CN103436523A - Method for establishing analysis immune library spectra based on random primer barcode technology - Google Patents
Method for establishing analysis immune library spectra based on random primer barcode technology Download PDFInfo
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- CN103436523A CN103436523A CN2013102308099A CN201310230809A CN103436523A CN 103436523 A CN103436523 A CN 103436523A CN 2013102308099 A CN2013102308099 A CN 2013102308099A CN 201310230809 A CN201310230809 A CN 201310230809A CN 103436523 A CN103436523 A CN 103436523A
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Abstract
The invention aims at the medical biotechnology, relates to a high-throughput sequencing method based on a primer barcode technology and an application thereof in study of immune genomics, and specifically relates to a T-cell high-throughput sequencing method based on a marking method of cDNA molecules. Specifically, the method comprises the following steps of extracting RNA from a sample, performing reverse transcription on the RNA to obtain cDNA, designing a specific primer according to the obtained cDNA, constituting a random primer barcode by a downstream primer of the designed specific primer, a random barcode and a sequencing joint in a series manner, adding excessive obtained random primer barcode in the obtained cDNA molecules, carrying out high-throughput sequencing by using the sequencing joint in the random primer barcode, carrying out VDJ comparison on sequencing data; analyzing a CDR3 sequence and establishing immune library spectra. The method reduces deflection and error generated in a PCR amplification process. The number of the cDNA molecules can be quantized according to the specific primer barcode corresponding to a given cDNA sequence.
Description
Technical field
The present invention is directed to the medical biotechnology theme, belong to the individuation Clinics of major disease, specifically a kind of high-flux sequence method based on barcode technology and for the research of immunogene group.
Background technology
The immunogene group is the genomics of researching human body major histocompatibility complex (MHC), φt cell receptor (TCR) and antibody, can be widely used in researchs such as instructing vaccine design, Personalized medicine.In the research of immunogene group, the T cell is the executive of body's immunity, and TCR is the molecule of T cell surface identification antigen, is also the first key molecule that the T cell produces immunne response.The T cell is in the thymus development process, and TCR passes through V(D) JC gene rearrangement and selection, formed the multifarious T cell bank of periphery, various antigen produces and replys to external world.
The CDR3 receptoire of TCR, since the nineties in last century, be widely used in the fundamental research of (evolution of T cell, growth, differentiation, propagation, subgroup, tolerance, aging etc.) under the organism physiology condition.Analyze the CDR3 receptoire of TCR, understand the situation that in T cells, tcr gene is reset, can be in order to estimate the diversity of body T cells, and can analyze the diversity of the CDR3 receptoire of the TCR between Different Individual, further study that the T cell occurs, growth course.The CDR3 receptoire difference of same individual different ages stage maincenter peripheral t CR, can be applied to inquire into age and the multifarious relation of TCR etc.In multiple situation, the comprehensive gene expression analysis in the CDR3 storehouse of individual TCR is to estimating immunne response highly significant, the expression frequency of CDR3 by research TCR, the relation of the T cell of screening and cloning propagation and infection, tumour, autoimmune disease, organ transplantation etc., the diagnosis and the treatment that can be these diseases provide basis and means.In the research of non-T cell tumour, can find and analyze by the CDR3 receptoire of TCR the characterization of molecules of antitumor T cell, for the recovery treatment of individual patients T cell and the development of tumor vaccine etc. provide basis; In ripe T cell tumour, can analyze the composition of the CDR3 of tumour T cell TCR, for T cell tumour patient finds the supplying methods such as diagnosis for the treatment of target and remaining pathology.The changing conditions of the CDR3 receptoire of TCR in performance analysis neoplastic disease human body simultaneously, can understand the immunological status of tumour patient and prognosis etc.
The T cell plays a key effect in limiting virus, bacterium and parasitic infection etc., the diversity of the CDR3 receptoire of TCR may affect the kill capability of CTL and regulate and control immunologic homeostasis, by studying in acute and persistent virus infection, the generation of different TCRCDR3 receptoires and maintaining, follow the trail of different T cell subsets, lay the foundation at the responsibility under Infection Status for improving the T cell.Comprehensive in-depth analysis to the CDR3 receptoire of Serum of Patients With Autoimmune Diseases TCR, can understand that self to reply the T cell be mechanism how to break self tolerance, simultaneously, the research formed by the CDR3 that self replys T cell TCR, the autoimmunization diagnosis and treatment that can be individuation provide practicable means.Along with the progress of stem-cell research, autologous and allogeneic stem cells is implanted in increasing disease and is applied in recent years.Detect the CDR3 receptoire of transplantation donor and receptor's T cell TCR, the CDR3 receptoire that rear different time sections TCR is transplanted in dynamic monitoring changes, and contribute to transplant rejection T cell derived in transplant patient's body and the assessment of immunologic reconstitution after transplanting etc. provides basis.In addition, to the multianalysis of the CDR3 receptoire of TCR, but investigate individual to susceptibility and the resistivity of disease, the disappearance of the CDR3 of some TCR or high expression level and body T cell are replied closely related to specific antigen (various disease).
DNA bar codes technique (DNA barcode) is to utilize one section emerging technology that conservative fragments is quick and precisely identified species in organism DNA, refer in organism and can represent these species, standard, that enough variations are arranged, easily amplification and relatively short DNA fragmentation.The DNA barcode has become the important tool of ecological study, not only for species, identifies, also helps the biologist further to understand the interaction occurred in the ecosystem simultaneously.Usually, when finding a kind of unknown species or species a part of, the researchist just describes the DNA barcode of its tissue, then with international data center in other barcodes compare; If be complementary with one of them, just the researchist can confirm the identity of this species.Desirable DNA barcdoing should meet following standard: (1) has enough variability to distinguish different species, has relative conservative property simultaneously; (2) must be that the DNA district of a segment standard differentiates different taxonomical groups as far as possible; (3) the target dna district should comprise enough phyletic evolution information with the position of species in categorizing system (section, genus etc.), location; (4) should be that the design of primers district of high conservative is so that the design of universal primer; (5) what the target dna district should be enough is short so that the amplification of the DNA of Partial digestion is arranged.The instrument that DNA barcoding identifies as biological " planting horizontal species-level " is noticeable.In the Genbank database, CO I sequence increases fast.Min etc. have analyzed the relation between CO I sequence and source genome nucleotide content thereof, result shows that the DNA barcoding sequence of 5 ends of 849 CO I genes has represented the important information of its complete chondriogen mtDNA that originates astoundingly exactly, that is to say the genome checked order for not, can predict fast the composition of complete genome group from DNA barcoding.
In addition, there are two defects in the current high throughput sequencing technologies for the research of immunogene group: the amplification deflection of 1) building the storehouse process; 2) sequencing error of low copy number sequence is larger.For above problem, the applicant has set up a kind of new high throughput sequencing technologies based on barcode technology.
The present invention is based on above-mentioned prior art, and for the deficiencies in the prior art improve the invention.
Summary of the invention
In view of this, the invention provides a kind of method of random primer barcode label cDNA molecule, and the method for the φt cell receptor high-flux sequence based under method is provided; Set up immune storehouse spectrum efficiency with aforesaid method high, error is little.
For achieving the above object, technical scheme of the present invention is:
The marking method of cDNA molecule, add excessive random primer bar code at the cDNA two ends, make random primer bar code corresponding to each mark of cDNA molecule two ends.
The cDNA molecule obtained by described marking method.
Further, described cDNA molecule, by the downstream primer of the Auele Specific Primer according to the cDNA molecular designing, random bar code and sequence measuring joints, the mode with series connection forms described random primer bar code.
The φt cell receptor high-flux sequence method of the marking method based on the cDNA molecule specifically comprises the following steps:
1) extract the RNA in sample;
2) RNA is carried out to reverse transcription, obtain cDNA;
3), according to gained cDNA design Auele Specific Primer, the downstream primer of the Auele Specific Primer of design, random bar code and sequence measuring joints form the random primer bar code in the mode of series connection;
4) by the excessive step 2 that adds of gained random primer bar code) in gained cDNA molecule;
5) utilize the sequence measuring joints in the random primer bar code to carry out high-flux sequence, sequencing data carries out the VDJ comparison, analyzes the CDR3 sequence.
Further, the sample source in step 1) is certainly by the patient of different steps after pathogen infection.
Further, the sample source in step 1) is certainly by the patient of different steps after hepatitis B virus infection.
Further, according to the result of the CDR3 sequence of analyzing same sample, obtain single complete immune storehouse spectrum.
Further, according to the result of the CDR3 sequence of analyzing different samples, obtain the complete immune storehouse spectrum of a plurality of set.
Further, described immune storehouse spectrum is chronic hepatitis B immunity storehouse spectrum.
Further, the platform compatibility of the sequence measuring joints in step 3) and Illumina.
Beneficial effect of the present invention is: the method the present invention relates to reduces the method that deflection and error occur in the pcr amplification process.When obtaining the cDNA library of pcr amplification, use is carried out reverse transcription reaction with stochastic sequence as the specificity downstream primer of bar code, thereby each cDNA template obtains a unique bar code sequence, through pcr amplification, degree of depth order-checking, then pass through the number of the bar code sequence of the uniqueness in the calculating raw sample, and given cDNA sequence corresponding to this specific bar code, make the quantity of cDNA molecule to quantize.
Embodiment
Below will be described in detail the preferred embodiments of the present invention.The experimental technique of unreceipted actual conditions in preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example, J. the work such as Pehanorm Brooker, Huang Peitang Deng Yi， Science Press, 2002) described in condition, or the condition of advising according to manufacturer is carried out.
One Chronic Hepatitis B sample is chosen
(1) choose one of chronic viral hepatitis B patients with mild: symptom is lighter, less, and as slight weak, slight abdominal distension, slight uncomfortable liver area etc., liver function has damage.Transaminase is 3 times (normal value is 40 units), and bilirubin is in 2 times of normal value left and right (normal value is 17.1 micromoles per liter), and albumin is more than or equal to 35 grams per liters (normal value is the 35-55 grams per liter).
(2) choose one of chronic viral hepatitis B moderate: sings and symptoms and liver function change between slight and severe.Transaminase surpasses 3 times of normal value, and bilirubin surpasses 4 times of normal value, albumin between 35 grams per liters.
(3) choose one of chronic viral hepatitis B severe: obvious or lasting hepatitis symptom is arranged, as weak, appetite is poor, abdominal distension, loose stool, urine Huang, hepatalgia, complexion are gloomy, liver palms, spider angioma etc., liver dysfunction is heavier, transaminase surpasses 5 times of normal values, bilirubin surpasses 5 times of normal values, and albumin equals 32 grams per liters.
The total RNA of two samples extracts
1. extract total RNA from the positive patients whole blood
The whole blood of getting-70 ℃ of preservations melts on ice, gets 3ml and adds the 15ml centrifuge tube, adds 6mlTRNzol-A+, and the 3ml trichloromethane, fully put upside down and mix 5 minutes.Centrifugal 10 minutes of 10,000g, in absorption, honest and upright and thrifty 2.5ml adds new 15ml centrifuge tube, adds 2.5ml Virahol and 250 μ l sodium-acetates, puts upside down and mixes rear placement 1 hour on ice.Centrifugal 20 minutes of 10,000g, 37 ℃ of oven dry after 70% washing with alcohol 2 times for precipitation, add 50 μ l TE and dissolve and be transferred to the 1.5ml centrifuge tube.Measure the OD260 value of extracting total RNA.Be total to about 120ml left and right according to 2 parts of positive whole blood samples, supplement and order 200ml TRNzol-A+ total RNA extraction reagent, 50ml, except RNase screw socket high speed centrifugation pipe, presses the preliminary experiment step, divides 3 batches, extracts 40ml whole blood RNA at every turn.
Three. reverse transcription prepares the cDNA molecule
Main agents is M-MLV Reverse Transcriptase(CatNo.:28025-013, Invtrogen company),
mRNA Mini Kits(CatNo.:70022, Qiagen company), DNA purification kit (Roche company).According to
mRNA Mini Kits test kit specification sheets, obtain mRNA by total RNA extracting and purifying of 24.1 μ g altogether.The mRNA of purifying of take is template, and according to the Auele Specific Primer of design, design obtains 9 sections cDNA molecules.
Four. the preparation of random primer bar code
The sequence that sequence measuring joints provides from the platform of Illumina, provided by Bo Ao biotech firm.Obtain 9 sections cDNA molecules according to above-mentioned design, design corresponding Auele Specific Primer, utilize minute other downstream primer, random bar code and sequence measuring joints to form the random primer bar code in the mode of series connection.The random primer bar code is obtained to 9 sections cDNA molecules, two ends separately with design and be connected, obtain 9 sections molecules of the cDNA by the random primer barcode label.) utilize the sequence measuring joints in the random primer bar code to carry out, utilizing the platform high-flux sequence of Illumina, sequencing data carries out the VDJ comparison, analyzes the CDR3 sequence.Obtain single complete immune storehouse spectrum and gather immune storehouse spectrum.
Finally explanation is, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although with reference to preferred embodiment, the present invention is had been described in detail, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not breaking away from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (10)
1.cDNA the marking method of molecule is characterized in that: add excessive random primer bar code at the cDNA two ends, make random primer bar code corresponding to each mark of cDNA molecule two ends.
2. the cDNA molecule obtained by marking method claimed in claim 1.
3. cDNA molecule according to claim 1 is characterized in that: by the downstream primer of the Auele Specific Primer according to the cDNA molecular designing, random bar code and sequence measuring joints, the mode with series connection forms described random primer bar code.
4. the φt cell receptor high-flux sequence method of the marking method based on the cDNA molecule, is characterized in that, specifically comprises the following steps:
1) extract the RNA in sample;
2) RNA is carried out to reverse transcription, obtain cDNA;
3), according to gained cDNA design Auele Specific Primer, the downstream primer of the Auele Specific Primer of design, random bar code and sequence measuring joints form the random primer bar code in the mode of series connection;
4) by the excessive step 2 that adds of gained random primer bar code) in gained cDNA molecule;
5) utilize the sequence measuring joints in the random primer bar code to carry out high-flux sequence, sequencing data carries out the VDJ comparison, analyzes the CDR3 sequence.
5. the φt cell receptor high-flux sequence method of the marking method based on the cDNA molecule according to claim 4 is characterized in that: the sample source in step 1) is from by the patient of different steps after pathogen infection.
6. the φt cell receptor high-flux sequence method of the marking method based on the cDNA molecule according to claim 5 is characterized in that: the sample source in step 1) is from by the patient of different steps after hepatitis B virus infection.
7. the φt cell receptor high-flux sequence method of the marking method based on the cDNA molecule according to claim 6, is characterized in that: according to the result of the CDR3 sequence of analyzing same sample, obtain single complete immune storehouse spectrum.
8. the φt cell receptor high-flux sequence method of the marking method based on the cDNA molecule according to claim 6, is characterized in that: according to the result of the CDR3 sequence of analyzing different samples, obtain the complete immune storehouse spectrum of a plurality of set.
9. the φt cell receptor high-flux sequence method of the marking method based on the cDNA molecule according to claim 8 or claim 9, it is characterized in that: described immune storehouse spectrum is chronic hepatitis B immunity storehouse spectrum.
10. the φt cell receptor high-flux sequence method of the marking method based on the cDNA molecule according to claim 4, is characterized in that: the described sequence measuring joints in step 3) and the platform compatibility of Illumina.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104263818A (en) * | 2014-09-02 | 2015-01-07 | 武汉凯吉盈科技有限公司 | Whole blood immune repertoire detection method based on high-flux sequencing technology |
| CN106557667A (en) * | 2015-09-28 | 2017-04-05 | 深圳华大基因科技有限公司 | The system for evaluating immune effect of vaccine |
| CN106987631A (en) * | 2017-04-01 | 2017-07-28 | 武汉赛云博生物科技有限公司 | A kind of immune group sequencing technologies for the adjoint diagnosis of PD 1/PD L1 blocking treatments |
| CN110675914A (en) * | 2019-09-17 | 2020-01-10 | 佛山市第一人民医院（中山大学附属佛山医院） | Method for screening tumor specific T cells and TCR |
-
2013
- 2013-06-09 CN CN2013102308099A patent/CN103436523A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104263818A (en) * | 2014-09-02 | 2015-01-07 | 武汉凯吉盈科技有限公司 | Whole blood immune repertoire detection method based on high-flux sequencing technology |
| CN104263818B (en) * | 2014-09-02 | 2016-06-01 | 武汉凯吉盈科技有限公司 | Based on the whole blood immunity group storehouse detection method of high throughput sequencing technologies |
| CN106557667A (en) * | 2015-09-28 | 2017-04-05 | 深圳华大基因科技有限公司 | The system for evaluating immune effect of vaccine |
| CN106557667B (en) * | 2015-09-28 | 2019-03-26 | 深圳华大基因科技有限公司 | The system for evaluating immune effect of vaccine |
| CN106987631A (en) * | 2017-04-01 | 2017-07-28 | 武汉赛云博生物科技有限公司 | A kind of immune group sequencing technologies for the adjoint diagnosis of PD 1/PD L1 blocking treatments |
| CN110675914A (en) * | 2019-09-17 | 2020-01-10 | 佛山市第一人民医院（中山大学附属佛山医院） | Method for screening tumor specific T cells and TCR |
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Application publication date: 20131211 |