WO2024119481A1 - 一种快速制备多重pcr测序文库的方法及其应用 - Google Patents

一种快速制备多重pcr测序文库的方法及其应用 Download PDF

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WO2024119481A1
WO2024119481A1 PCT/CN2022/137939 CN2022137939W WO2024119481A1 WO 2024119481 A1 WO2024119481 A1 WO 2024119481A1 CN 2022137939 W CN2022137939 W CN 2022137939W WO 2024119481 A1 WO2024119481 A1 WO 2024119481A1
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sequence
universal
primer
specific
sequencing
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PCT/CN2022/137939
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English (en)
French (fr)
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杨林
张艳艳
刘锋
夏军
陈芳
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深圳华大智造科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids

Definitions

  • the invention belongs to the technical field of multiplex PCR amplification, and in particular relates to a method for rapidly preparing a multiplex PCR sequencing 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. Both use multiple probes or primers to capture the gene region of interest at one time, combined with high-throughput sequencing technology, multiple samples are sequenced simultaneously, and sequence information of the target region is obtained.
  • the experimental process of the former is cumbersome and the probe cost is high, which limits its clinical application.
  • the latter is simple in experimental operation and has strong flexibility. It is suitable for screening and diagnosis of Mendelian genetic diseases, resequencing of GWAS candidate segments, resequencing of QTL positioning segments, and precision medicine research and application.
  • the high-throughput SNP detection service combines multiplex PCR and high-throughput sequencing technologies to design specific primers for the sites that need to be detected, perform multiplex PCR amplification in a single tube, and distinguish different samples with different label (barcode) primers. After mixing the samples, the amplicons are sequenced on the sequencing platform. The sequencing results use bioinformatics methods to distinguish different samples and finally obtain the SNP information of each site.
  • This method is suitable for genetic research with different purposes, such as disease genome research, tumor genome research, disease and gene association research, clinical molecular diagnosis, etc. In plant genome research, it can be used for QTL positioning and molecular breeding, and is very suitable for SNP analysis of large-scale samples.
  • the multiplex PCR experiment is simple to operate and the cost of a single test is very low, it requires repeated testing and optimization of multiple pairs of primers in the early stages of the experiment, which is time-consuming and laborious.
  • the complexity of the primer sequence makes it easy for the primers to form primer dimers.
  • the formation of primer dimers will rapidly consume the raw materials in the PCR reaction system, causing the PCR to quickly reach a plateau; the formed primer dimers will also be sequenced in subsequent sequencing, forming invalid data and affecting the efficiency of data utilization.
  • primers that are prone to forming primer dimers, which will seriously affect the amplification efficiency of the target amplification region corresponding to the primer, resulting in a low sequencing depth for the target, and ultimately affecting the uniformity of the entire amplification system.
  • specificity of the primers also greatly affects the performance of multiplex amplification.
  • the first aspect of the present invention aims to provide an oligonucleic acid combination.
  • the second aspect of the present invention aims to provide a kit for preparing multiplex PCR sequencing libraries.
  • the third aspect of the present invention is to provide a sequencing reagent kit.
  • the fourth aspect of the present invention aims to provide a sequencing system.
  • the fifth aspect of the present invention aims to provide a multiplex PCR amplification method.
  • the sixth aspect of the present invention aims to provide a method for preparing a multiplex PCR sequencing library.
  • the seventh aspect of the present invention aims to provide a sequencing method.
  • the purpose of the eighth aspect of the present invention is to provide a method for detecting gene loci in a target region.
  • the purpose of the ninth aspect of the present invention is to provide an application of the oligonucleic acid combination of the first aspect, the kit of the second aspect, the sequencing reagent set of the third aspect, or the sequencing system of the fourth aspect.
  • the present invention provides an oligonucleic acid combination.
  • 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 oligonucleic acid comprises: an upstream specific oligonucleic acid and a downstream specific oligonucleic acid;
  • the specific oligonucleic acid comprises: a specific sequence and a universal sequence
  • the upstream-specific oligonucleotide comprises: an upstream-specific sequence and a first universal sequence, wherein the upstream-specific sequence is located at the 5' end of the upstream-specific oligonucleotide, and the first universal sequence is located at the 3' end of the upstream-specific oligonucleotide;
  • the downstream specific oligonucleotide comprises: a downstream specific sequence and a second universal sequence, wherein the downstream specific sequence is located at the 5' end of the downstream specific oligonucleotide, and the second universal sequence is located at the 3' end of the downstream specific oligonucleotide;
  • the 3' end sequence of the first universal primer is complementary to a portion or 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 a portion or the entire sequence of the second universal sequence of the downstream specific oligonucleotide
  • the 3' end sequence of the second universal primer is complementary to a portion 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 portion or all of the second universal sequence of the downstream specific oligonucleotide.
  • an oligonucleotide combination comprising: a specific oligonucleotide, a second primer and a universal primer;
  • the universal primers include: a first universal primer;
  • the specific oligonucleotide comprises: a specific sequence and a universal sequence, wherein the specific sequence is located at the 5' end of the specific oligonucleotide, and the universal sequence is located at the 3' end of the specific oligonucleotide;
  • the specific sequence is a first specific sequence
  • the universal sequence is a first universal sequence
  • the second primer comprises: a second specific sequence and a second universal sequence, wherein the second specific sequence is located at the 3' end of the second primer, and the second universal sequence is located at the 5' end of the second primer;
  • the 3' end sequence of the first universal primer is complementary to a part or all of the first universal sequence of the specific oligonucleotide.
  • the oligonucleotide combination may or may not include a second universal primer:
  • the second primer further comprises a functional nucleic acid sequence.
  • the second primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.
  • the second primer further comprises a third tag sequence.
  • the third tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence.
  • the universal primer further comprises: a second universal primer; the 3' end of the second universal primer is identical to a partial sequence or the entire sequence of the second universal sequence of the second primer.
  • the first specific sequence is an upstream specific sequence
  • the second specific sequence is a downstream specific sequence
  • the first specific sequence is a downstream specific sequence
  • the second specific sequence is an upstream specific sequence
  • Oligonucleotide combination for the oligonucleotide combination of the first point above (i.e. “1. Oligonucleotide combination”) and the oligonucleotide combination comprising the second universal primer of the second point (i.e. “2. Oligonucleotide combination—(2) the oligonucleotide combination comprises the second universal primer”):
  • 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 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.
  • oligonucleotide combinations for the first and second points above i.e., "one, oligonucleotide combination” and "two, oligonucleotide combination":
  • 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.
  • 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.
  • 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 universal sequence of the 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 design of the specific sequence in the specific oligonucleic acid 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 sequence of the target region to be detected or is complementary to the positive chain, and the downstream specific sequence is identical to the downstream positive chain sequence of the target region to be detected or is complementary to the negative chain sequence.
  • the design of the second specific sequence of the second primer follows conventional primer design principles and is the same as a conventional primer sequence.
  • the specific oligonucleic acid comprises multiple groups of specific oligonucleic acids targeting different target areas to be detected.
  • the second primers include multiple sets of second primers targeting different target regions to be detected.
  • the first tag sequence, the second tag sequence and the third tag sequence may be the same or different.
  • Oligonucleotide combination for the oligonucleotide combination of the first point above (i.e. “1. Oligonucleotide combination”) and the oligonucleotide combination comprising the second universal primer of the second point (i.e. “2. Oligonucleotide combination—(2) the oligonucleotide combination comprises the second universal primer”):
  • the 5' end of the first universal primer contains a phosphate group, and 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; 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 first universal primer contains a phosphate group.
  • the first universal primer can be used to make the 5' end of the amplified product carry a phosphate group, thereby avoiding the high cost of special phosphorylation.
  • the second aspect of the present invention provides a kit for preparing a multiplex PCR sequencing library, comprising: multiple sets of oligonucleotide combinations according to the first aspect of the present invention.
  • the plurality of groups are at least two groups, preferably more than two groups.
  • the kit further comprises: at least one of DNA polymerase, Mg 2+ , PCR buffer, and dNTPs.
  • the kit comprises: a nucleic acid extraction reagent, wherein the nucleic acid extraction reagent is: a lysis reagent.
  • the kit comprises a nucleic acid extraction reagent combination selected from 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.
  • a nucleic acid extraction reagent combination selected from 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 third aspect of the present invention provides a sequencing reagent kit, comprising: the oligonucleic acid combination of the first aspect of the present invention and/or the reagent kit of 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: at least one of a1) to a3) and a sequencer:
  • the fifth aspect of the present invention provides a multiplex PCR amplification method, comprising the steps of using multiple sets of oligonucleotide combinations according to the first aspect of the present invention.
  • the multiplex PCR amplification method comprises the following steps: obtaining a biological sample, and performing an amplification reaction on the biological sample using multiple sets of oligonucleic acid combinations according to the first aspect of the present invention, wherein the amplification reaction is performed in the same system.
  • the plurality of groups are at least two groups, preferably more than two groups.
  • the same system is specifically not subjected to elution and/or purification treatment.
  • the biological sample is at least one of: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, nasal swabs, and nucleic acids.
  • the multiplex PCR amplification method does not include a cleavage reaction, and only performs an amplification reaction.
  • the multiplex PCR amplification method may further include a cleavage reaction.
  • the cleavage reaction and the amplification reaction are carried out in the same system.
  • no extraction and purification step is included after the cleavage reaction and before the amplification reaction.
  • an extraction and purification step is further included after the cleavage reaction and before the amplification reaction.
  • 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 sixth aspect of the present invention provides a method for preparing a multiplex PCR sequencing 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.
  • a seventh aspect of the present invention provides a sequencing method, comprising any one of b1) to b2):
  • the sequencing method comprises the following steps: preparing a library and sequencing;
  • the method for preparing a library is the method for preparing a multiplex PCR sequencing library according to the sixth aspect of the present invention.
  • the sequencing further includes the following steps: library quality inspection.
  • the eighth aspect of the present invention provides a method for detecting a target region gene locus, comprising any one of b1) to b3):
  • the method for detecting the gene loci in the target region comprises the following steps: obtaining a sequencing library of the target region; obtaining sequencing data; determining the gene loci in the target region;
  • the method for obtaining a sequencing library of a target region comprises b1) or b2):
  • the method for detecting the target region gene loci comprises the following steps: obtaining sequencing data; determining the target region gene loci;
  • the sequencing method is the sequencing method of the seventh aspect of the present invention.
  • the method for determining the target region gene loci comprises: comparing the obtained sequencing data with a reference genome to determine the bases of the target region gene loci.
  • the ninth aspect of the present invention provides use of the oligonucleic acid combination of the first aspect, the kit of the second aspect, the sequencing reagent set of the third aspect, or the sequencing system of the fourth aspect of the present invention.
  • the invention provides an oligonucleic acid combination, comprising: a specific oligonucleic acid and a universal primer; the 3' end of the specific oligonucleic acid is set as a universal sequence, the 5' end is set as a specific sequence, and the universal sequence at the 3' end of the specific oligonucleic acid is complementary to the 3' end of the universal primer, the 3' end of the universal primer is extended under the action of a polymerase, so as to obtain a product having a universal sequence at the 5' end and a specific sequence at the 3' end, the 3' end of the product can be complementary to a target region to be detected, the target region is amplified under the action of a polymerase, and a product containing the universal sequence and the target sequence to be detected is amplified; since the 3' end is a single fixed sequence, the formation of a complementary structure and the generation of a dimer can be effectively avoided, the problem that a traditional multiple PCR primer is prone to non-specific amplification
  • FIG1 is a schematic diagram of target-specific oligonucleotide design.
  • FIG. 2 is a schematic diagram of the reaction of target-specific oligonucleotides and universal primers.
  • FIG3 is a schematic diagram of primer amplification in the library preparation method of the present invention.
  • FIG. 4 is a flow chart of the preparation of the target amplification sequencing library of Example 1.
  • FIG5 is a flow chart of the preparation of the target amplification sequencing library of 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 present invention provides an oligonucleic acid combination.
  • 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 oligonucleic acid comprises: an upstream specific oligonucleic acid and a downstream specific oligonucleic acid;
  • the specific oligonucleic acid comprises: a specific sequence and a universal sequence
  • the upstream-specific oligonucleotide comprises: an upstream-specific sequence and a first universal sequence, wherein the upstream-specific sequence is located at the 5' end of the upstream-specific oligonucleotide, and the first universal sequence is located at the 3' end of the upstream-specific oligonucleotide;
  • the downstream specific oligonucleotide comprises: a downstream specific sequence and a second universal sequence, wherein the downstream specific sequence is located at the 5' end of the downstream specific oligonucleotide, and the second universal sequence is located at the 3' end of the downstream specific oligonucleotide;
  • the 3' end sequence of the first universal primer is complementary to a portion or 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 a portion or the entire sequence of the second universal sequence of the downstream specific oligonucleotide
  • the 3' end sequence of the second universal primer is complementary to a portion 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 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 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 part or all of the second universal sequence of the downstream specific oligonucleotide.
  • an oligonucleotide combination comprising: a specific oligonucleotide, a second primer and a universal primer;
  • the universal primers include: a first universal primer;
  • the specific oligonucleotide comprises: a specific sequence and a universal sequence, wherein the specific sequence is located at the 5' end of the specific oligonucleotide, and the universal sequence is located at the 3' end of the specific oligonucleotide;
  • the specific sequence is a first specific sequence
  • the universal sequence is a first universal sequence
  • the second primer comprises: a second specific sequence and a second universal sequence, wherein the second specific sequence is located at the 3' end of the second primer, and the second universal sequence is located at the 5' end of the second primer;
  • the 3' end sequence of the first universal primer is complementary to a part or all of the first universal sequence of the specific oligonucleotide.
  • the 5' end of a conventional primer is a universal sequence and the 3' end is a specific sequence.
  • the specific sequence at the 3' end is complementary to and extended with the target template, which is prone to non-specific amplification and dimers.
  • the 3' end of the specific oligonucleotide of the present application is a universal sequence and the 5' end is a specific sequence, and the universal sequence at the 3' end of the specific oligonucleotide is complementary to the 3' end of the universal primer.
  • the 3' end of the universal primer is extended under the action of a polymerase to obtain a product having a universal sequence at the 5' end and a specific sequence at the 3' end.
  • the 3' end of the product 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 the universal sequence and the target sequence to be detected.
  • the 3' end of the specific oligonucleotide having a universal sequence at the 3' end and a specific sequence at the 5' end in the present application is a single fixed sequence, dimers are not easily formed during the PCR amplification process, thereby achieving the purpose of one-step multiplex amplification.
  • the oligonucleotide combination may or may not include a second universal primer:
  • the second primer further comprises a functional nucleic acid sequence.
  • the 5' end of the second primer further comprises a functional nucleic acid sequence, that is, the 5' end of the second universal sequence of the second primer further comprises a functional nucleic acid sequence.
  • the second primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.
  • the 5' end of the second primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.
  • the second primer comprises one or more sequencing primer sequences or sequencing primer complementary sequences; further preferably, the second primer comprises two or more sequencing primer sequences or sequencing primer complementary sequences.
  • the 5' end of the second primer contains one or more sequencing primer sequences or sequencing primer complementary sequences; further preferably, the 5' end of the second primer contains two or more sequencing primer sequences or sequencing primer complementary sequences.
  • the second primer further comprises a third tag sequence for distinguishing different samples to facilitate subsequent mixed sequencing of multiple samples, such as a barcode sequence or an index sequence.
  • a third 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 primer also contains a third tag sequence for distinguishing different samples to facilitate subsequent multi-sample mixed sequencing, such as a barcode sequence or an index sequence.
  • a third tag sequence for distinguishing different samples to facilitate subsequent multi-sample mixed sequencing, such as a barcode sequence or an index sequence.
  • the third 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 third tag sequence is 5 to 20 bp.
  • the third tag sequence is located in the middle of the sequencing primer sequence or the sequencing primer complementary sequence; further preferably, the third tag sequence is located in the middle of two segments of the sequencing primer sequence or the sequencing primer complementary sequence.
  • the universal primer further comprises: a second universal primer; the 3' end of the second universal primer is identical to a partial sequence or the entire sequence of the second universal sequence of the second primer.
  • the 3' end of the second universal primer is identical to a portion or all of the second universal sequence of the second primer.
  • the first specific sequence is an upstream specific sequence
  • the second specific sequence is a downstream specific sequence
  • the first specific sequence is a downstream specific sequence
  • the second specific sequence is an upstream specific sequence
  • Oligonucleotide combination for the oligonucleotide combination of the first point above (i.e. “1. Oligonucleotide combination”) and the oligonucleotide combination comprising the second universal primer of the second point (i.e. “2. Oligonucleotide combination—(2) the oligonucleotide combination comprises the second universal primer”):
  • the second universal primer comprises a functional nucleic acid sequence.
  • the 5' end of 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 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 for subsequent mixed sequencing of multiple samples, for example, 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 to facilitate 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.
  • oligonucleotide combinations for the first and second points above i.e., "one, oligonucleotide combination” and "two, oligonucleotide combination":
  • the first universal primer comprises a functional nucleic acid sequence.
  • the 5' end of 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 5' end of the first universal primer comprises a sequencing primer sequence or a sequencing primer complementary sequence.
  • 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 to facilitate 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 universal sequence is 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 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 universal sequence of the specific oligonucleotide is blocked with a modification to prevent extension at the 3' end.
  • the blocking modification comprises: at least one of: phosphorylation modification, spacer modification, and amino modification; further preferably, the blocking modification comprises: phosphorylation modification.
  • the design of the specific sequence in the specific oligonucleic acid 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 sequence of the target region to be detected or is reverse complementary to the positive chain, and the downstream specific sequence is identical to the downstream positive chain sequence of the target region to be detected or is reverse complementary to the negative chain sequence.
  • the design of the second specific sequence of the second primer follows conventional primer design principles and is the same as a conventional primer sequence.
  • the upstream specific sequence of the upstream specific oligonucleic acid 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 oligonucleic acid is identical to the downstream positive strand sequence of the target region to be detected or is complementary to the negative strand sequence.
  • the first specific sequence of the specific oligonucleic acid is a downstream specific sequence
  • the first specific sequence of the specific oligonucleic acid is identical to the downstream positive strand sequence of the target region to be detected or is complementary to the negative strand sequence.
  • the first specific sequence of the specific oligonucleic acid is an upstream specific sequence
  • the first specific sequence of the specific oligonucleic acid is identical to the upstream negative strand sequence of the target region to be detected or is complementary to the positive strand sequence.
  • the second specific sequence of the second primer is an upstream specific sequence
  • the second specific sequence of the second primer is complementary to the upstream negative strand sequence or identical to the positive strand sequence of the target region to be detected.
  • the second specific sequence of the second primer is a downstream specific sequence
  • the second specific sequence of the second primer is complementary to the downstream positive strand sequence of the target region to be detected or is identical to the negative strand sequence.
  • the specific oligonucleic acid comprises multiple groups of specific oligonucleic acids targeting different target areas to be detected.
  • the first tag sequence, the second tag sequence and the third tag sequence may be the same or different.
  • the third tag sequence is different from the first tag sequence.
  • the second tag sequence is different from the first tag sequence.
  • the multiple tags can be set as a tag combination in the same primer.
  • Oligonucleotide combination for the oligonucleotide combination of the first point above (i.e. “1. Oligonucleotide combination”) and the oligonucleotide combination comprising the second universal primer of the second point (i.e. “2. Oligonucleotide combination—(2) the oligonucleotide combination comprises the second universal primer”):
  • the 5' end of the first universal primer contains a phosphate group, and 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; 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 first universal primer contains a phosphate group.
  • the first universal primer can be used to make the 5' end of the amplified product carry a phosphate group, thereby avoiding the high cost of special phosphorylation.
  • the oligonucleic acid combination for detecting the EGFR gene comprises: a specific oligonucleic acid and a universal primer;
  • the specific oligonucleic acid comprises: an upstream specific oligonucleic acid and a downstream specific oligonucleic acid;
  • 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.19 to SEQ ID NO.26, and the sequences of the downstream specific oligonucleotides are shown in SEQ ID NO.27 to SEQ ID NO.34;
  • the sequence of the first universal primer is shown in SEQ ID NO.43 to 50, and the sequence of the second universal primer is shown in SEQ ID NO.18.
  • the oligonucleic acid combination for detecting the EGFR gene comprises: a specific oligonucleic acid, a second primer and a universal primer;
  • the universal primers include: a first universal primer;
  • the sequence of the second primer is shown as SEQ ID NO.1 to SEQ ID NO.8, and the sequence of the specific oligonucleotide is shown as SEQ ID NO.9 to SEQ ID NO.16;
  • the sequences of the first universal primers are shown in SEQ ID NO.43 to 50.
  • the 3' end of the specific oligonucleic acid with a sequence as shown in SEQ ID NO. 9 to 16, 19 to 34 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 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 high cost of special phosphorylation.
  • the second aspect of the present invention provides a kit for preparing a multiplex PCR sequencing library, comprising: multiple sets of oligonucleotide combinations according to the first aspect of the present invention.
  • the plurality of groups are at least two groups, preferably more than two groups.
  • the kit further comprises: at least one of DNA polymerase, Mg 2+ , PCR buffer, and dNTPs; further preferably, the kit further comprises: DNA polymerase, Mg 2+ , PCR buffer, and dNTPs;.
  • the kit comprises: a nucleic acid extraction reagent, wherein the nucleic acid extraction reagent is: a lysis reagent.
  • the kit further comprises a nucleic acid extraction reagent combination, specifically a nucleic acid extraction reagent combination for use in 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.
  • a nucleic acid extraction reagent combination specifically a nucleic acid extraction reagent combination for use in 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: the oligonucleic acid combination of the first aspect of the present invention and/or the reagent kit of 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: at least one of a1) to a3) and a sequencer:
  • the fifth aspect of the present invention provides a multiplex PCR amplification method, comprising the steps of using multiple sets of oligonucleotide combinations according to the first aspect of the present invention.
  • the multiplex PCR amplification method comprises the following steps: obtaining a biological sample, and performing an amplification reaction on the biological sample using multiple sets of oligonucleic acid combinations according to the first aspect of the present invention, wherein the amplification reaction is performed in the same system.
  • the plurality of groups are at least two groups, preferably more than two groups.
  • the same system is specifically not subjected to elution and/or purification treatment.
  • the biological sample is at least one of: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, nasal swabs, and nucleic acids; further comprises: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, nasal swabs, or nucleic acids.
  • the nucleic acid may be a nucleic acid obtained by lysing the biological sample A, wherein the biological sample A is at least one of: cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, and nasal swabs; further is: 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 multiplex PCR amplification method does not include a cleavage reaction, and only performs an amplification reaction.
  • the multiplex PCR amplification method may further include a cleavage reaction.
  • the cleavage reaction precedes the amplification reaction.
  • the cleavage reaction and the amplification reaction are carried out in the same system.
  • the same system is specifically the same reaction container.
  • an extraction and purification step is also included after the cleavage reaction and before the amplification reaction to remove impurities such as salts and organic agents.
  • the extraction and purification further comprises: eluting or dissolving nucleic acids.
  • the cleavage reaction and the amplification reaction are performed in different systems.
  • 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, proteinase K, etc.
  • the number of cycles of the amplification is 20 to 30; further 23 to 27.
  • the amplification system further comprises: at least one of: DNA polymerase, Mg 2+ , PCR buffer, and dNTPs; further preferably, the amplification system further comprises: DNA polymerase, Mg 2+ , PCR buffer, and dNTPs;
  • the sixth aspect of the present invention provides a method for preparing a multiplex PCR sequencing library, comprising the steps of the multiplex PCR amplification method of the fifth aspect of the present invention to obtain a sequencing library.
  • the method further comprises the step of: purifying the amplified product.
  • the purification is performed using magnetic beads.
  • a seventh aspect of the present invention provides a sequencing method, comprising any one of b1) to b2):
  • the sequencing method comprises the following steps: preparing a library; sequencing;
  • the method for preparing a library is the method for preparing a multiplex PCR sequencing library according to the sixth aspect of the present invention.
  • the sequencing further includes the following steps: library quality inspection.
  • the present invention provides a method for detecting a target region gene locus, comprising any one of b1) to b3):
  • the method for detecting the gene loci in the target region comprises the following steps: obtaining a sequencing library of the target region; obtaining sequencing data; determining the gene loci in the target region;
  • the method for obtaining a sequencing library of a target region comprises b1) or b2):
  • the method for detecting the target region gene loci comprises the following steps: obtaining sequencing data; determining the target region gene loci;
  • a method for detecting EGFR gene loci comprises the following steps: obtaining a sequencing library of a target region; obtaining sequencing data; determining the gene loci of the target region;
  • the method for obtaining a sequencing library of a target region comprises b1) or b2):
  • the oligonucleic acid combination is the oligonucleic acid combination for detecting the EGFR gene according to the first aspect of the present invention.
  • the method for preparing a library of EGFR gene tumor hotspot genes comprises the following steps:
  • Example 2 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:
  • the panel includes 8 pairs of specific oligonucleotide sequences, and the amplicon size is 100 to 200 bp.
  • the design sequence of the specific oligonucleotide sequence is shown in Table 3; the universal primer includes universal primer F and universal primer R.
  • the nucleotide sequence of universal primer R is: (The double underlined part is SEQ ID NO.17, the bold part is SEQ ID NO.51, NNNNNNNNN is a tag sequence, which can be random or set in advance, and N is independently selected from A, T, C, and G); (the universal primer R is specifically shown in Table 13), the underlined part is complementary to the 3' end sequence of the upstream specific oligonucleotide (universal sequence); the nucleotide sequence of the universal primer F is: P-GAAC G ACATGGCTACGATCCGACTT (SEQ ID NO.18), P is phosphorylation modification, and the underlined part is complementary to the 3' end sequence of the downstream specific oligonucleotide.
  • a PCR tube configure the PCR system according to the reaction system shown in Table 4 (replace the specific oligonucleotide/primer pool (2 ⁇ M) shown in Table 1 with the specific oligonucleotide pool shown in Table 3), and carry out PCR reaction.
  • the reaction procedure is 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 ⁇ .
  • purify with 1.5X AMPure (Beckman Co.) magnetic beads and finally dissolve the purified product in 22 ⁇ L elution buffer to obtain the library.
  • the above-mentioned specific oligonucleotides were mixed at a concentration of 2 ⁇ M for each specific oligonucleotide to obtain a specific oligonucleotide pool with a total concentration of 2 ⁇ M;
  • the upstream specific oligonucleotide sequence was designed by the present invention, that is, the 5' to 3' ends were sequentially: a specific sequence and a universal sequence, wherein the underlined portion was complementary to the 3' end of the universal primer R;
  • the downstream specific primer sequence was designed by the present invention, that is, the 5' to 3' ends were sequentially: a specific sequence and a universal sequence, wherein the underlined portion was complementary to the 3' end of the universal primer F.
  • Example 3 Method for preparing a library of EGFR tumor hotspot genes without extracting blood samples
  • the method for preparing a library of EGFR gene tumor hotspot genes comprises the following steps:
  • the panel includes 8 pairs of specific oligonucleotide sequences, and the amplicon size is 100 to 200 bp.
  • the design sequence of the specific oligonucleotide sequence is shown in Table 3; the universal primer includes universal primer F and universal primer R.
  • the nucleotide sequence of universal primer R is: (The double underlined part is SEQ ID NO.17, the bold part is SEQ ID NO.51, NNNNNNNNN is the tag sequence, which can be random or set in advance, and N is independently selected from A, T, C, and G); (the universal primer R is specifically shown in Table 13), the underlined part is complementary to the 3' end sequence of the upstream specific oligonucleotide (universal sequence); the nucleotide sequence of the universal primer F is: P-GAAC GA CATGGCTACGATCCGACTT (SEQ ID NO.18), P is phosphorylation modification, and the underlined part is complementary to the 3' end sequence of the downstream specific oligonucleotide.
  • a PCR tube configure the PCR system according to the reaction system shown in Table 4 (replace the specific oligonucleotide/primer pool (2 ⁇ M) shown in Table 1 with the specific oligonucleotide pool shown in Table 3), and carry out PCR reaction.
  • the reaction procedure is 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 ⁇ .
  • purify with 1.5X AMPure (Beckman Co.) magnetic beads and finally dissolve the purified product in 22 ⁇ L elution buffer to obtain the library.
  • the method for preparing a library of EGFR gene tumor hotspot genes comprises the following steps:
  • the panel includes 8 pairs of primers, and the amplicon size is 100 to 200 bp.
  • the primer design sequences are shown in Table 5.
  • the universal primers include universal primer F and universal primer R.
  • the nucleotide sequence of universal primer R is: (The double underline is SEQ ID NO.17, the bold part is SEQ ID NO.51, NNNNNNNNN is a tag sequence, which can be random or set in advance, and N is independently selected from A, T, C, and G); (the universal primer R is specifically shown in Table 13), the underlined part is the same as the 5' end sequence of the conventional downstream specific primer (universal sequence); the nucleotide sequence of the universal primer F is: P- GAACGACATGGCTACGATCCGA CTT (SEQ ID NO.18), P is phosphorylation modification, and the underlined part is the same as the 5' end sequence of the conventional upstream specific amplification primer.
  • the PCR system was configured in a PCR tube according to the reaction system shown in Table 7, 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 ⁇ .
  • 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 upstream specific primer sequence was conventionally designed, that is, the 5' to 3' end was: universal sequence and specific sequence, of which the underlined part was the same as the 3' end of universal primer F
  • the downstream specific primer sequence was conventionally designed, that is, the 5' to 3' end was: universal sequence and specific sequence, of which the underlined part was the same as the 3' end of universal primer R.
  • the following effect examples are based on the MGISEQ-2000 platform of the sequencer of BGI; the reagents used are all 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, dimer ratio, alignment ratio, target region data ratio, uniformity 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:
  • Library preparation the library preparation method of Example 1, 2, 3 or Comparative Example 1;
  • the sequencing data statistics of the library obtained in Example 1 and Comparative Example 1 are shown in Table 8, and the mutation detection statistics of Example 1 are shown in Table 9: the unique alignment ratio and the target region data ratio in the sequencing data of the library obtained in Example 1 and Comparative Example 1 are equivalent, and the data utilization rate in Example 1 is higher than that in Comparative Example 1, the dimer ratio is lower than that in Comparative Example 1, and the operation step of the method in Example 1 is only one step; the detection value of the mutation frequency in Example 1 is equivalent to the theoretical value.
  • the sequencing data statistics of the libraries obtained in Example 2 and Comparative Example 1 are shown in Table 10, and the mutation detection statistics are shown in Table 11: the unique alignment ratio and the target region data ratio in the sequencing data of the libraries obtained in Example 2 and Comparative Example 1 are comparable, and the data utilization rate in Example 2 is higher than that in Comparative Example 1, and the dimer ratio is lower than that in Comparative Example 1, and the method of Example 2 has only one operation step; the detection value of the mutation frequency in Example 2 is comparable to the theoretical value.
  • the sequencing data statistics of the libraries obtained in Example 3 and Comparative Example 1 are shown in Table 12: the unique alignment ratio and target region data ratio in the sequencing data of the libraries obtained in Example 3 and Comparative Example 1 are comparable, and the data utilization rate in Example 3 is higher than that in Comparative Example 1, and the dimer ratio is lower than that in Comparative Example 1, and the method of Example 3 has only one operation step; at the same time, the unique alignment ratio, target region data ratio, data utilization rate, and dimer ratio in the sequencing data of the libraries obtained in Example 3 are comparable to those in Example 2, which shows that performing the cleavage reaction and the amplification reaction in the same system does not affect its performance.

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Abstract

一种快速制备多重PCR测序文库的方法及其应用。该寡核酸组合，包含：特异性寡核酸和通用引物；通过将特异性寡核酸的3'端定为通用序列，5'端定为特异性序列，并且位于特异性寡核酸的3端的通用序列与通用引物的3'端互补，通用引物的3'端在聚合酶的作用下的延伸，得到5'端含有通用序列，3'端含有特异性序列的产物，该产物3'端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，扩增得到含有通用序列和待测目标序列的产物；由于3'端是单一的固定序列，能够有效避免互补结构的形成和二聚体的产生。

Description

一种快速制备多重PCR测序文库的方法及其应用 技术领域
本发明属于多重PCR扩增技术领域，具体涉及一种快速制备多重PCR测序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。相对于全基因组测序，目标捕获测序技术在大样本量筛查的同时极大地降低成本。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。两者通过多重探针或者引物对感兴趣的基因区域一次性捕获，结合高通量测序技术，多样本同时测序，得到目标区域的序列信息。但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，适用于孟德尔遗传性疾病的筛查和诊断、GWAS候选区段重测序、QTL定位区段重测序、精准医疗研究与应用。
高通量SNP检测服务结合多重PCR和高通量测序技术，对需要检测的位点设计特异性引物，在单管内进行多重PCR扩增，不同的样本以不同的标签(barcode)引物区分。混合样本后，在测序平台上，对扩增子进行测序，测序结果使用生物信息学方法，区分不同样本，最终获得每个位点的SNP信息。该方法适用于不同目的遗传学研究，例如疾病基因组研究、肿瘤基因组研究、疾病与基因的关联研究、临床分子诊断等，在植物基因组研究中，可用于QTL定位及分子育种，非常适合大规模样本的SNP分析。
虽然多重PCR实验操作简单、单个检测成本很低，但其在实验前期需要对多对引物进行反复测试优化，费时费力。特别是在超高重的PCR中，引物序列的复杂性使得引物很容易形成引物二聚体。引物二聚体的形成会急剧消耗PCR反应体系中的原料，导致PCR很快达到平台期；形成的引物二聚体在后续的测序中也会被测序，形成无效数据，影响数据的利用效率。最严重的是那些容易形成引物二聚体的引物，会严重影响该引物对应的目标扩增区域的扩增效率，导致该目标测序深度低，最终影响整个扩增体系的均一性，此外，引物的特异性也极大地影响了多重扩增的性能。
在多重扩增中随着引物对数的增加，引物3'端多样性增加，提高了引物之间形成互补结构的可能性，在聚合酶的作用下形成二聚体。很多公司通过额外的酶处理对过多的引物二聚 体进行消除，如Life公司的Ampliseq技术，先采用一步特异性扩增，然后通过酶消化掉引物二聚体和扩增子中的特异性引物序列，最后对产物进行文库制备，整个过程繁琐。后来Paragon公司发明了Clean Plex二聚体消除技术，采用特定的酶只消除多重PCR过程中的引物二聚体，同样解决了引物二聚体生成的问题。得到的产物再采用通用引物进行通用扩增，完成文库的制备，但是整个过程操作步骤还是过多。
发明内容
本发明第一方面的目的，在于提供寡核酸组合。
本发明第二方面的目的，在于提供一种用于多重PCR测序文库制备的试剂盒。
本发明第三方面的目的，在于提供一种测序试剂套装。
本发明第四方面的目的，在于提供一种测序系统。
本发明第五方面的目的，在于提供一种多重PCR扩增方法。
本发明第六方面的目的，在于提供一种制备多重PCR测序文库的方法。
本发明第七方面的目的，在于提供一种测序方法。
本发明第八方面的目的，在于提供一种对目标区域基因位点检测的方法。
本发明第九方面的目的，在于提供第一方面的寡核酸组合、第二方面的试剂盒、第三方面的测序试剂套装或第四方面的测序系统的应用。
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，提供寡核酸组合。
一、寡核酸组合，包含：特异性寡核酸和通用引物；
所述通用引物包含：第一通用引物和第二通用引物；
所述特异性寡核酸包含：上游特异性寡核酸和下游特异性寡核酸；
所述特异性寡核酸包含：特异性序列和通用序列；
所述上游特异性寡核酸包含：上游特异性序列和第一通用序列，其中，所述上游特异性序列位于所述上游特异性寡核酸的5'端，所述第一通用序列位于所述上游特异性寡核酸的3'端；
所述下游特异性寡核酸包含：下游特异性序列和第二通用序列，其中，所述下游特异性序列位于所述下游特异性寡核酸的5'端，所述第二通用序列位于所述下游特异性寡核酸的3'端；
1)所述第一通用引物的3'端序列与所述上游特异性寡核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酸的第二通用序列的部分序列或全部序列互补。
二、寡核酸组合，包含：特异性寡核酸、第二引物和通用引物；
所述通用引物包含：第一通用引物；
所述特异性寡核酸包含：特异性序列和通用序列，其中，所述特异性序列位于所述特异性寡核酸的5'端，所述通用序列位于所述特异性寡核酸的3'端；
所述特异性序列为第一特异性序列，所述通用序列为第一通用序列；
所述第二引物包含：第二特异性序列和第二通用序列，其中，所述第二特异性序列位于所述第二引物的3'端，所述第二通用序列位于所述第二引物的5'端；
所述第一通用引物的3'端序列与所述特异性寡核酸的第一通用序列的部分序列或全部序列互补。
针对上述第二点的寡核酸组合(即“二、寡核酸组合”)，所述寡核酸组合包含或不包含第二通用引物：
(一)所述寡核酸组合不包含第二通用引物时：
优选地，所述第二引物还包含功能核酸序列。
优选地，所述第二引物包含测序引物序列或测序引物互补序列。
优选地，所述第二引物还包含第三标签序列。
优选地，所述第三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。
(二)所述寡核酸组合包含第二通用引物时：
优选地，所述通用引物进一步包含：第二通用引物；所述第二通用引物的3'端与所述第二引物的第二通用序列的部分序列或全部序列相同。
本发明的一个实施例，所述第一特异性序列为上游特异性序列，所述第二特异性序列为下游特异性序列；
本发明的另一个实施例，所述第一特异性序列为下游特异性序列，所述第二特异性序列为上游特异性序列。
针对上述第一点的寡核酸组合(即“一、寡核酸组合”)和第二点中包含第二通用引物的寡核酸组合(即“二、寡核酸组合--(二)所述寡核酸组合包含第二通用引物”)：
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。
针对上述第一点和第二点的寡核酸组合(即“一、寡核酸组合”和“二、寡核酸组合”)：
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。
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。
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，所述特异性寡核酸的通用序列的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，所述特异性寡核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序列相同或正链互补，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，所述第二引物的第二特异性序列的设计遵循常规的引物设计原则，与常规的引物序列相同。
优选地，所述特异性寡核酸包含多组靶向不同的待测目标区域的多条特异性寡核酸。
优选地，所述第二引物包含多组靶向不同的待测目标区域的多条第二引物。
优选地，所述第一标签序列、第二标签序列和第三标签序列可以是相同也可以是不同。
针对上述第一点的寡核酸组合(即“一、寡核酸组合”)和第二点中包含第二通用引物的寡核酸组合(即“二、寡核酸组合--(二)所述寡核酸组合包含第二通用引物”)：
优选地，1)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；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。
针对上述第二点中不包含第二通用引物的寡核酸组合(即“二、寡核酸组合--(一)所述寡核酸组合不包含第二通用引物”)：
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。
本发明的第二个方面，提供用于多重PCR测序文库制备的试剂盒，包含：本发明第一个方面的多组寡核酸组合。
其中，所述多组至少为两组，优选两组以上。
优选地，所述试剂盒还包含：DNA聚合酶、Mg 2+、PCR缓冲液、dNTPs中的至少一种。
优选地，所述试剂盒包含：核酸提取试剂，其中所述核酸提取试剂为：裂解试剂。
优选地，所述试剂盒包含核酸提取试剂组合，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。
本发明的第三个方面，提供一种测序试剂套装，包含：采用本发明第一个方面的寡核酸组合和/或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：a1)至a3)中至少一种和测序仪：
a1)本发明第一个方面的寡核酸组合；
a2)本发明第二个方面的试剂盒；
a3)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多组寡核酸组合的步骤。
优选地，所述多重PCR扩增方法包括如下步骤：获得生物样本，利用本发明第一个方面的多组寡核酸组合对生物样本进行扩增反应，其中所述扩增反应在同一体系中进行。
其中，所述多组至少为两组，优选两组以上。
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，所述生物样本是：细胞、组织、体液、微生物、唾液、尿液、痰液、粪便、咽拭子、鼻拭子、核酸中的至少一种。
优选地，所述生物样本为核酸时，所述多重PCR扩增方法不包含裂解反应，仅进行扩增反应。
优选地，所述生物样本是：细胞、组织、体液、微生物、唾液、尿液、痰液、粪便、咽拭子、鼻拭子中的至少一种时，所述多重PCR扩增方法还可以包含裂解反应。
优选地，所述裂解反应和所述扩增反应在同一体系中进行。
优选地，所述裂解反应后、扩增反应前不包含提取纯化步骤。
优选地，所述裂解反应后、扩增反应前还包含提取纯化步骤。
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。
本发明的第六个方面，提供一种制备多重PCR测序文库的方法，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b1)至b2)中任一种：
b1)本发明第五个方面的多重PCR扩增方法的步骤；
b2)本发明第六个方面的制备多重PCR测序文库的方法的步骤。
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制备多重PCR测序文库的方法。
优选地，所述测序前还包括如下步骤：文库质检。
本发明的第八个方面，提供一种对目标区域基因位点检测的方法，包含b1)至b3)中任一种：
b1)本发明第五个方面的多重PCR扩增方法的步骤；
b2)本发明第六个方面的制备多重PCR测序文库的方法的步骤；
b3)本发明第七个方面的测序方法。
优选地，所述对目标区域基因位点检测的方法包括如下步骤：获得目标区域的测序文库；获得测序数据；确定目标区域基因位点；
所述获得目标区域的测序文库的方法包含b1)或b2)：
b1)本发明第五个方面的多重PCR扩增方法的步骤；
b2)本发明第六个方面的制备多重PCR测序文库的方法的步骤。
优选地，所述对目标区域基因位点检测的方法包括如下步骤：获得测序数据；确定目标区域基因位点；
所述测序的方法为本发明第七个方面的测序方法。
优选地，所述确定目标区域基因位点的方法包含：将获得的测序数据与参考基因组进行比对，确定目标区域基因位点的碱基。
本发明第九个方面，提供本发明第一个方面的寡核酸组合、第二个方面的试剂盒、第三个方面的测序试剂套装或第四个方面的测序系统的应用。
本发明第一个方面的寡核酸组合和/或本发明第二个方面的试剂盒在c1)至c8)任一项中的应用；
c1)制备多重PCR测序文库；
c2)制备用于多重PCR测序文库制备的产品；
c3)测序；
c4)制备用于测序的产品；
c5)对目标区域基因位点检测；
c6)制备对目标区域基因位点检测的产品；
c7)多重PCR扩增；
c8)制备多重PCR扩增的产品。
本发明第三个方面的测序试剂套装和/或本发明第四个方面的测序系统在c3)至c6)任一项中的应用；
c3)测序；
c4)制备用于测序的产品；
c5)对目标区域基因位点检测；
c6)制备对目标区域基因位点检测的产品。
本发明的有益效果是：
本发明提供了一种寡核酸组合，包含：特异性寡核酸和通用引物；通过将特异性寡核酸的3'端定为通用序列，5'端定为特异性序列，并且位于特异性寡核酸的3'端的通用序列与通用引物的3'端互补，通用引物的3'端在聚合酶的作用下的延伸，得到5'端含有通用序列，3'端含有特异性序列的产物，该产物3'端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，扩增得到含有通用序列和待测目标序列的产物；由于3'端是单一的固定序列，能够有效避免互补结构的形成和二聚体的产生，解决了传统的多重PCR引物容易产生非特异性扩增和二聚体的问题，同时，能够实现一管中快速完成整个扩增和建库的过程；产物可直接进行后续的测序、克隆等应用。
附图说明
图1是目标特异性寡核酸设计示意图。
图2是目标特异性寡核酸和通用引物反应示意图。
图3是本发明文库制备方法的引物扩增示意图。
图4是实施例1的目标扩增测序文库的制备流程图。
图5是实施例2的目标扩增测序文库的制备流程图。
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，提供寡核酸组合。
一、寡核酸组合，包含：特异性寡核酸和通用引物；
所述通用引物包含：第一通用引物和第二通用引物；
所述特异性寡核酸包含：上游特异性寡核酸和下游特异性寡核酸；
所述特异性寡核酸包含：特异性序列和通用序列；
所述上游特异性寡核酸包含：上游特异性序列和第一通用序列，其中，所述上游特异性序列位于所述上游特异性寡核酸的5'端，所述第一通用序列位于所述上游特异性寡核酸的3'端；
所述下游特异性寡核酸包含：下游特异性序列和第二通用序列，其中，所述下游特异性序列位于所述下游特异性寡核酸的5'端，所述第二通用序列位于所述下游特异性寡核酸的3'端；
1)所述第一通用引物的3'端序列与所述上游特异性寡核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酸的第二通用序列的部分序列或全部序列互补。
二、寡核酸组合，包含：特异性寡核酸、第二引物和通用引物；
所述通用引物包含：第一通用引物；
所述特异性寡核酸包含：特异性序列和通用序列，其中，所述特异性序列位于所述特异性寡核酸的5'端，所述通用序列位于所述特异性寡核酸的3'端；
所述特异性序列为第一特异性序列，所述通用序列为第一通用序列；
所述第二引物包含：第二特异性序列和第二通用序列，其中，所述第二特异性序列位于所述第二引物的3'端，所述第二通用序列位于所述第二引物的5'端；
所述第一通用引物的3'端序列与所述特异性寡核酸的第一通用序列的部分序列或全部序列互补。
在制备多重PCR测序文库过程中，传统的引物(比如：第二引物)的5'端为通用序列，3'端为特异性序列，3'端的特异性序列与目标模板互补并延伸，其容易产生非特异性扩增和二聚体；而本申请的特异性寡核酸的3'端为通用序列，5'端为特异性序列，并且位于特异性寡核酸的3'端的通用序列与通用引物的3'端互补，通用引物的3'端在聚合酶的作用下的延伸，得到5'端含有通用序列，3'端含有特异性序列的产物，该产物3'端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，扩增得到含有通用序列和待测目标序列的产物；并且由于本申请中的3'端为通用序列，5'端为特异性序列的特异性寡核酸的3'端为单一固定序列，在PCR扩增过程中不易形成二聚体，从而实现一步法多重扩增的目的。
针对上述第二点的寡核酸组合(即“二、寡核酸组合”)，所述寡核酸组合包含或不包含第二通用引物：
(一)所述寡核酸组合不包含第二通用引物时：
优选地，所述第二引物还包含功能核酸序列。
优选地，所述第二引物的5'端还包含功能核酸序列，即在第二引物的第二通用序列的5'端还包含功能核酸序列。
优选地，所述第二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，所述第二引物还包含第三标签序列，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，所述第二引物的5'端还包含第三标签序列，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，所述第三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，所述第三标签序列的长度为5至20bp。
优选地，所述第三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，所述第三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。
(二)所述寡核酸组合包含第二通用引物时：
优选地，所述通用引物进一步包含：第二通用引物；所述第二通用引物的3'端与所述第二引物的第二通用序列的部分序列或全部序列相同。
优选地，所述第二通用引物的3'端与所述第二引物的第二通用序列的部分序列或全部序列相同。
本发明的一个实施例，所述第一特异性序列为上游特异性序列，所述第二特异性序列为下游特异性序列；
本发明的另一个实施例，所述第一特异性序列为下游特异性序列，所述第二特异性序列为上游特异性序列。
针对上述第一点的寡核酸组合(即“一、寡核酸组合”)和第二点中包含第二通用引物的寡核酸组合(即“二、寡核酸组合--(二)所述寡核酸组合包含第二通用引物”)：
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至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。
针对上述第一点和第二点的寡核酸组合(即“一、寡核酸组合”和“二、寡核酸组合”)：
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至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，所述通用序列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、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，所述特异性寡核酸的通用序列的3'端进行阻断修饰，从而防止在3'端进行延伸。
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，所述特异性寡核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序列相同或正链反向互补，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反向互补。
优选地，所述第二引物的第二特异性序列的设计遵循常规的引物设计原则，与常规的引物序列相同。
优选地，所述上游特异性寡核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，所述特异性寡核酸的第一特异性序列为下游特异性序列时，所述特异性寡核酸的第一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，所述特异性寡核酸的第一特异性序列为上游特异性序列时，所述特异性寡核酸的第一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，所述第二引物的第二特异性序列为上游特异性序列时，所述第二引物的第二特异性序列与待测目标区域上游负链序列互补或正链序列相同。
优选地，所述第二引物的第二特异性序列为下游特异性序列时，所述第二引物的第二特异性序列与待测目标区域下游正链序列互补或负链序列相同。
优选地，所述特异性寡核酸包含多组靶向不同的待测目标区域的多条特异性寡核酸。
优选地，所述第二引物包含多组靶向不同的待测目标区域的多条第二引物。
优选地，所述第一标签序列、第二标签序列和第三标签序列可以是相同也可以是不同。
优选地，所述第三标签序列与第一标签序列不同。
优选地，所述第二标签序列与第一标签序列不同。
优选的，所述多个标签可以做为标签组合设置于同一条引物中。
针对上述第一点的寡核酸组合(即“一、寡核酸组合”)和第二点中包含第二通用引物的寡核酸组合(即“二、寡核酸组合--(二)所述寡核酸组合包含第二通用引物”)：
优选地，1)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；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。
针对上述第二点中不包含第二通用引物的寡核酸组合：
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。
优选地，检测EGFR基因的寡核酸组合，包含：特异性寡核酸和通用引物；
所述特异性寡核酸包含：上游特异性寡核酸和下游特异性寡核酸；
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酸的序列如SEQ ID NO.19至SEQ ID NO.26所示，所述下游特异性寡核酸的序列如SEQ ID NO.27至SEQ ID NO.34所示；
所述第一通用引物的序列如SEQ ID NO.43至50所示，所述第二通用引物的序列如SEQ ID NO.18所示。
优选地，检测EGFR基因的寡核酸组合，包含：特异性寡核酸、第二引物和通用引物；
所述通用引物包含：第一通用引物；
所述第二引物的序列如SEQ ID NO.1至SEQ ID NO.8所示，所述特异性寡核酸的序列如SEQ ID NO.9至SEQ ID NO.16所示；
所述第一通用引物的序列如SEQ ID NO.43至50所示。
优选地，序列如SEQ ID NO.9至16、19至34所示的特异性寡核酸的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，提供用于多重PCR测序文库制备的试剂盒，包含：本发明第一个方面的多组寡核酸组合。
其中，所述多组至少为两组，优选两组以上。
优选地，所述试剂盒还包含：DNA聚合酶、Mg 2+、PCR缓冲液、dNTPs中的至少一种；进一步优选地，所述试剂盒还包含：DNA聚合酶、Mg 2+、PCR缓冲液和dNTPs；。
优选地，所述试剂盒包含：核酸提取试剂，其中所述核酸提取试剂为：裂解试剂。
优选地，所述试剂盒还包含核酸提取试剂组合，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：采用本发明第一个方面的寡核酸组合和/或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：a1)至a3)中至少一种和测序仪：
a1)本发明第一个方面的寡核酸组合；
a2)本发明第二个方面的试剂盒；
a3)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多组寡核酸组合的步骤。
优选地，所述多重PCR扩增方法包括如下步骤：获得生物样本，利用本发明第一个方面的多组寡核酸组合对生物样本进行扩增反应，其中所述扩增反应在同一体系中进行。
其中，所述多组至少为两组，优选两组以上。
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，所述生物样本是：细胞、组织、体液、微生物、唾液、尿液、痰液、粪便、咽拭子、鼻拭子、核酸中的至少一种；进一步是：细胞、组织、体液、微生物、唾液、尿液、痰液、粪便、咽拭子、鼻拭子或核酸。
优选地，所述核酸可以是通过裂解所述生物样本A得到的核酸，所述生物样本A是：细胞、组织、体液、微生物、唾液、尿液、痰液、粪便、咽拭子、鼻拭子中的至少一种；进一步是：细胞、组织、体液、微生物、唾液、尿液、痰液、粪便、咽拭子或鼻拭子。
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，所述生物样本为核酸时，所述多重PCR扩增方法不包含裂解反应，仅进行扩增反应。
优选地，所述生物样本是：细胞、组织、体液、微生物、唾液、尿液、痰液、粪便、咽拭子、鼻拭子中的至少一种时，所述多重PCR扩增方法还可以包含裂解反应。
优选地，所述裂解反应在所述扩增反应之前。
优选地，所述裂解反应和所述扩增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扩增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扩增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扩增反应在不同体系中进行。
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，所述扩增的循环数为20至30；进一步为23至27。
优选地，所述扩增的体系还包含：DNA聚合酶、Mg 2+、PCR缓冲液、dNTPs中的至少一种；进一步优选地，所述扩增的体系还包含：DNA聚合酶、Mg 2+、PCR缓冲液和dNTPs；
本发明的第六个方面，提供一种制备多重PCR测序文库的方法，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，所述方法还包括如下步骤：对扩增产物进行纯化。
优选地，所述纯化采用磁珠进行。
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。
本发明的第七个方面，提供一种测序方法，包含b1)至b2)中任一种：
b1)本发明第五个方面的多重PCR扩增方法的步骤；
b2)本发明第六个方面的制备多重PCR测序文库的方法的步骤。
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制备多重PCR测序文库的方法。
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，提供一种对目标区域基因位点检测的方法，包含b1)至b3)中任一种：
b1)本发明第五个方面的多重PCR扩增方法的步骤；
b2)本发明第六个方面的制备多重PCR测序文库的方法的步骤；
b3)本发明第七个方面的测序方法。
优选地，所述对目标区域基因位点检测的方法包括如下步骤：获得目标区域的测序文库；获得测序数据；确定目标区域基因位点；
所述获得目标区域的测序文库的方法包含b1)或b2)：
b1)本发明第五个方面的多重PCR扩增方法的步骤；
b2)本发明第六个方面的制备多重PCR测序文库的方法的步骤。
优选地，所述对目标区域基因位点检测的方法包括如下步骤：获得测序数据；确定目标区域基因位点；
所述测序的方法为本发明第七个方面的测序方法。
优选地，所述确定目标区域基因位点的方法包含：将获得的测序数据与参考基因组进行比对，确定目标区域基因位点的碱基。
一种EGFR基因位点的检测方法，包括如下步骤：获得目标区域的测序文库；获得测序数据；确定目标区域基因位点；
所述获得目标区域的测序文库的方法包含b1)或b2)：
b1)本发明第五个方面的多重PCR扩增方法的步骤；
b2)本发明第六个方面的制备多重PCR测序文库的方法的步骤；
所述寡核酸组合为本发明第一个方面的检测EGFR基因的寡核酸组合。
优选地，所述对目标区域基因位点检测的方法包括如下步骤：获得测序数据；确定目标区域基因位点；
所述测序的方法为本发明第七个方面的测序方法；
所述寡核酸组合为本发明第一个方面的检测EGFR基因的寡核酸组合。
优选地，所述确定目标区域基因位点的方法包含：将获得的测序数据与参考基因组进行比对，确定目标区域基因位点的碱基。
本发明第九个方面，提供本发明第一个方面的寡核酸组合、第二个方面的试剂盒、第三个方面的测序试剂套装或第四个方面的测序系统的应用。
本发明第一个方面的寡核酸组合和/或本发明第二个方面的试剂盒在c1)至c8)任一项中 的应用；
c1)制备多重PCR测序文库；
c2)制备用于多重PCR测序文库制备的产品；
c3)测序；
c4)制备用于测序的产品；
c5)对目标区域基因位点检测；
c6)制备对目标区域基因位点检测的产品；
c7)多重PCR扩增；
c8)制备多重PCR扩增的产品。
本发明第三个方面的测序试剂套装和/或本发明第四个方面的测序系统在c3)至c6)任一项中的应用；
c3)测序；
c4)制备用于测序的产品；
c5)对目标区域基因位点检测；
c6)制备对目标区域基因位点检测的产品。
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，示意图如图4所示，包括如下步骤：
(1)设计EGFR基因肿瘤热点基因捕获panel以及通用引物，该panel包含8对特异性寡核酸(EX1-EGFR_1R至EX1-EGFR_8R)/引物(EX1-EGFR_1F至EX1-EGFR_8F)，扩增子大小为100至200bp，特异性寡核酸/引物序列如表1所示；通用引物包含通用引物R，通用引物R的核苷酸序列为：
Figure PCTCN2022137939-appb-000001
Figure PCTCN2022137939-appb-000002
(双下划线部分为SEQ ID NO.17，加粗部分为SEQ ID NO.51，NNNNNNNNNNN为标签序列，可以是随机的，也可以是提前设定的，N独立选自A、T、C、G)；(通用引物R具体如表13所示)，下划线部分和特异性寡核酸3'端序列(通用序列)互补。
(2)PCR反应
在PCR管中按照表2所示反应体系配置PCR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1.5X 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特异性寡核酸/引物序列
Figure PCTCN2022137939-appb-000003
注：上述特异性寡核酸/引物按照每条特异性寡核酸/引物2μM浓度进行混合，得到总浓度为2μM的特异性寡核酸/引物池；引物序列为常规设计，即5'到3'端依次为：通用序列和特异性序列；特异性寡核酸序列为本发明设计，即5'到3'端依次为：特异性序列和通用序列，下划线部分和与通用引物R的3'端互补的序列。
表2 PCR反应体系
Figure PCTCN2022137939-appb-000004
实施例2 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，示意图如图5所示，包括如下步骤：
(1)设计EGFR基因肿瘤热点基因捕获panel以及通用引物，该panel包含8对特异性寡核酸序列，扩增子大小为100至200bp，特异性寡核酸序列设计序列如表3所示；通用引物包含通用引物F和通用引物R，通用引物R的核苷酸序列为：
Figure PCTCN2022137939-appb-000005
Figure PCTCN2022137939-appb-000006
(双下划线部分为SEQ ID NO.17，加粗部分为SEQ ID NO.51，NNNNNNNNNNN为标签序列，可以是随机的，也可以是提前设定的，N独立选自A、T、C、G)；(通用引物R具体如表13所示)，下划线部分和上游特异性寡核酸3'端序列(通用序列)互补；通用引物F的核苷酸序列为：P-GAAC G ACATGGCTACGATCCGACTT(SEQ ID NO.18)，P为磷酸化修饰，下划线部分和下游特异性寡核酸3'端序列互补。
(2)PCR反应
在PCR管中按照表4所示反应体系配置PCR体系(将表1所示特异性寡核酸/引物池(2μM)替换为表3所示特异性寡核酸池)，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1.5X 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3特异性寡核酸序列
Figure PCTCN2022137939-appb-000007
Figure PCTCN2022137939-appb-000008
注：上述特异性寡核酸按照每条特异性寡核酸2μM浓度进行混合，得到总浓度为2μM的特异性寡核酸池；上游特异性寡核酸序列为本发明设计，即5'到3'端依次为：特异性序列和通用序列，其中下划线部分和通用引物R的3'端互补；下游特异性引物序列为本发明设计，即5'到3'端依次为：特异性序列和通用序列，其中下划线部分与通用引物F的3'端互补。
表4 PCR反应体系
Figure PCTCN2022137939-appb-000009
实施例3免提取血液样本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，示意图如图5所示，包括如下步骤：
(1)设计EGFR基因肿瘤热点基因捕获panel以及通用引物，该panel包含8对特异性寡核酸序列，扩增子大小为100至200bp，特异性寡核酸序列设计序列如表3所示；通用引物包含通用引物F和通用引物R，通用引物R的核苷酸序列为：
Figure PCTCN2022137939-appb-000010
Figure PCTCN2022137939-appb-000011
(双下划线为SEQ ID NO.17，加粗部分为SEQ ID NO.51，NNNNNNNNNNN为标签序列，可以是随机的，也可以是提前设定的，N独立选自A、T、C、G)；(通用引物R具体如表13所示)，下划线部分和上游特异性寡核酸3'端序列(通用序列)互补；通用引物F的核苷酸序列为：P-GAAC GA CATGGCTACGATCCGACTT(SEQ ID NO.18)，P为磷酸化修饰，下划线部分和下游特异性寡核酸3'端序列互补。
(2)血液样本裂解
1μL血液样本加入25uL含有TE的溶液中，95℃裂解10分钟，10000g离心5分钟，取20μL上清液用于后续PCR反应。
在PCR管中按照表4所示反应体系配置PCR体系(将表1所示特异性寡核酸/引物池(2μM)替换为表3所示特异性寡核酸池)，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1.5X 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。
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以及通用引物，该panel包含8对引物，扩增子大小为100至200bp，引物设计序列如表5所示；通用引物包含通用引物F和通用引物R，通用引物R的核苷酸序列为：
Figure PCTCN2022137939-appb-000012
Figure PCTCN2022137939-appb-000013
(双下划线为SEQ ID NO.17，加粗部分为SEQ ID NO.51，NNNNNNNNNNN为标签序列，可以是随机的，也可以是提前设定的，N独立选自A、T、C、G)；(通用引物R具体如表13所示)，下划线部分和常规下游特异性引物5'端序列(通用序列)相同；通用引物F的核苷酸序列为：P- GAACGACATGGCTACGATCCGA CTT(SEQ ID NO.18)，P为磷酸化修饰，下划线部分和常规上游特异性扩增引物5'端序列相同。
(2)第一轮PCR
在PCR管中按照表6所示反应体系配置PCR体系，进行PCR反应，反应程序如下：94℃1min；94℃30s，58℃2min，72℃30s，10cycles；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，得到产物。
(3)第二轮PCR
在PCR管中按照表7所示反应体系配置PCR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，得到文库。
表5常规设计的特异性扩增引物
Figure PCTCN2022137939-appb-000014
注：上述引物按照每条引物10μM浓度进行混合，得到各引物浓度均为10μM的引物池；上游特异性引物序列为常规设计，即5'到3'端依次为：通用序列和特异性序列，其中下划线部分和通用引物F的3'端相同；下游特异性引物序列为常规设计，即5'到3'端依次为：通用序列和特异性序列，下划线部分和与通用引物R的3'端相同。
表6第一轮PCR反应体系
Figure PCTCN2022137939-appb-000015
表7第二轮PCR反应体系
Figure PCTCN2022137939-appb-000016
效果实施例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、二聚体比例、比对比例、目标区域数据比例、均一性等性能(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、2、3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8所示、实施例1的突变检测统计数据如表9所示：实施例1、对比例1得到的文库的测序数据中的唯一比对比例、目标区域数据比例相当，并且实施例1中的数据利用率高于对比例1、二聚体比例低于对比例1，并且实施例1的方法的操作步骤只有一步；实施例1的突变频率的检测值与理论值相当。
实施例2、对比例1的得到的文库的测序数据统计如表10所示、突变检测统计数据如表11所示：实施例2、对比例1得到的文库的测序数据中的唯一比对比例、目标区域数据比例相当，并且实施例2中的数据利用率高于对比例1、二聚体比例低于对比例1，并且实施例2的方法的操作步骤只有一步；实施例2的突变频率的检测值与理论值相当。
实施例3、对比例1的得到的文库的测序数据统计如表12所示：实施例3、对比例1得到的文库的测序数据中的唯一比对比例、目标区域数据比例相当，并且实施例3中的数据利用率高于对比例1、二聚体比例低于对比例1，并且实施例3的方法的操作步骤只有一步；同时，实施例3得到的文库的测序数据中的唯一比对比例、目标区域数据比例、数据利用率、二聚体比例相当与实施例2相当，可见，将裂解反应和扩增反应在同一体系中进行并不会影响其性能。
表8实施例1、对比例1得到的文库的测序数据统计
Figure PCTCN2022137939-appb-000017
表9突变检测统计
Figure PCTCN2022137939-appb-000018
表10实施例1、对比例1得到的文库的测序数据统计
Figure PCTCN2022137939-appb-000019
表11突变检测统计
Figure PCTCN2022137939-appb-000020
表12实施例3、对比例1得到的文库的测序数据统计
Figure PCTCN2022137939-appb-000021
表13通用引物R序列
Figure PCTCN2022137939-appb-000022
Figure PCTCN2022137939-appb-000023
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 (21)

  1. 寡核酸组合，包含：特异性寡核酸和通用引物；
    所述通用引物包含：第一通用引物和第二通用引物；
    所述特异性寡核酸包含：上游特异性寡核酸和下游特异性寡核酸；
    所述特异性寡核酸包含：特异性序列和通用序列；
    所述上游特异性寡核酸包含：上游特异性序列和第一通用序列，其中，所述上游特异性序列位于所述上游特异性寡核酸的5'端，所述第一通用序列位于所述上游特异性寡核酸的3'端；
    所述下游特异性寡核酸包含：下游特异性序列和第二通用序列，其中，所述下游特异性序列位于所述下游特异性寡核酸的5'端，所述第二通用序列位于所述下游特异性寡核酸的3'端；
    1)所述第一通用引物的3'端序列与所述上游特异性寡核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酸的第二通用序列的部分序列或全部序列互补。
  2. 寡核酸组合，包含：特异性寡核酸、第二引物和通用引物；
    所述通用引物包含：第一通用引物；
    所述特异性寡核酸包含：特异性序列和通用序列，其中，所述特异性序列位于所述特异性寡核酸的5'端，所述通用序列位于所述特异性寡核酸的3'端；
    所述特异性序列为第一特异性序列，所述通用序列为第一通用序列；
    所述第二引物包含：第二特异性序列和第二通用序列，其中，所述第二特异性序列位于所述第二引物的3'端，所述第二通用序列位于所述第二引物的5'端；
    所述第一通用引物的3'端序列与所述特异性寡核酸的第一通用序列的部分序列或全部序列互补。
  3. 根据权利要求2所述的寡核酸组合，其特征在于：
    所述通用引物进一步包含：第二通用引物；所述第二通用引物的3'端与所述第二引物的第二通用序列的部分序列或全部序列相同。
  4. 根据权利要求1或3所述的寡核酸组合，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。
  5. 根据权利要求2所述的寡核酸组合，其特征在于：
    所述第二引物还包含功能核酸序列；
    优选地，所述第二引物包含测序引物序列或测序引物互补序列；
    优选地，所述第二引物还包含第三标签序列；
    优选地，所述第三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。
  6. 根据权利要求1或2所述的寡核酸组合，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；
    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。
  7. 根据权利要求1或2所述的寡核酸组合，其特征在于：
    所述特异性寡核酸的通用序列的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。
  8. 根据权利要求1或2所述的寡核酸组合，其特征在于：
    所述第一通用序列和第二通用序列相同或者不同。
  9. 根据权利要求2所述的寡核酸组合，其特征在于：
    1)所述第一特异性序列为上游特异性序列，所述第二特异性序列为下游特异性序列；或
    2)所述第一特异性序列为下游特异性序列，所述第二特异性序列为上游特异性序列。
  10. 根据权利要求4-6中任一项所述的寡核酸组合，其特征在于：所述第一标签序列、第二标签序列和第三标签序列可以相同也可以不同。
  11. 一种试剂盒，包含权利要求1或2所述的寡核酸组合。
  12. 根据权利要求11所述的试剂盒，其特征在于：
    所述试剂盒还包含：DNA聚合酶、Mg2+、PCR缓冲液、dNTPs中的至少一种；
    优选地，所述试剂盒包含：核酸提取试剂，其中所述核酸提取试剂为：裂解试剂；
    优选地，所述试剂盒包含：核酸提取试剂组合。
  13. 一种测序试剂套装，包含：测序试剂盒和权利要求11所述的试剂盒。
  14. 一种多重PCR扩增方法，包括如下步骤：获得生物样本，利用权利要求1或2所述 的多组寡核酸组合对生物样本进行扩增反应，其中所述多重扩增反应在同一体系中进行，其中所述多组最少为两组或两组以上。
  15. 根据权利要求14所述的多重PCR扩增方法，其特征在于：所述同一体系具体为不进行洗脱和/或纯化处理。
  16. 根据权利要求14所述的多重PCR扩增方法，其特征在于：所述生物样本是：细胞、组织、体液、微生物、唾液、尿液、痰液、粪便、咽拭子、鼻拭子、核酸中的至少一种；
    优选地，所述生物样本是：细胞、组织、体液、微生物、唾液、尿液、痰液、粪便、咽拭子、鼻拭子中的至少一种时，所述多重PCR扩增方法还包含裂解反应；
    优选地，所述裂解反应和所述扩增反应在同一体系中进行。
  17. 根据权利要求16所述的多重PCR扩增方法，其特征在于：所述裂解反应后、扩增反应前还包含提取纯化步骤；
    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。
  18. 一种制备多重PCR测序文库的方法，包含权利要求14所述的多重PCR扩增方法的步骤，得到测序文库。
  19. 根据权利要求18所述的方法，其特征在于：
    所述方法还包括如下步骤：对扩增产物进行纯化；
    优选地，所述纯化采用磁珠进行。
  20. 一种对目标区域基因位点检测的方法，包括如下步骤：获得目标区域的测序文库；获得测序数据；确定目标区域基因位点；
    所述获得目标区域的测序文库的方法包含b1)或b2)：
    b1)权利要求14所述的多重PCR扩增方法的步骤；
    b2)权利要求18所述的制备多重PCR测序文库的方法的步骤。
  21. 根据权利要求20所述的方法，其特征在于：
    所述确定目标区域基因位点的方法包含：将获得的测序数据与参考基因组进行比对，确定目标区域基因位点的碱基。
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