WO2018095108A1 - 引物组合物、其用途、构建文库和确定核酸序列的方法 - Google Patents

引物组合物、其用途、构建文库和确定核酸序列的方法 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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English (en)
French (fr)
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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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    • 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

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  • 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

引物组合物及其用于构建文库、确定核酸序列的用途，其中该引物组合物包含：第一引物组，第一引物组包含第一正向引物和第一反向引物，第一正向引物包括目标区域特异性正向引物和第一接头；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；第二引物组，第二引物组包含第二正向引物和第二反向引物，第二正向引物包含第一退火，第二反向引物包含第二退火；第一接头与第二正向引物中的第一退火以及第二接头与第二反向引物中的第二退火相互退火互补，第一接头与第二正向引物以及第二接头与第二反向引物经PCR扩增后的产物包含完整的测序接头。

Description

引物组合物、其用途、构建文库和确定核酸序列的方法 技术领域
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。
背景技术
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***的平衡。免疫***是机体抵御病原菌入侵与免疫调节的重要***，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。
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。
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。
发明内容
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。
因此，本发明一方面提供一种引物组合物，包含：
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；
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；
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；
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；
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。
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。
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。
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：
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；
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；
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。
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：
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；
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；
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；
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。
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。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中
图1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。
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。
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。
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。
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。
具体实施方式
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。
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。
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：
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；
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；
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；
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；
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
根据本发明的实施例，所述PCR反应体系为：
组分 体积(μL)
QIAGEN多重PCR反应混合液 25
cDNA模板溶液 17
Q缓冲液5X 5
第一正向引物(2pmol/ul) 1
第二正向引物(12pmol/ul) 1
第二反向引物(14pmol/ul) 1
总体积 50
根据本发明的实施例，所述PCR反应程序为：
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。
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：
(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；
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。
(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；
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。
优选的，所述混合后在PCR管中进行PCR反应。
优选的，所述待测样品RNA为信使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)。
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。
(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。
优选的，所述混合后在PCR管中进行PCR反应。
优选的，所述PCR反应程序为：
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。
优选的，所述磁珠纯化采用AMPure XP DNA纯化试剂盒进行纯化。
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。
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：
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。
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：
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。
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。
根据本发明的实施例，参照图4，该方法包括以下步骤：
S101：构建待测样品的目标区域核酸测序文库
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。
S102：对目标区域核酸测序文库进行测序
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。
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。
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。
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。
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。
S103：确定待测样品目标区域核酸序列
基于所述测序结果，确定待测样品目标区域核酸的序列。
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。
根据本发明的实施例，参照图5，该方法包括以下步骤：
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
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。
S202：将多个待测样品的目标区域核酸测序文库混合
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。
S203：对混合文库进行测序
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。
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。
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。
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。
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。
S204：确定多个待测样品中每一个的目标区域核酸序列
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。
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。
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。
实施例一
设计引物
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。
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。
表1 2组引物组序列
Figure PCTCN2017100426-appb-000003
Figure PCTCN2017100426-appb-000004
样本文库制备
1、密度梯度分离人外周血单个核细胞
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。
2、RNA提取
采用Trizol regeant方法或者提取试剂盒等提取总RNA。
3、DNA消化
使用DNA消化酶将残留在RNA样本中的DNA去除。
4、cDNA 1st合成
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。
4.1在0.2ml PCR管内按以下体系配制(1个样品)。
组分 体积(μL)
已消化DNA后得到的RNA(20ng/ul) 10
第一反向引物(2pmol/ul) 1
dNTP mix(10mM) 1
经焦碳酸二乙酯处理水(DEPC水) 1
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。
组分 体积μL
5x1链缓冲液(5×1st strand buffer) 4
100mM M二硫苏糖醇(DTT) 1
RNA酶抑制剂(RNAseOUT)(40U/μL) 1
反转录酶III(SuperScriptTM III)(200U/μL) 1
其中，PCR管的总体积为20μL。轻微震荡混匀。
4.3在PCR仪上反应
50℃  50min
70℃  15min
4.4加入1μLRNase mix，轻微震荡并彻底混匀，37℃孵育30min。
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。
此步骤结束之后，可以将反应混合物储存于-20℃。
5、特殊设计的PCR条件富集目标区域并完成文库构建
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。
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。
Figure PCTCN2017100426-appb-000005
Figure PCTCN2017100426-appb-000006
PCR反应条件为：
Figure PCTCN2017100426-appb-000007
6、测序文库纯化
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。
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。
7、文库检测
Agilent 2100Bioanalyzer analysis system检测文库***片段大小及含量；Q-PCR精确定量文库的浓度。
高通量测序
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。
测序数据分析
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。
根据IMGT上的定义找出CDR3，并统计CDR3的长度和多样性。
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。
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。

Claims (14)

  1. 一种引物组合物，其特征在于，包含：
    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；
    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；
    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；
    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；
    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。
  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。
  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。
  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；
    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；
    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；
    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。
  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。
  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。
  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。
  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。
  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。
  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。
  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：
    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：
    (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；
    (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；
    (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；
    (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。
  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：
    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；
    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；
    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。
  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：
    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；
    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；
    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；
    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。
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CN110904204A (zh) * 2018-09-14 2020-03-24 上海吉凯基因化学技术有限公司 引物组合及其用途
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