CN107058484A - It is a kind of to detect T cell and the primer combination in B cell immune group storehouse and kit simultaneously applied to high-flux sequence - Google Patents
It is a kind of to detect T cell and the primer combination in B cell immune group storehouse and kit simultaneously applied to high-flux sequence Download PDFInfo
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Abstract
The invention belongs to molecular Biological Detection field, it is more particularly to a kind of to detect T cell (TCR) and the primer combination in B cell (BCR) immune group storehouse and kit simultaneously applied to high-flux sequence, the primer combination includes the Oligo of XCR 3 ' (dT) primer, the Oligo joints of XCR 5 ', the end connector primers of XCR 5 ', TCR alpha chain C areas primer, TCR beta chain C areas primer, IgA, IgG and IgM in BCR Heavy chain C areas primer, Kappa and Lambda in BCR Light chain C areas primer, and label upstream and downstream primer；The kit includes primer combination as claimed in claim 1 or 2.Obtain TCR in lymphocyte（Alpha chains or Beta chains）And BCR（Heavy chains or Light chains）Gene order total length information.
Description
Technical field
The invention belongs to biology field, more particularly to a kind of high-flux sequence that is applied to detects T cell simultaneously
Primer combination and kit with B cell immune group storehouse.
Background technology
Substantial amounts of V (variable region) on T, B cell locus, D (variable region), J (bonding pad) genetic fragments T, B cell by
Each species diversity restructuring can be produced in the formation of body.It is unique that the restructuring of this V-D-J genes imparts each T, B cell oneself
T, B-cell receptor (TCR, BCR) so that each TCR and BCR sequence can effectively turn into T, a B cell gram
Grand unique biomarker.
Because the maximum feature of TCR and BCR genes is the random restructuring of V, D, J genetic fragment, so, for unknown gene
Sequence the, it is difficult to 5 ' terminal sequences that a sense primer is used to recognize TCR, BCR gene be designed, so can not also utilize round pcr
Expand TCR, BCR gene and sequencing.
And another TCR sequence measurement, the method for Multiplex PCR, the partial sequence letter in tcr gene can only be sequenced
Breath, so that sequencing gene information is imperfect.In addition, the primer of Multiplex PCR method is according to known V, the design of J genes
, this sequencing result is confined to the known of wild type.But in cancer patient, the gene mutation of cancer cell is very normal
See, if the BCR or TCR of leukaemia cancer cell generate mutation, then the primer of known array is possible to None- identified
Gene after mutation, for testing result, is easy for causing false negative.Also, the immune group storehouse sequencing side of Multiplex PCR
Method, is only sequenced to TCR or BCR, has just needed tens pairs of primer, huge if detection TCR and BCR simultaneously
Primer quantity the efficiency and specificity of whole PCR amplification can be allowed to become very poor.
So, it can detect TCR and BCR simultaneously in an experimental implementation there is presently no a technology, utilize us
Method (single pair of primer method) can detect the TCR and BCR of multiple samples simultaneously, save experimental period and reagent, cost of labor.
The technology can be applied in the research of immunogene group, can not only detect the minimal residual after lymphoid malignancy treatment
Disease, additionally it is possible to judge the immunologic reconstitution after HSC transplanting, food hypersenstivity, the detection of immunity disease etc..
The content of the invention
In order to solve the above technical problems, the present invention, which provides a kind of high-flux sequence that is applied to, detects that T cell and B are thin simultaneously
The primer combination in born of the same parents' immune group storehouse and kit, φt cell receptor and B-cell receptor library structure are built based on high-flux sequence simultaneously
The cDNA joints and single pair of primer built, by the anti-sense primer of the sense primer from joint to C areas, while obtaining TCR and BCR bases
Because of the total length information of sequence.
Primer combination can effectively expand the complete sequence of tcr gene in the present invention, bis- generations of TCR sequencing library is built efficiency
Efficiently.Kit has provided the user simple and convenient application method, stabilised efficiency.TCR and BCR can be completed simultaneously builds storehouse,
Build object and the combination (different chains) in storehouse.
Solve a kind of of above technical problem and detect T cell and B cell immune group storehouse simultaneously applied to high-flux sequence
Primer is combined, it is characterised in that：The primer combination includes the Oligo of XCR 3 ' (dT) primer, the Oligo joints of XCR 5 ', XCR5 '
IgA, IgG in end connector primer, TCR-alpha chain C areas primer, TCR-beta chain C areas primer, BCR-Heavy chain C areas primer
And Kappa and Lambda in IgM, BCR-Light chain C areas primer, and label upstream and downstream primer；Every kind of primer sequence is such as
Under：
The Oligo of XCR 3 ' (dT) primer：5’TTTTTTTTTTTTTTTTTTTTGA 3’；
The Oligo joints of XCR 5 '：5’ATGCATCGGATCTTCAGCATGAACTTrGrGrG 3’；
The end connector primers of XCR 5 '：
5’GTCTCGTGGGCTGGGCGATGTGTATGAGAGACAGCATGCATCGGATCTTCAGCATGA 3’；
TCR-alpha chain C areas primer：
5’TCGTCGCCAGCGTCGGAAGTGTATAAGAGACAGTCTCAGCTGGTACATATCGATGTCAGGGT 3’；
TCR-beta chain C areas primer：
5’TCGTCGCCAGCGTCGGAAGTGTATAAGAGACAGTCGCAGCGTCAGATGTGTATAAGAGACAG 3’；
BCR-Heavy chain C areas primer：
IgA：5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGGCTCCTGGGTTCCGAAGCC 3’；
IgG：5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGCGCCTGAGAAGGACGACAC 3’；
IgM：5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGGGGAATTCTTCTGGGAGAC 3’；
BCR-Light chain C areas primer：
Kappa:
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTACCTTCCACTCTATATTGGCCTC 3’；
Lambda:
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGCCACTGTATCCGCTCCCGGG 3’；
Label sense primer：5’CAAGCAGAAGACGGCATACGAGAT[index1]GTCTCGTGGGCTGG 3’；
Label TCR anti-sense primers：5’AATGATACGGCGACCACCGAGATCTACAC[index2]TCGTCGCCAGCGTC
3’；
Label B CR anti-sense primers：5’AATGATACGGCGACCACCGAGATCTACAC[index3]TACGCGATATCGCT
3’；
Wherein, the index1 be ATCTATCG, TCAGGTGA, CACTAGTT, GAATTGCC, ATGTACAA,
One kind in GATTCAGT, CTGTTCGT or TATACGGC；Index2 be TAGCTACT, ATTATAGC, CCCGTACT,
One kind in GGGTATAA, AGCAGGTG, TATACGTA, CACCTAGT or GTTGCTAC；
Index3 be TAATCGCT, ATATTACC, CCTATACT, GGATGAAA, AGTCTTTG, TAGTGGTA,
One kind in CACGATGT or GTATGAAC；
A kind of kit for detecting T cell and B cell immune group storehouse simultaneously applied to high-flux sequence in the present invention, its
It is characterised by：The kit includes primer combination as claimed in claim 1 or 2.
Kit described in prioritization scheme in the present invention also includes PCR buffer solutions, Q5High-Fidelity 2X
Master Mix, remove nuclease water, AMPure XP Beads and 70% ethanol.
In further prioritization scheme, the kit includes：
(1) PCR buffer solutions：9.5μL；
(2) positive control RNA (1 μ g/ μ l)：10μL；
(3) the 10 μM of Oligo of TCR 3 ' (dT) primers：1.1μL；
(4) 10 μM of end connector primers of TCR 5 '：2.2μL；
(5) 10 μM of TCR C area's primers and 10 μM of BCR C areas primers：Each 1.1 μ L；
(6)Q5High-Fidelity 2X Master Mix：55μL；
(7) 10 μM of label sense primers：2.2μL；
(8) 10 μM of TCR labels anti-sense primers and 10 μM of BCR label anti-sense primers：Each 1.1 μ L；
(9) nuclease water is removed：105μL；
(10)AMPure XP Beads：88μL；
(11) 70% ethanol：165μL.
The PCR buffer solutions are made up of following system：
Kit be prepared as buy raw material, be then assembled into kit.According to required ratio, load test tube, encapsulation
Into kit.Such as one kit, which can be divided into, makees 96 samples, 48 samples, two kinds of specifications etc..
Using the primer combination in the present invention and kit, so that high-flux sequence detects that T cell and B cell are immune simultaneously
Group storehouse, detection method comprises the following steps：
(1) human blood sample 10mL is obtained in EDTA anticoagulant tubes；
(2) it is thin using lymphocyte separation medium Ficoll-1077 (Sigma Co., USA #10771) progress peripheral blood mononuclears
The separation of born of the same parents (PBMC)；
The effect of lymphocyte separation medium is that lymphocyte can be separated in whole blood, because T cell detects for us
Object, T cell belongs to one kind of lymphocyte, and the RNA that the cell mass after separation is obtained is to eliminate red blood cell, blood platelet etc.
The RNA's of cell.So building the total serum IgE that storehouse uses, to include T cell RNA template purity higher.
(3) PBMC total serum IgE is extracted using Trizol method, agents useful for same is RNAzol RT (MRC companies of U.S. #
RN190)；
Trizol method extracts RNA, and step is as follows：
Harvesting, is transferred in 1.5ml centrifuge tubes, adds 1ml Trizol, mixes, is stored at room temperature 5min.
0.2ml chloroforms are added, 15s is vibrated, 2min is stood.
0.5ml isopropanols are added, liquid in pipe is gently mixed, 10min is stored at room temperature.
4 DEG C of centrifugations, 12000g × 10min abandons supernatant.
The ethanol of 1ml 75% is added, gently washing precipitation.4 DEG C of centrifugations, 7500g × 5min abandons supernatant.
Natural air drying, adds 50ul DEPC H2O dissolvings, obtains lymphocyte total serum IgE.
(4) RNA reverse transcriptions are into cDNA, and simultaneously in the ends of cDNA 5 ' addition joint, and 5 ' ends are drawn when being expanded with PCR later
Thing is combined；
In reverse transcription, while adding joint, loss of the RNA during multistep reaction can be minimized.RNA is in operation
Middle stability extreme difference, is very easy to degraded, and a small amount of step can reduce degraded to the full extent, can be used for while also saving
The cDNA preparation times of amplification.
Joint is the nucleic acid linker at cDNA 5 ' ends, the Oligo joints of XCR 5 '.
(5)PCR1:Restructuring TCR and BCR is expanded by way of 1 sense primer, the anti-sense primer of 2 or more than 2
cDNA；
(6) PCR2 and purifying：The upper of Illumina high-flux sequence instrument is added for PCR1 products (the TCR sequences after amplification)
Machine joint and label, while more upper machine gene dosage is expanded again for increase；After PCR reactions terminate, DNA is carried out using magnetic bead
Purifying.
PCR primer is typically all containing excessive primer, Taq DNA enzymatics and dNTP.The presence of these compositions will be directly affected
To processes such as follow-up library quality inspection, sequencing reactions, purifying can remove the accessory substance of these influence subsequent experimentals.Meanwhile, it is pure
The process of change is also that the DNA fragmentation in the process of clip size screening, the present invention is in 700bp or so, using difference
The magnetic bead of volume is mixed with PCR primer, and volume ratios different magnetic bead/DNA can adsorb different size of fragment, using above-mentioned
Magnetic bead volume, can successfully remove machine text in mistake (error) fragment and primer dimer when PCR is expanded, the sequencing of let us
There was only our sequencing target DNA fragments in storehouse so that sequencing result is more accurate, reduce error.
As shown in figure 3, being only found that the peak of a fragment by library quality inspection.
(7) high-flux sequence is carried out：The cDNA library of gained is sequenced by Illumina MiSeq platforms, is sequenced
Pattern is PE300, and library denaturant concentration is 2nM, and upper machine concentration is 20pM, and passes through bioinformatic analysis high-flux sequence knot
Really；
Up to a million s' obtained by the end connectors of XCR 5 ' of the present invention, PCR primer and sequencing library preparation method
TCR and BCR sequences, the technology can be applied in the research of immunogene group, can not only detect that lymphoid malignancy is controlled
Minimal residual disease after treatment, additionally it is possible to judge the immunologic reconstitution after HSC transplanting, food hypersenstivity, the detection of immunity disease etc..
In heretofore described step (4) each RNA sample is mixed according to following ratio：
Reagent volume 1X (μ L),
RNA 8,
The Oligo of TCR 3 ' (dT) primer (10 μM) 1,
Hatch in 72 DEG C 3 minutes, then 4 DEG C 1 minute.
PCR reaction buffers are prepared according to following ratio
PCR reactions caching liquid and RNA samples are mixed, cDNA reverse transcriptions are started according to following response procedures
42 DEG C 60 minutes
70 DEG C 10 minutes
4 DEG C permanent
After reaction terminates, so that it may obtain and with the addition of total cDNA (such as Fig. 1) of joint at 5 ' ends.
Comprised the following steps that in the step (5)：Reaction system is prepared according to following ratio：
Reaction system is prepared according to above-mentioned reaction system, above-mentioned 5 sample reagent is mixed in PCR test tubes, mixes, centrifuge number
Second, the liquid residue on tube wall is centrifuged in ttom of pipe.Holding test tubes start fortune in PCR amplification instrument according to above-mentioned response procedures
OK.
The response procedures of PCR 1 are in the step (5):
95 DEG C 3 minutes；95 DEG C 30 seconds；65 DEG C 1 minute, 25 circulation；72 DEG C 1 minute；4 DEG C permanent.
Comprised the following steps that in the step (6)：
Reaction system is prepared according to following ratio：
The response procedures of PCR 2 are in the step (6):
94 DEG C 3 minutes；94 DEG C 30 seconds；55 DEG C 30 seconds, 18 circulation；72 DEG C 20 minutes, 72 DEG C 1 minute, 4 DEG C are permanent.
Specific purification step is as follows in the step (6)：
(1) 80 μ L AMPure XP Beads of addition enter PCR2 reaction products, mix.
(2) in incubation at room temperature 10 minutes.
C, magnetic bead-PCR2 product mixtures test tube is placed on magnetic frame, wait after all magnetic beads are adsorbed on magnetic frame,
All supernatants are sucked with pipettor, are abandoned.
(3) the addition ethanol of 150 μ L 70% is hatched 30 seconds on magnetic bead, all supernatants is sucked with pipettor, is abandoned.
(4) the 4th step is repeated 2 times.
(5) test tube cap is opened, is waited 5 minutes, treats that magnetic bead is air-dried, is residued in without any ethanol in test tube.
(6) test tube is removed from magnetic frame, and adds 50 μ L and remove nuclease water, suspension magnetic bead is blown and beaten using pipettor.
(7) tube back magnetic frame, all magnetic beads are waited all to be adsorbed in after magnetic frame, transfer supernatant is in new test tube
In.The PCR2 products after purifying are just contained in supernatant.
The present invention is based on high throughput sequencing technologies, and the library constructing method of single pair of primer is sequenced in TCR, by thin in acquisition T
After born of the same parents RNA, RNA is being carried out to while cDNA reverse transcriptions, to cDNA 5 ' one joint of end addition, so that by known to this
The joint design TCR amplification sense primers of sequence, then coordinate tcr gene 3 ' to hold C area's genes (constant region) design anti-sense primer, from
And reach the purpose for expanding whole TCR sequences gene.
Therefore it provides a kind of φt cell receptor and B-cell receptor detection means of being built based on high throughput sequencing technologies is simultaneously
It is very significant, it is effective to save experimental period and reagent, cost of labor.
The present invention provides the beneficial effect of the joint for building TCR and BCR libraries simultaneously based on high-flux sequence, primer and method
It is really：Obtain people TCR (Alpha chains or Beta chains) and BCR (Heavy chains or Light chains) complete genome sequence simultaneously；
The present invention is complete by being carried out to people's TCR and BCR gene sequencing result simultaneously on the basis of high-flux sequence platform
The bioinformatic analysis in face, obtains the gene Preference of TCR and BCR when VDJ is recombinated, and VDJ assortment of genes information is immunized
Group clone's kind of information, immune group diversity information, all CDR1, CDR2, CDR3 of immune group nucleotide sequence and amino acid
Abrupt information on sequence information, gene, etc..Exactly these factor quantity of formation are huge and immune group storehouse of wide variety.
Brief description of the drawings
RNA reverse transcription cDNA schematic diagrames in Fig. 1 present invention
Upper machine joint schematic diagram is sequenced to be expanded TCR and BCR cDNA using twice PCR in the present invention and being added in Fig. 2
Fig. 3 is sequencing library quality inspection result in the present invention.
Fig. 4 carries out bioinformatic analysis comparison for TCR sequencing results in the present invention, finds out the information (portion of every sequence
Point)
Fig. 5 carries out bioinformatic analysis comparison for BCR sequencing results in the present invention, finds out the information (portion of every sequence
Point)
Embodiment
With reference to embodiment, the present invention is described in further detail, and primer is by the U.S.
The synthesis of Invitrogen companies:
Embodiment 1
Using in the present invention primer combination and kit carry out high-flux sequence detection so that simultaneously detection T cell by
Body and B-cell receptor, specific steps comprise the following steps：
(1) human blood sample 10mL is obtained in EDTA anticoagulant tubes；
(2) peripheral blood mononuclear is carried out using lymphocyte separation medium Ficoll-1077 (Sigma Co., USA #10771)
The separation of cell (PBMC)；
(3) PBMC total serum IgE is extracted using Trizol method, agents useful for same is RNAzol RT (MRC companies of U.S. #
RN190)；
(4) utilize2.0Fluorometer (U.S. Thermo Fisher Scientific company #
Q32866), coordinateRNA HS Assay Kit kits (U.S. Thermo Fisher Scientific company #
Q32852 RNA concentration) is determined, reverse transcription RNA is subsequently used for；
(5) RNA reverse transcriptions are into cDNA, and 5 ' the end primer knots when the ends of cDNA 5 ' addition joint is expanded with PCR later
Close, comprise the following steps that,
The reagent used：
The Oligo of XCR 3 ' (dT) primer (10 μM)
5X reverse transcription buffers (250mM Tris-HCl (pH 8.3), 375mM KCl, 15mM MgCl2)
Dithiothreitol (DTT), DTT (20mM) U.S. Thermo Scientific#R0861
DNTP Mix (10mM) U.S. Invitrogen#18427088
RNAse Out (40U/ μ L) U.S. Invitrogen#10777019
The Oligo of XCR 5 ' joints (10 μM)
Superscript II RT (200U/ μ L) U.S. Invitrogen#18064022
Each RNA sample is mixed according to following ratio：
| Reagent | Volume 1X (μ L) |
| RNA | 8 |
| The Oligo of XCR 3 ' (dT) primer (10 μM) | 1 |
Hatch in 72 DEG C 3 minutes, then 4 DEG C 1 minute.
PCR reaction buffers are prepared according to following ratio
| Reagent | Volume 1X (μ L) |
| 5X reverse transcription buffers | 3.5 |
| DTT(20mM) | 1 |
| dNTP(10mM) | 1 |
| RNAse Out | 1 |
| The Oligo of XCR 5 ' joints (10 μM) | 1 |
| Superscript II RT | 1 |
PCR reactions caching liquid and RNA samples are mixed, RNA reverse transcriptions are started according to following response procedures
42 DEG C 60 minutes
70 DEG C 10 minutes
4 DEG C permanent
After reaction terminates, so that it may obtain and with the addition of total cDNA (such as Fig. 1) of joint at 5 ' ends
(6) PCR (PCR1).By way of " single pair of " primer, or by under 1 sense primer, 2 or more than 2
The mode of primer is swum, while expanding restructuring TCR and BCR cDNA, is comprised the following steps that：
The reagent used：
Q5High-Fidelity 2X Master Mix (U.S. NEB#M0492L)
The end connectors of XCR 5 ' primer (sense primer)
TCR C areas primer (anti-sense primer)
BCR C areas primer (anti-sense primer)
Remove nuclease water (U.S. Thermo Scientific AM9914G)
Reaction system is prepared according to following ratio：
The response procedures of PCR 1 are:
(7) PCR (PCR2).For PCR1 products (TCR and BCR sequences after amplification) addition Illumina high-flux sequences
The upper machine joint and label of instrument, while more upper machine gene dosage is expanded again for increase.Comprise the following steps that：
The reagent used：
Q5High-Fidelity 2X Master Mix (U.S. NEB#M0492L)
Label sense primer
Label TCR anti-sense primers
Label B CR anti-sense primers
Remove nuclease water (U.S. Thermo Scientific AM9914G)
Reaction system is prepared according to following ratio：
The response procedures of PCR 2 are:
(8) PCR2 product purifications.After above-mentioned PCR reactions terminate, DNA purifying is carried out using magnetic bead, is comprised the following steps that：
The reagent used：
Agencourt AMPure XP Beads (U.S. Beckman#A63882)
Specific purification step is as follows：
1. adding 80 μ L AMPure XP Beads enters PCR2 reaction products, mix.
2. in incubation at room temperature 10 minutes.
3. magnetic bead-PCR2 product mixtures test tube is placed after on magnetic frame, waiting all magnetic beads to be adsorbed on magnetic frame,
All supernatants are sucked with pipettor, are abandoned.
4. adding the ethanol of 150 μ L 75% on magnetic bead, hatch 30 seconds, all supernatants are sucked with pipettor, abandon.
5. repeat the 4th step 2 times.
6. opening test tube cap, wait 5 minutes, treat that magnetic bead is air-dried, residued in without any ethanol in test tube.
7. a test tube is removed from magnetic frame, and 50 μ L of addition remove nuclease water, utilize pipettor to blow and beat suspension magnetic bead.
8. a tube back magnetic frame, waits all magnetic beads to be all adsorbed in after magnetic frame, transfer supernatant is in new test tube
In.On
The PCR2 products after purifying are just contained in clear liquid.
(9) after library purifying terminates, Agilent 2100Bioanalyzer (Agilent companies of U.S. # are utilized
G2939AA the purity and size in library) are detected, the kit used is that (U.S. Agilent is public by Agilent DNA 1000Kit
Take charge of #5067-1504), testing result is as shown in figure 3, library size is in 768bp or so scope, and library purity is at a relatively high,
And have not seen other non-specific amplification sequences.
(10) utilize2.0Fluorometer (U.S. Thermo Fisher Scientific company #
Q32866), coordinateDsDNA HS Assay Kit kits (U.S. Thermo Fisher Scientific company #
Q32851 DNA library concentration) is determined, and send company to carry out high-flux sequence.The cDNA library of gained is passed through into IlluminaPlatform (Illumina companies of the U.S.) is sequenced, and sequencing pattern is PE300, and library denaturant concentration is 2nM, and upper machine is dense
Spend for 20pM, and pass through bioinformatic analysis high-flux sequence result.
Material and reagent explanation
Healthy volunteer's informed consent.No special illustrates that the reagent that the present invention is used is commercial goods, the present invention
The database that embodiment is used is disclosed online database.
Specifically, the termination of reverse transcription 5 ' header sequence of the present invention, primer sequence are following (5 ' -3 ')：
Reverse transcription step
The Oligo of XCR 3 ' (dT) primer：
5’TTTTTTTTTTTTTTTTTTTTGA 3’
The Oligo joints of XCR 5 '：
5’ATGCATCGGATCTTCAGCATGAACTTrGrGrG 3’
PCR1 steps
The end connector primers of XCR 5 '：
5’GTCTCGTGGGCTGGGCGATGTGTATGAGAGACAGCATGCATCGGATCTTCAGCATGA 3’
TCR-alpha chain C areas primer：
5’TCGTCGCCAGCGTCGGAAGTGTATAAGAGACAGTCTCAGCTGGTACATATCGATGTCAGGGT 3’
TCR-beta chain C areas primer：
5’TCGTCGCCAGCGTCGGAAGTGTATAAGAGACAGTCGCAGCGTCAGATGTGTATAAGAGACAG 3’
BCR-Heavy chain C areas primer：
IgA：
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGGCTCCTGGGTTCCGAAGCC 3’
IgG：
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGCGCCTGAGAAGGACGACAC 3’
IgM：
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGGGGAATTCTTCTGGGAGAC 3’
BCR-Light chain C areas primer：
Kappa:
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTACCTTCCACTCTATATTGGCCTC 3’
Lambda:
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGCCACTGTATCCGCTCCCGGG 3’。
PCR2 steps
Label sense primer：
5’CAAGCAGAAGACGGCATACGAGAT[index1]GTCTCGTGGGCTGG 3’
Label TCR anti-sense primers：
5’AATGATACGGCGACCACCGAGATCTACAC[index2]TCGTCGCCAGCGTC 3’
Label B CR anti-sense primers：
5’AATGATACGGCGACCACCGAGATCTACAC[index3]TACGCGATATCGCT 3’
RG=RNA nucleotides
In Tag primerUnderscoreSequence label is sequenced for Illumina in part, and [] interior sequence is replaced by following table sequence,
During for detecting multiple samples simultaneously, different index1-index2 or index1-index3 combinations and biological information are utilized
Learn φt cell receptor (TCR) and B-cell receptor (BCR) sequencing result that algorithm distinguishes multiple samples.
Design of primers：Joint sequence and TCR during for RNA reverse transcriptions in 5 ' end additions are divided in BCR C areas gene
Analysis, is analyzed primer dimer and stem ring mispairing using Oligo 7.36 and Primer Premier 6.0, at 5 ' ends
Manual splice is provided with sense primer, and reverse primer is designed for C downstream of gene, and amplification TCR transcribes subregion sequence with BCR total lengths
Row, wherein containing TCR and BCR immune groups storehouse FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 regions.
After the end connectors of PBMC RNA 5 ', primer and library construction using the present invention, high-flux sequence obtains about several
Million TCR and BCR sequences.
Sequencing result carries out bioinformatic analysis, and (analysis of biological information uses Bowtie 2aligner
(Ver.2.1.0), TCR and BCR derive from international immunogene information system according to storehouse matching www.imgt.org),
Partial analysis comparison result is as shown in Figure 4,5.
By bioinformatic analysis, the information of every TCR and BCR sequence, amino acid information, bar can be accurately known
Number and proportion.It is representative present invention obtains high-flux sequence sequence TCR and BCR by TCR and BCR comparative analyses
The statistic analysis result of clone, as a result as shown in Fig. 4 (TCR) and 5 (BCR).
The above results show that TCR and BCR genes can be covered by building TCR and BCR libraries simultaneously using the method for the present invention
Diversity information, improve the recall rate of low copy number immune cell clones and save time, reagent and cost of labor.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modifications, equivalent substitutions and improvements made within refreshing and principle etc., should be included in the scope of the protection.
Sequence table SEQ UENCE LISTING
<110>Sun Tao
<120>It is a kind of to detect T cell and the primer combination in B cell immune group storehouse and kit simultaneously applied to high-flux sequence
<160> 34
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>It is artificial synthesized
<220>
<223>The Oligo of XCR 3 ' (dT) primer
<400> 1
ttttttttttttttttttttga 22
<210> 2
<211> 32
<212> DNA
<213>It is artificial synthesized
<220>
<223>The Oligo joints of XCR 5 '
<400> 2
atgcatcggatcttcagcatgaacttrgrgrg 32
<210> 3
<211> 57
<212> DNA
<213>It is artificial synthesized
<220>
<223>The end connector primers of XCR 5 '
<400> 3
gtctcgtgggctgggcgatgtgtatgagagacagcatgcatcggatcttcagcatga 57
<210> 4
<211> 62
<212> DNA
<213>It is artificial synthesized
<220>
<223>TCR-alpha chain C areas primer
<400> 4
tcgtcgccagcgtcggaagtgtataagagacagtctcagctggtacatatcgatgtcagggt 62
<210> 5
<211> 62
<212> DNA
<213>It is artificial synthesized
<220>
<223>TCR-beta chain C areas primer
<400> 5
tcgtcgccagcgtcggaagtgtataagagacagtcgcagcgtcagatgtgtataagagacag 62
<210> 6
<211> 55
<212> DNA
<213>It is artificial synthesized
<220>
<223>BCR-Heavy chain C area primer-IgA
<400> 6
tacgcgatatcgctctgcgctaactagtcgtactaggctcctgggttccgaagcc 55
<210> 7
<211> 55
<212> DNA
<213>It is artificial synthesized
<220>
<223> IgG
<400> 7
tacgcgatatcgctctgcgctaactagtcgtactagcgcctgagaaggacgacac 55
<210> 8
<211> 55
<212> DNA
<213>It is artificial synthesized
<220>
<223> IgM
<400> 8
tacgcgatatcgctctgcgctaactagtcgtactaggggaattcttctgggagac 55
<210> 9
<211> 57
<212> DNA
<213>It is artificial synthesized
<220>
<223>BCR-Light chain C area primer-Kappa
<400> 9
tacgcgatatcgctctgcgctaactagtcgtactaccttccactctatattggcctc 57
<210> 10
<211> 56
<212> DNA
<213>It is artificial synthesized
<220>
<223> Lambda
<400> 10
tacgcgatatcgctctgcgctaactagtcgtactagccactgtatccgctcccggg 56
<210> 11
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 11
caagcagaagacggcatacgagatatctatcggtctcgtgggctgg 46
<210> 12
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 12
caagcagaagacggcatacgagattcaggtgagtctcgtgggctgg 46
<210> 13
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 13
caagcagaagacggcatacgagatcactagttgtctcgtgggctgg 46
<210> 14
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 14
caagcagaagacggcatacgagatgaattgccgtctcgtgggctgg 46
<210> 15
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 15
caagcagaagacggcatacgagat atgtacaagtctcgtgggctgg 46
<210> 16
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 16
caagcagaagacggcatacgagatgattcagtgtctcgtgggctgg 46
<210> 17
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 17
caagcagaagacggcatacgagatctgttcgtgtctcgtgggctgg 46
<210> 18
<211> 46
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label sense primer
<400> 18
caagcagaagacggcatacgagattatacggcgtctcgtgggctgg 46
<210> 19
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 19
aatgatacggcgaccaccgagatctacactagctacttcgtcgccagcgtc 51
<210> 20
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 20
aatgatacggcgaccaccgagatctacacattatagctcgtcgccagcgtc 51
<210> 21
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 21
aatgatacggcgaccaccgagatctacaccccgtacttcgtcgccagcgtc 51
<210> 22
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 22
aatgatacggcgaccaccgagatctacacgggtataatcgtcgccagcgtc 51
<210> 23
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 23
aatgatacggcgaccaccgagatctacacagcaggtgtcgtcgccagcgtc 51
<210> 24
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 24
aatgatacggcgaccaccgagatctacactatacgtatcgtcgccagcgtc 51
<210> 25
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 25
aatgatacggcgaccaccgagatctacaccacctagttcgtcgccagcgtc 51
<210> 26
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label TCR anti-sense primers
<400> 26
aatgatacggcgaccaccgagatctacacgttgctactcgtcgccagcgtc 51
<210> 27
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 27
aatgatacggcgaccaccgagatctacaccacgatgttacgcgatatcgct 51
<210> 28
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 28
aatgatacggcgaccaccgagatctacactaatcgcttacgcgatatcgct 51
<210> 29
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 29
aatgatacggcgaccaccgagatctacacatattacctacgcgatatcgct 51
<210> 30
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 30
aatgatacggcgaccaccgagatctacaccctatacttacgcgatatcgct 51
<210> 31
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 31
aatgatacggcgaccaccgagatctacac ggatgaaatacgcgatatcgct 51
<210> 32
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 32
aatgatacggcgaccaccgagatctacacagtctttgtacgcgatatcgct 51
<210> 33
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 33
aatgatacggcgaccaccgagatctacactagtggtatacgcgatatcgct 51
<210> 34
<211> 51
<212> DNA
<213>It is artificial synthesized
<220>
<223>Label B CR anti-sense primers
<400> 34
aatgatacggcgaccaccgagatctacacgtatgaactacgcgatatcgct 51
Claims (6)
1. a kind of primer combination for detecting T cell and B cell immune group storehouse simultaneously applied to high-flux sequence, it is characterised in that：
The primer combination includes the Oligo of XCR 3 ' (dT) primer, the Oligo joints of XCR 5 ', the end connector primers of XCR 5 ', TCR-
IgA, IgG and IgM in alpha chain C areas primer, TCR-beta chain C areas primer, BCR-Heavy chain C areas primer, BCR-Light
Kappa and Lambda in chain C areas primer, and label upstream and downstream primer；Every kind of primer sequence is as follows：
The Oligo of XCR 3 ' (dT) primer：5’TTTTTTTTTTTTTTTTTTTTGA 3’；
The Oligo joints of XCR 5 '：5’ATGCATCGGATCTTCAGCATGAACTTrGrGrG 3’；
The end connector primers of XCR 5 '：
5’GTCTCGTGGGCTGGGCGATGTGTATGAGAGACAGCATGCATCGGATCTTCAGCATGA 3’；
TCR-alpha chain C areas primer：
5’TCGTCGCCAGCGTCGGAAGTGTATAAGAGACAGTCTCAGCTGGTACATATCGATGTCAGGGT 3’；
TCR-beta chain C areas primer：
5’TCGTCGCCAGCGTCGGAAGTGTATAAGAGACAGTCGCAGCGTCAGATGTGTATAAGAGACAG 3’；
BCR-Heavy chain C areas primer：
IgA：5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGGCTCCTGGGTTCCGAAGCC 3’；
IgG：5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGCGCCTGAGAAGGACGACAC 3’；
IgM：5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGGGGAATTCTTCTGGGAGAC 3’；
BCR-Light chain C areas primer：
Kappa:
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTACCTTCCACTCTATATTGGCCTC 3’；
Lambda:
5’TACGCGATATCGCTCTGCGCTAACTAGTCGTACTAGCCACTGTATCCGCTCCCGGG 3’；
Label sense primer：5’CAAGCAGAAGACGGCATACGAGAT[index1]GTCTCGTGGGCTGG 3’；
Label TCR anti-sense primers：5’AATGATACGGCGACCACCGAGATCTACAC[index2]TCGTCGCCAGCGTC 3’；
Label B CR anti-sense primers：5’AATGATACGGCGACCACCGAGATCTACAC[index3]TACGCGATATCGCT 3’。
2. according to claim 2 a kind of drawing for T cell and B cell immune group storehouse is detected simultaneously applied to high-flux sequence
Thing is combined, it is characterised in that：The index1 be ATCTATCG, TCAGGTGA, CACTAGTT, GAATTGCC, ATGTACAA,
One kind in GATTCAGT, CTGTTCGT or TATACGGC；Index2 be TAGCTACT, ATTATAGC, CCCGTACT,
One kind in GGGTATAA, AGCAGGTG, TATACGTA, CACCTAGT or GTTGCTAC；
Index3 be TAATCGCT, ATATTACC, CCTATACT, GGATGAAA, AGTCTTTG, TAGTGGTA, CACGATGT or
One kind in GTATGAAC.
3. a kind of high-flux sequence that is applied to according to claim 1 or 2 detects T cell and B cell immune group storehouse simultaneously
Kit, it is characterised in that：The kit includes primer combination as claimed in claim 1 or 2.
4. a kind of examination for detecting T cell and B cell immune group storehouse simultaneously applied to high-flux sequence according to claim 3
Agent box, it is characterised in that：The kit also includes PCR buffer solutions, Q5High-Fidelity 2X Master Mix, stoning
Sour enzyme water, AMPure XP Beads and 70% ethanol.
5. a kind of examination for detecting T cell and B cell immune group storehouse simultaneously applied to high-flux sequence according to claim 4
Agent box, it is characterised in that：The kit includes：
(1) PCR buffer solutions：9.5μL；
(2) positive control RNA (1 μ g/ μ l)：10μL；
(3) the 10 μM of Oligo of TCR 3 ' (dT) primers：1.1μL；
(4) 10 μM of end connector primers of TCR 5 '：2.2μL；
(5) 10 μM of TCR C area's primers and 10 μM of BCR C areas primers：Each 1.1 μ L；
(6)Q5High-Fidelity 2X Master Mix：55μL；
(9) 10 μM of label sense primers：2.2μL；
(10) 10 μM of TCR labels anti-sense primers and 10 μM of BCR label anti-sense primers：Each 1.1 μ L；
(11) nuclease water is removed：105μL；
(12)AMPure XP Beads：88μL；
(13) 70% ethanol：165μL.
6. one kind according to claim 4 or 5 is applied to high-flux sequence and detects T cell and B cell immune group storehouse simultaneously
Kit, it is characterised in that：The PCR buffer solutions are made up of following system：
。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110938683A (en) * | 2018-09-21 | 2020-03-31 | 深圳华大生命科学研究院 | Method for evaluating library construction pollution degree of target area library and method for optimizing library construction |
| CN114480622A (en) * | 2022-01-29 | 2022-05-13 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Kit for predicting treatment sensitivity of psoriasis to methotrexate |
| CN117126921A (en) * | 2023-10-26 | 2023-11-28 | 立凌生物制药(苏州)有限公司 | Library construction method for detecting T cell and B cell immune repertoire, primer and kit thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110938683A (en) * | 2018-09-21 | 2020-03-31 | 深圳华大生命科学研究院 | Method for evaluating library construction pollution degree of target area library and method for optimizing library construction |
| CN114480622A (en) * | 2022-01-29 | 2022-05-13 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Kit for predicting treatment sensitivity of psoriasis to methotrexate |
| CN117126921A (en) * | 2023-10-26 | 2023-11-28 | 立凌生物制药(苏州)有限公司 | Library construction method for detecting T cell and B cell immune repertoire, primer and kit thereof |
| CN117126921B (en) * | 2023-10-26 | 2024-01-26 | 立凌生物制药(苏州)有限公司 | Library construction method for detecting T cell and B cell immune repertoire, primer and kit thereof |
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