WO2018095108A1 - Primer composition, use thereof, and methods for constructing library and for determining nucleic acid sequence - Google Patents

Primer composition, use thereof, and methods for constructing library and for determining nucleic acid sequence Download PDF

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WO2018095108A1
WO2018095108A1 PCT/CN2017/100426 CN2017100426W WO2018095108A1 WO 2018095108 A1 WO2018095108 A1 WO 2018095108A1 CN 2017100426 W CN2017100426 W CN 2017100426W WO 2018095108 A1 WO2018095108 A1 WO 2018095108A1
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primer
linker
sequencing
target region
library
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PCT/CN2017/100426
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French (fr)
Chinese (zh)
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曾晓静
高晓峘
韩颖鑫
张印新
李胜
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广州精科医学检验所有限公司
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Publication of WO2018095108A1 publication Critical patent/WO2018095108A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • 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
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the present invention relates to the field of biomedicine. Specifically, the present invention relates to a primer composition and use thereof, a method for constructing a nucleic acid sequencing library of a target region of a sample to be tested, a method for determining a nucleic acid sequence of a target region of a sample to be tested, and simultaneously determining a plurality of samples to be tested. Method of target region nucleic acid sequence.
  • Immunoglobulins, T cell receptors and HLA are the most active molecules in the human genome.
  • the diversity of immune macromolecules allows the body to recognize and eliminate countless foreign substances, remove some of the metabolites produced in the body, and even apoptosis or aging. Cells, and maintain the balance of the body's immune system.
  • the immune system is an important system for the body to resist the invasion and immune regulation of pathogens, and it is of great significance for its research. Due to the enormous diversity of immune macromolecules, research on immune macromolecules is challenging and difficult. Recently developed and continues to be popular immunohistochemistry techniques have been used to study immune pools.
  • the immunological pool refers to the sum of the diverse immune cells at a certain moment in an individual, and these sums reflect the individual genetic factors, the history of antigen exposure, the surrounding environment, and the individual's immune regulation.
  • the research on the immune group library focuses on the sequence of the variable region of the B cell receptor and the sequence of the variable region of the T cell receptor, mainly by designing primer enrichment in the V gene skeleton region and the conserved region of the C region or the J region.
  • the target sequence includes multiplex PCR, arm-PCR and 5' RACE, and then the bioinformatics method is used to analyze the sequence length, sequence diversity, sequence similarity and other multi-dimensionality of the target sequence.
  • the multiplex PCR method is to design a multi-primer enriched target fragment at a conserved position upstream and downstream, and the number of cycles used is often high, and one problem that cannot be avoided in the enrichment process is that the multiple primers are amplified in the PCR process due to the efficiency of each pair of primers.
  • the inconsistency brings the partial distortion of the product of the enrichment, which is different from the real situation, and it is difficult to correct the distortion of this part from the level of bioinformatics. Therefore, it is necessary to take measures to reduce the number of cycles of multiple primer PCR. .
  • PCR amplification enrichment target region needs to add an exogenous sequence between the positive and negative primers to achieve annealing complementary, more cycles, increase the amount of data in subsequent sequencing, increase the cost of sequencing, and on the other hand, PCR amplification is performed by means of an auxiliary device such as a high-throughput chip.
  • the library tag can only be added to the sample during PCR enrichment, and once the sample is contaminated with each other during the experiment, the contaminated sample cannot be identified.
  • the object of the present invention is to continuously optimize the primer design, the PCR reaction system and the PCR thermal cycle program, etc., and propose that compared with the existing primers and methods, the preference of the multiple primers can be effectively reduced, so that the sequencing data is more precise and unnecessary.
  • the specific device assists the PCR amplification of the primer set enriched in the target region, and the method of constructing the nucleic acid sequencing library of the target region of the sample to be tested is completed in one step using the primer set.
  • the invention provides a primer composition comprising:
  • the first primer set comprising a first forward primer and a first reverse primer, the first forward primer comprising a target region-specific forward primer and a first linker;
  • the first reverse primer comprises a target region-specific reverse primer, a reverse library tag and a second linker, the reverse library tag being located between the 5' end of the target region-specific reverse primer and the second linker;
  • the second primer set comprising a second forward primer and a second reverse primer, the second forward primer comprising a first annealing, and the second reverse primer comprising a second annealing;
  • the first junction and the first annealing in the second forward primer are annealed to each other, and the PCR-amplified product of the first linker and the second forward primer comprises a first complete sequencing linker;
  • the second linker and the second annealing in the second reverse primer are annealed to each other, and the product of the second linker and the second reverse primer after PCR amplification comprises a second complete sequencing linker.
  • Another aspect of the invention provides the use of the above primer composition for use in immunohistochemistry studies.
  • Another aspect of the present invention provides a method for constructing a nucleic acid sequencing library of a target region of a sample to be tested, comprising: using the above primer set, performing PCR amplification on a nucleic acid sample containing a target region of the sample to be obtained, so as to obtain an amplification product, the expansion
  • the product of the formation constitutes a nucleic acid sequencing library of the target region of the sample to be tested.
  • Another aspect of the present invention also provides a method for determining a nucleic acid sequence of a target region of a sample to be tested, comprising the steps of:
  • Another aspect of the present invention also provides a method for simultaneously determining a target region nucleic acid sequence of a plurality of samples to be tested, comprising the steps of:
  • sequencing results including a target region nucleic acid library sequence of the plurality of samples to be tested and the positive/negative library tag;
  • target region nucleic acid sequencing library sequences of the plurality of samples to be tested based on the positive/negative library tag, and determining a target region nucleic acid sequence of each of the plurality of samples to be tested.
  • the primer composition designed by the invention does not need to add any exogenous sequence as a PCR binding position, but directly uses all or part of the linker of the sequencing platform as an annealing complementary region of the primer, thereby improving the efficiency of sequencing data and making the sequencing result more complete. Accurate, reducing the cost of sequencing and reducing the difficulty of data analysis. And the product is mainly enriched by the second forward/reverse primer, and the second forward/reverse primer is a single primer, which does not introduce amplification preference, and can effectively reduce the preference of multiple primers.
  • the method for constructing a library of the present invention adds a sample text to each sample at the time of reverse transcription as compared with the prior art method. The library label avoids mutual contamination between samples during subsequent experiments.
  • the sequencing results are accurate, reliable and repeatable.
  • Figure 1 is a schematic illustration of reverse transcription of mRNA by a first reverse primer in accordance with one embodiment of the present invention.
  • FIG. 2 is a schematic diagram showing PCR of cDNA1st by a first forward primer and a second primer set in an embodiment of the present invention.
  • FIG. 3 is a schematic diagram showing PCR of cDNA 1st by a first forward primer and a second primer set in another embodiment of the present invention.
  • FIG. 4 is a schematic flow chart of a method for determining a nucleic acid sequence of a target region of a sample to be tested according to an embodiment of the present invention.
  • FIG. 5 is a schematic flow chart of a method for simultaneously determining a target region nucleic acid sequence of a plurality of samples to be tested according to an embodiment of the present invention.
  • first”, “second”, “third”, “fourth” are used for descriptive purposes only, and are not to be construed as indicating or implying relative importance or implicitly indicating the indicated technical features. quantity. Thus, features defining “first”, “second”, “third”, “fourth” may include one or more of the features, either explicitly or implicitly. Further, in the description of the present invention, the meaning of "a plurality” is two or more unless otherwise specified.
  • the invention provides a primer composition comprising:
  • the first primer set comprising a first forward primer and a first reverse primer, the first forward primer comprising a target region-specific forward primer and a first linker;
  • the first reverse primer comprises a target region-specific reverse primer, a reverse library tag and a second linker, the reverse library tag being located between the 5' end of the target region-specific reverse primer and the second linker;
  • the second primer set comprising a second forward primer and a second reverse primer, the second forward primer comprising a first annealing, and the second reverse primer comprising a second annealing;
  • the first junction and the first annealing in the second forward primer are annealed to each other, and the PCR-amplified product of the first linker and the second forward primer comprises a first complete sequencing linker;
  • the second linker and the second annealing in the second reverse primer are annealed to each other, and the product of the second linker and the second reverse primer after PCR amplification comprises a second complete sequencing linker.
  • Primer compositions designed using the present invention need not be added in accordance with embodiments of the present invention
  • Any exogenous sequence acts as a PCR binding site, but directly uses all or part of the linker as an annealing complementary region of the primer, which improves the efficiency of sequencing data, makes the sequencing result more accurate, reduces the sequencing cost, and reduces the data analysis. Difficulty.
  • the product is mainly enriched by the second forward/reverse primer, and the second forward/reverse primer is a single primer, which does not introduce amplification preference, and can effectively reduce the preference of multiple primers.
  • the first forward primer further comprises a forward library tag located between the 5' end of the target region specific forward primer and the first linker.
  • the library tag is used to distinguish different sample libraries, and after PCR amplification, the PCR products of the plurality of samples are mixed and sequenced, and the sample sources of each sequence are distinguished based on the difference of the library tags.
  • the index/barcode tag has a library tag length of 6-12 bp.
  • the forward/reverse library tags may be the same or different.
  • the positive/negative library tags are identical.
  • the first joint may be the same as or different from the first annealing.
  • the first joint is the same as the first annealing.
  • the second joint may be the same as or different from the second annealing.
  • the second joint is the same as the second annealing.
  • the first annealing and the second annealing are each 16-25 bp in length.
  • the PCR-amplified product of the first linker and the second forward primer comprises a first complete sequencing linker.
  • the second forward primer is the first complete sequencing linker.
  • the PCR-amplified product of the second linker and the second reverse primer comprises a second complete sequencing linker.
  • the second reverse primer is the second complete sequencing linker.
  • the PCR-amplified product of the first linker and the second forward primer comprises a P5 sequencing linker of an illumina sequencing platform, and the second linker and the second reverse primer PCR
  • the amplified product contained the P7 sequencing linker of the illumina sequencing platform.
  • the first complete sequencing linker is a P5 sequencing linker of an illumina sequencing platform.
  • the second complete sequencing linker is a P7 sequencing linker for the illumina sequencing platform.
  • the PCR-amplified product of the first linker and the second forward primer comprises a P7 sequencing linker of an illumina sequencing platform
  • the product of the second linker and the second reverse primer after PCR amplification comprises illumina P5 sequencing linker for sequencing platform.
  • the first complete sequencing linker is a P7 sequencing linker of an illumina sequencing platform
  • the second complete sequencing linker is a P5 sequencing linker of an illumina sequencing platform.
  • the PCR-amplified product of the first linker and the second forward primer comprises a P1 sequencing linker of an Ion Torrent/Proton sequencing platform, the second linker and the second counter
  • the product after PCR amplification of the primers contained the A-sequencing linker of the Ion Torrent/Proton sequencing platform.
  • the first complete sequencing linker is a P1 sequencing linker of the Ion Torrent/Proton sequencing platform
  • the second complete sequencing linker is an A-sequence linker of the Ion Torrent/Proton sequencing platform.
  • the PCR-amplified product of the first linker and the second forward primer comprises an A-sequencing linker of a 454 sequencing platform, and the second linker and the second reverse primer PCR
  • the amplified product contains the B measurement of the 454 sequencing platform Order connector.
  • the first complete sequencing linker is an A-sequencing linker of the 454 sequencing platform
  • the second complete sequencing linker is a B-sequence linker of the 454 sequencing platform.
  • the first forward primer is used in an Ion Torrentt/Proton sequencing platform when it does not contain a forward library tag.
  • the first forward primer comprises a forward library tag
  • it is used in an illumina sequencing platform or a 454 sequencing platform.
  • the target region-specific forward primer and the target region-specific reverse primer are 18-25 bp in length.
  • the target region-specific forward primer comprises a specific primer for a V gene conserved region of a B cell receptor or a T cell receptor, the target region specific reverse primer comprising a B cell receptor Or a specific primer for the conserved region of the C gene of the T cell receptor.
  • the invention provides the use of the above primer composition in immunobanking studies.
  • the present invention provides a method for constructing a nucleic acid sequencing library of a target region of a sample to be tested, comprising: using the above primer set, performing PCR amplification on a nucleic acid containing a target region of the sample to be obtained, to obtain an amplification product,
  • the amplification product constitutes a nucleic acid sequencing library of the target region of the sample to be tested.
  • the first forward primer and the second forward primer have a molar ratio of 1:1 to 1:10, and the first forward primer and the second forward primer are combined with the second
  • the reverse primer has a molar ratio of 1:1.
  • the PCR reaction system is:
  • the PCR reaction procedure is:
  • the PCR amplification reaction using the primer set of the present invention can complete the enrichment of the nucleic acid sample in only 10-25 cycles, with less cycle, short reaction time and high efficiency.
  • the method of constructing a sample region nucleic acid sequencing library of a sample to be tested further comprises the steps of:
  • each target area it is designed to amplify the target area core Acid-amplified target region-specific forward/reverse primers, first and second adaptors, second forward primers, second reverse primers, and forward/reverse library tags, target region-specific positive/reverse primers,
  • the first linker and the second linker and the positive/negative library tag are synthesized to obtain a first forward primer and a first reverse primer;
  • the invention adopts all or part of the complete sequencing linker as the annealing complementary region of the first forward primer and the second forward primer, the first reverse primer and the second reverse primer, and the sequencing result is more accurate without introducing the foreign sequence. , reducing the cost of sequencing and reducing the difficulty of subsequent data analysis.
  • the sample library tag is introduced into the cDNA 1st by reverse transcription during reverse transcription, and the binding site of the second reverse primer is introduced into the cDNA 1st, and the second reverse primer can be used in the subsequent PCR reaction.
  • the amplification reaction is carried out. That is, the present invention adds a sample library label to each sample at the time of reverse transcription, thereby avoiding mutual contamination between samples in subsequent experimental procedures.
  • the PCR reaction is carried out in a PCR tube after the mixing.
  • the sample RNA to be tested is messenger RNA (mRNA).
  • mRNA messenger RNA
  • the cDNA synthesis reagent comprises a mixture of deoxyribonucleoside triphosphate (dNTP mi), 5x1 chain buffer (5 ⁇ 1st strand buffer), dithiothreitol (DTT), and RNase inhibitor (RNAseOUT). , Reverse transcriptase III (SuperScriptTM III), RNase mix.
  • dNTP mi deoxyribonucleoside triphosphate
  • DTT dithiothreitol
  • RNAseOUT RNase inhibitor
  • Reverse transcriptase III SuperScriptTM III
  • RNase mix Reverse transcriptase mix.
  • the synthesizing step comprises: first detecting the genomic RNA to be tested, and first reversing Primer and dNTP mix were mixed for RNA denaturation; after denaturation, 5 ⁇ 1st strand buffer, DTT, RNAseOUT, SuperScriptTM III were added to carry out PCR reaction.
  • the reaction procedure was 50°C for 50min and 70°C for 15min. After the PCR reaction was completed, the reaction was added. The RNase mix was incubated at 37 ° C for 30 min to obtain cDNA 1 st.
  • the PCR reaction is carried out in a PCR tube after the mixing.
  • the PCR reaction procedure is:
  • the PCR product is recovered and subjected to magnetic bead purification, and the magnetic beads are added in a volume of 0.8-1 volume of the PCR product, and the purified product is a nucleic acid library.
  • the magnetic bead purification is purified using an AMPure XP DNA purification kit.
  • the method for constructing a nucleic acid sequencing library of a target region of a sample to be tested does not require an additional step of sequencing a linker, a step of purifying the product, and does not require a specific device such as a single tube or a high-throughput chip to assist the PCR reaction, and uses a common PCR device.
  • the product enrichment process can be completed, the operation is simple, the cost is low, the sample loss is reduced, the efficiency is high, the cycle number is small, the time is short, the human error is reduced, the sequencing result is accurate, reliable, and the repeatability is good.
  • the method for constructing a nucleic acid sequencing library of a sample region to be tested of the present invention may further comprise the following steps:
  • the first primer set obtained by the synthesis is subjected to pre-PCR amplification and gel electrophoresis detection to verify whether the first primer set meets the requirements, and whether a single band conforming to the design size can be amplified is used as a criterion.
  • the method of constructing a sample region nucleic acid sequencing library of a sample to be tested further comprises the steps of:
  • the constructed nucleic acid library is subjected to library quality detection, and the mass concentration, fragment size distribution, and molar concentration can be detected using, for example, an Agilent 2100 Bioanalyzer or a Caliper Bioanalyzer, or an ABI StepOnerPlus Real-Time PCR System. After passing the test, it can be sequenced on the machine.
  • the present invention also provides a method of determining a nucleic acid sequence of a target region of a sample to be tested.
  • the method comprises the following steps:
  • a target region nucleic acid sequencing library of the sample to be tested is constructed.
  • the target region nucleic acid sequencing library of the sample to be tested is sequenced to obtain sequencing results.
  • the PCR-amplified product of the first linker and the second forward primer comprises a P5 sequencing linker of an illumina sequencing platform, the second linker and the second reverse primer
  • the sequencing is performed using an Illumina sequencing platform.
  • the PCR-amplified product of the first linker and the second forward primer comprises a P7 sequencing linker of an illumina sequencing platform
  • the product of the second linker and the second reverse primer after PCR amplification comprises The sequencing of the P5 sequencing linker of the illumina sequencing platform was performed using an Illumina sequencing platform.
  • the PCR-amplified product of the first linker and the second forward primer comprises a P1 sequencing linker of an Ion Torrent/Proton sequencing platform, the second linker and the first
  • the product after PCR amplification of the two reverse primers contains the A-sequence linker of the Ion Torrent/Proton sequencing platform, the sequencing was performed using the Ion Torrent/Proton sequencing platform.
  • the PCR-amplified product of the first linker and the second forward primer comprises an A-sequence linker of a 454 sequencing platform, the second linker and the second reverse
  • the sequencing is performed using a 454 sequencing platform.
  • the sequence of the nucleic acid of the target region of the sample to be tested is determined.
  • the present invention further provides for simultaneously determining a plurality of to-be-tested Method of nucleic acid sequence of a target region of a sample.
  • the method comprises the following steps:
  • a plurality of target region nucleic acid sequencing libraries of the samples to be tested are mixed to obtain a hybrid library.
  • the hybrid library is sequenced to obtain sequencing results including a target region nucleic acid library sequence of the plurality of samples to be tested and the positive/negative library tag.
  • the PCR-amplified product of the first linker and the second forward primer comprises a P5 sequencing linker of an illumina sequencing platform, the second linker and the second reverse primer
  • the sequencing is performed using an Illumina sequencing platform.
  • the PCR-amplified product of the first linker and the second forward primer comprises a P7 sequencing linker of an illumina sequencing platform
  • the product of the second linker and the second reverse primer after PCR amplification comprises The sequencing of the P5 sequencing linker of the illumina sequencing platform was performed using an Illumina sequencing platform.
  • the PCR-amplified product of the first linker and the second forward primer comprises a P1 sequencing linker of an Ion Torrent/Proton sequencing platform, the second linker and the first
  • the product after PCR amplification of the two reverse primers contains the A-sequence linker of the Ion Torrent/Proton sequencing platform, the sequencing was performed using the Ion Torrent/Proton sequencing platform.
  • the PCR-amplified product of the first linker and the second forward primer comprises an A-sequence linker of a 454 sequencing platform, the second linker and the second reverse
  • the PCR amplified product of the primer contained the B-sequencing linker of the 454 sequencing platform, which was sequenced using the 454 sequencing platform.
  • a sequence of a target region nucleic acid sequencing library of the plurality of samples to be tested is distinguished based on the positive/negative library tag, and a target region nucleic acid sequence of each of the plurality of samples to be tested is determined.
  • the first reverse primer is designed in the conserved region of the C gene, and the first reverse primer is ligated to the reverse library tag at the 5' end of the C gene-specific primer, and the 5' end of the reverse library tag is ligated to the second linker, see The first reverse primer in Figure 1.
  • the first forward primer includes a specific primer and a first linker of the conserved region of the V gene, see the first forward primer in Figure 2, or a specific primer including a conserved region of the V gene, a forward library tag, and a first linker, see The first forward primer in Figure 3. Both positive/negative library tags are used to distinguish between different samples.
  • the second forward primer comprises a first anneal, as shown in Figure 2, and the second reverse primer comprises a second anneal, as shown in Figure 3.
  • the main function is the linker at the upper and lower ends, that is, the second forward primer and the second reverse primer.
  • the present invention does not introduce a foreign sequence as a PCR binding site, but uses a partial sequence of the sequencing linker, that is, the 3' end 16-25 bp of the complete sequencing linker as the first forward primer and the second forward primer, A reverse primer and a second reverse primer anneal the complementary region. It is precisely because of this special design that the method does not require end repair, adding 'A' and adding a linker, directly performing the target product enrichment and completing the library construction. .
  • two sample primer set sequences were designed as follows.
  • Table 1 sets of primer set sequences
  • a sterile blood collection tube containing an anticoagulant for blood collection (the anticoagulant is usually EDTA), using a lymphocyte separation solution such as Ficoll-Paque PLUS lymphocyte separation solution or Percoll lymphocyte separation solution for density gradient PBMC were isolated.
  • a lymphocyte separation solution such as Ficoll-Paque PLUS lymphocyte separation solution or Percoll lymphocyte separation solution for density gradient PBMC were isolated.
  • Total RNA was extracted using a Trizol regeant method or an extraction kit or the like.
  • DNA remaining in the RNA sample is removed using DNA digestive enzymes.
  • RNA is reverse transcribed using reverse transcriptase and reverse transcription primers to obtain a cDNA 1 strand, and an RNA digestive enzyme is added to digest the RNA.
  • the first reverse primer reverse transcribes mRNA.
  • the total volume of the PCR tube was 20 ⁇ L. Mix gently with a slight shake.
  • reaction mixture can be stored at -20 °C.
  • the primer used in the PCR includes a first forward primer (V region forward primer) and a second primer set, wherein the first forward primer and the second forward primer have a molar ratio of 1:1 to 1:10.
  • the molar ratio of the sum of the forward primer to the second forward primer to the second reverse primer is 1:1.
  • the cDNA 1st obtained by the synthesis was PCR-enriched as a template.
  • the following mixed solution was used to prepare a PCR reaction system in a 200 ⁇ L PCR tube.
  • the PCR reaction conditions are:
  • the constructed sequencing library can be purified by three methods, namely magnetic bead purification, purification column purification and agarose gel electrophoresis recovery and purification.
  • the PCR product was transferred to a 1.5 mL centrifuge tube, and the amplified sample was purified using AMPure XP DNA Purification Kit (SPRI beads).
  • the Agilent 2100 Bioanalyzer analysis system detects library insert size and content; Q-PCR accurately quantifies library concentration.
  • the constructed sequencing libraries were sequenced on a high-throughput sequencing platform including Illumina Hiseq and Miseq sequencing platforms, Roche 454 sequencing platform and Life Technologies' Ion Torrent sequencing platform.
  • the data obtained by sequencing is subjected to mass filtration and length filtration to remove contamination and linker sequences; the statistical results include: length of the measured sequence (Reads), and data yield. Then, the obtained sequences were subjected to alignment analysis, and the V, (D,) J genes were identified and determined according to the definition of the V and (D,) J genes on IMGT, and the diversity of V, (D,) J genes was counted. Sex.
  • the description of the terms “one embodiment”, “specific example” and the like means that a specific feature, structure, material or characteristic described in connection with the embodiment or example is included in at least one embodiment of the invention or In the example.
  • the schematic representation of the above terms does not necessarily mean the same embodiment or example.
  • the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

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Abstract

A primer composition and uses thereof for constructing a library and for determining a nucleic acid sequence. The primer composition comprises: a first primer combination comprising a first forward primer and a first reverse primer, the first forward primer comprising a target region-specific forward primer and a first adaptor, and the first reverse primer comprising a target region-specific reverse primer, a reverse library tag, and a second adaptor; a second primer combination comprising a second forward primer and a second reverse primer, the second forward primer comprising a first annealing component and the second reverse primer comprising a second annealing component, and the first adaptor and the first annealing component in the second forward primer and the second adaptor and the second annealing component in the second reverse primer being complementary to each other in annealing, and a product obtained by PCR amplification of the first adaptor and the second forward primer and the second adaptor and the second reverse primer comprising a complete sequencing adaptor.

Description

引物组合物、其用途、构建文库和确定核酸序列的方法Primer composition, use thereof, library construction and method for determining nucleic acid sequence 技术领域Technical field
本发明涉及生物医学领域，具体的，本发明涉及引物组合物及其用途、构建待测样品目标区域核酸测序文库的方法、确定待测样品目标区域核酸序列的方法、同时确定多个待测样品的目标区域核酸序列的方法。The present invention relates to the field of biomedicine. Specifically, the present invention relates to a primer composition and use thereof, a method for constructing a nucleic acid sequencing library of a target region of a sample to be tested, a method for determining a nucleic acid sequence of a target region of a sample to be tested, and simultaneously determining a plurality of samples to be tested. Method of target region nucleic acid sequence.
背景技术Background technique
免疫球蛋白、T细胞受体和HLA等是人类基因组中最活跃的分子，免疫大分子的多样性使得机体能识别并清除无数的外来物质、清除体内产生的部分代谢物甚至凋亡或者衰老的细胞，且保持着机体免疫系统的平衡。免疫系统是机体抵御病原菌入侵与免疫调节的重要系统，对其研究具有很重要的意义。由于免疫大分子巨大的多样性，使得对免疫大分子的研究充满挑战和困难。最近发展起来并持续受欢迎的免疫组库技术被用于研究免疫组库。免疫组库即是指多样性的免疫细胞在一个个体内某一时刻的总和，而这些总和反应了个体遗传因素、抗原接触史、周围环境和个体的免疫调控。目前对免疫组库的研究集中于B细胞受体可变区的序列和T细胞受体可变区的序列，主要通过在V基因骨架区和C区或J区的保守区区域设计引物富集目的序列，采用的方法包括多重PCR、arm-PCR和5’RACE，然后采用生物信息学的方法对目的序列进行序列长度，序列多样性、序列异同性等多维度的分析。 Immunoglobulins, T cell receptors and HLA are the most active molecules in the human genome. The diversity of immune macromolecules allows the body to recognize and eliminate countless foreign substances, remove some of the metabolites produced in the body, and even apoptosis or aging. Cells, and maintain the balance of the body's immune system. The immune system is an important system for the body to resist the invasion and immune regulation of pathogens, and it is of great significance for its research. Due to the enormous diversity of immune macromolecules, research on immune macromolecules is challenging and difficult. Recently developed and continues to be popular immunohistochemistry techniques have been used to study immune pools. The immunological pool refers to the sum of the diverse immune cells at a certain moment in an individual, and these sums reflect the individual genetic factors, the history of antigen exposure, the surrounding environment, and the individual's immune regulation. At present, the research on the immune group library focuses on the sequence of the variable region of the B cell receptor and the sequence of the variable region of the T cell receptor, mainly by designing primer enrichment in the V gene skeleton region and the conserved region of the C region or the J region. The target sequence includes multiplex PCR, arm-PCR and 5' RACE, and then the bioinformatics method is used to analyze the sequence length, sequence diversity, sequence similarity and other multi-dimensionality of the target sequence.
目前来说，若要研究这些巨大多样性的区域，必须将这些区域从基因组或者total RNA水平富集并分离出来。多重PCR方法是在上下游保守的位置设计多重引物富集目的片段，使用的循环数常常较高，而该富集过程中无法回避的一个问题就是多重引物在PCR过程由于每对引物扩增效率不一致而带来的偏好性，使得富集的产物部分失真，与真实情况存在一定的差异，并且很难从生物信息学水平来矫正这部分的失真，因此需要采取方法降低多重引物PCR的循环数。For the time being, to study these vastly diverse regions, these regions must be enriched and separated from genomic or total RNA levels. The multiplex PCR method is to design a multi-primer enriched target fragment at a conserved position upstream and downstream, and the number of cycles used is often high, and one problem that cannot be avoided in the enrichment process is that the multiple primers are amplified in the PCR process due to the efficiency of each pair of primers. The inconsistency brings the partial distortion of the product of the enrichment, which is different from the real situation, and it is difficult to correct the distortion of this part from the level of bioinformatics. Therefore, it is necessary to take measures to reduce the number of cycles of multiple primer PCR. .
目前的PCR扩增富集目标区域一方面需要在正反向引物之间增加一段外源序列来实现退火互补，循环次数多，后续测序时数据量增加，增加了测序成本，另外一方面还需借助高通量芯片等辅助设备进行PCR扩增。此外，现有技术只能在PCR富集时才能将文库标签加入样品中，一旦实验过程中出现样品间相互污染，即无法识别被污染的样品。The current PCR amplification enrichment target region needs to add an exogenous sequence between the positive and negative primers to achieve annealing complementary, more cycles, increase the amount of data in subsequent sequencing, increase the cost of sequencing, and on the other hand, PCR amplification is performed by means of an auxiliary device such as a high-throughput chip. In addition, in the prior art, the library tag can only be added to the sample during PCR enrichment, and once the sample is contaminated with each other during the experiment, the contaminated sample cannot be identified.
发明内容Summary of the invention
本发明的目的在于，通过不断优化引物设计，PCR反应体系和PCR热循环程序等，提出一种与现有引物及方法相比，能有效降低多重引物的偏好性，使得测序数据更精准，无需特定设备辅助即可进行PCR扩增富集目标区域的引物组，及使用所述引物组一步完成构建待测样品目标区域核酸测序文库的方法。The object of the present invention is to continuously optimize the primer design, the PCR reaction system and the PCR thermal cycle program, etc., and propose that compared with the existing primers and methods, the preference of the multiple primers can be effectively reduced, so that the sequencing data is more precise and unnecessary. The specific device assists the PCR amplification of the primer set enriched in the target region, and the method of constructing the nucleic acid sequencing library of the target region of the sample to be tested is completed in one step using the primer set.
因此，本发明一方面提供一种引物组合物，包含：Accordingly, in one aspect, the invention provides a primer composition comprising:
第一引物组，所述第一引物组包含第一正向引物和第一反向引物，所述第一正向引物包括目标区域特异性正向引物和第一接头； a first primer set, the first primer set comprising a first forward primer and a first reverse primer, the first forward primer comprising a target region-specific forward primer and a first linker;
所述第一反向引物包括目标区域特异性反向引物、反向文库标签和第二接头，所述反向文库标签位于目标区域特异性反向引物5’端和第二接头之间；The first reverse primer comprises a target region-specific reverse primer, a reverse library tag and a second linker, the reverse library tag being located between the 5' end of the target region-specific reverse primer and the second linker;
第二引物组，所述第二引物组包含第二正向引物和第二反向引物，所述第二正向引物包含第一退火，所述第二反向引物包含第二退火；a second primer set, the second primer set comprising a second forward primer and a second reverse primer, the second forward primer comprising a first annealing, and the second reverse primer comprising a second annealing;
所述第一接头与所述第二正向引物中的第一退火相互退火互补，所述第一接头与所述第二正向引物经PCR扩增后的产物包含第一完整测序接头；The first junction and the first annealing in the second forward primer are annealed to each other, and the PCR-amplified product of the first linker and the second forward primer comprises a first complete sequencing linker;
所述第二接头与所述第二反向引物中的第二退火相互退火互补，所述第二接头与所述第二反向引物经PCR扩增后的产物包含第二完整测序接头。The second linker and the second annealing in the second reverse primer are annealed to each other, and the product of the second linker and the second reverse primer after PCR amplification comprises a second complete sequencing linker.
本发明另一方面提供上述引物组合物，在免疫组库研究中的用途。Another aspect of the invention provides the use of the above primer composition for use in immunohistochemistry studies.
本发明另一方面还提供一种构建待测样品目标区域核酸测序文库的方法，包括采用上述引物组，对待测样品包含目标区域的核酸样本进行PCR扩增，以便获得扩增产物，所述扩增产物构成所述待测样品目标区域核酸测序文库。Another aspect of the present invention provides a method for constructing a nucleic acid sequencing library of a target region of a sample to be tested, comprising: using the above primer set, performing PCR amplification on a nucleic acid sample containing a target region of the sample to be obtained, so as to obtain an amplification product, the expansion The product of the formation constitutes a nucleic acid sequencing library of the target region of the sample to be tested.
本发明另一方面还提供一种确定待测样品目标区域核酸序列的方法，包括以下步骤：Another aspect of the present invention also provides a method for determining a nucleic acid sequence of a target region of a sample to be tested, comprising the steps of:
采用上述构建待测样品目标区域核酸测序文库的方法，构建待测样品的目标区域核酸测序文库；Constructing a target region nucleic acid sequencing library of the sample to be tested by using the above method for constructing a nucleic acid sequencing library of the target region of the sample to be tested;
对所述待测样品的目标区域核酸测序文库进行测序，以便获得 测序结果；Sequencing the target region nucleic acid sequencing library of the sample to be tested to obtain Sequencing results;
以及基于所述测序结果，确定待测样品目标区域核酸的序列。And determining a sequence of the nucleic acid of the target region of the sample to be tested based on the sequencing result.
本发明另一方面还提供一种同时确定多个待测样品的目标区域核酸序列的方法，包括以下步骤：Another aspect of the present invention also provides a method for simultaneously determining a target region nucleic acid sequence of a plurality of samples to be tested, comprising the steps of:
针对所述多个待测样品中的每一个，分别独立地采用上述构建待测样品目标区域核酸测序文库的方法，构建待测样品的目标区域核酸测序文库，其中，所述多个待测样品的正/反文库标签互不相同，所述多个为至少2个；Determining, by each of the plurality of samples to be tested, a method for constructing a nucleic acid sequencing library of a target region of the sample to be tested, and constructing a target region nucleic acid sequencing library of the sample to be tested, wherein the plurality of samples to be tested The positive/negative library tags are different from each other, and the plurality of the plurality of tags are at least two;
将所述多个待测样品的目标区域核酸测序文库混合，以获得混合文库；Mixing the target region nucleic acid sequencing libraries of the plurality of samples to be tested to obtain a mixed library;
对所述混合文库进行测序，以获得测序结果，所述测序结果包括所述多个待测样品的目标区域核酸文库序列和所述正/反文库标签；Sequencing the hybrid library to obtain sequencing results, the sequencing result including a target region nucleic acid library sequence of the plurality of samples to be tested and the positive/negative library tag;
以及基于所述正/反文库标签对所述多个待测样品的目标区域核酸测序文库序列进行区分，并确定所述多个待测样品的每一个的目标区域核酸序列。And distinguishing the target region nucleic acid sequencing library sequences of the plurality of samples to be tested based on the positive/negative library tag, and determining a target region nucleic acid sequence of each of the plurality of samples to be tested.
利用本发明设计的引物组合物无需添加任何外源序列作为PCR结合位置，而是直接使用测序平台的接头的全部或部分作为引物的退火互补区，提高了测序数据的有效率，使得测序结果更加精准，减少了测序成本，降低了数据分析的难度。并且通过第二正/反向引物对产物起到主要富集作用，第二正/反向引物为单一引物，不会引入扩增偏好，能够有效降低多重引物的偏好性。此外本发明的构建文库的方法与现有方法相比，在反转录时即给每个样品加了样品文 库标签，避免了后续实验过程中样品间的相互污染。并且无需单管或高通量芯片等特定设备辅助PCR反应，使用普通的PCR设备即可完成产物的富集过程，操作简单，成本低、减低样品损失，效率高，循环次数少，耗时短，减少人为操作误差，测序结果准确、可靠，可重复性好。The primer composition designed by the invention does not need to add any exogenous sequence as a PCR binding position, but directly uses all or part of the linker of the sequencing platform as an annealing complementary region of the primer, thereby improving the efficiency of sequencing data and making the sequencing result more complete. Accurate, reducing the cost of sequencing and reducing the difficulty of data analysis. And the product is mainly enriched by the second forward/reverse primer, and the second forward/reverse primer is a single primer, which does not introduce amplification preference, and can effectively reduce the preference of multiple primers. In addition, the method for constructing a library of the present invention adds a sample text to each sample at the time of reverse transcription as compared with the prior art method. The library label avoids mutual contamination between samples during subsequent experiments. Moreover, it is not necessary to assist the PCR reaction by a specific device such as a single tube or a high-throughput chip, and the product enrichment process can be completed by using ordinary PCR equipment, the operation is simple, the cost is low, the sample loss is reduced, the efficiency is high, the number of cycles is small, and the time is short. To reduce human error, the sequencing results are accurate, reliable and repeatable.
附图说明DRAWINGS
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments herein
图1是本发明一实施例中第一反向引物对mRNA进行反转录的示意图。BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic illustration of reverse transcription of mRNA by a first reverse primer in accordance with one embodiment of the present invention.
图2是本发明一实施例中第一正向引物和第二引物组对cDNA1st进行PCR的示意图。2 is a schematic diagram showing PCR of cDNA1st by a first forward primer and a second primer set in an embodiment of the present invention.
图3是本发明另一实施例中第一正向引物和第二引物组对cDNA 1st进行PCR的示意图。3 is a schematic diagram showing PCR of cDNA 1st by a first forward primer and a second primer set in another embodiment of the present invention.
图4是本发明一实施例中确定待测样品目标区域核酸序列的方法的流程示意图。4 is a schematic flow chart of a method for determining a nucleic acid sequence of a target region of a sample to be tested according to an embodiment of the present invention.
图5是本发明一实施例中同时确定多个待测样品的目标区域核酸序列的方法的流程示意图。5 is a schematic flow chart of a method for simultaneously determining a target region nucleic acid sequence of a plurality of samples to be tested according to an embodiment of the present invention.
具体实施方式detailed description
下面详细描述本发明的实施例。下面通过参考附图描述的实施例是是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。 Embodiments of the present invention are described in detail below. The embodiments described below with reference to the accompanying drawings are intended to be illustrative of the invention and are not to be construed as limiting.
需要说明的是，术语“第一”、“第二”、“第三”、“第四”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”、“第四”的特征可以明示或者隐含地包括一个或更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。It should be noted that the terms "first", "second", "third", "fourth" are used for descriptive purposes only, and are not to be construed as indicating or implying relative importance or implicitly indicating the indicated technical features. quantity. Thus, features defining "first", "second", "third", "fourth" may include one or more of the features, either explicitly or implicitly. Further, in the description of the present invention, the meaning of "a plurality" is two or more unless otherwise specified.
根据本发明的一个方面，本发明提供一种引物组合物，该引物组合物包含：According to one aspect of the invention, the invention provides a primer composition comprising:
第一引物组，所述第一引物组包含第一正向引物和第一反向引物，所述第一正向引物包含目标区域特异性正向引物和第一接头；a first primer set, the first primer set comprising a first forward primer and a first reverse primer, the first forward primer comprising a target region-specific forward primer and a first linker;
所述第一反向引物包括目标区域特异性反向引物、反向文库标签和第二接头，所述反向文库标签位于目标区域特异性反向引物5’端和第二接头之间；The first reverse primer comprises a target region-specific reverse primer, a reverse library tag and a second linker, the reverse library tag being located between the 5' end of the target region-specific reverse primer and the second linker;
第二引物组，所述第二引物组包含第二正向引物和第二反向引物，所述第二正向引物包含第一退火，所述第二反向引物包含第二退火；a second primer set, the second primer set comprising a second forward primer and a second reverse primer, the second forward primer comprising a first annealing, and the second reverse primer comprising a second annealing;
所述第一接头与所述第二正向引物中的第一退火相互退火互补，所述第一接头与所述第二正向引物经PCR扩增后的产物包含第一完整测序接头；The first junction and the first annealing in the second forward primer are annealed to each other, and the PCR-amplified product of the first linker and the second forward primer comprises a first complete sequencing linker;
所述第二接头与所述第二反向引物中的第二退火相互退火互补，所述第二接头与所述第二反向引物经PCR扩增后的产物包含第二完整测序接头。The second linker and the second annealing in the second reverse primer are annealed to each other, and the product of the second linker and the second reverse primer after PCR amplification comprises a second complete sequencing linker.
根据本发明的实施例，利用本发明设计的引物组合物无需添加 任何外源序列作为PCR结合位置，而是直接使用接头中的全部或部分作为引物的退火互补区，提高了测序数据的有效率，使得测序结果更加精准，减少了测序成本，降低了数据分析的难度。并且通过第二正/反向引物对产物起到主要富集作用，第二正/反向引物为单一引物，不会引入扩增偏好，能够有效降低多重引物的偏好性。Primer compositions designed using the present invention need not be added in accordance with embodiments of the present invention Any exogenous sequence acts as a PCR binding site, but directly uses all or part of the linker as an annealing complementary region of the primer, which improves the efficiency of sequencing data, makes the sequencing result more accurate, reduces the sequencing cost, and reduces the data analysis. Difficulty. And the product is mainly enriched by the second forward/reverse primer, and the second forward/reverse primer is a single primer, which does not introduce amplification preference, and can effectively reduce the preference of multiple primers.
根据本发明的实施例，所述第一正向引物还包括正向文库标签，所述正向文库标签位于目标区域特异性正向引物5’端和第一接头之间。According to an embodiment of the invention, the first forward primer further comprises a forward library tag located between the 5' end of the target region specific forward primer and the first linker.
所述文库标签用于区分不同样品文库，能够在进行PCR扩增后，将多个样本的PCR产物进行混合测序，进而基于文库标签的不同，对各序列的样本来源进行区分。例如index/barcode标签，所述文库标签的长度为6-12bp。The library tag is used to distinguish different sample libraries, and after PCR amplification, the PCR products of the plurality of samples are mixed and sequenced, and the sample sources of each sequence are distinguished based on the difference of the library tags. For example, the index/barcode tag has a library tag length of 6-12 bp.
根据本发明的实施例，所述正/反文库标签可以相同也可以不同。优选的，正/反文库标签相同。According to an embodiment of the invention, the forward/reverse library tags may be the same or different. Preferably, the positive/negative library tags are identical.
根据本发明的实施例，所述第一接头与所述第一退火可以相同也可以不同，优选的，所述第一接头与第一退火相同。According to an embodiment of the invention, the first joint may be the same as or different from the first annealing. Preferably, the first joint is the same as the first annealing.
根据本发明的实施例，所述第二接头与所述第二退火可以相同也可以不同，优选的，所述第二接头与第二退火相同。According to an embodiment of the invention, the second joint may be the same as or different from the second annealing. Preferably, the second joint is the same as the second annealing.
根据本发明的实施例，所述第一退火和第二退火的长度均为16-25bp。According to an embodiment of the invention, the first annealing and the second annealing are each 16-25 bp in length.
根据本发明的实施例，所述第一接头与所述第二正向引物PCR扩增后的产物包含第一完整测序接头。优选的，所述第二正向引物即为第一完整的测序接头。 According to an embodiment of the invention, the PCR-amplified product of the first linker and the second forward primer comprises a first complete sequencing linker. Preferably, the second forward primer is the first complete sequencing linker.
根据本发明的实施例，所述第二接头与所述第二反向引物PCR扩增后的产物包含第二完整测序接头。优选的，所述第二反向引物即为第二完整的测序接头。According to an embodiment of the invention, the PCR-amplified product of the second linker and the second reverse primer comprises a second complete sequencing linker. Preferably, the second reverse primer is the second complete sequencing linker.
根据本发明的一个具体示例，所述第一接头与所述第二正向引物PCR扩增后的产物包含illumina测序平台的P5测序接头，所述第二接头与所述第二反向引物PCR扩增后的产物包含illumina测序平台的P7测序接头。优选的，所述第一完整测序接头为illumina测序平台的P5测序接头。所述第二完整测序接头为illumina测序平台的P7测序接头。According to a specific example of the present invention, the PCR-amplified product of the first linker and the second forward primer comprises a P5 sequencing linker of an illumina sequencing platform, and the second linker and the second reverse primer PCR The amplified product contained the P7 sequencing linker of the illumina sequencing platform. Preferably, the first complete sequencing linker is a P5 sequencing linker of an illumina sequencing platform. The second complete sequencing linker is a P7 sequencing linker for the illumina sequencing platform.
或者所述第一接头与所述第二正向引物PCR扩增后的产物包含illumina测序平台的P7测序接头，所述第二接头与所述第二反向引物PCR扩增后的产物包含illumina测序平台的P5测序接头。优选的，所述第一完整测序接头为illumina测序平台的P7测序接头，所述第二完整测序接头为illumina测序平台的P5测序接头。Or the PCR-amplified product of the first linker and the second forward primer comprises a P7 sequencing linker of an illumina sequencing platform, and the product of the second linker and the second reverse primer after PCR amplification comprises illumina P5 sequencing linker for sequencing platform. Preferably, the first complete sequencing linker is a P7 sequencing linker of an illumina sequencing platform, and the second complete sequencing linker is a P5 sequencing linker of an illumina sequencing platform.
根据本发明的一个具体示例，所述第一接头与所述第二正向引物PCR扩增后的产物包含Ion Torrent/Proton测序平台的P1测序接头，所述第二接头与所述第二反向引物PCR扩增后的产物包含Ion Torrent/Proton测序平台的A测序接头。优选的，所述第一完整测序接头为Ion Torrent/Proton测序平台的P1测序接头，所述第二完整测序接头为Ion Torrent/Proton测序平台的A测序接头。According to a specific example of the present invention, the PCR-amplified product of the first linker and the second forward primer comprises a P1 sequencing linker of an Ion Torrent/Proton sequencing platform, the second linker and the second counter The product after PCR amplification of the primers contained the A-sequencing linker of the Ion Torrent/Proton sequencing platform. Preferably, the first complete sequencing linker is a P1 sequencing linker of the Ion Torrent/Proton sequencing platform, and the second complete sequencing linker is an A-sequence linker of the Ion Torrent/Proton sequencing platform.
根据本发明的一个具体示例，所述第一接头与所述第二正向引物PCR扩增后的产物包含454测序平台的A测序接头，所述第二接头与所述第二反向引物PCR扩增后的产物包含454测序平台的B测 序接头。优选的，所述第一完整测序接头为454测序平台的A测序接头，所述第二完整测序接头为454测序平台的B测序接头。According to a specific example of the present invention, the PCR-amplified product of the first linker and the second forward primer comprises an A-sequencing linker of a 454 sequencing platform, and the second linker and the second reverse primer PCR The amplified product contains the B measurement of the 454 sequencing platform Order connector. Preferably, the first complete sequencing linker is an A-sequencing linker of the 454 sequencing platform, and the second complete sequencing linker is a B-sequence linker of the 454 sequencing platform.
根据本发明的一个具体示例，当所述第一正向引物不包含正向文库标签时，用于Ion Torrentt/Proton测序平台。According to a specific example of the invention, the first forward primer is used in an Ion Torrentt/Proton sequencing platform when it does not contain a forward library tag.
根据本发明的一个具体示例，当所述第一正向引物包含正向文库标签时，用于illumina测序平台或454测序平台。According to a specific example of the invention, when the first forward primer comprises a forward library tag, it is used in an illumina sequencing platform or a 454 sequencing platform.
根据本发明的实施例，所述目标区域特异性正向引物和所述目标区域特异性反向引物的长度为18-25bp。According to an embodiment of the invention, the target region-specific forward primer and the target region-specific reverse primer are 18-25 bp in length.
根据本发明的实施例，所述目标区域特异性正向引物包括B细胞受体或T细胞受体的V基因保守区的特异性引物，所述目标区域特异性反向引物包括B细胞受体或T细胞受体的C基因保守区的特异性引物。According to an embodiment of the invention, the target region-specific forward primer comprises a specific primer for a V gene conserved region of a B cell receptor or a T cell receptor, the target region specific reverse primer comprising a B cell receptor Or a specific primer for the conserved region of the C gene of the T cell receptor.
根据本发明的一个方面，本发明提供上述引物组合物在免疫组库研究中的用途。According to one aspect of the invention, the invention provides the use of the above primer composition in immunobanking studies.
根据本发明的另一方面，本发明提供一种构建待测样品目标区域核酸测序文库的方法，包括利用上述引物组，对待测样品包含目标区域的核酸进行PCR扩增，以获得扩增产物，所述扩增产物构成所述待测样品目标区域核酸测序文库。According to another aspect of the present invention, the present invention provides a method for constructing a nucleic acid sequencing library of a target region of a sample to be tested, comprising: using the above primer set, performing PCR amplification on a nucleic acid containing a target region of the sample to be obtained, to obtain an amplification product, The amplification product constitutes a nucleic acid sequencing library of the target region of the sample to be tested.
根据本发明的实施例，所述第一正向引物和第二正向引物的摩尔比为1:1-1:10，所述第一正向引物与第二正向引物之和与第二反向引物的摩尔比为1:1。According to an embodiment of the invention, the first forward primer and the second forward primer have a molar ratio of 1:1 to 1:10, and the first forward primer and the second forward primer are combined with the second The reverse primer has a molar ratio of 1:1.
根据本发明的实施例，所述PCR反应体系为： According to an embodiment of the invention, the PCR reaction system is:
组分Component 体积(μL)Volume (μL)
QIAGEN多重PCR反应混合液QIAGEN multiplex PCR reaction mixture 2525
cDNA模板溶液cDNA template solution 1717
Q缓冲液5X Q Buffer 5X 55
第一正向引物(2pmol/ul)First forward primer (2pmol/ul) 11
第二正向引物(12pmol/ul)Second forward primer (12pmol/ul) 11
第二反向引物(14pmol/ul)Second reverse primer (14pmol/ul) 11
总体积total capacity 5050
根据本发明的实施例，所述PCR反应程序为：According to an embodiment of the invention, the PCR reaction procedure is:
Figure PCTCN2017100426-appb-000001
Figure PCTCN2017100426-appb-000001
采用本发明的引物组进行PCR扩增反应，仅需10-25个循环即可完成核酸样本的富集，循环少，反应时间短，效率高。The PCR amplification reaction using the primer set of the present invention can complete the enrichment of the nucleic acid sample in only 10-25 cycles, with less cycle, short reaction time and high efficiency.
根据本发明的实施例，构建待测样品目标区域核酸测序文库的方法进一步包括以下步骤：According to an embodiment of the present invention, the method of constructing a sample region nucleic acid sequencing library of a sample to be tested further comprises the steps of:
(1)针对每一个目标区域，均设计适用于扩增所述目标区域核 酸扩增的目标区域特异性正/反向引物，第一接头和第二接头、第二正向引物、第二反向引物和正/反文库标签，将目标区域特异性正/反向引物、第一接头和第二接头以及正/反文库标签，进行合成，得到第一正向引物和第一反向引物；(1) For each target area, it is designed to amplify the target area core Acid-amplified target region-specific forward/reverse primers, first and second adaptors, second forward primers, second reverse primers, and forward/reverse library tags, target region-specific positive/reverse primers, The first linker and the second linker and the positive/negative library tag are synthesized to obtain a first forward primer and a first reverse primer;
本发明采用完整测序接头的全部或部分作为第一正向引物和第二正向引物、第一反向引物和第二反向引物的退火互补区，无需引入外源序列，得到测序结果更加精准，减少了测序成本，降低了后续数据分析的难度。The invention adopts all or part of the complete sequencing linker as the annealing complementary region of the first forward primer and the second forward primer, the first reverse primer and the second reverse primer, and the sequencing result is more accurate without introducing the foreign sequence. , reducing the cost of sequencing and reducing the difficulty of subsequent data analysis.
(2)将待测样品的RNA、第一反向引物和cDNA合成试剂混合进行cDNA 1st合成；(2) mixing the RNA of the sample to be tested, the first reverse primer and the cDNA synthesis reagent for cDNA 1st synthesis;
现有技术需要在PCR时才能将文库标签加入样品中，若实验过程出现样品间相互污染，即无法识别。而本发明在反转录时将样本文库标签通过反转录引入cDNA 1st，同时把第二反向引物的结合位点引入了cDNA 1st中，后续PCR反应中只要使用第二反向引物就可以进行扩增反应。也即本发明在反转录时即将每个样品加了样本文库标签，避免了在后续实验过程样品间的相互污染。In the prior art, it is necessary to add a library tag to a sample at the time of PCR, and if the samples are mutually contaminated during the experiment, they are not recognized. In the present invention, the sample library tag is introduced into the cDNA 1st by reverse transcription during reverse transcription, and the binding site of the second reverse primer is introduced into the cDNA 1st, and the second reverse primer can be used in the subsequent PCR reaction. The amplification reaction is carried out. That is, the present invention adds a sample library label to each sample at the time of reverse transcription, thereby avoiding mutual contamination between samples in subsequent experimental procedures.
优选的，所述混合后在PCR管中进行PCR反应。Preferably, the PCR reaction is carried out in a PCR tube after the mixing.
优选的，所述待测样品RNA为信使RNA(mRNA)。Preferably, the sample RNA to be tested is messenger RNA (mRNA).
优选的，所述cDNA合成试剂包括脱氧核糖核苷三磷酸混合液(dNTP mi)、5x1链缓冲液(5×1st strand buffer)、二硫苏糖醇(DTT)、RNA酶抑制剂(RNAseOUT)、反转录酶III(SuperScriptTM III)、RNA酶混合物(RNase mix)。Preferably, the cDNA synthesis reagent comprises a mixture of deoxyribonucleoside triphosphate (dNTP mi), 5x1 chain buffer (5×1st strand buffer), dithiothreitol (DTT), and RNase inhibitor (RNAseOUT). , Reverse transcriptase III (SuperScriptTM III), RNase mix.
优选的，所述合成步骤包括：先将待测基因组RNA、第一反向 引物以及dNTP mix混合，进行RNA变性；变性结束后，再加入5×1st strand buffer、DTT、RNAseOUT、SuperScriptTM III，进行PCR反应，反应程序为50℃50min，70℃15min；PCR反应结束后，加入RNase mix，37℃孵育30min，得到cDNA 1st。Preferably, the synthesizing step comprises: first detecting the genomic RNA to be tested, and first reversing Primer and dNTP mix were mixed for RNA denaturation; after denaturation, 5×1st strand buffer, DTT, RNAseOUT, SuperScriptTM III were added to carry out PCR reaction. The reaction procedure was 50°C for 50min and 70°C for 15min. After the PCR reaction was completed, the reaction was added. The RNase mix was incubated at 37 ° C for 30 min to obtain cDNA 1 st.
(3)将合成得到的cDNA 1st与第一正向引物、第二引物组以及PCR反应试剂混合进行PCR扩增，所述第一正向引物和第二正向引物的摩尔比为1:1-1:10，所述第一正向引物与第二正向引物之和与第二反向引物的摩尔数为1:1。(3) mixing the synthesized cDNA 1st with the first forward primer, the second primer set, and the PCR reaction reagent for PCR amplification, and the molar ratio of the first forward primer to the second forward primer is 1:1. -1:10, the number of moles of the first forward primer and the second forward primer and the second reverse primer is 1:1.
优选的，所述混合后在PCR管中进行PCR反应。Preferably, the PCR reaction is carried out in a PCR tube after the mixing.
优选的，所述PCR反应程序为：Preferably, the PCR reaction procedure is:
Figure PCTCN2017100426-appb-000002
Figure PCTCN2017100426-appb-000002
(4)PCR反应结束后，回收PCR产物，进行磁珠纯化，按照PCR产物体积的0.8-1倍体积加入磁珠，纯化产物即为核酸文库。(4) After the completion of the PCR reaction, the PCR product is recovered and subjected to magnetic bead purification, and the magnetic beads are added in a volume of 0.8-1 volume of the PCR product, and the purified product is a nucleic acid library.
优选的，所述磁珠纯化采用AMPure XP DNA纯化试剂盒进行纯化。 Preferably, the magnetic bead purification is purified using an AMPure XP DNA purification kit.
本发明提供的构建待测样品目标区域核酸测序文库的方法，无需额外的测序接头连接、连接产物纯化的步骤，也无需单管或高通量芯片等特定设备辅助PCR反应，使用普通的PCR设备即可完成产物的富集过程，操作简单，成本低、减低样品损失，效率高，循环次数少，耗时短，减少人为操作误差，测序结果准确、可靠，可重复性好。The method for constructing a nucleic acid sequencing library of a target region of a sample to be tested does not require an additional step of sequencing a linker, a step of purifying the product, and does not require a specific device such as a single tube or a high-throughput chip to assist the PCR reaction, and uses a common PCR device. The product enrichment process can be completed, the operation is simple, the cost is low, the sample loss is reduced, the efficiency is high, the cycle number is small, the time is short, the human error is reduced, the sequencing result is accurate, reliable, and the repeatability is good.
根据本发明的一些具体示例，本发明的构建待测样品目标区域核酸测序文库的方法还可以包括以下步骤：According to some specific examples of the present invention, the method for constructing a nucleic acid sequencing library of a sample region to be tested of the present invention may further comprise the following steps:
将合成得到的第一引物组进行预PCR扩增以及凝胶电泳检测，以验证第一引物组是否符合要求，其中以是否能扩增出符合设计大小的单一条带为判定标准。The first primer set obtained by the synthesis is subjected to pre-PCR amplification and gel electrophoresis detection to verify whether the first primer set meets the requirements, and whether a single band conforming to the design size can be amplified is used as a criterion.
根据本发明的一些具体示例，构建待测样品目标区域核酸测序文库的方法进一步包括以下步骤：According to some specific examples of the present invention, the method of constructing a sample region nucleic acid sequencing library of a sample to be tested further comprises the steps of:
将构建好的核酸文库进行文库质量检测，可以使用例如Agilent2100Bioanalyzer或Caliper Bioanalyzer，或ABI StepOnerPlus Real-Time PCR System进行质量浓度、片段大小分布和摩尔浓度的检测。经检测合格后，即可上机测序。The constructed nucleic acid library is subjected to library quality detection, and the mass concentration, fragment size distribution, and molar concentration can be detected using, for example, an Agilent 2100 Bioanalyzer or a Caliper Bioanalyzer, or an ABI StepOnerPlus Real-Time PCR System. After passing the test, it can be sequenced on the machine.
根据本发明的另一方面，本发明还提供一种确定待测样品目标区域核酸序列的方法。According to another aspect of the present invention, the present invention also provides a method of determining a nucleic acid sequence of a target region of a sample to be tested.
根据本发明的实施例，参照图4，该方法包括以下步骤：According to an embodiment of the invention, referring to Figure 4, the method comprises the following steps:
S101：构建待测样品的目标区域核酸测序文库S101: constructing a target region nucleic acid sequencing library of the sample to be tested
根据上述构建待测样品目标区域核酸测序文库的方法，构建待测样品的目标区域核酸测序文库。 According to the above method for constructing a nucleic acid sequencing library of a target region of a sample to be tested, a target region nucleic acid sequencing library of the sample to be tested is constructed.
S102：对目标区域核酸测序文库进行测序S102: Sequencing a target region nucleic acid sequencing library
对所述待测样品的目标区域核酸测序文库进行测序，以便获得测序结果。The target region nucleic acid sequencing library of the sample to be tested is sequenced to obtain sequencing results.
根据本发明的一些实施例，当所述第一接头与所述第二正向引物PCR扩增后的产物包含illumina测序平台的P5测序接头，所述第二接头与所述第二反向引物PCR扩增后的产物包含illumina测序平台的P7测序接头时，利用Illumina测序平台进行所述测序。According to some embodiments of the present invention, the PCR-amplified product of the first linker and the second forward primer comprises a P5 sequencing linker of an illumina sequencing platform, the second linker and the second reverse primer When the PCR-amplified product contains the P7 sequencing linker of the illumina sequencing platform, the sequencing is performed using an Illumina sequencing platform.
或者当所述第一接头与所述第二正向引物PCR扩增后的产物包含illumina测序平台的P7测序接头，所述第二接头与所述第二反向引物PCR扩增后的产物包含illumina测序平台的P5测序接头时，利用Illumina测序平台进行所述测序。Or the PCR-amplified product of the first linker and the second forward primer comprises a P7 sequencing linker of an illumina sequencing platform, and the product of the second linker and the second reverse primer after PCR amplification comprises The sequencing of the P5 sequencing linker of the illumina sequencing platform was performed using an Illumina sequencing platform.
根据本发明的另一些实施例，当所述第一接头与所述第二正向引物PCR扩增后的产物包含Ion Torrent/Proton测序平台的P1测序接头，所述第二接头与所述第二反向引物PCR扩增后的产物包含Ion Torrent/Proton测序平台的A测序接头时，利用Ion Torrent/Proton测序平台进行所述测序。According to further embodiments of the present invention, the PCR-amplified product of the first linker and the second forward primer comprises a P1 sequencing linker of an Ion Torrent/Proton sequencing platform, the second linker and the first When the product after PCR amplification of the two reverse primers contains the A-sequence linker of the Ion Torrent/Proton sequencing platform, the sequencing was performed using the Ion Torrent/Proton sequencing platform.
根据本发明的另一些实施例，当所述第一接头与所述第二正向引物PCR扩增后的产物包含454测序平台的A测序接头，所述第二接头与所述第二反向引物PCR扩增后的产物包含454测序平台的B测序接头时，利用454测序平台进行所述测序。According to further embodiments of the present invention, the PCR-amplified product of the first linker and the second forward primer comprises an A-sequence linker of a 454 sequencing platform, the second linker and the second reverse When the primer-amplified product contains the B-sequencing linker of the 454 sequencing platform, the sequencing is performed using a 454 sequencing platform.
S103：确定待测样品目标区域核酸序列S103: determining a nucleic acid sequence of a target region of the sample to be tested
基于所述测序结果，确定待测样品目标区域核酸的序列。Based on the sequencing result, the sequence of the nucleic acid of the target region of the sample to be tested is determined.
根据本发明的再一方面，本发明还提供一种同时确定多个待测 样品的目标区域核酸序列的方法。According to still another aspect of the present invention, the present invention further provides for simultaneously determining a plurality of to-be-tested Method of nucleic acid sequence of a target region of a sample.
根据本发明的实施例，参照图5，该方法包括以下步骤：According to an embodiment of the invention, referring to Figure 5, the method comprises the following steps:
S201：分别独立地构建多个待测样品中的每一个目标区域核酸测序文库S201: independently constructing each target region nucleic acid sequencing library independently of each sample to be tested
针对所述多个待测样品中的每一个，分别独立地根据上述构建待测样品目标区域核酸测序文库的方法，构建待测样品的目标区域核酸测序文库，其中，所述多个待测样品的正/反文库标签相互不同，所述多个为至少2个。Constructing, for each of the plurality of samples to be tested, a target region nucleic acid sequencing library of the sample to be tested, according to the method for constructing the nucleic acid sequencing library of the target region of the sample to be tested, wherein the plurality of samples to be tested are The positive/negative library tags are different from each other, and the plurality of the plurality are at least two.
S202：将多个待测样品的目标区域核酸测序文库混合S202: mixing a target region nucleic acid sequencing library of a plurality of samples to be tested
将多个待测样品的目标区域核酸测序文库混合，以便获得混合文库。A plurality of target region nucleic acid sequencing libraries of the samples to be tested are mixed to obtain a hybrid library.
S203：对混合文库进行测序S203: Sequencing the mixed library
对所述混合文库进行测序，以便获得测序结果，所述测序结果包括所述多个待测样品的目标区域核酸文库序列和所述正/反文库标签。The hybrid library is sequenced to obtain sequencing results including a target region nucleic acid library sequence of the plurality of samples to be tested and the positive/negative library tag.
根据本发明的一些实施例，当所述第一接头与所述第二正向引物PCR扩增后的产物包含illumina测序平台的P5测序接头，所述第二接头与所述第二反向引物PCR扩增后的产物包含illumina测序平台的P7测序接头时，利用Illumina测序平台进行所述测序。According to some embodiments of the present invention, the PCR-amplified product of the first linker and the second forward primer comprises a P5 sequencing linker of an illumina sequencing platform, the second linker and the second reverse primer When the PCR-amplified product contains the P7 sequencing linker of the illumina sequencing platform, the sequencing is performed using an Illumina sequencing platform.
或者当所述第一接头与所述第二正向引物PCR扩增后的产物包含illumina测序平台的P7测序接头，所述第二接头与所述第二反向引物PCR扩增后的产物包含illumina测序平台的P5测序接头时，利用Illumina测序平台进行所述测序。 Or the PCR-amplified product of the first linker and the second forward primer comprises a P7 sequencing linker of an illumina sequencing platform, and the product of the second linker and the second reverse primer after PCR amplification comprises The sequencing of the P5 sequencing linker of the illumina sequencing platform was performed using an Illumina sequencing platform.
根据本发明的另一些实施例，当所述第一接头与所述第二正向引物PCR扩增后的产物包含Ion Torrent/Proton测序平台的P1测序接头，所述第二接头与所述第二反向引物PCR扩增后的产物包含Ion Torrent/Proton测序平台的A测序接头时，利用Ion Torrent/Proton测序平台进行所述测序。According to further embodiments of the present invention, the PCR-amplified product of the first linker and the second forward primer comprises a P1 sequencing linker of an Ion Torrent/Proton sequencing platform, the second linker and the first When the product after PCR amplification of the two reverse primers contains the A-sequence linker of the Ion Torrent/Proton sequencing platform, the sequencing was performed using the Ion Torrent/Proton sequencing platform.
根据本发明的另一些实施例，当所述第一接头与所述第二正向引物PCR扩增后的产物包含454测序平台的A测序接头，所述第二接头与所述第二反向引物PCR扩增后的产物包含454测序平台的B测序接头，利用454测序平台进行所述测序。According to further embodiments of the present invention, the PCR-amplified product of the first linker and the second forward primer comprises an A-sequence linker of a 454 sequencing platform, the second linker and the second reverse The PCR amplified product of the primer contained the B-sequencing linker of the 454 sequencing platform, which was sequenced using the 454 sequencing platform.
S204：确定多个待测样品中每一个的目标区域核酸序列S204: determining a target region nucleic acid sequence of each of the plurality of samples to be tested
基于所述正/反文库标签对所述多个待测样品的目标区域核酸测序文库的序列进行区分，并确定所述多个待测样品的每一个的目标区域核酸序列。A sequence of a target region nucleic acid sequencing library of the plurality of samples to be tested is distinguished based on the positive/negative library tag, and a target region nucleic acid sequence of each of the plurality of samples to be tested is determined.
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面示例仅用于解释本发明，而不能理解为对本发明的限制。除另有交待，以下实施例中涉及的未特别交待的试剂、序列(接头、标签和引物)、软件及仪器，都是常规市售产品或者开源的。The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be construed as limiting. Unless otherwise stated, the reagents, sequences (linkers, tags and primers), software and instruments not specifically addressed in the following examples are conventionally commercially available or open source.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、 材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Moreover, the specific features, structures, Materials or features may be combined in any suitable manner in any one or more embodiments or examples.
实施例一Embodiment 1
设计引物Design primer
以IMGT数据库的参考序列为依据，设计BCR、TCR的特异性引物。第一反向引物在C基因的保守区设计，同时所述第一反向引物在C基因特异引物的5'端连接反向文库标签，反向文库标签的5'端连接第二接头，见图1中第一反向引物。第一正向引物包括V基因保守区的特异性引物和第一接头，见图2中第一正向引物，或者包括V基因保守区的特异性引物、正向文库标签和第一接头，见图3中第一正向引物。正/反文库标签均用于区分不同的样本。第二正向引物包含第一退火，见图2所示，第二反向引物包含第二退火，见图3所示。Specific primers for BCR and TCR were designed based on the reference sequence of the IMGT database. The first reverse primer is designed in the conserved region of the C gene, and the first reverse primer is ligated to the reverse library tag at the 5' end of the C gene-specific primer, and the 5' end of the reverse library tag is ligated to the second linker, see The first reverse primer in Figure 1. The first forward primer includes a specific primer and a first linker of the conserved region of the V gene, see the first forward primer in Figure 2, or a specific primer including a conserved region of the V gene, a forward library tag, and a first linker, see The first forward primer in Figure 3. Both positive/negative library tags are used to distinguish between different samples. The second forward primer comprises a first anneal, as shown in Figure 2, and the second reverse primer comprises a second anneal, as shown in Figure 3.
在进行PCR反应时，主要起作用的是上下游两端的接头，也就是第二正向引物和第二反向引物。本发明并没有引入外源的一段序列作为PCR结合位置，而是使用了测序接头的部分序列，即完整测序接头的3'端16-25bp作为第一正向引物和第二正向引物、第一反向引物和第二反向引物退火互补区，正是由于这个特殊的设计使得本方法不需要进行末端修复，加‘A’和加接头等过程，直接进行目的产物富集同时完成文库构建。举例如下，设计2个样本引物组序列。When performing the PCR reaction, the main function is the linker at the upper and lower ends, that is, the second forward primer and the second reverse primer. The present invention does not introduce a foreign sequence as a PCR binding site, but uses a partial sequence of the sequencing linker, that is, the 3' end 16-25 bp of the complete sequencing linker as the first forward primer and the second forward primer, A reverse primer and a second reverse primer anneal the complementary region. It is precisely because of this special design that the method does not require end repair, adding 'A' and adding a linker, directly performing the target product enrichment and completing the library construction. . For example, two sample primer set sequences were designed as follows.
表1 2组引物组序列 Table 1 2 sets of primer set sequences
Figure PCTCN2017100426-appb-000003
Figure PCTCN2017100426-appb-000003
Figure PCTCN2017100426-appb-000004
Figure PCTCN2017100426-appb-000004
样本文库制备Sample library preparation
1、密度梯度分离人外周血单个核细胞1. Density gradient separation of human peripheral blood mononuclear cells
使用含抗凝剂的无菌采血管进行采血(抗凝剂一般有乙二胺四乙酸EDTA)，利用淋巴细胞分离液，比如Ficoll-Paque PLUS淋巴细胞分离液或Percoll淋巴细胞分离液进行密度梯度分离PBMC。 Use a sterile blood collection tube containing an anticoagulant for blood collection (the anticoagulant is usually EDTA), using a lymphocyte separation solution such as Ficoll-Paque PLUS lymphocyte separation solution or Percoll lymphocyte separation solution for density gradient PBMC were isolated.
2、RNA提取2, RNA extraction
采用Trizol regeant方法或者提取试剂盒等提取总RNA。Total RNA was extracted using a Trizol regeant method or an extraction kit or the like.
3、DNA消化3, DNA digestion
使用DNA消化酶将残留在RNA样本中的DNA去除。DNA remaining in the RNA sample is removed using DNA digestive enzymes.
4、cDNA 1st合成4, cDNA 1st synthesis
使用反转录酶和反转录引物对RNA进行反转录，得到cDNA 1链，并加入RNA消化酶，消化RNA。如图1所示，第一反向引物对mRNA进行反转录。RNA is reverse transcribed using reverse transcriptase and reverse transcription primers to obtain a cDNA 1 strand, and an RNA digestive enzyme is added to digest the RNA. As shown in Figure 1, the first reverse primer reverse transcribes mRNA.
4.1在0.2ml PCR管内按以下体系配制(1个样品)。4.1 Prepared in a 0.2 ml PCR tube according to the following system (1 sample).
组分Component 体积(μL)Volume (μL)
已消化DNA后得到的RNA(20ng/ul)RNA obtained after digesting DNA (20ng/ul) 1010
第一反向引物(2pmol/ul)First reverse primer (2pmol/ul) 11
dNTP mix(10mM)dNTP mix (10mM) 11
经焦碳酸二乙酯处理水(DEPC水)Treated water by diethyl pyrocarbonate (DEPC water) 11
4.2将混合物置于65℃5分钟以使RNA变性，变性结束，放置冰上1min，瞬时离心后，将以下混合溶液加至4.1中0.2ml PCR管。4.2 The mixture was placed at 65 ° C for 5 minutes to denature the RNA, the denaturation was completed, and placed on ice for 1 min. After centrifugation, the following mixed solution was added to a 0.2 ml PCR tube in 4.1.
组分Component 体积μLVolume μL
5x1链缓冲液(5×1st strand buffer)5x1 chain buffer (5 × 1st strand buffer) 44
100mM M二硫苏糖醇(DTT)100 mM M dithiothreitol (DTT) 11
RNA酶抑制剂(RNAseOUT)(40U/μL)RNase inhibitor (RNAseOUT) (40U/μL) 11
反转录酶III(SuperScriptTM III)(200U/μL)Reverse Transcriptase III (SuperScriptTM III) (200U/μL) 11
其中，PCR管的总体积为20μL。轻微震荡混匀。Among them, the total volume of the PCR tube was 20 μL. Mix gently with a slight shake.
4.3在PCR仪上反应4.3 Reaction on the PCR machine
50℃  50min50 ° C 50 min
70℃  15min70 ° C 15 min
4.4加入1μLRNase mix，轻微震荡并彻底混匀，37℃孵育30min。4.4 Add 1 μL of RNase mix, shake gently and mix thoroughly, incubate for 30 min at 37 °C.
4.5按照说明书，使用AMpure XP纯化cDNA1链样品，以18ul无核酸酶水回收cDNA1链样品。4.5 Purification of the cDNA1 chain sample using AMpure XP according to the instructions, and recovery of the cDNA1 chain sample with 18 ul of nuclease-free water.
此步骤结束之后，可以将反应混合物储存于-20℃。After the end of this step, the reaction mixture can be stored at -20 °C.
5、特殊设计的PCR条件富集目标区域并完成文库构建5. Specially designed PCR conditions enrich the target region and complete library construction
PCR使用的引物包括第一正向引物(V区正向引物)及第二引物组，其中第一正向引物和第二正向引物的摩尔比为1:1-1:10，所述第一正向引物与第二正向引物之和与第二反向引物的摩尔比为1:1。此步PCR结束后，目的片段两端含有完整的测序接头，文库构建结束。见图2和图3，第一正向引物和第二引物组对cDNA 1st进行PCR。The primer used in the PCR includes a first forward primer (V region forward primer) and a second primer set, wherein the first forward primer and the second forward primer have a molar ratio of 1:1 to 1:10. The molar ratio of the sum of the forward primer to the second forward primer to the second reverse primer is 1:1. After the end of this step of PCR, the ends of the target fragment contain the complete sequencing linker, and the library construction is completed. 2 and 3, the first forward primer and the second primer set were subjected to PCR for cDNA 1st.
5.1将合成得到的cDNA 1st作为模板PCR富集。将以下混合溶液在200μL的PCR管中配制PCR反应体系。5.1 The cDNA 1st obtained by the synthesis was PCR-enriched as a template. The following mixed solution was used to prepare a PCR reaction system in a 200 μL PCR tube.
Figure PCTCN2017100426-appb-000005
Figure PCTCN2017100426-appb-000005
Figure PCTCN2017100426-appb-000006
Figure PCTCN2017100426-appb-000006
PCR反应条件为：The PCR reaction conditions are:
Figure PCTCN2017100426-appb-000007
Figure PCTCN2017100426-appb-000007
6、测序文库纯化6. Sequencing library purification
构建的测序文库可用三种方法进行纯化，分别为磁珠纯化、纯化柱纯化和琼脂糖胶电泳回收纯化。The constructed sequencing library can be purified by three methods, namely magnetic bead purification, purification column purification and agarose gel electrophoresis recovery and purification.
PCR反应结束后，将PCR产物转移至1个1.5mL离心管中,用AMPure XP DNA纯化试剂盒(SPRI beads)纯化扩增后的样品。After the end of the PCR reaction, the PCR product was transferred to a 1.5 mL centrifuge tube, and the amplified sample was purified using AMPure XP DNA Purification Kit (SPRI beads).
7、文库检测7, library testing
Agilent 2100Bioanalyzer analysis system检测文库插入片段大小及含量；Q-PCR精确定量文库的浓度。The Agilent 2100 Bioanalyzer analysis system detects library insert size and content; Q-PCR accurately quantifies library concentration.
高通量测序 High-throughput sequencing
将构建的测序文库在高通量测序平台上进行测序，高通量测序平台包括Illumina Hiseq及Miseq测序平台，Roche 454测序平台及Life Technologies的Ion Torrent测序平台等。The constructed sequencing libraries were sequenced on a high-throughput sequencing platform including Illumina Hiseq and Miseq sequencing platforms, Roche 454 sequencing platform and Life Technologies' Ion Torrent sequencing platform.
测序数据分析Sequencing data analysis
将测序得到的数据进行质量过滤和长度过滤去除污染及接头序列；统计结果包括：测定的序列(Reads)长度、数据产量。然后将得到的序列进行比对分析，按照IMGT上V、(D、)J基因的定义将V、(D、)J基因找出并确定位置，并统计V、(D、)J基因的多样性。The data obtained by sequencing is subjected to mass filtration and length filtration to remove contamination and linker sequences; the statistical results include: length of the measured sequence (Reads), and data yield. Then, the obtained sequences were subjected to alignment analysis, and the V, (D,) J genes were identified and determined according to the definition of the V and (D,) J genes on IMGT, and the diversity of V, (D,) J genes was counted. Sex.
根据IMGT上的定义找出CDR3，并统计CDR3的长度和多样性。Find CDR3 according to the definition on IMGT and count the length and diversity of CDR3.
在本说明书的描述中，参考术语“一个实施例”、“具体示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。In the description of the present specification, the description of the terms "one embodiment", "specific example" and the like means that a specific feature, structure, material or characteristic described in connection with the embodiment or example is included in at least one embodiment of the invention or In the example. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.
以上实施方式仅用以说明本发明的技术方案而非限制，尽管参照以上较佳实施方式对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或等同替换都不应脱离本发明技术方案的精神和范围。 The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to be limiting, and the present invention will be described in detail with reference to the preferred embodiments of the present invention. Neither should the spirit and scope of the technical solutions of the present invention be deviated.

Claims (14)

  1. 一种引物组合物，其特征在于，包含：A primer composition comprising:
    第一引物组，所述第一引物组包含第一正向引物和第一反向引物，所述第一正向引物包含目标区域特异性正向引物和第一接头；a first primer set, the first primer set comprising a first forward primer and a first reverse primer, the first forward primer comprising a target region-specific forward primer and a first linker;
    所述第一反向引物包括目标区域特异性反向引物、反向文库标签和第二接头，所述反向文库标签位于目标区域特异性反向引物5’端和第二接头之间；The first reverse primer comprises a target region-specific reverse primer, a reverse library tag and a second linker, the reverse library tag being located between the 5' end of the target region-specific reverse primer and the second linker;
    第二引物组，所述第二引物组包含第二正向引物和第二反向引物，所述第二正向引物包含第一退火，所述第二反向引物包含第二退火；a second primer set, the second primer set comprising a second forward primer and a second reverse primer, the second forward primer comprising a first annealing, and the second reverse primer comprising a second annealing;
    所述第一接头与所述第二正向引物中的第一退火相互退火互补，所述第一接头与所述第二正向引物经PCR扩增后的产物包含第一完整测序接头；The first junction and the first annealing in the second forward primer are annealed to each other, and the PCR-amplified product of the first linker and the second forward primer comprises a first complete sequencing linker;
    所述第二接头与所述第二反向引物中的第二退火相互退火互补，所述第二接头与所述第二反向引物PCR扩增后的产物包含第二完整测序接头。The second linker and the second annealing in the second reverse primer are annealed to each other, and the second linker and the second reverse primer are PCR amplified products comprising a second complete sequencing linker.
  2. 如权利要求1所述的引物组合物，其特征在于，所述第一正向引物还包括正向文库标签，所述正向文库标签位于目标区域特异性正向引物5’端和第一接头之间。The primer composition according to claim 1, wherein said first forward primer further comprises a forward library tag located at a 5' end of the target region-specific forward primer and a first linker between.
  3. 如权利要求1所述的引物组合物，其特征在于，所述第一退火和第二退火的长度均为16-25bp。The primer composition according to claim 1, wherein the first annealing and the second annealing are each 16-25 bp in length.
  4. 如权利要求1所述的引物组合物，其特征在于，所述第一接头与所述第二正向引物经PCR扩增后的产物包含illumina测序平台的P5测序接头，所述第二接头与所述第二反向引物经PCR扩增后 的产物包含illumina测序平台的P7测序接头；The primer composition according to claim 1, wherein the PCR-amplified product of the first linker and the second forward primer comprises a P5 sequencing linker of an illumina sequencing platform, and the second linker The second reverse primer is amplified by PCR The product contains a P7 sequencing linker for the illumina sequencing platform;
    或者所述第一接头与所述第二正向引物经PCR扩增后的产物包含illumina测序平台的P7测序接头，所述第二接头与所述第二反向引物经PCR扩增后的产物包含illumina测序平台的P5测序接头；Or the PCR-amplified product of the first linker and the second forward primer comprises a P7 sequencing linker of an illumina sequencing platform, and the second linker and the second reverse primer are PCR-amplified products a P5 sequencing adaptor comprising an illumina sequencing platform;
    或者所述第一接头与所述第二正向引物经PCR扩增后的产物包含Ion Torrent/Proton测序平台的P1测序接头，所述第二接头与所述第二反向引物经PCR扩增后的产物包含Ion Torrent/Proton测序平台的A测序接头；Or the PCR-amplified product of the first linker and the second forward primer comprises a P1 sequencing linker of an Ion Torrent/Proton sequencing platform, and the second linker and the second reverse primer are PCR amplified The latter product comprises an A-sequencing linker of the Ion Torrent/Proton sequencing platform;
    或者所述第一接头与所述第二正向引物经PCR扩增后的产物包含454测序平台的A测序接头，所述第二接头与所述第二反向引物经PCR扩增后的产物包含454测序平台的B测序接头。Or the PCR-amplified product of the first linker and the second forward primer comprises an A-sequence linker of a 454 sequencing platform, and the product of the second linker and the second reverse primer after PCR amplification A B-sequencing linker comprising the 454 sequencing platform.
  5. 如权利要求1所述的引物组合物，其特征在于，当第一正向引物不包含正向文库标签时，用于Ion Torrentt/Proton测序平台。The primer composition of claim 1 for use in an Ion Torrentt/Proton sequencing platform when the first forward primer does not comprise a forward library tag.
  6. 如权利要求1所述的引物组合物，其特征在于，当所述第一正向引物包含正向文库标签时，用于illumina测序平台或454测序平台。The primer composition according to claim 1, wherein when the first forward primer comprises a forward library tag, it is used in an illumina sequencing platform or a 454 sequencing platform.
  7. 如权利要求1所述的引物组合物，其特征在于，所述目标区域特异性正向引物包括B细胞受体或T细胞受体的V基因保守区的特异性引物，所述目标区域特异性反向引物包括B细胞受体或T细胞受体的C基因保守区的特异性引物。The primer composition according to claim 1, wherein said target region-specific forward primer comprises a specific primer for a V gene conserved region of a B cell receptor or a T cell receptor, said target region specificity The reverse primer includes a specific primer for the conserved region of the C gene of the B cell receptor or T cell receptor.
  8. 权利要求1所述的引物组合物在免疫组库研究中的用途。Use of the primer composition of claim 1 in an immunobank study.
  9. 一种构建待测样品目标区域核酸测序文库的方法，其特征在于，利用权利要求1-7任一项所述的引物组，对待测样品包含目标区域的核酸进行PCR扩增，以获得扩增产物，所述扩增产物构成所 述待测样品目标区域核酸测序文库。A method for constructing a nucleic acid sequencing library of a target region of a sample to be tested, characterized in that, by using the primer set according to any one of claims 1 to 7, a sample containing a nucleic acid of a target region is subjected to PCR amplification to obtain amplification. Product, the amplification product constitutes The nucleic acid sequencing library of the target region of the sample to be tested is described.
  10. 根据权利要求9所述的方法，其特征在于，所述第一正向引物和所述第二正向引物的摩尔比为1:1-1:10，所述第一正向引物与所述第二正向引物之和与所述第二反向引物的摩尔比为1:1。The method according to claim 9, wherein a molar ratio of said first forward primer to said second forward primer is 1:1 to 1:10, said first forward primer and said The molar ratio of the sum of the second forward primer to the second reverse primer is 1:1.
  11. 根据权利要求9所述的方法，其特征在于，所述PCR扩增的反应程序为：The method according to claim 9, wherein the PCR amplification reaction procedure is:
    Figure PCTCN2017100426-appb-100001
    Figure PCTCN2017100426-appb-100001
  12. 根据权利要求9所述的方法，其特征在于，进一步包括以下步骤：The method of claim 9 further comprising the steps of:
    (1)针对每一个目标区域，均设计适用于扩增所述目标区域核酸扩增的目标区域特异性正/反向引物，第一接头和第二接头、第二正/反向引物和正/反文库标签，将目标区域特异性正/反向引物、第一接头和第二接头以及正/反文库标签，进行合成，得到第一正向引物和第一反向引物；(1) For each target region, a target region-specific positive/reverse primer suitable for amplifying the nucleic acid amplification of the target region is designed, the first linker and the second linker, the second forward/reverse primer, and the positive/reverse/ The reverse library tag, the target region-specific positive/reverse primer, the first linker and the second linker, and the positive/negative library tag are synthesized to obtain a first forward primer and a first reverse primer;
    (2)将待测基因组RNA、第一反向引物和cDNA合成试剂混合，以进行cDNA 1st合成； (2) mixing the genomic RNA to be tested, the first reverse primer and the cDNA synthesis reagent for cDNA 1st synthesis;
    (3)将合成得到的cDNA 1st与第一正向引物、第二引物组以及PCR反应试剂混合进行PCR扩增，所述第一正向引物和第二正向引物的摩尔比为1:1-1:10，所述第一正向引物与第二正向引物之和与第二反向引物的摩尔数为1:1；(3) mixing the synthesized cDNA 1st with the first forward primer, the second primer set, and the PCR reaction reagent for PCR amplification, and the molar ratio of the first forward primer to the second forward primer is 1:1. -1:10, the sum of the first forward primer and the second forward primer and the second reverse primer are 1:1;
    (4)PCR反应结束后，回收PCR产物，进行磁珠纯化，按照PCR产物体积的0.8-1倍体积加入磁珠，纯化产物即为核酸文库。(4) After the completion of the PCR reaction, the PCR product is recovered and subjected to magnetic bead purification, and the magnetic beads are added in a volume of 0.8-1 volume of the PCR product, and the purified product is a nucleic acid library.
  13. 一种确定待测样品目标区域核酸序列的方法，其特征在于，包括以下步骤：A method for determining a nucleic acid sequence of a target region of a sample to be tested, characterized in that it comprises the following steps:
    根据权利要求9-12任一项所述的方法，构建待测样品的目标区域核酸测序文库；The method according to any one of claims 9 to 12, wherein a target region nucleic acid sequencing library of the sample to be tested is constructed;
    对所述待测样品的目标区域核酸测序文库进行测序，以获得测序结果；Sequencing the target region nucleic acid sequencing library of the sample to be tested to obtain a sequencing result;
    以及基于所述测序结果，确定待测样品目标区域核酸的序列。And determining a sequence of the nucleic acid of the target region of the sample to be tested based on the sequencing result.
  14. 一种同时确定多个待测样品的目标区域核酸序列的方法，其特征在于，包括以下步骤：A method for simultaneously determining a target region nucleic acid sequence of a plurality of samples to be tested, comprising the steps of:
    针对所述多个待测样品中的每一个，分别独立地根据权利要求9-12任一项所述的方法，构建待测样品的目标区域核酸测序文库，其中，所述多个待测样品的正/反文库标签互不相同，所述多个为至少2个；Determining, according to the method of any one of claims 9-12, a target region nucleic acid sequencing library of the sample to be tested, wherein the plurality of samples to be tested are independently for each of the plurality of samples to be tested The positive/negative library tags are different from each other, and the plurality of the plurality of tags are at least two;
    将所述多个待测样品的目标区域核酸测序文库混合，以获得混合文库；Mixing the target region nucleic acid sequencing libraries of the plurality of samples to be tested to obtain a mixed library;
    对所述混合文库进行测序，以获得测序结果，所述测序结果包括所述多个待测样品的目标区域核酸文库序列和所述正/反文库标签； Sequencing the hybrid library to obtain sequencing results, the sequencing result including a target region nucleic acid library sequence of the plurality of samples to be tested and the positive/negative library tag;
    以及基于所述正/反文库标签对所述多个待测样品的目标区域核酸测序文库序列进行区分，并确定所述多个待测样品的每一个的目标区域核酸序列。 And distinguishing the target region nucleic acid sequencing library sequences of the plurality of samples to be tested based on the positive/negative library tag, and determining a target region nucleic acid sequence of each of the plurality of samples to be tested.
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