to or complementary to the two segments of the target region to be detected.
  • the design of the specific sequence in the specific oligonucleotide follows conventional primer design principles, but is reverse complementary to the conventional primer sequence, for example: the upstream specific sequence is identical to the upstream negative chain (i.e., antisense chain, cDNA second chain (when constructing RNA targeting library)) sequence of the target region to be detected or complementary to the positive chain (i.e., sense chain, cDNA first chain (when constructing RNA targeting library)) sequence, and the downstream specific sequence is identical to the downstream positive chain (i.e., sense chain, cDNA first chain (when constructing RNA targeting library)) sequence of the target region to be detected or complementary to the negative chain (i.e., antisense chain, cDNA second chain (when constructing RNA targeting library)) sequence.
  • the upstream specific sequence is identical to the upstream negative chain (i.e., antisense chain, cDNA second chain (when constructing RNA targeting library)) sequence of the target region to be detected or complementary to the positive chain (i.e.,
  • the upstream specific sequence of the upstream specific oligonucleotide is identical to the upstream negative strand sequence of the target region to be detected or is complementary to the positive strand sequence.
  • downstream specific sequence of the downstream specific oligonucleotide is identical to the downstream positive strand sequence of the target region to be detected or is complementary to the negative strand sequence.
  • the 5' end of the first universal primer contains a phosphate group
  • the 5' end of the amplified product can be provided with a phosphate group by the first universal primer, thereby avoiding the high cost of special phosphorylation; and/or
  • the 5' end of the second universal primer contains a phosphate group.
  • the second universal primer can be used to make the 5' end of the amplified product carry a phosphate group, thereby avoiding the excessively high cost of special phosphorylation.
  • the 5' end of the second universal primer contains a phosphate group.
  • the specific oligonucleotide and universal primer are DNA fragments or their analogs.
  • the upstream specific sequence of the upstream specific oligonucleotide has 15 to 45 nucleotides.
  • the first universal sequence of the upstream specific oligonucleotide has 15-45 nucleotides.
  • the upstream specific oligonucleotide has 30 to 90 nucleotides.
  • the number of nucleotides in the downstream specific sequence of the downstream specific oligonucleotide is 15-45.
  • the second universal sequence of the downstream specific oligonucleotide has 15-45 nucleotides.
  • the number of nucleotides in the downstream specific oligonucleotide is 30-90.
  • the first universal primer has 15-90 nucleotides.
  • the second universal primer has 15-90 nucleotides.
  • the specific oligonucleotides include multiple pairs of specific oligonucleotides that can bind to different positions of the target region to be detected; and further include multiple pairs of specific oligonucleotides targeting different target regions to be detected.
  • the plurality of pairs are two or more pairs.
  • the plurality of groups are two or more groups.
  • the universal primers comprise a plurality of pairs of universal primers.
  • the first universal primer comprises one or more first universal primers; further comprises multiple first universal primers.
  • the second universal primer comprises one or more second universal primers; and further comprises one second universal primer.
  • the oligonucleotide combination for detecting the EGFR gene comprises: a specific oligonucleotide and a universal primer;
  • the specific oligonucleotides include: an upstream specific oligonucleotide and a downstream specific oligonucleotide;
  • the universal primers include: a first universal primer and a second universal primer;
  • sequences of the upstream specific oligonucleotides are shown in SEQ ID NO.1 to SEQ ID NO.8, and the sequences of the downstream specific oligonucleotides are shown in SEQ ID NO.9 to SEQ ID NO.16;
  • the upstream specific oligonucleotides whose sequences are shown in SEQ ID NO.1 to SEQ ID NO.8 each independently contain one or more cleavage sites that can be cleaved by a specific cleavage agent;
  • the downstream specific oligonucleotides whose sequences are shown in SEQ ID NO.9 to SEQ ID NO.16 each independently contain one or more cleavage sites that can be cleaved by a specific cleavage agent;
  • the sequence of the first universal primer is shown as SEQ ID NO.53, and the sequence of the second universal primer is shown as SEQ ID NO.54.
  • the 3’ end of the specific oligonucleotide with a sequence as shown in SEQ ID NO.1 to 16 is subjected to blocking modification.
  • the oligonucleotide combination for detecting the SARS-COV-2 gene comprises: a specific oligonucleotide and a universal primer;
  • the specific oligonucleotides include: an upstream specific oligonucleotide and a downstream specific oligonucleotide;
  • the universal primers include: a first universal primer and a second universal primer;
  • sequences of the upstream specific oligonucleotides are shown in SEQ ID NOs. 17, 19, 21, 23, and 25, and the sequences of the downstream specific oligonucleotides are shown in SEQ ID NOs. 18, 20, 22, 24, and 26;
  • the upstream specific oligonucleotides whose sequences are shown in SEQ ID NO. 17, 19, 21, 23, and 25 each independently contain one or more cleavage sites that can be cleaved by a specific cleavage agent;
  • the downstream specific oligonucleotides whose sequences are shown in SEQ ID NO. 18, 20, 22, 24, and 26 each independently contain one or more cleavage sites that can be cleaved by a specific cleavage agent;
  • the sequence of the first universal primer is shown as SEQ ID NO.53, and the sequence of the second universal primer is shown as SEQ ID NO.54.
  • the 3’ end of the specific oligonucleotide with a sequence as shown in SEQ ID NO.17 to 26 is subjected to blocking modification.
  • oligonucleotide combination for detecting EGFR gene and/or oligonucleotide combination for detecting SARS-COV-2 gene For the above oligonucleotide combination for detecting EGFR gene and/or oligonucleotide combination for detecting SARS-COV-2 gene:
  • the specific cleavage agent comprises an enzymatic cleavage agent.
  • the enzymatic cleavage agent comprises a ribonuclease (e.g., ribonuclease H), a glycosylase (e.g., uracil-DNA glycosylase (UDG), a USER enzyme (e.g., a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII), 3-methyladenine DNA glycosylase, 3-methyladenine DNA glycosylase II, pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase, thymine mismatch-DNA glycosylase (e.g., hypoxanthine-DNA At least one of glycosylase, uracil DNA glycosylase (UDG), 5-hydroxymethyluracil DNA glycosylase (HmUDG), 5-hydroxymethylcytosine DNA glycosylase, 1,N6-vinylidene adenine DNA glycosylase); further comprising at least one of
  • the enzymatic cleavage agent is selected from at least one of RNase H and UDG; and is further selected from RNase H.
  • the ribonuclease H comprises at least one of ribonuclease HI and ribonuclease HII.
  • the specific cleavage agents in the upstream specific oligonucleotide and the downstream specific oligonucleotide may be the same or different.
  • the multiple specific cleavage agents in the upstream specific oligonucleotide may be the same or different.
  • the multiple specific cleavage agents in the downstream specific oligonucleotide may be the same or different.
  • the cleavage site comprises at least one of c1) to c2); further comprises c1):
  • rNTPs ribonucleotides
  • rATP ribonucleotide
  • rGTP ribonuclease
  • rUTP ribonuclease
  • its corresponding specific cleavage agent may also be uracil-DNA glycosylase (UDG) or USER enzyme (such as a mixture of uracil-DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII);
  • dUTP uracil deoxyribonucleotide
  • the corresponding specific cutting agent is uracil-DNA glycosylase (UDG) or USER Enzymes (e.g., a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII).
  • the cleavage sites in the upstream specific oligonucleotide and the downstream specific oligonucleotide may be the same or different.
  • the multiple cleavage sites in the upstream specific oligonucleotide may be the same or different.
  • the multiple cleavage sites in the downstream specific oligonucleotide may be the same or different.
  • the location of the cutting site of the specific oligonucleotide whose sequence is shown in SEQ ID NO.1 ⁇ 16 is as shown in Table 1 and the notes.
  • the location of the cutting site of the specific oligonucleotide with a sequence as shown in SEQ ID NO.17 to 26 is as shown in Table 4 and the notes.
  • the blocking modification comprises: at least one of: phosphorylation modification, spacer modification, and amino modification; further preferably, the blocking modification comprises: phosphorylation modification.
  • the 5' end of the second universal primer contains a phosphate group, and the 5' end of the amplified product can be provided with a phosphate group by the second universal primer, thereby avoiding the excessively high cost of special phosphorylation.
  • the second aspect of the present invention provides a kit comprising: the oligonucleotide combination of the first aspect of the present invention.
  • the kit further comprises: an enzyme for extending the universal primer.
  • the enzyme used for extending the universal primer comprises at least one of a reverse transcriptase and a DNA polymerase; further comprises a reverse transcriptase and a DNA polymerase; which is used to extend the universal primer (the first universal primer and the second universal primer) with specific oligonucleotides (the upstream specific oligonucleotide and the downstream specific oligonucleotide) as templates to form a double-stranded oligonucleotide (preferably an incompletely complementary double-stranded oligonucleotide).
  • the kit further comprises: a specific cutting agent (preferably the specific cutting agent in the first aspect of the present invention), which is used to cut the specific oligonucleotides (upstream specific oligonucleotide and downstream specific oligonucleotide) in the double-stranded oligonucleotide (preferably incompletely complementary double-stranded oligonucleotide) (e.g., DNA double strands) formed by extending universal primers (first universal primer and second universal primer) with specific oligonucleotides (upstream specific oligonucleotide and downstream specific oligonucleotide) as templates.
  • a specific cutting agent preferably the specific cutting agent in the first aspect of the present invention
  • the kit further comprises: a PCR reaction mixture, which is used for PCR reaction.
  • the PCR reaction mixture comprises: at least one of: DNA polymerase, Mg 2+ , PCR buffer, and dNTPs; further preferably, the PCR reaction mixture also comprises: DNA polymerase, Mg 2+ , PCR buffer and dNTPs; for the components already contained in the kit, the PCR reaction mixture can reduce the components accordingly, for example: when the enzyme used for the universal primer extension comprises DNA polymerase, the PCR reaction mixture may not comprise DNA polymerase.
  • the kit further comprises: a reverse transcription reaction mixture, which is used for reverse transcription reaction (ie, reverse transcribing RNA into cDNA) when constructing a targeted RNA library.
  • a reverse transcription reaction mixture which is used for reverse transcription reaction (ie, reverse transcribing RNA into cDNA) when constructing a targeted RNA library.
  • the reverse transcription reaction mixture comprises: at least one of reverse transcriptase, DNA polymerase, reverse transcription buffer, reverse transcription primer, and dNTPs; preferably, the reverse transcription reaction mixture comprises: reverse transcriptase, DNA polymerase, reverse transcription buffer, dNTPs and reverse transcription primer; for the components already contained in the kit, the reverse transcription reaction mixture can reduce the components accordingly: for example: when the enzyme for extending the universal primer comprises reverse transcriptase, the reverse transcription reaction mixture may not comprise reverse transcriptase; when the enzyme for extending the universal primer or the PCR reaction mixture comprises DNA polymerase, the reverse transcription reaction mixture may not comprise DNA polymerase; when the PCR reaction mixture comprises dNTPs, the reverse transcription reaction mixture may not comprise dNTPs.
  • the DNA polymerase is a thermostable DNA polymerase.
  • the kit comprises: a nucleic acid extraction reagent combination.
  • the nucleic acid extraction reagent combination is specifically a nucleic acid extraction reagent combination for any one of the following methods: alkaline lysis method, phenol chloroform extraction method, chelating resin method, centrifugal column membrane adsorption method and magnetic bead method.
  • the nucleic acid extraction reagent combination comprises: at least one of: a lysis solution, a washing solution, an eluent, and a nucleic acid adsorbent; further preferably, the nucleic acid extraction reagent combination comprises: a lysis solution, a washing solution, an eluent, and a nucleic acid adsorbent.
  • the nucleic acid adsorbent comprises at least one of magnetic beads and adsorption membranes.
  • the third aspect of the present invention provides a sequencing reagent kit, comprising: a reagent kit using the second aspect of the present invention.
  • the sequencing reagent set further comprises: a sequencing reagent kit.
  • a fourth aspect of the present invention provides a sequencing system, comprising: a sequencer and the sequencing reagent set of the third aspect of the present invention.
  • the fifth aspect of the present invention provides a multiplex PCR amplification method, comprising the step of using the oligonucleotide combination of the first aspect of the present invention.
  • the multiplex PCR amplification method comprises the following steps: performing a multiplex PCR reaction on nucleic acid using the oligonucleotide combination of the first aspect of the present invention.
  • reaction steps include:
  • Amplification reaction was performed using PCR reaction primers.
  • the system of the multiplex PCR reaction comprises: an enzyme for extending the universal primer (preferably the enzyme for extending the universal primer in the second aspect of the present invention), a specific cleavage agent (preferably the specific cleavage agent in the second aspect of the present invention); under the action of the enzyme for extending the universal primer (e.g., reverse transcriptase and/or polymerase), the specific oligonucleotide and the universal primer form a double-stranded DNA containing the universal primer sequence and the specific oligonucleotide sequence (the 3' end of the specific oligonucleotide is preferably modified with a blocking group and cannot be extended, so the double-stranded DNA is an incompletely complementary double-stranded DNA).
  • an enzyme for extending the universal primer preferably the enzyme for extending the universal primer in the second aspect of the present invention
  • a specific cleavage agent preferably the specific cleavage agent in the second aspect of the present invention
  • the enzyme for extending the universal primer e.g
  • the short chain is a specific oligonucleotide
  • the other chain (herein referred to as the long chain) contains a universal primer sequence and a sequence complementary to the specific oligonucleotide)
  • the cleavage site in the short chain is cut under the action of a specific cutting agent (when the 3' end of the specific oligonucleotide is not modified with a blocking group, it has a similar effect), and is separated from the long chain during the subsequent PCR high temperature process
  • the long chain can be complementary to the target region to be tested, and the target region is amplified under the action of a polymerase to obtain a product containing a universal primer sequence (such as a sequencing adapter sequence and a sample tag sequence) and a target sequence to be tested.
  • a universal primer sequence such as a sequencing adapter sequence and a sample tag sequence
  • the extension reaction and amplification reaction are carried out in the same system.
  • the same system is specifically not subjected to elution and/or purification treatment.
  • the concentration of the specific cleavage agent in the multiplex PCR reaction system is lower than the concentration of the enzyme used for universal primer extension, so that the extension reaction precedes the cleavage reaction.
  • the multiplex PCR reaction system further comprises: a PCR reaction mixture (preferably the PCR reaction mixture in the second aspect of the present invention).
  • the nucleic acid is from any one or more of the following biological samples: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, and nasal swabs.
  • the body fluid comprises at least one of tissue fluid, lymph fluid, blood and cerebrospinal fluid.
  • the microorganism comprises at least one of bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, and spirochetes.
  • the nucleic acid is obtained by a cleavage reaction of the biological sample.
  • the cleavage reaction precedes the multiplex PCR reaction.
  • the cleavage reaction and the multiplex PCR reaction are performed in the same system.
  • the same system is specifically the same reaction container.
  • the extraction and purification step is not included after the cleavage reaction and before the multiplex PCR reaction.
  • an extraction and purification step is further included to remove impurities such as salts and organic agents.
  • the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid.
  • the extraction and purification further comprises: eluting or dissolving nucleic acids.
  • the cleavage reaction and the multiplex PCR reaction are performed in different systems, that is, not in the same system.
  • the same system is specifically the same reaction container.
  • the cleavage reaction further comprises: an extraction and purification step to remove impurities such as salts and organic agents.
  • the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid.
  • the extraction and purification further comprises: eluting or dissolving nucleic acids.
  • the lysis method comprises at least one of a physical method, a chemical method and a biological method.
  • the physical method comprises: at least one of a boiling method, a glass bead method, an ultrasonic method, a grinding method, a freeze-thaw method, and a homogenization method.
  • the chemical method comprises at least one of a surfactant method (SDS method) and an alkaline lysis method.
  • SDS method surfactant method
  • alkaline lysis method alkaline lysis method
  • the biological method comprises: an enzymatic method, such as cleavage by enzymes such as lysozyme and proteinase K.
  • a reverse transcription reaction is further included after the cleavage reaction and before the multiplex PCR reaction, that is, reverse transcription of RNA into cDNA.
  • the reverse transcription reaction and the multiplex PCR reaction are performed in the same system or in different systems; further preferably, the reverse transcription reaction and the multiplex PCR reaction are performed in the same system.
  • the same system is specifically carried out in the same reaction system; that is, there is no need to first carry out a reverse transcription reaction in one reaction system and then carry out a multiplex PCR reaction in other reaction systems.
  • the 3' end sequences of the specific oligonucleotides and universal primers designed in the present application are universal and have low diversity.
  • 1) the 3' ends of the specific oligonucleotides and universal primers are single sequences with low diversity, and the possibility of binding to the template is low. Even if the non-specific products generated by binding to the template are generated, they will not accumulate during the subsequent PCR amplification process; 2) the 3' ends of the specific oligonucleotides and universal primers are single sequences with low diversity, and the possibility of forming dimers between each other is small, and the probability of generating dimers is small.
  • the process of generating long-chain primers from specific short-chain oligonucleotides is compatible with the reverse transcription process; first, N6 reverse transcribes RNA to generate cDNA, and at the same time, short-chain oligonucleotides (specific oligonucleotides and universal primers) generate long-chain primers containing the universal primer sequence and the target sequence to be tested, and then the cDNA target region is amplified under the action of the polymerase to obtain a product containing the universal primer sequence and the target sequence to be tested, which is compatible with the reverse transcription reaction and the PCR reaction.
  • the multiplex PCR reaction system further comprises: a reverse transcription reaction mixture (preferably the reverse transcription reaction mixture in the second aspect of the present invention).
  • the sixth aspect of the present invention provides a method for constructing a targeted library, comprising the steps of the multiplex PCR amplification method of the fifth aspect of the present invention to obtain a sequencing library.
  • a circularization reaction may be further included before obtaining the sequencing library, that is, circularizing the linear library obtained by amplification.
  • the method further comprises the step of: purifying the sequencing library.
  • the purification is performed using magnetic beads.
  • the seventh aspect of the present invention provides a sequencing method, comprising: the steps of the method for constructing a targeted library according to the sixth aspect of the present invention.
  • the sequencing method comprises the following steps: preparing a library; sequencing;
  • the method for preparing a library is the method for constructing a targeted library according to the sixth aspect of the present invention.
  • the sequencing further includes the following steps: library quality inspection.
  • An eighth aspect of the present invention provides a method for obtaining gene information of a target region, comprising: the steps of the sequencing method of the seventh aspect of the present invention.
  • the method for obtaining the target region gene information comprises the following steps: sequencing to obtain sequencing data; obtaining the target region gene information; the information comprises at least one of sequence information and mutation information;
  • the sequencing method is the sequencing method of the seventh aspect of the present invention.
  • the method for obtaining the target region gene information further comprises the following steps: comparing the obtained sequencing data with the reference genome to determine the mutation information of the target region gene.
  • a method for obtaining EGFR gene information comprising the following steps: sequencing to obtain sequencing data; obtaining EGFR gene information; the information comprising at least one of sequence information and mutation information;
  • the sequencing method is the sequencing method of the seventh aspect of the present invention.
  • the oligonucleotide combination is the oligonucleotide combination for detecting the EGFR gene in the first aspect of the present invention.
  • the method for obtaining EGFR gene information further comprises the following steps: The data were compared with the reference genome to obtain mutation information of the EGFR gene.
  • the present invention provides the use of the oligonucleotide combination of the first aspect, the kit of the second aspect, the sequencing reagent set of the third aspect and/or the sequencing system of the fourth aspect in any one of items c1) to c8);
  • Example 1 Method for preparing a library of EGFR gene tumor hotspot genes
  • the method for preparing a library of EGFR gene tumor hotspot genes comprises the following steps:
  • an EGFR gene tumor hotspot gene capture panel and universal tag primers comprising 8 pairs of specific oligonucleotides (EGFR_1F to EGFR_8F, EGFR_1R to EGFR_8R), the amplicon size being 100 to 200 bp, and the sequences of the specific oligonucleotides being shown in Table 1;
  • the universal tag primers comprising universal tag primer F and universal tag primer R, the nucleotide sequence of universal tag primer F being: TGTGAGCCAAGGAGTTATCGGACCTAATTGTCTTCCTAAGAC CGCTTGGCCTCCGACTT (SEQ ID NO.53, wherein the bold portion (SEQ ID NO.55) is the tag sequence of the universal tag primer F of this embodiment, the tag sequence may be random or pre-set, such as the tag sequences shown in SEQ ID NOs.56 to 62 in Table 2); the underlined portion of the universal tag primer F is complementary to the 3' end sequence (universal sequence) of the up
  • an mPCR (multiplex PCR) system was configured according to the reaction system shown in Table 3, and the mPCR reaction was performed.
  • the reaction procedure was as follows: 94°C, 2 min, 60°C, 5 min; 94°C 30 s, 58°C 2 min, 72°C 30 s, 27 cycles; 72°C 5 min; 12°C ⁇ .
  • the cells were purified with 1.5X AMPure (Beckman Co.) magnetic beads, and the purified products were dissolved in 22 ⁇ L elution buffer to obtain the library.
  • the above-mentioned specific oligonucleotides were mixed at a concentration of 20 ⁇ M each to obtain a specific oligonucleotide pool with a total concentration of 20 ⁇ M; the 3’ end of the above-mentioned specific oligonucleotides was blocked with phosphate (phos) to prevent 3’ end extension, the underlined bases are ribonucleotides (cleavage sites), which can be recognized and hydrolyzed by RNase HII; the 5’ to 3’ ends of the upstream specific oligonucleotides (EGFR_1F ⁇ EGFR_8F) are: specific sequence and universal sequence (bold part), and the bold part is the sequence complementary to the 3’ end of the universal tag primer F; the 5’ to 3’ ends of the downstream specific oligonucleotides (EGFR_1R ⁇ EGFR_8R) are: specific sequence and universal sequence (bold part), and the bold part is the sequence complementary to the 3’ end of the universal tag
  • HawkZ05 Fast DNA Polymerase is from Roche, catalog number 07731264103.
  • RNase HII enzyme is from NEB, catalog number M0288S.
  • Example 2 Method for preparing a library of SARS-COV-2 genes
  • the method for preparing the library of the SARS-COV-2 gene is shown in FIG3 , and includes the following steps:
  • SARS-COV-2 gene capture panel and universal tag primers comprising 5 pairs of specific oligonucleotides (N1-F, N1-R, N2-F, N2-R, N3-F, N3-R, Orf1ab-F, Orf1ab-R, RDRP-F, RDRP-R), the sequences of the specific oligonucleotides are shown in Table 4;
  • the universal tag primers comprise universal tag primer F and universal tag primer R, the nucleotide sequence of universal tag primer F is: TGTGAGCCAAGGAGTTATCGGACCTAATTGTCTTCCTAAGAC CGCTTGGCCTCCGACTT (wherein, the bold portion (SEQ ID NO.
  • the tag sequence of the universal tag primer F of this embodiment is the tag sequence of the universal tag primer F of this embodiment, the tag sequence can be random or pre-set, such as the sequence in Table 2 as SEQ ID NO.56-62 ); the underlined portion of universal tag primer F is complementary to the 3' end sequence (universal sequence) of the upstream specific oligonucleotide; the nucleotide sequence of universal tag primer R is: Phos-GAACG ACATGGCTACGATCCGACTT , Phos is phosphorylated, and the underlined portion is complementary to the 3' end sequence (universal sequence) of the downstream specific oligonucleotide.
  • RT-mPCR Reverse transcript multiplex PCR
  • the reaction procedure was as follows: 25°C for 10 min; 45°C for 15 min; 94°C for 5 min; 94°C for 30 s, 58°C for 2 min, 72°C for 30 s, 40 cycles; 72°C for 5 min; 12°C ⁇ .
  • the DNA was purified with 1.5X AMPure (Beckman Co.) magnetic beads, and the purified product was dissolved in 22 ⁇ L elution buffer to obtain the library.
  • the above-mentioned specific oligonucleotides were mixed at a concentration of 10 ⁇ M each to obtain a specific oligonucleotide pool with a total concentration of 10 ⁇ M; the 3’ end of the above-mentioned specific oligonucleotides was blocked with phosphate (phos) to prevent 3’ end extension, the underlined bases are ribonucleotides (cleavage sites), which can be recognized and hydrolyzed by RNase HII; the 5’ to 3’ ends of the upstream specific oligonucleotides (N1-F, N2-F, N3-F, Orf1ab-F, RDRP-F) are: specific sequence and universal sequence (bold part), the bold part is the sequence complementary to the 3’ end of the universal tag primer F; the 5’ to 3’ ends of the downstream specific oligonucleotides (N1-R, N2-R, N3-R, Orf1ab-R, RDRP-
  • Reverse transcription system (2 ⁇ One-step Mix, One-step enzyme Mix) was from Novozymes, HiScript II One Step RT-PCR Kit, Catalog No. P611-01.
  • RNase HII enzyme was from NEB, Catalog No. M0288S
  • RNA inhibitor was from NEB, Catalog No. M0314S
  • T4 DNA Polymerase was from NEB, Catalog No. M0203S.
  • the method for preparing a library of EGFR gene tumor hotspot genes comprises the following steps:
  • an mPCR (multiplex PCR) system was prepared according to the reaction system shown in Table 7, and an mPCR reaction was performed.
  • the reaction procedure was as follows: Next: 94°C for 5 min; 94°C for 30 s, 58°C for 2 min, 72°C for 30 s, 27 cycles; 72°C for 5 min; 12°C ⁇ .
  • the product was purified with 1.5X AMPure (Beckman Co.) magnetic beads, and the purified product was dissolved in 22 ⁇ L elution buffer to obtain the library.
  • the above primers were mixed at a concentration of 20 ⁇ M for each primer to obtain a primer pool with a total concentration of 20 ⁇ M; the 5' end of the downstream primer was phosphorylated; the 5' to 3' ends of the upstream primers (EGFR_1F ⁇ EGFR_8F) were: a universal sequence (bold part) and a specific sequence, and the bold part was the same as the sequence of the universal tag primer F in Example 1; the 5' to 3' ends of the downstream primers (EGFR_1R ⁇ EGFR_8R) were: a universal sequence (bold part) and a specific sequence, and the bold part was the same as the sequence of the universal tag primer R in Example 1, that is, the above primers were the same as the sequence of the product obtained by the reverse transcription reaction of the specific oligonucleotides and the universal tag primers in Example 1.
  • the method for preparing a library of the SARS-COV-2 gene comprises the following steps:
  • a SARS-COV-2 gene capture panel and universal tag primers comprising 5 pairs of specific oligonucleotides (N1-F, N1-R, N2-F, N2-R, N3-F, N3-R, Orf1ab-F, Orf1ab-R, RDRP-F, RDRP-R), the sequences of the specific oligonucleotides are shown in Table 8;
  • the universal tag primers comprise universal tag primer F and universal tag primer R, the nucleotide sequence of universal tag primer F is: TGTGAGCCAAGGAGTTATCGGACCTAATTGTCTTCCTAAGAC CGCTTGGCCTCCGACTT (wherein, the bold portion (SEQ ID NO.55) is the tag sequence of the universal tag primer F of this comparative example, the tag sequence can be random or pre-set, such as the sequence in Table 2 such as SEQ ID NO.56-62 ); the underlined portion of the universal tag primer F is the same as the 5' end sequence of
  • the RT reaction system was prepared in a PCR tube according to the reaction system shown in Table 9, and the RT reaction was performed.
  • the reaction procedure was as follows: 25°C for 10 min, 42°C for 20 min, and 85°C for 10 min to obtain the RT reaction product.
  • the first round of PCR reaction system was configured in a PCR tube according to the reaction system shown in Table 10, and the PCR reaction was carried out.
  • the reaction procedure was as follows: 94°C for 1 min; 94°C for 30 s, 58°C for 2 min, 72°C for 30 s, 15 cycles; 72°C for 5 min; 12°C ⁇ .
  • 1.5X AMPure (Beckman Co.) magnetic beads were used for purification, and the purified product was dissolved in 22 ⁇ L elution buffer to obtain the first round of PCR product.
  • the second round of PCR reaction system was configured in a PCR tube according to the reaction system shown in Table 11, and the PCR reaction was carried out.
  • the reaction procedure was as follows: 94°C for 1 min; 94°C for 30 s, 58°C for 2 min, 72°C for 30 s, 25 cycles; 72°C for 5 min; 12°C ⁇ .
  • the DNA was purified with 1.0X AMPure (Beckman Co.) magnetic beads, and the purified product was dissolved in 22 ⁇ L elution buffer to obtain the library.
  • the above primers were mixed at a concentration of 10 ⁇ M each to obtain a primer pool with a total concentration of 10 ⁇ M; the 5’ to 3’ ends of the upstream primers (N1-F, N2-F, N3-F, Orf1ab-F, RDRP-F) were: universal sequence (bold part) and specific sequence, and the bold part was the same sequence as the 3’ end of universal tag primer F; the 5’ to 3’ ends of the downstream specific oligonucleotides (N1-R, N2-R, N3-R, Orf1ab-R, RDRP-R) were: universal sequence (bold part) and specific sequence, and the bold part was the same sequence as the 3’ end of universal tag primer R.
  • the reverse transcription system is from Novozymes, M-MLV(H-)Reverse Transcriptase, catalog number R021-01.
  • the following effect examples are based on the MGISEQ-2000 platform of the sequencer of BGI; the reagents used are all derived from the library construction kit and the double-end sequencing kit (hereinafter referred to as the PE100 kit) used in conjunction with the sequencer, and the sequencing read length of PE100 is used in the following effect examples; the sequencer and reagent operations used in the process refer to the method of use of the platform, and finally data analysis is performed, including data utilization, unique alignment ratio, target region data ratio and other performance (analysis method reference: Campbell, Nathan R., Stephanie A. Harmon, and Shawn R. Narum. "Genotyping-in-Thousands by sequencing (GT-seq): A cost-effective SNP genotyping method based on custom amplicon sequencing.” Molecular ecology resources 15.4 (2015): 855-867.), as follows:
  • Example 12 The sequencing data statistics of the library obtained in Example 1 and Comparative Example 1 are shown in Table 12, and the mutation detection statistics of Example 1 are shown in Table 13: the data utilization rate, unique alignment ratio, and target area data ratio in the sequencing data of the library obtained in Example 1 and Comparative Example 1 are equivalent; the detection values of the mutation frequencies of Example 1 and Comparative Example 1 are equivalent to the theoretical values; that is, the method of the present invention (Example 1) can achieve the performance of the control method (Comparative Example 1).
  • the method of the present invention only requires one-step PCR to complete the preparation of the library, but the method of the present invention does not require the advance synthesis of long-chain primers containing complete sequencing adapters and sample tag sequences, which overcomes the following disadvantages: 1) decreased primer accuracy; 2) increased primer cost; the method of the present invention can achieve long-chain primer synthesis, and can complete the enrichment of the target area and the preparation of the library in one step ( Figure 4).
  • the following effect examples are based on the MGISEQ-2000 platform of the sequencer of BGI; the reagents used are all derived from the library construction kit and the double-end sequencing kit (hereinafter referred to as the PE100 kit) used in conjunction with the sequencer, and the sequencing read length of PE100 is used in the following effect examples; the sequencer and reagent operations used in the process refer to the method of use of the platform, and finally data analysis is performed, including data utilization, unique alignment ratio, target region data ratio and other performance (analysis method reference: Campbell, Nathan R., Stephanie A. Harmon, and Shawn R. Narum. "Genotyping-in-Thousands by sequencing (GT-seq): A cost-effective SNP genotyping method based on custom amplicon sequencing.” Molecular ecology resources 15.4 (2015): 855-867.), as follows:
  • the sequencing data statistics of the libraries obtained in Example 2 and Comparative Example 2 are shown in Table 14: the data utilization rate, unique alignment ratio, and target region data ratio in the sequencing data of the libraries obtained in Example 2 and Comparative Example 2 are comparable; that is, the method of the present invention (Example 2) can achieve the performance of the control method (Comparative Example 2), and the data quality is comparable; however, the method of the present invention only requires one-step PCR to complete the preparation of the RNA targeted library, can achieve long-chain primer synthesis, and is compatible with the RT system, and can complete reverse transcription, enrichment of the target region, and preparation of the library in one-step reaction ( FIG. 4 ).

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Abstract

本发明提供一种寡核苷酸组合，包含特异性寡核苷酸和通用引物，通过特异性寡核苷酸和通用引物生成包含通用引物序列和与特异性寡核苷酸互补的序列的长链引物，长链引物在PCR过程中实现目标区域的富集和产物上通用序列的添加，实现一步法构建靶向文库。

Description

一种一步法构建靶向文库的方法及其应用 技术领域
本发明属于文库构建技术领域，具体涉及一种一步法构建靶向文库的方法及其应用。
背景技术
随着测序技术的发展，对基因组候选区段的重测序需求日益增加，人们对序列的关注超过了少数的SNP，候选区段的范围可能在5kb～10M之间。使用传统sanger法或全基因组测序价格昂贵，使用目标区域捕获测序很好地解决了这一难题。利用PCR技术或探针捕获技术选取特定基因或区域进行扩增，然后进行高通量测序，快速获得基因组信息的方法。相较于全基因组测序，它具有定向、高覆盖度和低成本等优点，因此，在肿瘤学、人类遗传学、微生物学、植物学等领域中有广泛的应用。目标区域捕获技术可大致分为两种：一种基于杂交的捕获测序技术，另外一种基于多重PCR的捕获技术。前者的实验流程繁琐，探针成本较高，限制了其在临床上的应用，后者实验操作简单，灵活性强，成本较低适应性更广。
多重PCR的靶向测序文库是一种通过特定PCR技术选取目标基因或区域进行扩增，并加上测序接头和样本标签序列的文库制备方法。目前主要的技术路线有两种：基于两步PCR的方法和基于PCR扩增和接头连接的方法。这两种方法都需要多步反应，操作相对复杂、时间较长，限制了其在临床检测中的应用。因此，需要开发更简单的建库技术，以扩大靶向测序技术在临床检测中的应用范围。
对于第一种基于两步PCR的技术路线，可以将第二步PCR需要添加的完整测序接头和样本标签序列整合到第一步PCR中特异性引物的5’端，第一轮PCR完成后文库的5’端就加上了对应的接头和样本标签序列。但相对于常规的两步PCR的方案，有两个弊端，1)引物合成的长度非常长，引物准确性会下降，此外引物的成本会更高；2)每一条特异性引物都需要对应的样本标签序列，有多少标签序列就需要对应多少套特异性引物，因此引物的总成本会急剧增加。
此外，基于多重PCR的靶向文库制备方式除了可以对DNA进行靶向扩增，还可以对RNA进行靶向文库制备。但相对于DNA靶向文库制备，RNA靶向文库制备需要一步额外的逆转录过程。通常地，先采用N6引物对所有RNA进行逆转录得到一链cDNA产物，然后再通过特异性引物对cDNA进行扩增和富集，整体的操作是比较复杂的。
Thermo公司的Ampliseq和Paragon genomics公司的Clean plex建库的方法是目前常用的建库方法，但是，其都包含多个步骤，建库操作繁琐，因此，需要开发更简单的建库方法。
发明内容
本发明第一方面的目的，在于提供寡核苷酸组合。
本发明第二方面的目的，在于提供一种试剂盒。
本发明第三方面的目的，在于提供一种多重PCR扩增方法。
本发明第四方面的目的，在于提供一种构建靶向文库的方法。
为了实现上述目的，本发明所采取的技术方案是：
本发明的第一个方面，提供寡核苷酸组合，包含：特异性寡核苷酸和通用引物；
所述通用引物包含：第一通用引物和第二通用引物；
所述特异性寡核苷酸包含：上游特异性寡核苷酸和下游特异性寡核苷酸：其中，
所述上游特异性寡核苷酸从5’端到3’端依次包含：上游特异性序列和第一通用序列；
所述下游特异性寡核苷酸从5’端到3’端依次包含：下游特异性序列和第二通用序列；
1)所述第一通用引物的3’端序列与所述上游特异性寡核苷酸的第一通用序列的部分序列或全部序列互 补，所述第二通用引物的3’端序列与所述下游特异性寡核苷酸的第二通用序列的部分序列或全部序列互补；或
2)所述第二通用引物的3’端序列与所述上游特异性寡核苷酸的第一通用序列的部分序列或全部序列互补，所述第一通用引物的3’端序列与所述下游特异性寡核苷酸的第二通用序列的部分序列或全部序列互补；
所述上游特异性寡核苷酸的序列包含一个或多个可以被特异性切割剂切割的切割位点；
所述下游特异性寡核苷酸的序列包含一个或多个可以被特异性切割剂切割的切割位点。
优选地，所述特异性切割剂包含酶促切割剂。
优选地，所述酶促切割剂包含核糖核酸酶(例如：核糖核酸酶H)、糖基化酶(例如：尿嘧啶-DNA糖基化酶(UDG)、USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)、3-甲基腺嘌呤DNA糖基化酶、3-甲基腺嘌呤DNA糖基化酶II、嘧啶水合物-DNA糖基化酶、FaPy-DNA糖基化酶、胸腺嘧啶错配-DNA糖基化酶(例如：次黄嘌呤-DNA糖基化酶、尿嘧啶DNA糖基化酶(UDG)、5-羟甲基尿嘧啶DNA糖基化酶(HmUDG)、5-羟甲基胞嘧啶DNA糖基化酶、1,N6-亚乙烯基腺嘌呤DNA糖基化酶))中的至少一种。
优选地，所述酶促切割剂选自核糖核酸酶H、UDG中的至少一种；进一步选自核糖核酸酶H。
优选地，所述核糖核酸酶H包含核糖核酸酶HⅠ、核糖核酸酶HII中至少一种。
优选地，所述上游特异性寡核苷酸和下游特异性寡核苷酸中所述的特异性切割剂可以相同或不同。
优选地，所述上游特异性寡核苷酸中的多个特异性切割剂可以相同或不同。
优选地，所述下游特异性寡核苷酸中的多个特异性切割剂可以相同或不同。
优选地，所述切割位点包含c1)～c2)中至少一种：
c1)核糖核苷酸(rNTP，例如：rATP、rGTP、tCTP、rUTP)；
c2)尿嘧啶脱氧核糖核苷酸(dUTP)。
优选地，所述切割位点包含c1)。
优选地，所述上游特异性寡核苷酸和下游特异性寡核苷酸中所述的切割位点可以相同或不同。
优选地，所述上游特异性寡核苷酸中的多个切割位点可以相同或不同。
优选地，所述下游特异性寡核苷酸中的多个切割位点可以相同或不同。
优选地，所述第一通用引物包含功能核酸序列。
优选地，所述第一通用引物包含测序引物序列或测序引物互补序列。
优选地，所述第一通用引物包含一段或多段测序引物序列或测序引物互补序列；进一步优选地，所述第一通用引物包含两段或两段以上测序引物序列或测序引物互补序列。
优选地，所述第一通用引物还包含第一标签序列。
优选地，所述第一标签序列位于所述测序引物序列或测序引物互补序列的中间；进一步优选地，所述第一标签序列位于两段所述测序引物序列或测序引物互补序列的中间。
优选地，所述第二通用引物包含功能核酸序列。
优选地，所述第二通用引物包含测序引物序列或测序引物互补序列。
优选地，所述第二通用引物包含一段或多段测序引物序列或测序引物互补序列；进一步优选地，所述第二通用引物包含两段或两段以上测序引物序列或测序引物互补序列。
优选地，所述第二通用引物还包含第二标签序列。
优选地，所述第二标签序列位于所述测序引物序列或测序引物互补序列的中间；进一步优选地，所述第二标签序列位于两段所述测序引物序列或测序引物互补序列的中间。
优选地，所述第一标签序列、第二标签序列相同或不同。
优选地，所述第一通用序列、第二通用序列各自独立选自部分测序接头序列、全部测序接头序列、测序引物结合序列或任意固定序列(比如包含酶切位点)。
优选地，所述第一通用序列与所述第二通用序列相同或不同；进一步优选地，所述第一通用序列与所述第二通用序列不同。
优选地，所述上游特异性寡核苷酸的3’端进行阻断修饰；和/或
所述下游特异性寡核苷酸的3’端进行阻断修饰。
优选地，所述阻断修饰包含：磷酸化修饰、间臂修饰、氨基修饰中的至少一种；进一步优选地，所述阻断修饰包含：磷酸化修饰。
优选地，所述上游特异性寡核苷酸和所述下游特异性寡核苷酸的阻断修饰相同或不同。
优选地，所述特异性寡核苷酸中的上游特异性序列和下游特异性序列分别与待测目标区域的两个区段相同或互补。
优选地，所述第一通用引物的5’端含有磷酸基团；和/或
所述第二通用引物的5’端含有磷酸基团。
优选地，所述上游特异性寡核苷酸的序列包含多个可以被特异性切割剂切割的切割位点，切割位点数量大于等于1/10碱基，例如：可以是切割位点的个数大于等于n个；或者是n～N个；或者是3～9个；其中，n＝N/10，N为上游特异性寡核苷酸的核苷酸个数，n为整数。
优选地，所述下游特异性寡核苷酸的序列包含多个可以被特异性切割剂切割的切割位点，切割位点数量大于等于1/10碱基，例如：可以是切割位点的个数大于等于n个；或者是n～N个；或者是3～9个；其中，n＝N/10，N为下游特异性寡核苷酸的核苷酸个数，n为整数。
优选地，所述可以被特异性切割剂切割的切割位点的个数为整数。
优选地，所述上游特异性寡核苷酸的上游特异性序列的核苷酸个数为15～45。
优选地，所述上游特异性寡核苷酸的第一通用序列的核苷酸个数为15～45。
优选地，所述上游特异性寡核苷酸的核苷酸个数为30～90。
优选地，所述下游特异性寡核苷酸的下游特异性序列的核苷酸个数为15～45。
优选地，所述下游特异性寡核苷酸的第二通用序列的核苷酸个数为15～45。
优选地，所述下游特异性寡核苷酸的核苷酸个数为30～90。
优选地，所述第一通用引物的核苷酸个数为15～90。
优选地，所述第二通用引物的核苷酸个数为15～90。
优选地，所述多个可以被特异性切割剂切割的切割位点在所述上游特异性寡核苷酸中随机分布。
优选地，所述多个可以被特异性切割剂切割的切割位点在所述下游特异性寡核苷酸中随机分布。
优选地，所述特异性寡核苷酸包含多对特异性寡核苷酸；进一步包含多组靶向不同的待测目标区域的多对特异性寡核苷酸。
优选地，所述通用引物包含多对通用引物。
本发明的第二个方面，提供一种试剂盒，包含：本发明第一个方面的寡核苷酸组合。
优选地，所述试剂盒还包含：逆转录酶和DNA聚合酶中的至少一种；进一步包含逆转录酶和DNA聚合酶。
优选地，所述试剂盒还包含：本发明第一个方面中的特异性切割剂。
优选地，所述试剂盒还包含：逆转录引物。
优选地，所述试剂盒包含：核酸提取试剂组合。
优选地，所述核酸提取试剂组合是用于选自以下任意一种方法的核酸提取试剂组合：碱裂解法、酚氯仿抽提法、螯合树脂法、离心柱膜吸附法以及磁珠法。
优选地，所述核酸提取试剂组合包含：裂解液、洗涤液、洗脱液、核酸吸附物中的至少一种；进一步优选地，所述核酸提取试剂组合包含：裂解液、洗涤液、洗脱液和核酸吸附物。
优选地，所述核酸吸附物包含磁珠、吸附膜中的至少一种。
本发明的第三个方面，提供一种多重PCR扩增方法，包含采用本发明第一个方面的寡核苷酸组合的步骤。
优选地，所述多重PCR扩增方法包括如下步骤：利用本发明第一个方面的寡核苷酸组合对核酸进行多重PCR反应。
优选地，所述反应的步骤包括：
通用引物延伸反应，并切割互补链，生成PCR反应引物；
利用PCR反应引物进行扩增反应。
优选地，所述延伸反应和扩增反应在同一体系中进行。
优选地，所述核酸为RNA时，所述多重PCR反应前还包含：逆转录反应。
优选地，所述逆转录反应与所述多重PCR反应在同一体系或不在同一体系中进行；进一步优选地，所述逆转录反应与所述多重PCR反应在同一体系中进行。
优选地，所述同一体系具体为在同一反应体系中进行。
本发明的第四个方面，提供一种构建靶向文库的方法，包含本发明第三个方面的多重PCR扩增方法的步骤，得到测序文库。
优选地，得到测序文库前还可以包括环化反应，即对扩增得到的线性文库进行环化。
优选地，所述方法还包括如下步骤：对测序文库进行纯化。
优选地，所述纯化采用磁珠进行。
本发明的有益效果是：
本发明提供了寡核苷酸组合，包含：特异性寡核苷酸和通用引物；其中，特异性寡核苷酸的5’端到3’端依次包含：特异性序列和通用序列，特异性寡核苷酸包含一个或多个可以被特异性切割剂切割的切割位点；通用引物的3’端包含与特异性寡核苷酸的通用序列互补的序列；从而可以通过特异性寡核苷酸和通用引物(短链寡核苷酸)生成包含通用引物序列和与特异性寡核苷酸互补的序列的长链引物，长链引物在PCR过程中实现目标区域的富集和产物上通用引物序列(比如测序接头序列和样本标签序列)的添加，进而实现一步法构建靶向文库，具有操作简单，时间短，设计的引物/特异性寡核苷酸短，准确性高，成本低等优点；同时，特异性寡核苷酸和通用引物的3’端序列是通用的，多样性较低，在RNA靶向文库制备的过程中1)RT反应中，特异性寡核苷酸和通用引物的3’端序列多样性低，引物和模板结合的可能性较低，并且哪怕和模板结合的产生的非特异性产物，在后续PCR扩增过程中也不会积累；2)RT过程中，特异性寡核苷酸和通用引物的3’端序列多样性低，互相之间形成二聚体的可能性较小，产生二聚体的概率小；通过短链寡核苷酸(特异性寡核苷酸和通用引物)生成包含通用引物序列和与特异性寡核苷酸互补的序列的长链引物并兼容RNA逆转录过程，实现逆转录步骤、长链引物生成、PCR目标区域的富集和通用引物序列(比如测序接头序列和样本标签序列)的添加；在一管中实现逆转录、扩增和文库制备的过程；产物可直接进行后续的测序、克隆等应用。
附图说明
图1是特异性寡核苷酸和通用引物反应结构示意图。
图2是实施例1的构建靶向文库的方法的流程图。
图3是实施例2的构建靶向文库的方法的流程图。
图4是实施例2的构建靶向文库的方法相比对比例2的流程优势图。
具体实施方式
在本发明的描述中，术语“第一”、“第二”、“第三”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”、“第三”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
本发明的第一个方面，提供寡核苷酸组合，包含：特异性寡核苷酸和通用引物；
所述通用引物包含：第一通用引物和第二通用引物；
所述特异性寡核苷酸包含：上游特异性寡核苷酸和下游特异性寡核苷酸：其中，
所述上游特异性寡核苷酸从5’端到3’端依次包含：上游特异性序列和第一通用序列；
所述下游特异性寡核苷酸从5’端到3’端依次包含：下游特异性序列和第二通用序列；
1)所述第一通用引物的3’端序列与所述上游特异性寡核苷酸的第一通用序列的部分序列或全部序列互补，所述第二通用引物的3’端序列与所述下游特异性寡核苷酸的第二通用序列的部分序列或全部序列互补；或
2)所述第二通用引物的3’端序列与所述上游特异性寡核苷酸的第一通用序列的部分序列或全部序列互补，所述第一通用引物的3’端序列与所述下游特异性寡核苷酸的第二通用序列的部分序列或全部序列互补；
所述上游特异性寡核苷酸的序列包含一个或多个可以被特异性切割剂切割的切割位点；
所述下游特异性寡核苷酸的序列包含一个或多个可以被特异性切割剂切割的切割位点。
优选地，所述第一通用引物的3’端序列与所述上游特异性寡核苷酸的第一通用序列的部分序列或全部序列互补，所述第二通用引物的3’端序列与所述下游特异性寡核苷酸的第二通用序列的部分序列或全部序列互补。
优选地，所述第一通用引物的3’端序列与所述上游特异性寡核苷酸的第一通用序列的全部序列互补，所述第二通用引物的3’端序列与所述下游特异性寡核苷酸的第二通用序列的全部序列互补。
在构建靶向文库的过程中，一般有两种：基于两步PCR的方法和基于PCR扩增和接头连接的方法，其中，基于两步PCR的方法可以是：1)先进行第一步PCR扩增目的基因，得到产物再进行第二步PCR添加测序接头序列和样本标签序列等通用引物序列(需要多步反应，操作相对复杂、时间较长)；或2)将第二步PCR需要添加的测序接头序列和样本标签序列等通用引物序列引入第一步PCR的特异性引物的5’端(引物长，准确性下降，成本增加)；
而本申请通过设计特异性寡核苷酸和通用引物，其中，特异性寡核苷酸的5’端到3’端依次包含：特异性序列和通用序列，特异性寡核苷酸包含一个或多个可以被特异性切割剂切割的切割位点；通用引物的3’端包含与特异性寡核苷酸的通用序列互补的序列；从而使得特异性寡核苷酸与通用引物在逆转录酶和/或聚合酶的作用下形成含有通用引物序列和特异性寡核苷酸序列的双链DNA(图1，特异性寡核苷酸的3’端优选修饰有阻断基团时，无法延伸，因此，双链DNA为不完全互补的双链DNA，其中，一条链(在这里称为短链)为特异性寡核苷酸，另一条链(在这里称为长链)包含通用引物序列和与特异性寡核苷酸互补的序列)，短链中的切割位点在特异性切割剂的作用下切割(特异性寡核苷酸的3’端未修饰阻断基团时，具有类似的效果)，并且在后续PCR高温过程中与长链分离，长链可以和待测目标区域互补，在聚合酶的作用下对目标区域进行扩增，扩增得到含有通用引物序列和待测目标序列的产物，即靶向文库，从而实现一步反应完成目标区域的捕获和通用引物序列(比如测序接头序列和样本标签序列)的添加，即通过短链寡核苷酸(特异性寡核苷酸和通用引物)生成包含通用引物序列和待测目标序列的长链引物，实现目标区域的富集和通用引物序列(比如测序接头序列和样本标签序列)的添加，实现一步法构建靶向文库，具有操作简单，时间短，设计的引物/特异性寡核苷酸短，准确性高，成本低等优点。
同时，在RNA靶向文库制备RT-mPCR(Reverse transcript multiplex PCR)的过程中，RT反应和mPCR 反应是两个独立的反应，通常需要先经过随机引物进行逆转录，然后再加入多重PCR引物进行目标区域PCR扩增，步骤较为复杂；如果采用一步法进行RT-mPCR，在反应的开始就需要加入特异性引物，其存在如下缺点：多条特异性引物的3’端序列多样性较强，在低温的RT过程中1)会和模板产生非特异性结合，产生非特异性产物，并在后续的PCR过程中指数积累；2)特异性引物序列多样性较高，易互补结合并在逆转录酶的作用下产生二聚体，在后续PCR过程中指数积累，导致反应终止；而本申请发明了一种特异性引物可以兼容逆转录反应而不产生非特异性和二聚体的方法，申请方法特异性寡核苷酸和通用引物的3’端序列是通用的，多样性较低，在RT过程中1)特异性寡核苷酸和通用引物的3’端是单一序列，多样性低，和模板互相结合的可能性较低，并且哪怕和模板结合的产生的非特异性产物，在后续PCR扩增过程中也不会积累；2)特异性寡核苷酸和通用引物的3’端都是单一的序列，多样性低，互相之间形成二聚体的可能性较小，产生二聚体的概率小，因此在特异性短链寡核苷酸生成长链引物的过程可以兼容逆转录过程；首先，N6逆转录RNA生成cDNA，同时短链寡核苷酸(特异性寡核苷酸和通用引物)生成包含通用引物序列和待测目标序列的长链引物，然后在聚合酶的作用下对cDNA目标区域进行扩增，扩增得到含有通用引物序列和待测目标序列的产物，即靶向文库，实现逆转录步骤、长链引物生成和目标区域的富集和通用引物序列(比如测序接头序列和样本标签序列)的添加，实现一步反应完成RT和mPCR反应，即实现一步法对RNA构建靶向文库。
优选地，所述特异性切割剂包含酶促切割剂。
优选地，所述酶促切割剂包含核糖核酸酶(例如：核糖核酸酶H)、糖基化酶(例如：尿嘧啶-DNA糖基化酶(UDG)、USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)、3-甲基腺嘌呤DNA糖基化酶、3-甲基腺嘌呤DNA糖基化酶II、嘧啶水合物-DNA糖基化酶、FaPy-DNA糖基化酶、胸腺嘧啶错配-DNA糖基化酶(例如：次黄嘌呤-DNA糖基化酶、尿嘧啶DNA糖基化酶(UDG)、5-羟甲基尿嘧啶DNA糖基化酶(HmUDG)、5-羟甲基胞嘧啶DNA糖基化酶、1,N6-亚乙烯基腺嘌呤DNA糖基化酶))中的至少一种；进一步包含核糖核酸酶(例如：核糖核酸酶H)、糖基化酶(例如：尿嘧啶-DNA糖基化酶(UDG)、USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)中的至少一种。
优选地，所述酶促切割剂选自核糖核酸酶H、UDG中的至少一种；进一步选自核糖核酸酶H。
优选地，所述核糖核酸酶H包含核糖核酸酶HⅠ、核糖核酸酶HII中至少一种。
优选地，所述上游特异性寡核苷酸和下游特异性寡核苷酸中所述的特异性切割剂可以相同或不同。
优选地，所述上游特异性寡核苷酸中的多个特异性切割剂可以相同或不同。
优选地，所述下游特异性寡核苷酸中的多个特异性切割剂可以相同或不同。
优选地，所述切割位点包含c1)～c2)中至少一种；进一步包含c1)：
c1)核糖核苷酸(rNTP，例如：rATP、rGTP、tCTP、rUTP)，其对应的特异性切割剂为核糖核酸酶(例如：核糖核酸酶H)，当rNTP为rUTP时，其对应的特异性切割剂也可以是尿嘧啶-DNA糖基化酶(UDG)或USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)；
c2)尿嘧啶脱氧核糖核苷酸(dUTP)，其对应的特异性切割剂是尿嘧啶-DNA糖基化酶(UDG)或USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)。
优选地，所述上游特异性寡核苷酸和下游特异性寡核苷酸中所述的切割位点可以相同或不同。
优选地，所述上游特异性寡核苷酸中的多个切割位点可以相同或不同。
优选地，所述下游特异性寡核苷酸中的多个切割位点可以相同或不同。
优选地，所述第一通用引物包含功能核酸序列。
优选地，所述第一通用引物的5’端包含功能核酸序列。
优选地，所述第一通用引物包含测序引物序列或测序引物互补序列。
优选地，所述第一通用引物的5’端包含测序引物序列或测序引物互补序列。
优选地，所述第一通用引物包含一段或多段测序引物序列或测序引物互补序列；进一步优选地，所述第一通用引物包含两段或两段以上测序引物序列或测序引物互补序列。
优选地，所述第一通用引物的5’端包含一段或多段测序引物序列或测序引物互补序列；进一步优选地，所述第一通用引物的5’端包含两段或两段以上测序引物序列或测序引物互补序列。
优选地，所述第一通用引物还包含第一标签序列，用于区分不同样本，以便后续的多样本混合测序。例如可以是barcode序列或index序列。
优选地，所述第一通用引物的5’端还包含第一标签序列，用于区分不同样本，以便后续的多样本混合测序。例如可以是barcode序列或index序列。
优选地，所述第一标签序列可以为唯一分子标签(UMI)，用于统计样本中核酸分子的拷贝数。
优选地，所述第一标签序列的长度为5～20bp。
优选地，所述第一标签序列位于所述测序引物序列或测序引物互补序列的中间；进一步优选地，所述第一标签序列位于两段所述测序引物序列或测序引物互补序列的中间。
优选地，所述第二通用引物包含功能核酸序列。
优选地，所述第二通用引物的5’端包含功能核酸序列。
优选地，所述第二通用引物包含测序引物序列或测序引物互补序列。
优选地，所述第二通用引物的5’端包含测序引物序列或测序引物互补序列。
优选地，所述第二通用引物包含一段或多段测序引物序列或测序引物互补序列；进一步优选地，所述第二通用引物包含两段或两段以上测序引物序列或测序引物互补序列。
优选地，所述第二通用引物的5’端包含一段或多段测序引物序列或测序引物互补序列；进一步优选地，所述第二通用引物的5’端包含两段或两段以上测序引物序列或测序引物互补序列。
优选地，所述第二通用引物还包含第二标签序列，用于区分不同样本，以便后续的多样本混合测序。例如可以是barcode序列或index序列。
优选地，所述第二通用引物的5’端还包含第二标签序列，用于区分不同样本，以便后续的多样本混合测序。例如可以是barcode序列或index序列。
优选地，所述第二标签序列可以为唯一分子标签(UMI)，用于统计样本中核酸分子的拷贝数。
优选地，所述第二标签序列的长度为5～20bp。
优选地，所述第二标签序列位于所述测序引物序列或测序引物互补序列的中间；进一步优选地，所述第二标签序列位于两段所述测序引物序列或测序引物互补序列的中间。
优选地，所述第一标签序列、第二标签序列相同或不同。
优选地，所述第一标签序列、第二标签序列不同。
优选地，所述第一通用序列、第二通用序列各自独立选自部分测序接头序列、全部测序接头序列、测序引物结合序列或任意固定序列(比如包含酶切位点)。
优选地，所述测序接头序列可以是任何一种测序平台的任何一种测序接头。
优选地，所述第一通用序列与所述第二通用序列相同或不同；进一步优选地，所述第一通用序列与所述第二通用序列不同。
优选地，所述上游特异性寡核苷酸的3’端进行阻断修饰；和/或
所述下游特异性寡核苷酸的3’端进行阻断修饰。
进一步优选地，所述上游特异性寡核苷酸的3’末端进行阻断修饰；和/或
所述下游特异性寡核苷酸的3’末端进行阻断修饰。
优选地，所述阻断修饰包含：磷酸化修饰、间臂修饰、氨基修饰中的至少一种；进一步优选地，所述 阻断修饰包含：磷酸化修饰。
优选地，所述上游特异性寡核苷酸和所述下游特异性寡核苷酸的阻断修饰相同或不同。
优选地，所述特异性寡核苷酸中的上游特异性序列和下游特异性序列分别与待测目标区域的两个区段相同或互补。
优选地，所述特异性寡核苷酸中的特异性序列的设计遵循常规的引物设计原则，但是和常规的引物序列反向互补，比如：上游特异性序列与待测目标区域上游负链(即反义链、cDNA第二链(构建RNA靶向文库时))序列相同或正链(即有义链、cDNA第一链(构建RNA靶向文库时))序列互补，下游特异性序列与待测目标区域的下游正链(即有义链、cDNA第一链(构建RNA靶向文库时))序列相同或负链(即反义链、cDNA第二链(构建RNA靶向文库时))序列互补。
优选地，所述上游特异性寡核苷酸的上游特异性序列与待测目标区域的上游负链序列相同或正链序列互补。
优选地，所述下游特异性寡核苷酸的下游特异性序列与待测目标区域的下游正链序列相同或负链序列互补。
优选地，所述第一通用引物的5’端含有磷酸基团，通过第一通用引物可以使得扩增的产物的5’端带有磷酸基团，从而避免专门进行磷酸化的过高成本；和/或
所述第二通用引物的5’端含有磷酸基团，通过第二通用引物可以使得扩增的产物的5’端带有磷酸基团，从而避免专门进行磷酸化的过高成本。
优选地，所述第二通用引物的5’端含有磷酸基团。
优选地，所述特异性寡核苷酸、通用引物为DNA片段或其类似物。
优选地，所述上游特异性寡核苷酸的序列包含多个可以被特异性切割剂切割的切割位点，切割位点数量大于等于1/10碱基，例如：可以是切割位点的个数大于等于n个；或者是n～N个；或者是3～9个；其中，n＝N/10，N为上游特异性寡核苷酸的核苷酸个数。
优选地，所述下游特异性寡核苷酸的序列包含多个可以被特异性切割剂切割的切割位点，切割位点数量大于等于1/10碱基，例如：可以是切割位点的个数大于等于n个；或者是n～N个；或者是3～9个；其中，n＝N/10，N为下游特异性寡核苷酸的核苷酸个数。
优选地，所述上游特异性寡核苷酸的上游特异性序列的核苷酸个数为15～45。
优选地，所述上游特异性寡核苷酸的第一通用序列的核苷酸个数为15～45。
优选地，所述上游特异性寡核苷酸的核苷酸个数为30～90。
优选地，所述下游特异性寡核苷酸的下游特异性序列的核苷酸个数为15～45。
优选地，所述下游特异性寡核苷酸的第二通用序列的核苷酸个数为15～45。
优选地，所述下游特异性寡核苷酸的核苷酸个数为30～90。
优选地，所述第一通用引物的核苷酸个数为15～90。
优选地，所述第二通用引物的核苷酸个数为15～90。
优选地，所述特异性寡核苷酸包含多对特异性寡核苷酸，可结合至待测目标区域的不同位置；进一步包含多组靶向不同的待测目标区域的多对特异性寡核苷酸。
优选地，所述多对为两对以上。
优选地，所述多组为两组以上。
优选地，所述通用引物包含多对通用引物。
优选地，所述第一通用引物包含一条或多条第一通用引物；进一步包含多条第一通用引物。
优选地，所述第二通用引物包含一条或多条第二通用引物；进一步包含一条第二通用引物。
优选地，检测EGFR基因的寡核苷酸组合，包含：特异性寡核苷酸和通用引物；
所述特异性寡核苷酸包含：上游特异性寡核苷酸和下游特异性寡核苷酸；
所述通用引物包含：第一通用引物和第二通用引物；
所述上游特异性寡核苷酸的序列如SEQ ID NO.1～SEQ ID NO.8所示，所述下游特异性寡核苷酸的序列如SEQ ID NO.9～SEQ ID NO.16所示；
序列如SEQ ID NO.1～SEQ ID NO.8所示的上游特异性寡核苷酸各自独立包含一个或多个可以被特异性切割剂切割的切割位点；
序列如SEQ ID NO.9～SEQ ID NO.16所示的下游特异性寡核苷酸各自独立包含一个或多个可以被特异性切割剂切割的切割位点；
所述第一通用引物的序列如SEQ ID NO.53所示，所述第二通用引物的序列如SEQ ID NO.54所示。
优选地，序列如SEQ ID NO.1～16所示的特异性寡核苷酸的3’端进行阻断修饰。
优选地，检测SARS-COV-2基因的寡核苷酸组合，包含：特异性寡核苷酸和通用引物；
所述特异性寡核苷酸包含：上游特异性寡核苷酸和下游特异性寡核苷酸；
所述通用引物包含：第一通用引物和第二通用引物；
所述上游特异性寡核苷酸的序列如SEQ ID NO.17、19、21、23、25所示，所述下游特异性寡核苷酸的序列如SEQ ID NO.18、20、22、24、26所示；
序列如SEQ ID NO.17、19、21、23、25所示的上游特异性寡核苷酸各自独立包含一个或多个可以被特异性切割剂切割的切割位点；
序列如SEQ ID NO.18、20、22、24、26所示的下游特异性寡核苷酸各自独立包含一个或多个可以被特异性切割剂切割的切割位点；
所述第一通用引物的序列如SEQ ID NO.53所示，所述第二通用引物的序列如SEQ ID NO.54所示。
优选地，序列如SEQ ID NO.17～26所示的特异性寡核苷酸的3’端进行阻断修饰。
对于上述检测EGFR基因的寡核苷酸组合和/或检测SARS-COV-2基因的寡核苷酸组合：
优选地，所述特异性切割剂包含酶促切割剂。
优选地，所述酶促切割剂包含核糖核酸酶(例如：核糖核酸酶H)、糖基化酶(例如：尿嘧啶-DNA糖基化酶(UDG)、USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)、3-甲基腺嘌呤DNA糖基化酶、3-甲基腺嘌呤DNA糖基化酶II、嘧啶水合物-DNA糖基化酶、FaPy-DNA糖基化酶、胸腺嘧啶错配-DNA糖基化酶(例如：次黄嘌呤-DNA糖基化酶、尿嘧啶DNA糖基化酶(UDG)、5-羟甲基尿嘧啶DNA糖基化酶(HmUDG)、5-羟甲基胞嘧啶DNA糖基化酶、1,N6-亚乙烯基腺嘌呤DNA糖基化酶))中的至少一种；进一步包含核糖核酸酶(例如：核糖核酸酶H)、糖基化酶(例如：尿嘧啶-DNA糖基化酶(UDG)、USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)中的至少一种。
优选地，所述酶促切割剂选自核糖核酸酶H、UDG中的至少一种；进一步选自核糖核酸酶H。
优选地，所述核糖核酸酶H包含核糖核酸酶HⅠ、核糖核酸酶HII中至少一种。
优选地，所述上游特异性寡核苷酸和下游特异性寡核苷酸中所述的特异性切割剂可以相同或不同。
优选地，所述上游特异性寡核苷酸中的多个特异性切割剂可以相同或不同。
优选地，所述下游特异性寡核苷酸中的多个特异性切割剂可以相同或不同。
优选地，所述切割位点包含c1)～c2)中至少一种；进一步包含c1)：
c1)核糖核苷酸(rNTP，例如：rATP、rGTP、tCTP、rUTP)，其对应的特异性切割剂为核糖核酸酶(例如：核糖核酸酶H)，当rNTP为rUTP时，其对应的特异性切割剂也可以是尿嘧啶-DNA糖基化酶(UDG)或USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)；
c2)尿嘧啶脱氧核糖核苷酸(dUTP)，其对应的特异性切割剂是尿嘧啶-DNA糖基化酶(UDG)或USER 酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)。
优选地，所述上游特异性寡核苷酸和下游特异性寡核苷酸中所述的切割位点可以相同或不同。
优选地，所述上游特异性寡核苷酸中的多个切割位点可以相同或不同。
优选地，所述下游特异性寡核苷酸中的多个切割位点可以相同或不同。
优选地，序列如SEQ ID NO.1～16所示的特异性寡核苷酸的切割位点的位置如表1及附注所示。
优选地，序列如SEQ ID NO.17～26所示的特异性寡核苷酸的切割位点的位置如表4及附注所示。
优选地，所述阻断修饰包含：磷酸化修饰、间臂修饰、氨基修饰中的至少一种；进一步优选地，所述阻断修饰包含：磷酸化修饰。
优选地，所述第二通用引物的5’端含有磷酸基团，通过第二通用引物可以使得扩增的产物的5’端带有磷酸基团，从而避免专门进行磷酸化的过高成本。
本发明的第二个方面，提供一种试剂盒，包含：本发明第一个方面的寡核苷酸组合。
优选地，所述试剂盒还包含：用于所述通用引物延伸的酶。
优选地，所述用于所述通用引物延伸的酶包含逆转录酶和DNA聚合酶中的至少一种；进一步包含逆转录酶和DNA聚合酶；其用于通用引物(第一通用引物和第二通用引物)以特异性寡核苷酸(上游特异性寡核苷酸和下游特异性寡核苷酸)为模板延伸形成双链寡核苷酸(优选为不完全互补的双链寡核苷酸)。
优选地，所述试剂盒还包含：特异性切割剂(优选为本发明第一个方面中的特异性切割剂)，其用于切割通用引物(第一通用引物和第二通用引物)以特异性寡核苷酸(上游特异性寡核苷酸和下游特异性寡核苷酸)为模板延伸形成的双链寡核苷酸(优选为不完全互补的双链寡核苷酸)(例如DNA双链)中的特异性寡核苷酸(上游特异性寡核苷酸和下游特异性寡核苷酸)。
优选地，所述试剂盒还包含：PCR反应混合液，其用于PCR反应。
优选地，所述PCR反应混合液包含：DNA聚合酶、Mg2+、PCR缓冲液、dNTPs中的至少一种；进一步优选地，所述PCR反应混合液还包含：DNA聚合酶、Mg2+、PCR缓冲液和dNTPs；对于试剂盒已经包含的组分，所述PCR反应混合液可以相应地减少该组分，比如：所述用于所述通用引物延伸的酶包含DNA聚合酶时，所述PCR反应混合液可以不包含DNA聚合酶。
优选地，所述试剂盒还包含：逆转录反应混合液，其用于构建RNA的靶向文库时的逆转录反应(即将RNA逆转录为cDNA)。
优选地，所述逆转录反应混合液包含：逆转录酶、DNA聚合酶、逆转录缓冲液、逆转录引物、dNTPs中的至少一种；优选地，所述逆转录反应混合液包含：逆转录酶、DNA聚合酶、逆转录缓冲液、dNTPs和逆转录引物；对于试剂盒中已经包含的组分，所述逆转录反应混合液可以相应地减少该组分：比如：所述用于所述通用引物延伸的酶包含逆转录酶时，所述逆转录反应混合液可以不包含逆转录酶；所述用于所述通用引物延伸的酶或所述PCR反应混合液包含DNA聚合酶时，所述逆转录反应混合液可以不包含DNA聚合酶；所述PCR反应混合液包含dNTPs时，所述逆转录反应混合液可以不包含dNTPs。
优选地，所述DNA聚合酶为耐高温DNA聚合酶。
优选地，所述试剂盒包含：核酸提取试剂组合。
优选地，所述核酸提取试剂组合具体是用于选自以下任意一种方法的核酸提取试剂组合：碱裂解法、酚氯仿抽提法、螯合树脂法、离心柱膜吸附法以及磁珠法。
优选地，所述核酸提取试剂组合包含：裂解液、洗涤液、洗脱液、核酸吸附物中的至少一种；进一步优选地，所述核酸提取试剂组合包含：裂解液、洗涤液、洗脱液和核酸吸附物。
优选地，所述核酸吸附物包含磁珠、吸附膜中的至少一种。
本发明的第三个方面，提供一种测序试剂套装，包含：采用本发明第二个方面的试剂盒。
优选地，所述测序试剂套装还包含：测序试剂盒。
本发明的第四个方面，提供一种测序系统，包含：测序仪和本发明第三个方面的测序试剂套装。
本发明的第五个方面，提供一种多重PCR扩增方法，包含采用本发明第一个方面的寡核苷酸组合的步骤。
优选地，所述多重PCR扩增方法包括如下步骤：利用本发明第一个方面的寡核苷酸组合对核酸进行多重PCR反应。
优选地，所述反应的步骤包括：
通用引物延伸反应，并切割互补链，生成PCR反应引物；
利用PCR反应引物进行扩增反应。
优选地，所述多重PCR反应的体系包含：用于所述通用引物延伸的酶(优选为本发明第二个方面中的用于所述通用引物延伸的酶)、特异性切割剂(优选为本发明第二个方面中的特异性切割剂)；特异性寡核苷酸与通用引物在用于所述通用引物延伸的酶(例如：逆转录酶和/或聚合酶)的作用下形成含有通用引物序列和特异性寡核苷酸序列的双链DNA(特异性寡核苷酸的3’端优选修饰有阻断基团，无法延伸，因此，双链DNA为不完全互补的双链DNA，其中，一条链(在这里称为短链)为特异性寡核苷酸，另一条链(在这里称为长链)包含通用引物序列和与特异性寡核苷酸互补的序列)，短链中的切割位点在特异性切割剂的作用下切割(特异性寡核苷酸的3’端未修饰阻断基团时，具有类似的效果)，并且在后续PCR高温过程中与长链分离，长链可以和待测目标区域互补，在聚合酶的作用下对目标区域进行扩增，扩增得到含有通用引物序列(比如测序接头序列和样本标签序列)和待测目标序列的产物。
优选地，所述延伸反应和扩增反应在同一体系中进行。
优选地，所述同一体系具体为不进行洗脱和/或纯化处理。
优选地，所述多重PCR反应的体系中所述特异性切割剂的浓度小于用于所述通用引物延伸的酶的浓度，从而使延伸反应先于切割反应。
优选地，所述多重PCR反应的体系还包含：PCR反应混合液(优选为本发明第二个方面中的PCR反应液)。
优选地，所述核酸来自以下任意一种或多种生物样本：细胞、组织、体液、微生物、唾液、尿液、痰液、粪便、咽拭子、鼻拭子。
优选地，所述体液包含组织液、淋巴液、血液、脑脊液中的至少一种。
优选地，所述微生物包含细菌、病毒、真菌、放线菌、立克次氏体、支原体、衣原体、螺旋体中的至少一种。
优选地，所述核酸通过所述生物样本发生裂解反应得到。
优选地，所述裂解反应在所述多重PCR反应之前。
优选地，所述裂解反应和所述多重PCR反应在同一体系中进行。
优选地，所述同一体系具体为同一反应容器。
优选地，所述裂解反应后和所述多重PCR反应前不包含：提取纯化步骤。
优选地，所述裂解反应后和所述多重PCR反应前还包含：提取纯化步骤，去除盐类，有机剂等杂质。
优选地，所述提取纯化进一步包含：沉淀核酸或吸附核酸。
优选地，所述提取纯化后还包含：洗脱或溶解核酸。
优选地，所述裂解反应和所述多重PCR反应在不同体系中进行，即不在同一体系中进行。
优选地，所述同一体系具体为同一反应容器。
优选地，所述裂解反应后还包含：提取纯化步骤，去除盐类，有机剂等杂质。
优选地，所述提取纯化进一步包含：沉淀核酸或吸附核酸。
优选地，所述提取纯化后还包含：洗脱或溶解核酸。
优选地，所述裂解的方法包含：物理方式、化学方式、生物方式中的至少一种。
优选地，所述物理方式包含：煮沸法、玻璃珠法、超声波法、研磨法、冻融法、匀浆法中的至少一种。
优选地，所述化学方式包含：表面活性剂法(SDS法)、碱裂解法中的至少一种。
优选地，所述生物方式包含：酶法，比如通过溶菌酶、蛋白酶K等酶裂解。
优选地，所述核酸为RNA时，所述裂解反应后和所述多重PCR反应前还包含逆转录反应，即将RNA逆转录为cDNA。
优选地，所述逆转录反应与所述多重PCR反应在同一体系或不在同一体系中进行；进一步优选地，所述逆转录反应与所述多重PCR反应在同一体系中进行。
优选地，所述同一体系具体为在同一反应体系中进行；即无需先在一个反应体系中进行逆转录反应，然后再在其他反应体系中进行多重PCR反应。
本申请设计的特异性寡核苷酸和通用引物的3’端序列是通用的，多样性较低，在逆转录反应过程中1)特异性寡核苷酸和通用引物的3’端是单一序列，多样性低，和模板互相结合的可能性较低，并且哪怕和模板结合的产生的非特异性产物，在后续PCR扩增过程中也不会积累；2)特异性寡核苷酸和通用引物的3’端都是单一的序列，多样性低，互相之间形成二聚体的可能性较小，产生二聚体的概率小，因此在特异性短链寡核苷酸生成长链引物的过程可以兼容逆转录过程；首先，N6逆转录RNA生成cDNA，同时短链寡核苷酸(特异性寡核苷酸和通用引物)生成包含通用引物序列和待测目标序列的长链引物，然后在聚合酶的作用下对cDNA目标区域进行扩增，扩增得到含有通用引物序列和待测目标序列的产物，兼容逆转录反应和PCR反应。
优选地，所述核酸为RNA时，所述多重PCR反应的体系还包含：逆转录反应混合液(优选为本发明第二个方面中的逆转录反应混合液)。
本发明的第六个方面，提供一种构建靶向文库的方法，包含本发明第五个方面的多重PCR扩增方法的步骤，得到测序文库。
优选地，得到测序文库前还可以包括环化反应，即对扩增得到的线性文库进行环化。
优选地，所述方法还包括如下步骤：对测序文库进行纯化。
优选地，所述纯化采用磁珠进行。
本发明的第七个方面，提供一种测序方法，包含：本发明第六个方面的构建靶向文库的方法的步骤。
优选地，所述测序方法包括如下步骤：制备文库；测序；
所述制备文库的方法为本发明第六个方面的构建靶向文库的方法。
优选地，所述测序前还包括如下步骤：文库质检。
本发明第八个方面，提供一种获得目标区域基因信息的方法，包含：本发明第七个方面的测序方法的步骤。
优选地，所述获得目标区域基因信息的方法包括如下步骤：测序，获得测序数据；获得目标区域基因信息；所述信息包含序列信息、突变信息中的至少一种；
所述测序的方法为本发明第七个方面的测序方法。
优选地，所述信息包含突变信息时，所述获得目标区域基因信息的方法还包含如下步骤：将获得的测序数据与参考基因组进行比对，确定目标区域基因的突变信息。
一种获得EGFR基因信息的方法，包括如下步骤：测序，获得测序数据；获得EGFR基因信息；所述信息包含序列信息、突变信息中的至少一种；
所述测序的方法为本发明第七个方面的测序方法；
所述寡核苷酸组合为本发明第一个方面中的检测EGFR基因的寡核苷酸组合。
优选地，所述信息包含突变信息时，所述获得EGFR基因信息的方法还包含如下步骤：将获得的测序 数据与参考基因组进行比对，获得EGFR基因的突变信息。
本发明第九个方面，提供本发明第一个方面的寡核苷酸组合、第二个方面的试剂盒、第三个方面的测序试剂套装和/或第四个方面的测序系统在c1)～c8)任一项中的应用；
c1)构建靶向文库；
c2)制备用于构建靶向文库的产品；
c3)测序；
c4)制备用于测序的产品；
c5)获得目标区域基因信息；
c6)制备用于获得目标区域基因信息的产品；
c7)多重PCR扩增；
c8)制备用于多重PCR扩增的产品。
以下通过具体的实施例对本发明的内容作进一步详细的说明。
应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。本实施例中所使用的材料、试剂等，如无特别说明，为从商业途径得到的试剂和材料。
实施例1EGFR基因肿瘤热点基因的文库制备方法
EGFR基因肿瘤热点基因的文库制备方法，示意图如图2所示，包括如下步骤：
(1)设计EGFR基因肿瘤热点基因捕获panel以及通用标签引物，该panel包含8对特异性寡核苷酸(EGFR_1F～EGFR_8F，EGFR_1R～EGFR_8R)，扩增子大小为100～200bp，特异性寡核苷酸的序列如表1所示；通用标签引物包含通用标签引物F和通用标签引物R，通用标签引物F的核苷酸序列为：TGTGAGCCAAGGAGTTATCGGACCTAATTGTCTTCCTAAGACCGCTTGGCCTCCGACTT(SEQ IDNO.53，其中，加粗部分(SEQ ID NO.55)为本实施例的通用标签引物F的标签序列，标签序列可以是随机的，也可以是提前设定的，比如表2中序列如SEQ ID NO.56～62所示的标签序列)；通用标签引物F的下划线部分和上游特异性寡核苷酸3’端序列(通用序列)互补；通用标签引物R的核苷酸序列为：Phos-GAACGACATGGCTACGATCCGACTT(SEQ ID NO.54)，Phos为磷酸化修饰，下划线部分和下游特异性寡核苷酸3’端序列(通用序列)互补。
(2)PCR反应
在PCR管中按照表3所示反应体系配置mPCR(multiplex PCR)体系，进行mPCR反应，反应程序如下：94℃，2min，60℃,5min；94℃30s，58℃2min，72℃30s，27cycles；72℃5min；12℃∞，反应完后用1.5X AMPure(贝克曼公司)磁珠进行纯化，最后将纯化产物溶于22μL洗脱缓冲液，得到文库。
表1特异性寡核苷酸

注：上述特异性寡核苷酸按照每条特异性寡核苷酸20μM浓度进行混合，得到总浓度为20μM的特异性寡核苷酸池；上述特异性寡核苷酸的3’端采用磷酸(phos)封闭，防止3’端延伸，下划线碱基为核糖核苷酸(切割位点)，可以被Rnase HII识别并水解；上游特异性寡核苷酸(EGFR_1F～EGFR_8F)的5’到3’端依次为：特异性序列和通用序列(加粗部分)，加粗部分为与通用标签引物F的3’端互补的序列；下游特异性寡核苷酸(EGFR_1R～EGFR_8R)的5’到3’端依次为：特异性序列和通用序列(加粗部分)，加粗部分为与通用标签引物R的3’端互补的序列。
表2通用标签引物F可选的标签序列
表3 mPCR反应体系

注：HawkZ05 Fast DNA Polymerase来自罗氏公司，货号07731264103。Rnase HII酶来自NEB公司，货号M0288S。
实施例2新冠SARS-COV-2基因的文库制备方法
新冠SARS-COV-2基因的文库制备方法，示意图如图3所示，包括如下步骤：
(1)设计新冠SARS-COV-2基因捕获panel以及通用标签引物，该panel包含5对特异性寡核苷酸(N1-F、N1-R、N2-F、N2-R、N3-F、N3-R、Orf1ab-F、Orf1ab-R、RDRP-F、RDRP-R)，特异性寡核苷酸的序列如表4所示；通用标签引物包含通用标签引物F和通用标签引物R，通用标签引物F的核苷酸序列为：TGTGAGCCAAGGAGTTATCGGACCTAATTGTCTTCCTAAGACCGCTTGGCCTCCGACTT(其中，加粗部分(SEQ ID NO.55)为本实施例的通用标签引物F的标签序列，标签序列可以是随机的，也可以是提前设定的，比如表2中序列如SEQ ID NO.56～62所示的标签序列)；通用标签引物F的下划线部分和上游特异性寡核苷酸3’端序列(通用序列)互补；通用标签引物R的核苷酸序列为：Phos-GAACGACATGGCTACGATCCGACTT，Phos为磷酸化修饰，下划线部分和下游特异性寡核苷酸3’端序列(通用序列)互补。
(2)PCR反应
在PCR管中按照表5所示反应体系配置RT-mPCR(Reverse transcript multiplex PCR)体系，进行RT-mPCR反应，反应程序如下：25℃10min；45℃15min；94℃5min；94℃30s，58℃2min，72℃30s，40cycles；72℃5min；12℃∞，反应完后用1.5X AMPure(贝克曼公司)磁珠进行纯化，最后将纯化产物溶于22μL洗脱缓冲液，得到文库。
表4特异性寡核苷酸

注：上述特异性寡核苷酸按照每条特异性寡核苷酸10μM浓度进行混合，得到总浓度为10μM的特异性寡核苷酸池；上述特异性寡核苷酸的3’端采用磷酸(phos)封闭，防止3’端延伸，下划线碱基为核糖核苷酸(切割位点)，可以被Rnase HII识别并水解；上游特异性寡核苷酸(N1-F、N2-F、N3-F、Orf1ab-F、RDRP-F)的5’到3’端依次为：特异性序列和通用序列(加粗部分)，加粗部分为与通用标签引物F的3’端互补的序列；下游特异性寡核苷酸(N1-R、N2-R、N3-R、Orf1ab-R、RDRP-R)的5’到3’端依次为：特异性序列和通用序列(加粗部分)，加粗部分为与通用标签引物R的3’端互补的序列。
表5 RT-mPCR反应体系
注：逆转录体系(2×One-step Mix、One-step enzyme Mix)来自诺唯赞公司，HiScript II One Step RT-PCR Kit，货号P611-01。Rnase HII酶来自NEB公司，货号M0288S，RNA inhibitor来自NEB公司，货号M0314S，T4DNA Polymerase来自NEB公司，货号M0203S。
对比例1 EGFR基因肿瘤热点基因的文库制备方法
EGFR基因肿瘤热点基因的文库制备方法，包括如下步骤：
(1)设计EGFR基因肿瘤热点基因捕获panel，该panel包含8对引物(EGFR_1F～EGFR_8F，EGFR_1R～EGFR_8R)，扩增子大小为100～200bp，引物的序列如表6所示。
(2)PCR反应
在PCR管中按照表7所示反应体系配置mPCR(multiplex PCR)体系，进行mPCR反应，反应程序如 下：94℃5min；94℃30s，58℃2min，72℃30s，27cycles；72℃5min；12℃∞，反应完后用1.5X AMPure(贝克曼公司)磁珠进行纯化，最后将纯化产物溶于22μL洗脱缓冲液，得到文库。
表6引物

注：上述引物按照每条引物20μM浓度进行混合，得到总浓度为20μM的引物池；下游引物的5’端进行磷酸化修饰；上游引物(EGFR_1F～EGFR_8F)的5’到3’端依次为：通用序列(加粗部分)和特异性序列，加粗部分与实施例1中的通用标签引物F序列相同；下游引物(EGFR_1R～EGFR_8R)的5’到3’端依次为：通用序列(加粗部分)和特异性序列，加粗部分与实施例1中的通用标签引物R的序列相同，即上述引物与实施例1中的特异性寡核苷酸与通用标签引物逆转录反应得到的产物的序列相同。
表7 mPCR体系
对比例2新冠SARS-COV-2基因的文库制备方法
新冠SARS-COV-2基因的文库制备方法，包括如下步骤：
(1)设计新冠SARS-COV-2基因捕获panel以及通用标签引物，该panel包含5对特异性寡核苷酸(N1-F、N1-R、N2-F、N2-R、N3-F、N3-R、Orf1ab-F、Orf1ab-R、RDRP-F、RDRP-R)，特异性寡核苷酸的序列如表8所示；通用标签引物包含通用标签引物F和通用标签引物R，通用标签引物F的核苷酸序列为：TGTGAGCCAAGGAGTTATCGGACCTAATTGTCTTCCTAAGACCGCTTGGCCTCCGACTT(其中，加粗部分(SEQ ID NO.55)为本对比例的通用标签引物F的标签序列，标签序列可以是随机的，也可以是提前设定的，比如表2中序列如SEQ ID NO.56～62所示的标签序列)；通用标签引物F的下划线部分和上游特异性寡核苷酸5’端序列(通用序列)相同；通用标签引物R的核苷酸序列为：Phos-GAACGACATGGCTACGATCCGACTT，Phos为磷酸化修饰，下划线部分和下游特异性寡核苷酸5’端序列(通用序列)相同。
(2)RT反应
在PCR管中按照表9所示反应体系配置RT反应体系，进行RT反应，反应程序如下：25℃10min，42℃20min，85℃，10min，得到RT反应产物。
(3)第一轮PCR
在PCR管中按照表10所示反应体系配置第一轮PCR反应体系，进行PCR反应，反应程序如下：94℃1min；94℃30s，58℃2min，72℃30s，15cycles；72℃5min；12℃∞，反应完后用1.5X AMPure(贝克曼公司)磁珠进行纯化，最后将纯化产物溶于22μL洗脱缓冲液，得到第一轮PCR产物。
(4)第二轮PCR
在PCR管中按照表11所示反应体系配置第二轮PCR反应体系，进行PCR反应，反应程序如下：94℃1min；94℃30s，58℃2min，72℃30s，25cycles；72℃5min；12℃∞，反应完后用1.0X AMPure(贝克曼公司)磁珠进行纯化，最后将纯化产物溶于22μL洗脱缓冲液，得到文库。
表8引物
注：上述引物按照每引物10μM浓度进行混合，得到总浓度为10μM的引物池；上游引物(N1-F、N2-F、N3-F、Orf1ab-F、RDRP-F)的5’到3’端依次为：通用序列(加粗部分)和特异性序列，加粗部分为与通用标签引物F的3’端相同的序列；下游特异性寡核苷酸(N1-R、N2-R、N3-R、Orf1ab-R、RDRP-R)的5’到3’端依次为：通用序列(加粗部分)和特异性序列，加粗部分为与通用标签引物R的3’端相同的序列。
表9 RT反应体系

注：逆转录体系来自诺唯赞公司，M-MLV(H-)Reverse Transcriptase，货号R021-01。
表10第一轮PCR反应体系
表11第二轮PCR反应体系
效果实施例1 EGFR基因肿瘤热点基因的高通量测序方法
下述效果实施例是基于华大基因的测序仪MGISEQ-2000平台；使用的试剂均来源于该测序仪配套使用的建库试剂盒以及双端测序试剂盒(以下简称PE100试剂盒)，下述效果实施例中采用的是PE100的测序读长；过程中用到的测序仪、试剂操作等过程参照该平台的使用方法，最后进行数据分析，包括数据利用率、唯一比对比例、目标区域数据比例等性能(分析方法参考文献：Campbell,Nathan R.,Stephanie A.Harmon,and Shawn R.Narum."Genotyping‐in‐Thousands by sequencing(GT‐seq):A cost effective SNP genotyping method based on custom amplicon sequencing."Molecular ecology resources 15.4(2015):855-867.)，具体如下：
1)文库制备：实施例1或对比例1的文库制备方法；
2)文库质检：得到的产物进行定量和条带大小质检；
3)上机测序：得到的文库在华大智造MGISEQ-2000平台上进行上机测序，上机类型PE100；
4)数据分析：得到的下机数据采用BWA软件比对到人参考基因组(hg19)，使用samtools对比对率、特异性、均一性进行统计，最后采用GATK软件进行突变分析。
实施例1、对比例1得到的文库的测序数据统计如表12所示、实施例1的突变检测统计数据如表13所示：实施例1、对比例1得到的文库的测序数据中的数据利用率、唯一比对比例、目标区域数据比例相当；实施例1、对比例1的突变频率的检测值与理论值相当；即采用本发明的方法(实施例1)可以达到对照方法(对比例1)的性能，与对照方法相同，本发明方法只需要一步PCR即可完成文库的制备，但是本发明方法无需提前合成包含完整测序接头和样本标签序列的长链引物，克服了以下弊端：1)引物准确性下降；2)引物成本增加；本发明方法可以实现长链引物合成，并且可以在一步完成目标区域的富集和文库的制备(图4)。
表12测序数据统计
表13突变检测统计
效果实施例2新冠SARS-COV-2基因的高通量测序方法
下述效果实施例是基于华大基因的测序仪MGISEQ-2000平台；使用的试剂均来源于该测序仪配套使用的建库试剂盒以及双端测序试剂盒(以下简称PE100试剂盒)，下述效果实施例中采用的是PE100的测序读长；过程中用到的测序仪、试剂操作等过程参照该平台的使用方法，最后进行数据分析，包括数据利用率、唯一比对比例、目标区域数据比例等性能(分析方法参考文献：Campbell,Nathan R.,Stephanie A.Harmon,and Shawn R.Narum."Genotyping‐in‐Thousands by sequencing(GT‐seq):A cost effective SNP genotyping method based on custom amplicon sequencing."Molecular ecology resources 15.4(2015):855-867.)，具体如下：
1)文库制备：实施例2或对比例2的文库制备方法；
2)文库质检：得到的产物进行定量和条带大小质检；
3)上机测序：得到的文库在华大智造MGISEQ-2000平台上进行上机测序，上机类型PE100；
4)数据分析：得到的下机数据采用BWA软件比对到wuhan-01参考毒株，使用samtools进行统计。
实施例2、对比例2得到的文库的测序数据统计如表14所示：实施例2、对比例2得到的文库的测序数据中的数据利用率、唯一比对比例、目标区域数据比例相当；即采用本发明的方法(实施例2)可以达到对照方法(对比例2)的性能，数据质量相当；但是本发明方法只需要一步PCR即可完成RNA靶向文库的制备，可以实现长链引物合成，并且可以兼容RT体系，可以在一步反应完成逆转录、目标区域的富集和文库的制备(图4)。
表14测序数据统计

上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (15)

  1. 寡核苷酸组合，包含：特异性寡苷核酸和通用引物；
    所述通用引物包含：第一通用引物和第二通用引物；
    所述特异性寡核苷酸包含：上游特异性寡核苷酸和下游特异性寡核苷酸：其中，
    所述上游特异性寡核苷酸从5’端到3’端依次包含：上游特异性序列和第一通用序列；
    所述下游特异性寡核苷酸从5’端到3’端依次包含：下游特异性序列和第二通用序列；
    1)所述第一通用引物的3’端序列与所述上游特异性寡核苷酸的第一通用序列的部分序列或全部序列互补，所述第二通用引物的3’端序列与所述下游特异性寡核苷酸的第二通用序列的部分序列或全部序列互补；或
    2)所述第二通用引物的3’端序列与所述上游特异性寡核苷酸的第一通用序列的部分序列或全部序列互补，所述第一通用引物的3’端序列与所述下游特异性寡核苷酸的第二通用序列的部分序列或全部序列互补；
    所述上游特异性寡核苷酸的序列包含一个或多个可以被特异性切割剂切割的切割位点；
    所述下游特异性寡核苷酸的序列包含一个或多个可以被特异性切割剂切割的切割位点。
  2. 根据权利要求1所述的寡核苷酸组合，其特征在于：
    所述特异性切割剂包含酶促切割剂；
    优选地，所述酶促切割剂包含核糖核酸酶(例如：核糖核酸酶H)、糖基化酶(例如：尿嘧啶-DNA糖基化酶(UDG)、USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)、3-甲基腺嘌呤DNA糖基化酶、3-甲基腺嘌呤DNA糖基化酶II、嘧啶水合物-DNA糖基化酶、FaPy-DNA糖基化酶、胸腺嘧啶错配-DNA糖基化酶(例如：次黄嘌呤-DNA糖基化酶、尿嘧啶DNA糖基化酶(UDG)、5-羟甲基尿嘧啶DNA糖基化酶(HmUDG)、5-羟甲基胞嘧啶DNA糖基化酶、1,N6-亚乙烯基腺嘌呤DNA糖基化酶))中的至少一种；
    优选地，所述上游特异性寡核苷酸和下游特异性寡核苷酸中所述的特异性切割剂可以相同或不同；
    优选地，所述上游特异性寡核苷酸中的多个特异性切割剂可以相同或不同；
    优选地，所述下游特异性寡核苷酸中的多个特异性切割剂可以相同或不同。
  3. 根据权利要求2所述的寡核苷酸组合，其特征在于：
    所述切割位点包含c1)～c2)中至少一种：
    c1)核糖核苷酸(rNTP，例如：rATP、rGTP、tCTP、rUTP)；
    c2)尿嘧啶脱氧核糖核苷酸(dUTP)；
    优选地，所述上游特异性寡核苷酸和下游特异性寡核苷酸中所述的切割位点可以相同或不同；
    优选地，所述上游特异性寡核苷酸中的多个切割位点可以相同或不同；
    优选地，所述下游特异性寡核苷酸中的多个切割位点可以相同或不同。
  4. 根据权利要求3所述的寡核苷酸组合，其特征在于：
    所述酶促切割剂选自核糖核酸酶H、UDG中的至少一种；
    优选地，所述核糖核酸酶H包含核糖核酸酶HⅠ、核糖核酸酶HII中至少一种。
  5. 根据权利要求1所述的寡核苷酸组合，其特征在于：
    所述上游特异性寡核苷酸的序列包含多个可以被特异性切割剂切割的切割位点，例如：可以是切割位点的个数大于等于n个；或者是n～N个；或者是3～9个；其中，n＝N/10，N为上游特异性寡核苷酸的核苷酸个数；和/或
    所述下游特异性寡核苷酸的序列包含多个可以被特异性切割剂切割的切割位点，例如：可以是切割位点的个数大于等于n个；或者是n～N个；或者是3～9个；其中，n＝N/10，N为下游特异性寡核苷酸的核苷酸个数；
    优选地，所述上游特异性寡核苷酸的上游特异性序列的核苷酸个数为15～45；
    优选地，所述上游特异性寡核苷酸的第一通用序列的核苷酸个数为15～45；
    优选地，所述上游特异性寡核苷酸的核苷酸个数为30～90；
    优选地，所述下游特异性寡核苷酸的下游特异性序列的核苷酸个数为15～45；
    优选地，所述下游特异性寡核苷酸的第二通用序列的核苷酸个数为15～45；
    优选地，所述下游特异性寡核苷酸的核苷酸个数为30～90；
    优选地，所述多个可以被特异性切割剂切割的切割位点在所述上游特异性寡核苷酸中随机分布；
    优选地，所述多个可以被特异性切割剂切割的切割位点在所述下游特异性寡核苷酸中随机分布。
  6. 根据权利要求1所述的寡核苷酸组合，其特征在于：
    所述第一通用引物包含功能核酸序列；
    优选地，所述第一通用引物包含测序引物序列或测序引物互补序列；
    优选地，所述第一通用引物还包含第一标签序列。
  7. 根据权利要求1所述的寡核苷酸组合，其特征在于：
    所述第二通用引物包含功能核酸序列；
    优选地，所述第二通用引物包含测序引物序列或测序引物互补序列；
    优选地，所述第二通用引物还包含第二标签序列；
    优选地，所述第一标签序列、第二标签序列相同或不同。
  8. 根据权利要求1所述的寡核苷酸组合，其特征在于：
    所述第一通用序列、第二通用序列各自独立选自部分测序接头序列、全部测序接头序列、测序引物结合序列、任意固定序列(比如包含酶切位点)；
    优选地，所述第一通用序列与所述第二通用序列相同或不同。
  9. 根据权利要求1～8任一项所述的寡核苷酸组合，其特征在于：
    所述上游特异性寡核苷酸的3’端进行阻断修饰；和/或
    所述下游特异性寡核苷酸的3’端进行阻断修饰；
    优选地，所述阻断修饰包含：磷酸化修饰、间臂修饰、氨基修饰中的至少一种；
    优选地，所述上游特异性寡核苷酸和所述下游特异性寡核苷酸的阻断修饰相同或不同；
    优选地，所述特异性寡核苷酸中的上游特异性序列和下游特异性序列分别与待测目标区域的两个区段相同或互补；
    优选地，所述第一通用引物的5’端含有磷酸基团；和/或
    所述第二通用引物的5’端含有磷酸基团；
    优选地，所述特异性寡核苷酸包含多对特异性寡核苷酸；进一步包含多组靶向不同的待测目标区域的多对特异性寡核苷酸；
    优选地，所述通用引物包含多对通用引物。
  10. 一种试剂盒，包含权利要求1～9任一项所述的寡核苷酸组合；
    所述试剂盒还包含：逆转录酶和DNA聚合酶中的至少一种；
    优选地，所述试剂盒还包含：权利要求1～9任一项中的特异性切割剂；
    优选地，所述试剂盒还包含：逆转录引物。
  11. 根据权利要求10所述的试剂盒，其特征在于：
    所述试剂盒包含：核酸提取试剂组合；
    优选地，所述核酸提取试剂组合是用于选自以下任意一种方法的核酸提取试剂组合：碱裂解法、酚氯仿抽提法、螯合树脂法、离心柱膜吸附法以及磁珠法；
    优选地，所述核酸提取试剂组合包含：裂解液、洗涤液、洗脱液、核酸吸附物中的至少一种；
    优选地，所述核酸吸附物包含磁珠、吸附膜中的至少一种。
  12. 一种多重PCR扩增方法，其特征在于：利用权利要求1～9任一项所述的的寡核苷酸组合对核酸进行多重PCR反应；所述反应的步骤包括：
    通用引物延伸反应，并切割互补链，生成PCR反应引物；
    利用PCR反应引物进行扩增反应；
    优选地，所述延伸反应和扩增反应在同一体系中进行。
  13. 根据权利要求12所述的多重PCR扩增方法，其特征在于：
    所述核酸为RNA时，所述多重PCR反应前还包含：逆转录反应；
    优选地，所述逆转录反应与所述多重PCR反应在同一体系或不在同一体系中进行；
    优选地，所述同一体系具体为在同一反应体系中进行。
  14. 一种构建靶向文库的方法，包含权利要求12～13任一项所述的多重PCR扩增方法的步骤，得到测序文库。
  15. 根据权利要求14所述的方法，其特征在于：
    得到测序文库前还可以包括环化反应；
    优选地，所述方法还包括如下步骤：对测序文库进行纯化；
    优选地，所述纯化采用磁珠进行。
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