CN107058484B - Primer combination and kit applied to high-throughput sequencing and simultaneous detection of T cell and B cell immune repertoire - Google Patents
Primer combination and kit applied to high-throughput sequencing and simultaneous detection of T cell and B cell immune repertoire Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology detection, and particularly relates to a primer combination and a kit which are applied to high-throughput sequencing and are used for simultaneously detecting a T Cell (TCR) and a B Cell (BCR) immune repertoire, wherein the primer combination comprises an XCR 3 ' Oligo (dT) primer, an XCR5 ' Oligo joint, an XCR5 ' end joint primer, a TCR-alpha chain C area primer, a TCR-beta chain C area primer, IgA, IgG and IgM in the BCR-Heavy chain C area primer, Kappa and Lambda in the BCR-Light chain C area primer, and a label upstream primer and a label downstream primer; the kit comprises the primer combination according to claim 1 or 2. Full-length information of the gene sequences of TCR (Alpha chain or Beta chain) and BCR (Heavy chain or Light chain) in lymphocytes was obtained.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a primer combination and a kit for simultaneously detecting T cells and B cell immune repertoires by high-throughput sequencing.
Background
T, B A large number of V (variable region), D (variable region), J (joining region) gene segments at the cellular locus produced diverse recombination in the formation of T, B cellular receptors. This recombination of the V-D-J genes conferred unique T, B cell receptors (TCR, BCR) to each T, B cell, making the sequence of each TCR and BCR effective as a unique biomarker for a T, B cell clone.
Since the biggest characteristic of the TCR and BCR genes is the random recombination of V, D, J gene segments, aiming at unknown gene sequences, it is difficult to design an upstream primer for identifying the 5' end sequences of the TCR and BCR genes, so that the PCR technology cannot be used for amplifying the TCR and BCR genes and sequencing.
The other TCR sequencing method, the multiplex primer PCR method, can only sequence partial sequence information in the TCR gene, thus leading to incomplete sequencing gene information. In addition, the primers of the multiplex primer PCR method are designed according to the known V and J genes, and the sequencing result is only limited to the known genes of wild types. However, in cancer patients, gene mutation of cancer cells is very common, and if BCR or TCR of leukemia cancer cells is mutated, primers with known sequences may not recognize the mutated gene, and false negative results are easy to be caused for detection results. Furthermore, the sequencing method of the multiple primer PCR immune repertoire only sequences TCR or BCR, dozens of pairs of primers are already needed, and if TCR and BCR are detected simultaneously, the large number of primers can cause the amplification efficiency and specificity of the whole PCR to be poor.
Therefore, at present, no technology can simultaneously detect TCR and BCR in one experimental operation, and TCR and BCR of a plurality of samples can be simultaneously detected by using the method (single-pair primer method), so that the experimental time, the reagents and the labor cost are saved. The technology can be applied to the research of immune genomics, can detect the minimal residual disease after the treatment of lymphoid malignant tumor, and can judge the immune reconstruction, food allergy, immune disease detection and the like after HSC transplantation.
Disclosure of Invention
In order to solve the technical problems, the invention provides a primer combination and a kit which are applied to high-throughput sequencing and are used for simultaneously detecting T cell and B cell immune repertoires.
The primer combination can effectively amplify the complete sequence of the TCR gene, so that the construction efficiency of the TCR next-generation sequencing library is high. The kit provides a simple and convenient use method for users, and has stable efficiency. The library construction of TCR and BCR, the object and combination (different chains) of the library construction can be completed simultaneously.
The primer combination for simultaneously detecting T cells and B cell immune repertoires by high-throughput sequencing is characterized by comprising the following steps of: the primer combination comprises an XCR 3 ' Oligo (dT) primer, an XCR5 ' Oligo joint, an XCR5 ' end joint primer, a TCR-alpha chain C area primer, a TCR-beta chain C area primer, IgA, IgG and IgM in a BCR-Heavy chain C area primer, Kappa and Lambda in the BCR-Light chain C area primer and a label upstream and downstream primer; each primer sequence is as follows:
XCR 3' oligo (dT) primer: 5 'TTTTTTTTTTTTTTTTTTTTGA 3';
XCR 5' Oligo linker: 5 'ATGCATCGGTATTCAGCATGAACTTrGrGrG 3';
XCR 5' end linker primer:
5’GTCTCGTGGGCTGGGCGATGTGTATGAGAGACAGCATGCATCGGATCTTCAGCATGA 3’；
TCR-alpha chain C region primers:
5’TCGTCGCCAGCGTCGGAAGTGTATAAGAGACAGTCTCAGCTGGTACATATCGATGTCAGGGT 3’；
TCR-beta chain C region primers:
5’TCGTCGCCAGCGTCGGAAGTGTATAAGAGACAGTCGCAGCGTCAGATGTGTATAAGAGACAG 3’；
BCR-Heavy chain C region primer:
IgA：5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGGCTCCTGGGTTCCGAAGCC 3’；
IgG：5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGCGCCTGAGAAGGACGACAC 3’；
IgM：5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGGGGAATTCTTCTGGGAGAC 3’；
BCR-Light chain C region primer:
Kappa:
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTACCTTCCACTCTATATTGGCCTC 3’；
Lambda:
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGCCACTGTATCCGCTCCCGGG 3’；
a label upstream primer: 5'CAAGCAGAAGACGGCATACGAGAT[index1]GTCTCGTGGGCTGG 3’；
Tag TCR downstream primers: 5'AATGATACGGCGACCACCGAGATCTACAC[index2]TCGTCGCCAGCGTC
3’；
A label BCR downstream primer: 5'AATGATACGGCGACCACCGAGATCTACAC[index3]TACGCGATATCGCT
3’；
Wherein the index1 is one of ATCTATCG, TCAGGTGA, CACTAGTT, GAATTGCC, ATGTACAA, GATTCAGT, CTGTTCGT or TATACGGC; index2 is one of TAGCTACT, ATTATAGC, CCCGTACT, GGGTATAA, AGCAGGTG, TATACGTA, CACCTAGT or GTTGCTAC;
index3 is TAATCGCT, ATATTACC, CCTATACT, GGATGAAA, AGTCTTTG, TAGTGGTA, CACGATGT, or GTATGAAC;
the invention relates to a kit for simultaneously detecting T cell and B cell immune repertoire by high-throughput sequencing, which is characterized in that: the kit comprises the primer combination according to claim 1 or 2.
In the optimization scheme of the invention, the kit also comprises PCR buffer solution, Q5High-Fidelity 2XMaster Mix, enucleated enzyme water, AMPure XP Beads and 70% ethanol.
In a further optimized scheme, the kit comprises:
(1) PCR buffer solution: 9.5 mu L;
(2) positive control RNA (1. mu.g/. mu.l): 10 mu L of the solution;
(3)10 μ M TCR 3' oligo (dT) primer: 1.1 μ L;
(4)10 μ M TCR 5' end linker primer: 2.2 mu L;
(5)10 μ M TCR C region primer and 10 μ M BCR C region primer: 1.1. mu.L each;
(6)Q5High-Fidelity 2X Master Mix：55μL；
(7)10 μ M tag upstream primer: 2.2 mu L;
(8)10 μ M TCR tag downstream primer and 10 μ M BCR tag downstream primer: 1.1. mu.L each;
(9) enucleated enzyme water: 105 mu L of the solution;
(10)AMPure XP Beads：88μL；
(11) 70% ethanol: 165. mu.L.
The PCR buffer consists of the following systems:
the kit is prepared by purchasing raw materials and then assembling the raw materials into the kit. According to the required proportion, the mixture is filled into a test tube and packaged into a kit. For example, a kit can be divided into 96 samples, 48 samples and the like.
The primer combination and the kit are utilized to detect the T cell and B cell immune repertoire simultaneously by high-throughput sequencing, and the detection method comprises the following steps:
(1) obtaining a human blood sample of 10mL in an EDTA anticoagulant tube;
(2) peripheral Blood Mononuclear Cells (PBMC) were isolated using lymphocyte isolate Ficoll-1077 (Sigma #10771, USA);
the lymphocyte separating medium is useful for separating lymphocytes from whole blood because T cells are the subject of our assay, T cells are a kind of lymphocytes, and RNA obtained from the separated cell population is RNA from which cells such as red blood cells and platelets are removed. Therefore, the total RNA contained T cell RNA template used for library construction has higher purity.
(3) Extracting total RNA of PBMC by Trizol method with RNAzol RT (MRC # RN 190);
the Trizol method is used for extracting RNA and comprises the following steps:
the cells were harvested, transferred to a 1.5ml centrifuge tube, added to 1ml Trizol, mixed well and allowed to stand at room temperature for 5 min.
0.2ml of chloroform was added thereto, and the mixture was shaken for 15 seconds and allowed to stand for 2 min.
0.5ml of isopropanol was added, the liquid in the tube was gently mixed and allowed to stand at room temperature for 10 min.
Centrifugation is carried out at 4 ℃ for 12000g × 10min, and the supernatant is discarded.
1ml of 75% ethanol was added and the precipitate was washed gently. Centrifuge at 4 ℃ for 7500g × 5min, discard the supernatant.
Air-drying, adding 50ul DEPC H2O, and dissolving to obtain total RNA of lymphocyte.
(4) RNA is reversely transcribed into cDNA, and meanwhile, a joint is added at the 5 'end of the cDNA for 5' end primer combination in the following PCR amplification;
addition of the linker simultaneously during reverse transcription minimizes loss of RNA during the multi-step reaction. RNA has extremely poor stability in operation and is very easy to degrade, a small number of steps can reduce degradation to the maximum extent, and simultaneously, the preparation time of cDNA which can be used for amplification is saved.
The linker is a nucleic acid linker at the 5 'end of cDNA, and XCR 5' Oligo linker.
(5) PCR1, amplifying recombinant TCR and BCRcDNA by means of 1 upstream primer and 2 or more downstream primers;
(6) PCR2 and purification: adding an upper machine joint and a label of an Illumina high-throughput sequencer to a PCR1 product (amplified TCR sequence), and amplifying more upper machine gene quantity again; after the PCR reaction, DNA was purified using magnetic beads.
The PCR product generally contains an excess of primers, Taq DNase and dNTPs. The existence of the components directly influences the subsequent library quality detection, sequencing reaction and other processes, and the purification can remove the byproducts influencing the subsequent experiments. Meanwhile, the purification process is a process of screening the size of the fragment, the DNA fragment in the invention is about 700bp, magnetic beads with different volumes can be used for mixing with PCR products, the different volume ratios of the magnetic beads to the DNA can adsorb the fragments with different sizes, the wrong (error) fragment and primer dimer during PCR amplification can be successfully removed by using the volume of the magnetic beads, so that the sequencing on computer library only has the sequencing target DNA fragment, the sequencing result is more accurate, and the error is reduced.
As shown in FIG. 3, only one peak of the fragment was found by quality inspection of the library.
(7) High throughput sequencing was performed: sequencing the obtained cDNA library by an Illumina MiSeq platform, wherein the sequencing mode is PE300, the denaturation concentration of the library is 2nM, the loading concentration is 20pM, and analyzing a high-throughput sequencing result by bioinformatics;
millions of TCR and BCR sequences obtained by the XCR 5' end connector, the PCR primer and the sequencing library preparation method can be applied to the research of immune genomics, can detect the minimal residual disease after the treatment of lymphoid malignant tumor, and can judge the immune reconstruction, the food allergy, the detection of immune diseases and the like after HSC transplantation.
In step (4) of the present invention, each RNA sample was mixed in the following ratio:
the reagent volume was 1X (μ L),
TCR 3' oligo (dT) primer (10. mu.M) 1,
incubate at 72 ℃ for 3 minutes, followed by 4 ℃ for 1 minute.
PCR reaction buffer was prepared in the following proportions
Mix the PCR buffer with the RNA sample and begin reverse transcription of cDNA according to the following reaction protocol
60 minutes at 42 DEG C
10 minutes at 70 DEG C
Permanent at 4 deg.C
After the reaction is completed, the total cDNA to which the linker has been added at the 5' end can be obtained (see FIG. 1).
The specific steps in the step (5) are as follows: the reaction system was prepared according to the following proportions:
and preparing a reaction system according to the reaction system, mixing the 5 samples of reagent in a PCR test tube, uniformly mixing, centrifuging for several seconds, and centrifuging the liquid residue on the tube wall to the bottom of the tube. The tube was placed in a PCR amplification apparatus and run according to the above reaction protocol.
The PCR1 reaction procedure in the step (5) is as follows:
3 minutes at 95 ℃; 30 seconds at 95 ℃; 1 minute at 65 ℃ and 25 cycles; 1 minute at 72 ℃; permanent at 4 ℃.
The specific steps in the step (6) are as follows:
the reaction system was prepared according to the following proportions:
the PCR2 reaction program in the step (6) is as follows:
3 minutes at 94 ℃; 30 seconds at 94 ℃; 30 seconds at 55 ℃ and 18 cycles; 20 minutes at 72 ℃,1 minute at 72 ℃ and permanent at 4 ℃.
The specific purification steps in step (6) are as follows:
(1) add 80. mu.L of AMPure XP Beads to PCR2 reaction product and mix well.
(2) Incubate at room temperature for 10 minutes.
C. Place the magnetic bead-PCR 2 product mixture tube on a magnetic rack, wait for all magnetic beads to adsorb on the magnetic rack, pipette off all supernatants, and discard.
(3) Add 150. mu.L 70% ethanol to the beads, incubate for 30 seconds, pipette off all supernatants, and discard.
(4) The fourth step was repeated 2 times.
(5) And opening the test tube cover, waiting for 5 minutes, and allowing the magnetic beads to air dry without any ethanol remaining in the test tube.
(6) The test tube was removed from the magnetic stand, 50. mu.L of the enucleated enzyme water was added thereto, and the suspended magnetic beads were blown by a pipette.
(7) And (4) putting the test tube back to the magnetic frame, and transferring the supernatant into a new test tube after all the magnetic beads are adsorbed on the magnetic frame. The supernatant contained the purified PCR2 product.
The invention is based on high-throughput sequencing technology, and a library construction method of a TCR sequencing single pair primer, wherein after T cell RNA is obtained, a joint is added to the 5 'end of cDNA while reverse transcription from RNA to cDNA is carried out, so that a TCR amplification upstream primer is designed through the joint with a known sequence, and a downstream primer is designed by matching with a C region gene (invariant region) at the 3' end of a TCR gene, thereby achieving the purpose of amplifying the whole TCR sequence full-length gene.
Therefore, it is very meaningful to provide a detection means for simultaneously constructing a T cell receptor and a B cell receptor based on a high-throughput sequencing technology, and the experimental time, the reagent and the labor cost are effectively saved.
The invention provides a joint for simultaneously constructing TCR (T cell receptor) and BCR (B cell receptor) libraries based on high-throughput sequencing, and the primers and the method have the beneficial effects that: simultaneously obtaining the complete gene sequences of human TCR (Alpha chain or Beta chain) and BCR (Heavy chain or Light chain);
on the basis of a high-throughput sequencing platform, comprehensive bioinformatics analysis is carried out on the sequencing results of the human TCR and BCR genes at the same time, so that the gene preference of the TCR and the BCR during VDJ recombination, VDJ gene combination information, immune group clone variety information, immune group diversity information, the nucleic acid sequence and amino acid sequence information of all CDRs 1, CDRs 2 and CDRs 3 of an immune group, mutation information on the genes and the like are obtained. It is these factors that form a vast and diverse repertoire of immunizations.
Drawings
FIG. 1 schematic diagram of reverse transcribed cDNA of RNA of the present invention
FIG. 2 is a schematic diagram of the amplification of TCR and BCR cDNA by two PCR and the addition of a sequencing machine adapter in the present invention
FIG. 3 shows the quality inspection results of the sequencing library of the present invention.
FIG. 4 shows the bioinformatics analysis and alignment of the sequencing results of the TCR of the invention to find out the information (part) of each sequence
FIG. 5 shows the sequence results of the BCR sequencing of the present invention, which are analyzed and compared in bioinformatics to find out the information (part) of each sequence
Detailed Description
The present invention will be described in further detail with reference to specific embodiments, in which primers are synthesized by Invitrogen corporation, USA:
example 1
The primer combination and the kit are utilized to carry out high-throughput sequencing detection, so that the T cell receptor and the B cell receptor are simultaneously detected, and the specific steps comprise the following steps:
firstly, obtaining 10mL of human blood sample in an EDTA anticoagulant tube;
(II) separating Peripheral Blood Mononuclear Cells (PBMC) by using lymphocyte separating medium Ficoll-1077 (Sigma company #10771 in the United states);
thirdly, extracting the total RNA of PBMC by using a Trizol method, wherein the used reagent is RNAzol RT (American MRC company # RN 190);
(IV) utilization of
2.0Fluorometer (Thermo Fisher Scientific # Q32866, USA), in combination
RNA HS Assay Kit (Thermo Fisher Scientific # Q32852, USA) for determining RNA concentration, and then for reverse transcription RNA;
(V) reverse transcription of RNA into cDNA, and adding a linker at the 5 'end of the cDNA for 5' end primer binding during the subsequent PCR amplification,
reagents used:
XCR 3' oligo (dT) primer (10. mu.M)
5 Xreverse transcription buffer (250mM Tris-HCl (pH 8.3),375mM KCl,15mM MgCl2)
Dithiothreitol, DTT (20mM) U.S. Thermo Scientific # R0861
dNTP Mix (10mM) U.S. Invitrogen #18427088
RNAse Out (40U/. mu.L) U.S. Invitrogen #10777019
XCR 5' Oligo linker (10. mu.M)
Superscript II RT (200U/. mu.L) U.S. Invitrogen #18064022
Each RNA sample was mixed in the following ratio:
| reagent | Volume 1X (μ L) |
| |
8 |
| XCR 3' oligo (dT) primer (10. mu.M) | 1 |
Incubate at 72 ℃ for 3 minutes, followed by 4 ℃ for 1 minute.
PCR reaction buffer was prepared in the following proportions
| Reagent | Volume 1X (μ L) |
| 5X reverse transcription buffer solution | 3.5 |
| DTT(20mM) | 1 |
| dNTP(10mM) | 1 |
| RNAse Out | 1 |
| XCR 5' Oligo linker (10. mu.M) | 1 |
| |
1 |
Mixing the buffer solution of PCR reaction with the RNA sample, and starting reverse transcription of RNA according to the following reaction procedure
60 minutes at 42 DEG C
10 minutes at 70 DEG C
Permanent at 4 deg.C
After the reaction is completed, the total cDNA to which the linker has been added at the 5' end can be obtained (see FIG. 1)
(VI) PCR (PCR 1). The method comprises the following steps of simultaneously amplifying recombinant TCR and BCR cDNA by a single pair of primers or by 1 upstream primer, 2 or more than 2 downstream primers:
reagents used:
q5High-Fidelity 2X Master Mix (U.S. NEB # M0492L)
XCR 5' end adapter primer (upstream primer)
TCR C region primer (downstream primer)
BCR C region primer (downstream primer)
Enucleated enzyme water (U.S. Thermo Scientific AM9914G)
The reaction system was prepared according to the following proportions:
the PCR1 reaction program is:
(VII) PCR (PCR 2). And adding an upper machine joint and a label of an Illumina high-throughput sequencer to a PCR1 product (amplified TCR and BCR sequences), and amplifying again to increase more upper machine gene quantity. The method comprises the following specific steps:
reagents used:
q5High-Fidelity 2X Master Mix (U.S. NEB # M0492L)
Label upstream primer
Tag TCR downstream primer
Label BCR downstream primer
Enucleated enzyme water (U.S. Thermo Scientific AM9914G)
The reaction system was prepared according to the following proportions:
the PCR2 reaction program is:
(eight) PCR2 product purification. After the PCR reaction is finished, DNA purification is carried out by using magnetic beads, and the method specifically comprises the following steps:
reagents used:
agencourt AMPure XP Beads (U.S. Beckman # A63882)
The specific purification steps are as follows:
1. add 80. mu.L of AMPure XP Beads to PCR2 reaction product and mix well.
2. Incubate at room temperature for 10 minutes.
3. Place the magnetic bead-PCR 2 product mixture tube on a magnetic rack, wait for all magnetic beads to adsorb on the magnetic rack, pipette off all supernatants, and discard.
4. Add 150. mu.L of 75% ethanol to the beads, incubate for 30 seconds, pipette off all supernatants, and discard.
5. The fourth step was repeated 2 times.
6. And opening the test tube cover, waiting for 5 minutes, and allowing the magnetic beads to air dry without any ethanol remaining in the test tube.
7. The test tube was removed from the magnetic stand, 50. mu.L of the enucleated enzyme water was added thereto, and the suspended magnetic beads were blown by a pipette.
8. And (4) putting the test tube back to the magnetic frame, and transferring the supernatant into a new test tube after all the magnetic beads are adsorbed on the magnetic frame. On the upper part
The purified PCR2 product was contained in the supernatant.
(nine) after the purification of the library, the purity and size of the library were measured by Agilent 2100Bioanalyzer (Agilent # G2939AA), the Kit used was Agilent DNA 1000Kit (Agilent #5067-1504), and the results of the measurement are shown in FIG. 3, where the library size was in the range of 768bp, the purity of the library was quite high, and no other non-specific amplification sequences were observed.
(ten) utilization of
2.0Fluorometer (Thermo Fisher Scientific # Q32866, USA), in combination
The dsDNA HS Assay Kit (Thermo Fisher Scientific # Q32851, USA) determines the DNA library concentration and sends it to the company for high throughput sequencing. Passing the obtained cDNA library through Illumina
The platform (Illumina, USA) was used for sequencing with PE300 as sequencing mode, 2nM for library denaturation concentration, 20pM for loading concentration, and bioinformatics analysis of high throughput sequencing results.
Description of materials and reagents
Healthy volunteer subjects gave informed consent. Specifically, the reagents adopted in the invention are all commercially available products, and the databases adopted in the embodiment of the invention are all public online databases.
Specifically, the reverse transcription 5 ' end linker sequence and primer sequence of the present invention are as follows (5 ' -3 '):
reverse transcription step
XCR 3' oligo (dT) primer:
5’TTTTTTTTTTTTTTTTTTTTGA 3’
XCR 5' Oligo linker:
5’ATGCATCGGATCTTCAGCATGAACTTrGrGrG 3’
PCR1 step
XCR 5' end linker primer:
5’GTCTCGTGGGCTGGGCGATGTGTATGAGAGACAGCATGCATCGGATCTTCAGCATGA 3’
TCR-alpha chain C region primers:
5’TCGTCGCCAGCGTCGGAAGTGTATAAGAGACAGTCTCAGCTGGTACATATCGATGTCAGGGT 3’
TCR-beta chain C region primers:
5’TCGTCGCCAGCGTCGGAAGTGTATAAGAGACAGTCGCAGCGTCAGATGTGTATAAGAGACAG 3’
BCR-Heavy chain C region primer:
IgA：
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGGCTCCTGGGTTCCGAAGCC 3’
IgG：
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGCGCCTGAGAAGGACGACAC 3’
IgM：
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGGGGAATTCTTCTGGGAGAC 3’
BCR-Light chain C region primer:
Kappa:
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTACCTTCCACTCTATATTGGCCTC 3’
Lambda:
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGCCACTGTATCCGCTCCCGGG 3’。
PCR2 step
A label upstream primer:
5’CAAGCAGAAGACGGCATACGAGAT[index1]GTCTCGTGGGCTGG 3’
tag TCR downstream primers:
5’AATGATACGGCGACCACCGAGATCTACAC[index2]TCGTCGCCAGCGTC 3’
a label BCR downstream primer:
5’AATGATACGGCGACCACCGAGATCTACAC[index3]TACGCGATATCGCT 3’
rG ═ RNA nucleotides
In the labeled primerUnderliningPart of the sequence is an Illumina sequencing tag sequence]The internal sequences can be replaced by the following table sequences, and when a plurality of samples are detected simultaneously, T Cell Receptor (TCR) and B Cell Receptor (BCR) sequencing results of the plurality of samples are distinguished by using different index1-index2 or index1-index3 combinations and bioinformatics algorithms.
Designing a primer: analyzing a joint sequence added at the 5 'end during RNA reverse transcription and a gene of TCR in a BCR C region, analyzing Primer dimer and stem-loop mismatch by adopting Oligo 7.36 and Primer Premier 6.0, arranging an upstream Primer at the 5' end artificial joint, designing a reverse Primer aiming at the downstream of the C gene, and amplifying sequences of TCR and BCR full-length transcription sub-regions, wherein the sequences comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 regions of a TCR and BCR immune repertoire.
After the PBMC RNA 5' end connector, the primers and the library are constructed, about millions of TCR and BCR sequences are obtained through high-throughput sequencing.
Sequencing results are subjected to bioinformatics analysis (the bioinformatics analysis adopts Bowtie 2aligner (Ver.2.1.0), and TCR and BCR database matching is from the international immune gene information system
www.imgt.org) The results of partial analysis are shown in FIGS. 4 and 5.
Through bioinformatics analysis, the information, the amino acid information, the number and the proportion of each TCR sequence and each BCR sequence can be accurately known. Through comparative analysis of TCR and BCR, the invention obtains the statistical analysis result of representative clones of high-throughput sequencing sequences TCR and BCR, and the result is shown in FIGS. 4(TCR) and 5 (BCR).
The results show that the method for simultaneously constructing the TCR library and the BCR library can cover the diversity information of the TCR gene and the BCR gene, improve the detection rate of low copy number immune cell cloning and save time, reagents and labor cost.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
SEQUENCE LISTING
<110> extra large
<120> primer combination and kit applied to high-throughput sequencing and simultaneous detection of T cell and B cell immune repertoire
<160>34
<170>PatentIn version 3.3
<210>1
<211>22
<212>DNA
<213> Artificial Synthesis
<220>
<223> XCR 3' oligo (dT) primer
<400>1
<210>2
<211>32
<212>DNA
<213> Artificial Synthesis
<220>
<223> XCR 5' Oligo linker
<400>2
atgcatcggatcttcagcatgaacttrgrgrg 32
<210>3
<211>57
<212>DNA
<213> Artificial Synthesis
<220>
<223> XCR 5' end linker primer
<400>3
gtctcgtgggctgggcgatgtgtatgagagacagcatgcatcggatcttcagcatga 57
<210>4
<211>62
<212>DNA
<213> Artificial Synthesis
<220>
<223> TCR-alpha chain C region primer
<400>4
tcgtcgccagcgtcggaagtgtataagagacagtctcagctggtacatatcgatgtcagggt 62
<210>5
<211>62
<212>DNA
<213> Artificial Synthesis
<220>
<223> TCR-beta chain C region primer
<400>5
tcgtcgccagcgtcggaagtgtataagagacagtcgcagcgtcagatgtgtataagagacag 62
<210>6
<211>55
<212>DNA
<213> Artificial Synthesis
<220>
<223> BCR-Heavy chain C region primer-IgA
<400>6
tacgcgatatcgctctgcgctaactagtcgtactaggctcctgggttccgaagcc 55
<210>7
<211>55
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<213> Artificial Synthesis
<220>
<223>IgG
<400>7
tacgcgatatcgctctgcgctaactagtcgtactagcgcctgagaaggacgacac 55
<210>8
<211>55
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<220>
<223>IgM
<400>8
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<210>9
<211>57
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<220>
<223> BCR-Light chain C region primer-Kappa
<400>9
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<210>10
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<220>
<223>Lambda
<400>10
tacgcgatatcgctctgcgctaactagtcgtactagccactgtatccgctcccggg 56
<210>11
<211>46
<212>DNA
<213> Artificial Synthesis
<220>
<223> upstream primer of tag
<400>11
caagcagaagacggcatacgagatatctatcggtctcgtgggctgg 46
<210>12
<211>46
<212>DNA
<213> Artificial Synthesis
<220>
<223> upstream primer of tag
<400>12
caagcagaagacggcatacgagattcaggtgagtctcgtgggctgg 46
<210>13
<211>46
<212>DNA
<213> Artificial Synthesis
<220>
<223> upstream primer of tag
<400>13
caagcagaagacggcatacgagatcactagttgtctcgtgggctgg 46
<210>14
<211>46
<212>DNA
<213> Artificial Synthesis
<220>
<223> upstream primer of tag
<400>14
caagcagaagacggcatacgagatgaattgccgtctcgtgggctgg 46
<210>15
<211>46
<212>DNA
<213> Artificial Synthesis
<220>
<223> upstream primer of tag
<400>15
caagcagaagacggcatacgagat atgtacaagtctcgtgggctgg 46
<210>16
<211>46
<212>DNA
<213> Artificial Synthesis
<220>
<223> upstream primer of tag
<400>16
caagcagaagacggcatacgagatgattcagtgtctcgtgggctgg 46
<210>17
<211>46
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<213> Artificial Synthesis
<220>
<223> upstream primer of tag
<400>17
caagcagaagacggcatacgagatctgttcgtgtctcgtgggctgg 46
<210>18
<211>46
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<213> Artificial Synthesis
<220>
<223> upstream primer of tag
<400>18
caagcagaagacggcatacgagattatacggcgtctcgtgggctgg 46
<210>19
<211>51
<212>DNA
<213> Artificial Synthesis
<220>
<223> tag TCR downstream primer
<400>19
aatgatacggcgaccaccgagatctacactagctacttcgtcgccagcgtc51
<210>20
<211>51
<212>DNA
<213> Artificial Synthesis
<220>
<223> tag TCR downstream primer
<400>20
aatgatacggcgaccaccgagatctacacattatagctcgtcgccagcgtc 51
<210>21
<211>51
<212>DNA
<213> Artificial Synthesis
<220>
<223> tag TCR downstream primer
<400>21
aatgatacggcgaccaccgagatctacaccccgtacttcgtcgccagcgtc 51
<210>22
<211>51
<212>DNA
<213> Artificial Synthesis
<220>
<223> tag TCR downstream primer
<400>22
aatgatacggcgaccaccgagatctacacgggtataatcgtcgccagcgtc 51
<210>23
<211>51
<212>DNA
<213> Artificial Synthesis
<220>
<223> tag TCR downstream primer
<400>23
aatgatacggcgaccaccgagatctacacagcaggtgtcgtcgccagcgtc 51
<210>24
<211>51
<212>DNA
<213> Artificial Synthesis
<220>
<223> tag TCR downstream primer
<400>24
aatgatacggcgaccaccgagatctacactatacgtatcgtcgccagcgtc 51
<210>25
<211>51
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<213> Artificial Synthesis
<220>
<223> tag TCR downstream primer
<400>25
aatgatacggcgaccaccgagatctacaccacctagttcgtcgccagcgtc 51
<210>26
<211>51
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<213> Artificial Synthesis
<220>
<223> tag TCR downstream primer
<400>26
aatgatacggcgaccaccgagatctacacgttgctactcgtcgccagcgtc 51
<210>27
<211>51
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<213> Artificial Synthesis
<220>
<223> tag BCR downstream primer
<400>27
aatgatacggcgaccaccgagatctacaccacgatgttacgcgatatcgct 51
<210>28
<211>51
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<213> Artificial Synthesis
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<223> tag BCR downstream primer
<400>28
aatgatacggcgaccaccgagatctacactaatcgcttacgcgatatcgct 51
<210>29
<211>51
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<213> Artificial Synthesis
<220>
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<400>29
aatgatacggcgaccaccgagatctacacatattacctacgcgatatcgct 51
<210>30
<211>51
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<213> Artificial Synthesis
<220>
<223> tag BCR downstream primer
<400>30
aatgatacggcgaccaccgagatctacaccctatacttacgcgatatcgct 51
<210>31
<211>51
<212>DNA
<213> Artificial Synthesis
<220>
<223> tag BCR downstream primer
<400>31
aatgatacggcgaccaccgagatctacac ggatgaaatacgcgatatcgct 51
<210>32
<211>51
<212>DNA
<213> Artificial Synthesis
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<223> tag BCR downstream primer
<400>32
aatgatacggcgaccaccgagatctacacagtctttgtacgcgatatcgct 51
<210>33
<211>51
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<213> Artificial Synthesis
<220>
<223> tag BCR downstream primer
<400>33
aatgatacggcgaccaccgagatctacactagtggtatacgcgatatcgct 51
<210>34
<211>51
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<220>
<223> tag BCR downstream primer
<400>34
aatgatacggcgaccaccgagatctacacgtatgaactacgcgatatcgct 51
Claims (6)
1. A primer combination applied to high-throughput sequencing and simultaneous detection of T cell and B cell immune repertoires is characterized in that: the primer combination comprises an XCR 3 ' Oligo (dT) primer, an XCR5 ' Oligo joint, an XCR5 ' end joint primer, a TCR-alpha chain C area primer, a TCR-beta chain C area primer, IgA, IgG and IgM in a BCR-Heavy chain C area primer, Kappa and Lambda in the BCR-Light chain C area primer and a label upstream and downstream primer; each primer sequence is as follows:
XCR 3' oligo (dT) primer: 5 'TTTTTTTTTTTTTTTTTTTTGA 3';
XCR 5' Oligo linker: 5 'ATGCATCGGTATTCAGCATGAACTTrGrGrG 3';
XCR 5' end linker primer:
5’GTCTCGTGGGCTGGGCGATGTGTATGAGAGACAGCATGCATCGGATCTTCAGCATGA 3’；
TCR-alpha chain C region primers:
5’TCGTCGCCAGCGTCGGAAGTGTATAAGAGACAGTCTCAGCTGGTACATATCGATGTCAGGGT 3’；
TCR-beta chain C region primers:
5’TCGTCGCCAGCGTCGGAAGTGTATAAGAGACAGTCGCAGCGTCAGATGTGTATAAGAGACAG 3’；
BCR-Heavy chain C region primer:
IgA：5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGGCTCCTGGGTTCCGAAGCC 3’；
IgG：5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGCGCCTGAGAAGGACGACAC 3’；
IgM：5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGGGGAATTCTTCTGGGAGAC 3’；
BCR-Light chain C region primer:
Kappa:
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTACCTTCCACTCTATATTGGCCTC 3’；
Lambda:
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGCCACTGTATCCGCTCCCGGG 3’；
a label upstream primer: 5'CAAGCAGAAGACGGCATACGAGAT[index1]GTCTCGTGGGCTGG 3’；
Tag TCR downstream primers: 5'AATGATACGGCGACCACCGAGATCTACAC[index2]TCGTCGCCAGCGTC 3’；
A label BCR downstream primer: 5'AATGATACGGCGACCACCGAGATCTACAC[index3]TACGCGATATCGCT 3’。
2. The primer combination for high-throughput sequencing and simultaneous detection of T cell and B cell immune repertoires according to claim 1, wherein: the index1 is one of ATCTATCG, TCAGGTGA, CACTAGTT, GAATTGCC, ATGTACAA, GATTCAGT, CTGTTCGT or TATACGGC; index2 is one of TAGCTACT, ATTATAGC, CCCGTACT, GGGTATAA, AGCAGGTG, TATACGTA, CACCTAGT or GTTGCTAC;
index3 is one of TAATCGCT, ATATTACC, CCTATACT, GGATGAAA, AGTCTTTG, TAGTGGTA, CACGATGT, or GTATGAAC.
3. A kit for simultaneously detecting T cells and B cell immune repertoires by high-throughput sequencing is characterized in that: the kit comprises the primer combination according to claim 1 or 2.
4. The kit for simultaneous detection of T cell and B cell immune repertoires by high-throughput sequencing according to claim 3, wherein: the kit also comprises PCR buffer solution, Q5High-Fidelity 2X Master Mix, enucleated enzyme water, AMPure XP Beads and 70% ethanol.
5. The kit for simultaneous detection of T cell and B cell immune repertoire by high-throughput sequencing according to claim 4, wherein: the kit comprises:
(1) PCR buffer solution: 9.5 mu L;
(2) positive control RNA (1. mu.g/. mu.l): 10 mu L of the solution;
(3)10 μ M TCR 3' oligo (dT) primer: 1.1 μ L;
(4)10 μ M TCR 5' end linker primer: 2.2 mu L;
(5)10 μ M TCR C region primer and 10 μ M BCR C region primer: 1.1. mu.L each;
(6)Q5 High-Fidelity 2X Master Mix：55μL；
(9)10 μ M tag upstream primer: 2.2 mu L;
(10)10 μ M TCR tag downstream primer and 10 μ M BCR tag downstream primer: 1.1. mu.L each;
(11) enucleated enzyme water: 105 mu L of the solution;
(12)AMPure XP Beads：88μL；
(13) 70% ethanol: 165. mu.L.
6. The kit for simultaneous detection of T-cell and B-cell immune repertoires by high-throughput sequencing according to claim 4 or 5, wherein: the PCR buffer consists of the following systems:
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| CN103710454A (en) * | 2013-12-31 | 2014-04-09 | 南方科技大学 | Method for TCR or BCR high-throughput sequencing and method for correcting multiple PCR primer deviation by using tag sequence |
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