CN109136329A - A kind of construction method of unimolecule label TCR immune group library high-throughput sequencing library - Google Patents
A kind of construction method of unimolecule label TCR immune group library high-throughput sequencing library Download PDFInfo
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- CN109136329A CN109136329A CN201810918681.8A CN201810918681A CN109136329A CN 109136329 A CN109136329 A CN 109136329A CN 201810918681 A CN201810918681 A CN 201810918681A CN 109136329 A CN109136329 A CN 109136329A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
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- 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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- 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
Abstract
The invention belongs to field of biotechnology, and in particular to a kind of construction method of unimolecule label TCR immune group library high-throughput sequencing library.Utilize SMART technology and specific primer, it specific can capture entire V (D) the J Variable Area for having no low abundance TCR- α and TCR- β transcript in preference amplification source of people RNA, amplification TCR- α or TCR- β can also be individually captured, or captures amplification TCR- α and TCR- β simultaneously.The present invention can utmostly eliminate the base mistake generated in amplification or sequencing procedure, restore molecule amount and gene expression abundance in original sample, and method is simple, and the library of foundation is suitable for multiple microarray datasets.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of unimolecule label TCR immune group library high-flux sequence text
The construction method in library.
Background technique
Second generation sequencing technologies (NGS) have been increasingly being applied to human T cell receptor (TCR, T cell
Receptor) the research in immune group library.But, when generating TCR NGS sequencing library using conventional method, or only include
A part of the variable region TCR, or need to use complicated multiplex PCR, Preference it can expand some region.Therefore how complete
It is whole and without preference capture and expand the complete variable region V (D) J in TCR transcript, have to the research in immune group library great
Meaning.
Clontech under TakaraHumanTCR a/b Profiling kit utilizes SMART skill
The strategy of art and similar 5 ' RACE can capture and variable without complete V (D) J in preference amplification TCR- α and TCR- β transcript
Area.Substantially steps are as follows: 1, obtaining the T cell of total serum IgE or purifying；2, using TCR dT Primer andv4
Oligonucleotide carries out First-strand cDNA synthesis；3, PCR1 expands the entire variable region TCR- α and TCR- β and absolutely
Most of constant region cDNA sequence；4, PCR2 utilizes the entire variable region of half-nest type primer amplification and portion constant area and adjunction head；
5, purifying sorting however.The technology carries out RT using dT Primer, RT can only be carried out with the RNA with Poly A tail, to low rich
The transcript of degree has a limitation, while not having the measure of unimolecule label correction, thus this method have it is to be optimized.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of unimolecule label TCR immune group library high-flux sequence
The construction method in library utilizes unimolecule label technique, it is therefore an objective to most on the basis of existing TCR immune group library banking process
Big degree eliminates the base mistake generated in amplification or sequencing procedure, so that it is rich to restore molecule amount and expression in original sample
Degree；The present invention is using TCR- α and TCR- β full length gene in primer amplified RNA sample simultaneously, can analyze comprehensively this two
The rearrangement and expressing information of a gene.
The construction method of unimolecule label TCR immune group library high-throughput sequencing library of the invention, according to the following steps into
Row:
(1) sample total serum IgE is extracted；
(2) using total serum IgE as Material synthesis First-strand cDNA, while template switch is carried out；
(3) PCR1 reaction system is added afterwards in step (2), carries out hemi-nested amplification, specific amplification target area；
(4) product purification is carried out afterwards in step (3)；
(5) PCR2 reaction system is added afterwards in step (4), carries out connector connection reaction and amplification；
(6) magnetic bead is added after step (5) amplified production and carries out product sorting purifying, product after purification is that TCR exempts from
Epidemic disease group library text library.
Wherein, the sample is source of people blood, tissue or cell.
The synthesis First-strand cDNA is synthesized using TCR α and/or β specific RT primer.
The template switch is to carry out template switch using the TS-3G primer with unimolecule label, wherein unimolecule mark
Label are the merger bases of 1-20, and annexing base is original base or modified base, the modification include thio, methylation,
LNA and hypoxanthine.
The synthesis and template switch reaction condition is 25-50 DEG C, and the reaction time is 30-90min.
The PCR1 reaction system includes buffer, and archaeal dna polymerase, dNTPs, Specific PCR primers and P5 connector draw
Object；Wherein Specific PCR primers be TCR α and/or β specificity heminested PCR primer, primer be original base composition or by
Modified base composition, the modification include thio, methylation, LNA and hypoxanthine；3 ' the end part of P5 adapter-primer
Base is complementary with the connector after the TS-3G Primed template conversion with unimolecule label, and 5 ' ends include P5 binding sequence and Read 1
Sequencing sequence.
The PCR2 reaction system includes buffer, archaeal dna polymerase, dNTPs, P7 adapter-primer and P5 adapter-primer；
Wherein P7 adapter-primer includes P7 binding sequence, and Index sequence i7 and Read2 read sequence；The P5 adapter-primer and step
Suddenly the PCR1 reaction system center tap primer of (3) is identical, or for comprising P5 binding sequence, Index sequence i5 and Read 1 is surveyed
The connector of sequence sequence.
The TCR immune group library text library size is 500-1000bp.
The immune group library text library TCR is used for Roche, Illumina, ThermoFisher, Pacific
Biosciences, Hua Da gene, Oxford Nanopore Technologies, Hua Yinkang, big desert gene high-flux sequence are flat
Platform.
Compared with prior art, the features of the present invention and beneficial effect are:
(1) the present invention provides a kind of TCR immune group library text base construction method, using SMART technology and specific primer,
It specific can capture the variable region entire V (D) J for having no low abundance TCR- α and TCR- β transcript in preference amplification source of people RNA
Domain (can individually capture amplification TCR- α or TCR- β, can also capture amplification TCR- α and TCR- β simultaneously).
(2) present invention is built during library, the dosage and reaction temperature of each reagent component of control reaction and time, and is taken
A kind of mode being simple and efficient carries out the building of high-throughput sequencing library to it, the unimolecule label of institute's band can error correction, can be with
Keep data analysis more accurate, to carry out the analysis of the β immune group library α and/or TCR.
(3) present invention takes turns PCR amplifications using 2, more acurrate, easier.
(4) banking process provided by the invention can be used for different microarray datasets, and applicability is wide, easy to spread.
Detailed description of the invention
Fig. 1 is the schematic diagram of building unimolecule label TCR immune group library high-throughput sequencing library of the invention；
Fig. 2 is the flow chart for establishing unimolecule label TCR immune group library high-throughput sequencing library of the invention；
Fig. 3 is 2% agarose gel electrophoresis detection library figure；
Wherein: M:100bp DNAladder (precious bioengineering (Dalian) Co., Ltd)；1:TCR α/β immune group library text
Library.
Specific embodiment
Now by taking Illumina platform as an example, the present invention is further described in conjunction with the embodiments.
Embodiment 1
The method of the building unimolecule label TCR immune group library high-throughput sequencing library of the present embodiment, first progress primer
Design:
TCR α/β specific RT primer sequence are as follows:
TCR α (TAC-RT1): 5'-GTCTAGCACAGTTTTGTC-3 '
TCR β (TBC-RT1): 5'-GTATCTGGAGTCATTGA-3；
TS-3G primer sequence with unimolecule label are as follows:
TS-3G:5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNX-3'；
TCR α/β Specific PCR primers sequence are as follows:
TCR α (TAC-RT2):
5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCACTGGATTTAGAGTC-3'；
TCR β (TBC-RT2):
5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGCTTCTGATGGCTCAAAC AC-3'；
P5 adapter-primer (TS-Primer1) sequence are as follows:
5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC-3'；
P7 adapter-primer (TS-Index) sequence are as follows:
5'-CAAGCAGAAGACGGCATACGAGAT[Index-I7]GTGACTGGAGTTCAGACGTGT-3'。
Specifically TCR- α and TCR- β immune group library library construction is carried out using human total rna to follow the steps below:
(1) sample total serum IgE is extracted；
The extraction of total serum IgE is carried out according to blood/tissue/cell total rna extracts kit extraction step；
Then RT pretreatment is carried out:
Reaction system is prepared by table 1, is mixed, is set 72 DEG C of processing 3min in PCR instrument, set immediately on ice after reaction；
Table 1RNA pretreatment system
| Component | Volume (uL) |
| Total RNA | 5ug |
| RT1(TAC&TBC) | 1 |
| NF water | It mends to 9 |
(2) using total serum IgE as Material synthesis First-strand cDNA, while template switch is carried out；
RT reaction reagent is added by table 2 in the pretreated 9 μ l reactant of step (1) and carries out First-strand cDNA conjunction
At and template switch；
Table 2First-strand cDNA synthesis and template switch system
| Component | Volume (uL) |
| 5×First-strand Buffer | 4 |
| 20mM DTT | 2 |
| 10mM dNTP | 2 |
| TS-3G (unimolecule label) | 1 |
| RNase Inhibitor | 1 |
| RT enzyme | 1 |
| Total volume | 20 |
Wink is mixed from rear, 25-50 DEG C of reaction 30-90min in PCR instrument；
(3) PCR1 reaction system is added afterwards in step (2), carries out hemi-nested amplification, specific amplification target area；By table
3 prepare PCR1 reaction system:
Table 3PCR1 reaction system
| Component | Volume (uL) |
| RT product | 5 |
| 2×KAPAHiFi HotStart ReadyMix | 25 |
| RT2(TAC&TBC) | 1 |
| TS-Primer1 | 1 |
| NF water | 18 |
| Total volume | 50 |
According to following table, PCR amplification program is executed:
(4) product purification is carried out afterwards in step (3)；
It is purified after PCR using AMPure XP beads, is eluted with 24 μ L NF- water；
(5) PCR2 reaction system is added afterwards in step (4), carries out connector connection reaction and amplification；
PCR2 reaction system is prepared by table 4:
Table 4PCR2 reaction system
| Component | Volume (uL) |
| Amplified production after purification | 23 |
| 2×KAPAHiFi HotStart ReadyMix | 25 |
| P5 adapter-primer | 1 |
| P7 adapter-primer | 1 |
| Total volume | 50 |
According to following table, PCR amplification program is executed:
(6) it is washed using the sorting purifying of AMPure XP beads clip size with 27 μ L NF- water after PCR2
De-, product after purification is TCR immune group library text library.
Quality inspection is carried out to the immune group library text library TCR:
(1) wherein 5 μ L purified products progress, 1.5% agarose gel electrophoresis detection is taken:
Library electrophoresis detection analysis result is shown in Fig. 3, and library is left in 600bp or so and 900bp between 500bp-1000bp
Right main band occur, band is limpid in sight, consistent with expected results.
(2) 2 μ L purified products are taken to carry out Qubit quantitative:
| Sample | Qubit concentration (ng/ul) |
| TCR immune group library text library | 8.34 |
The library constructed as the result is shown reaches confidential and seeks concentration, can be used for machine sequencing.
(3) machine is sequenced on: carrying out library denaturation, dilution and sequencing according to MiSeq sequenator operating process.
Upper machine data statistics see the table below:
| Metric | Sample 1 |
| Read pairs total | 2,711,426 |
| read pairs from gene TRAC | 1,258,862 |
| read pairs from gene TRBC | 1,238,536 |
| reads usable for clonotype calls TRAC | 95.41% |
| reads usable for clonotype calls TRBC | 96.78% |
Meanwhile used unimolecule label corrects similar following table data, avoids and misreads, keeps result more accurate.
Shown in table data as above, in nucleic acid sequence, wherein a base of a chain becomes bases G by C, lead to amino
Acid encoding becomes E by D, if we will be considered that table data are made of two chains without molecular label, result is caused to be missed
It reads.But because there is molecular label, and the data of same molecular label come from same chain, it can be appreciated that come from same point
4 chains of subtab " AACGTG ", 3 bases in that position are C, and the base of only 1 chain is G, so we can
It is misread with excluding G's as a result, avoiding result.
Claims (9)
1. a kind of construction method of unimolecule label TCR immune group library high-throughput sequencing library, it is characterised in that according to following step
It is rapid to carry out:
(1) sample total serum IgE is extracted；
(2) using total serum IgE as Material synthesis First-strand cDNA, while template switch is carried out；
(3) PCR1 reaction system is added afterwards in step (2), carries out hemi-nested amplification, specific amplification target area；
(4) product purification is carried out afterwards in step (3)；
(5) PCR2 reaction system is added afterwards in step (4), carries out connector connection reaction and amplification；
(6) magnetic bead is added after step (5) amplified production and carries out product sorting purifying, product after purification is TCR immune group
Library text library.
2. a kind of construction method of unimolecule label TCR immune group library according to claim 1 high-throughput sequencing library,
It is characterized in that the sample is source of people blood, tissue or cell.
3. a kind of construction method of unimolecule label TCR immune group library according to claim 1 high-throughput sequencing library,
It is characterized in that the synthesis First-strand cDNA is synthesized using TCR α and/or β specific RT primer.
4. a kind of construction method of unimolecule label TCR immune group library according to claim 1 high-throughput sequencing library,
It is characterized in that the template switch is to carry out template switch using the TS-3G primer with unimolecule label, wherein unimolecule mark
Label are the merger bases of 1-20, and annexing base is original base or modified base, the modification include thio, methylation,
LNA and hypoxanthine.
5. a kind of construction method of unimolecule label TCR immune group library according to claim 1 high-throughput sequencing library,
It is characterized in that the synthesis and template switch reaction condition is 25-50 DEG C, the reaction time is 30-90min.
6. a kind of construction method of unimolecule label TCR immune group library according to claim 1 high-throughput sequencing library,
It is characterized in that the PCR1 reaction system includes buffer, archaeal dna polymerase, dNTPs, Specific PCR primers and P5 connector draw
Object；Wherein Specific PCR primers be TCR α and/or β specificity heminested PCR primer, primer be original base composition or by
Modified base composition, the modification include thio, methylation, LNA and hypoxanthine；3 ' the end part of P5 adapter-primer
Base is complementary with the connector after the TS-3G Primed template conversion with unimolecule label, and 5 ' ends include P5 binding sequence and Read 1
Sequencing sequence.
7. a kind of construction method of unimolecule label TCR immune group library according to claim 1 high-throughput sequencing library,
It is characterized in that the PCR2 reaction system includes buffer, archaeal dna polymerase, dNTPs, P7 adapter-primer and P5 adapter-primer；
Wherein P7 adapter-primer includes P7 binding sequence, and Index sequence i7 and Read2 read sequence；The P5 adapter-primer and step
Suddenly the PCR1 reaction system center tap primer of (3) is identical, or for comprising P5 binding sequence, Index sequence i5 and Read 1 is surveyed
The connector of sequence sequence.
8. a kind of construction method of unimolecule label TCR immune group library according to claim 1 high-throughput sequencing library,
It is characterized in that the TCR immune group library text library size is 500-1000bp.
9. a kind of construction method of unimolecule label TCR immune group library according to claim 1 high-throughput sequencing library,
It is characterized in that the immune group library text library TCR is used for Roche, Illumina, ThermoFisher, Pacific
Biosciences, Hua Da gene, Oxford Nanopore Technologies, Hua Yinkang, big desert gene high-flux sequence are flat
Platform.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110241459A (en) * | 2019-05-31 | 2019-09-17 | 南方医科大学南方医院 | A kind of immune group library method screening sample room and being reacted with independent sample crossover |
| CN110257476A (en) * | 2019-05-31 | 2019-09-20 | 南方医科大学南方医院 | A kind of construction method of immune group library high-throughput sequencing library that screening sample room cross reaction |
| CN110734958A (en) * | 2019-10-13 | 2020-01-31 | 湖南大地同年生物科技有限公司 | Construction method of high-throughput sequencing library of monomolecular label immune repertoire |
| CN111575343A (en) * | 2019-02-18 | 2020-08-25 | 北京全谱医学检验实验室有限公司 | Construction method and kit of immune repertoire sequencing library |
| CN111662970A (en) * | 2020-06-29 | 2020-09-15 | 武汉菲沙基因信息有限公司 | Three-generation library construction sequencing method for BCR immune repertoire full-length amplification |
| CN114277091A (en) * | 2021-09-17 | 2022-04-05 | 广东省人民医院 | Method for constructing high-quality immune repertoire library |
| CN114807123A (en) * | 2021-01-29 | 2022-07-29 | 深圳华大基因科技服务有限公司 | Method for designing and connecting amplification primer of DNA molecule |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106319064A (en) * | 2016-09-13 | 2017-01-11 | 北京天科雅生物科技有限公司 | Multi-PCR-primer and method for constructing human TCRA library on basis of high-throughput sequencing |
| CN107747134A (en) * | 2017-10-20 | 2018-03-02 | 重庆天科雅生物科技有限公司 | A kind of method for building people TCRalphaCDR3 areas library |
| CN107779495A (en) * | 2017-10-31 | 2018-03-09 | 中国医学科学院肿瘤医院 | The construction method and kit of T cell antigen receptor diversity sequencing library |
-
2018
- 2018-08-13 CN CN201810918681.8A patent/CN109136329A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106319064A (en) * | 2016-09-13 | 2017-01-11 | 北京天科雅生物科技有限公司 | Multi-PCR-primer and method for constructing human TCRA library on basis of high-throughput sequencing |
| CN107747134A (en) * | 2017-10-20 | 2018-03-02 | 重庆天科雅生物科技有限公司 | A kind of method for building people TCRalphaCDR3 areas library |
| CN107779495A (en) * | 2017-10-31 | 2018-03-09 | 中国医学科学院肿瘤医院 | The construction method and kit of T cell antigen receptor diversity sequencing library |
Non-Patent Citations (2)
| Title |
|---|
| THOMAS J JACKSON ET AL.: "Evaluating bias-reducing protocols for RNA sequencing library preparation", 《BMC GENOMICS》 * |
| 万谅等: "高通量测序技术在可选择性多聚腺苷酸化研究中的应用", 《生命科学》 * |
Cited By (10)
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|---|---|---|---|---|
| CN111575343A (en) * | 2019-02-18 | 2020-08-25 | 北京全谱医学检验实验室有限公司 | Construction method and kit of immune repertoire sequencing library |
| CN110241459A (en) * | 2019-05-31 | 2019-09-17 | 南方医科大学南方医院 | A kind of immune group library method screening sample room and being reacted with independent sample crossover |
| CN110257476A (en) * | 2019-05-31 | 2019-09-20 | 南方医科大学南方医院 | A kind of construction method of immune group library high-throughput sequencing library that screening sample room cross reaction |
| CN110734958A (en) * | 2019-10-13 | 2020-01-31 | 湖南大地同年生物科技有限公司 | Construction method of high-throughput sequencing library of monomolecular label immune repertoire |
| CN111662970A (en) * | 2020-06-29 | 2020-09-15 | 武汉菲沙基因信息有限公司 | Three-generation library construction sequencing method for BCR immune repertoire full-length amplification |
| CN111662970B (en) * | 2020-06-29 | 2023-05-05 | 武汉菲沙基因信息有限公司 | Three-generation library-building sequencing method for full-length amplification of BCR immune repertoire |
| CN114807123A (en) * | 2021-01-29 | 2022-07-29 | 深圳华大基因科技服务有限公司 | Method for designing and connecting amplification primer of DNA molecule |
| CN114807123B (en) * | 2021-01-29 | 2023-12-01 | 深圳华大基因科技服务有限公司 | Design and connection method of amplification primers of DNA molecules |
| CN114277091A (en) * | 2021-09-17 | 2022-04-05 | 广东省人民医院 | Method for constructing high-quality immune repertoire library |
| CN114277091B (en) * | 2021-09-17 | 2024-02-27 | 广东省人民医院 | Method for constructing high-quality immune repertoire library |
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