CN105087789A - Method for detecting BCR and TCR immune repertoire in blood plasma cfDNA - Google Patents
Method for detecting BCR and TCR immune repertoire in blood plasma cfDNA Download PDFInfo
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Abstract
The invention provides a method for detecting BCR and TCR immune repertoire in blood plasma cfDNA. The method includes: blood plasma cfDNA extraction, BCR H chain overall length multiple PCR amplification, TCR beta chain CDR3 multiple PCR amplification, PCR amplification after the products of the two previous amplifications are mixed in an equal-volume manner, high-throughput sequencing, and immune repertoire precise information analysis. The primers of the three amplifications are respectively shown in SEQ ID No. 1-28, SEQ ID No. 29-77 and SEQ ID No. 78-79. The invention further provides a kit, which contains the primers, for detecting the BCR and TCR in the blood plasma cfDNA. By the method and the kit which are promising in application prospect in the field of non-invasive detection, the BCR and TCR in the blood plasma cfDNA can be detected at the same time, precise information analysis and precise quantification of immune repertoire subcloning are achieved.
Description
Technical field
The invention belongs to sequencing technologies field, immune group storehouse, be specifically related to a kind of method detecting BCR and TCR immune group storehouse in blood plasma cfDNA.
Background technology
Have benefited from fast development and the widespread use of high throughput sequencing technologies of future generation, DNA and RNA order-checking platform plays outstanding pushing effect in all respects of genomics.Meanwhile, this emerging technical field has also been applied to the immune group storehouse order-checking of T cell and B-cell receptor.
The immune group storehouse order-checking of conventional T cell and B-cell receptor, mainly based on 2 large strategies, first: based on cell DNA by TCR/BCR gene rearrangement rule, at variable region (variable) gene of TCR (alpha/beta/gamma/delta chain) and BCR (heavy/light chain), upstream primer is set, at joining region (joining) J gene or constant region (constant) C gene, downstream primer is set, the PCR primer of object chain is gone out by pcr amplification, but unhomogeneity when it often exists amplification, thus affect the accuracy of result; Second: based on cell RNA, adopt 5 ' RACE technology, carry out the pcr amplification of second time without preference (bias) based on the joint sequence introduced.But RNA sample process is complicated, repeatability, not as DNA detection result, is not a kind of desirable starting material.
Circulate in blood plasma dissociative DNA (cfDNA), and its source is mainly discharged in blood by apoptosis.Tumour patient blood middle reaches become study hotspot from the gene test diagnosis of ctDNA (Cell-freeCirculatingTumorDNA) in recent years, and the relevant IG-seq based on blood plasma ctDNA detects in development just fast, it is using blood plasma cfDNA/ctDNA as the first raw material, increase for specific immunity subclone, for risk assessment and the prediction of relative disease, the bright prospects of this " liquid biopsy " mark of cfDNA application are expanded.But the analysis and research of indivedual specificity clone, there is limitation, it completely comprehensively can not represent the progression of disease situation of patient.Simultaneously for the optimization of DNA cloning unhomogeneity adjustment, the introducing of current molecular label coding techniques, not only can distinguish order-checking and increase mistake and the rare sudden change in canonical sequence, and can molecular method quantification accurately be carried out, thus the maximized data analysis achieved without Preference.
Summary of the invention
The invention provides a kind of method detecting BCR and TCR immune group storehouse in blood plasma cfDNA to overcome the deficiencies in the prior art.
The method in BCR and TCR immune group storehouse in detection blood plasma cfDNA provided by the invention, comprises the following steps:
(1) blood plasma cfDNA extracts;
(2) BCRH chain total length multiplexed PCR amplification and TCR β chain CDR3 multiplexed PCR amplification;
(3) pcr amplification is carried out again after two kinds of amplified production mixing in step (2);
(4) high-flux sequence;
(5) immune group storehouse accurate information is analyzed.
The schema of the inventive method is shown in Fig. 1.
In the inventive method, the upstream primer totally 22 that BCRH chain total length multiplexed PCR amplification adopts in step (2), sequence respectively as shown in SEQIDNO.1-22, downstream primer totally 6, sequence is respectively as shown in SEQIDNO.23-28;
The upstream primer totally 45 that TCR β chain CDR3 multiplexed PCR amplification adopts, sequence is respectively as shown in SEQIDNO.29-73, and downstream primer totally 4, sequence is respectively as shown in SEQIDNO.74-77.
Further, in step (2), the concentration of each bar upstream primer is identical, and the concentration of each bar downstream primer is identical, and the single primer concentration in downstream is 20 times of the single primer concentration in upstream.
Preferably, in step (2), BCRH chain total length multiplexed PCR amplification condition is: 98 DEG C of denaturation 45s; 98 DEG C of sex change 15s, 60 DEG C of annealing 30s, 72 DEG C extend 30s, totally 8 circulations; 72 DEG C of ends extend 60s; 4 DEG C of maintenances.
Preferably, in step (2), TCR β chain CDR3 multiplexed PCR amplification condition is: 98 DEG C of denaturation 45s; 98 DEG C of sex change 15s, 54 DEG C of annealing 30s, 72 DEG C extend 30s, totally 8 circulations; 72 DEG C of ends extend 60s; 4 DEG C of maintenances.
Two kinds of amplified productions described in step (3) of the inventive method refer to the product of clip size after the fragment of 200-250bp reclaims purifying in product after the fragment of 300-350bp reclaims purifying of clip size in BCRH chain total length multiplexed PCR amplification product and TCR β chain CDR3 multiplexed PCR amplification product respectively.
Preferably, a pcr amplification is carried out again by after two kinds of amplified production equal-volume mixing of step (2) in step (3); The primer sequence adopted is as shown in SEQIDNO.78-79.
In step (3), PCR response procedures is: 98 DEG C of denaturation 45s; 98 DEG C of sex change 15s, 65 DEG C of annealing 30s, 72 DEG C extend 30s, totally 15 circulations; 72 DEG C of ends extend 60s; 4 DEG C of maintenances.
In detection blood plasma cfDNA of the present invention BCR and TCR immune group storehouse method in, the immune group storehouse accurate information analysis of step (5) comprises the following steps:
1) determine the position of 7 randomized bases N based on known joint sequence, 7 randomized bases can form 4
7plant unique tags, the sequencing sequence 1 of paired sequencing sequence and the unique tags sequence of sequencing sequence 2 are joined end to end, form an index of 14bp, and using this 14bp as paired sequencing sequence index carry out external sort, to reach the object be brought together by all sequencing sequences deriving from same DNA profiling;
2) central cluster is carried out to the sequencing sequence having same index gathered together, according to the Hamming distance between insertion sequence, large bunch of same index is had to be gathered into several tuftlets by each, in each tuftlet, any two are no more than 3 to the Hamming distance of paired sequencing sequence, have same index but from the object of the sequencing sequence of different DNA profiling to reach to distinguish;
3) each the order-checking base for the sequencing sequence in dup bunch (the serial repeated fragment that each DNA profiling amplification is formed) of each DNA profiling carries out mutual comparison, if the concordance rate of certain base type in sequencing sequence reaches 80%, then remember this base base type for this reason of new sequencing sequence, otherwise be designated as N, so just, obtain the new sequencing sequence representing original DNA template sequence, correct by this method, effectively can remove the random mistake introduced in order-checking and PCR, improve the accuracy detected;
4) data filter is carried out to new sequencing sequence, the sequencing sequence that removal joint sequence and comparison quality are less than 30;
5) according to 4) in the sequencing sequence that obtains and IMGT database the embryonal system reference sequences of V, D and J district gene fragment compare and annotate, simultaneously according to the uniquely tagged of unique tags to each template, statistics is carried out quantitatively to each immune subclone;
6) according to 5) result carry out Sequence structure analysis, immune group storehouse expression pattern analysis, Biomarker analyze.
Above-mentioned steps 2) described in Hamming distance distance values be no more than the key point that 3 are information analysis of the present invention, in order to the subclone that differentiation as much as possible is different, need the tolerance of compression Hamming distance as far as possible, to obtain the subclone type of more comprehensively BCR and TCR.
On the other hand, the invention provides a kind of system detecting BCR and TCR immune group storehouse in blood plasma cfDNA, comprise following operating unit:
(1) blood plasma cfDNA extraction unit;
(2) BCRH chain total length multiplexed PCR amplification and TCR β chain CDR3 multiplexed PCR amplification unit;
(3) a pcr amplification unit is carried out again after two kinds of amplified production mixing in step (2);
(4) high-flux sequence unit;
(5) immune group storehouse accurate information analytical unit.
The upstream primer totally 22 that BCRH chain total length multiplexed PCR amplification adopts in described unit (2), sequence respectively as shown in SEQIDNO.1-22, downstream primer totally 6, sequence is respectively as shown in SEQIDNO.23-28;
The upstream primer totally 45 that TCR β chain CDR3 multiplexed PCR amplification adopts, sequence is respectively as shown in SEQIDNO.29-73, and downstream primer totally 4, sequence is respectively as shown in SEQIDNO.74-77.
Carry out a pcr amplification again after two kinds of amplified production equal-volume mixing by unit (2) in unit (3); The primer sequence adopted is as shown in SEQIDNO.78-79.
The immune group storehouse accurate information analysis of unit (5) comprises the following steps:
1) determine the position of 7 randomized bases N based on known joint sequence, 7 randomized bases can form 4
7plant unique tags, the sequencing sequence 1 of paired sequencing sequence and the unique tags sequence of sequencing sequence 2 are joined end to end, form an index of 14bp, and using this 14bp as paired sequencing sequence index carry out external sort, to reach the object be brought together by all sequencing sequences deriving from same DNA profiling;
2) central cluster is carried out to the sequencing sequence having same index gathered together, according to the Hamming distance between insertion sequence, large bunch of same index is had to be gathered into several tuftlets by each, in each tuftlet, any two are no more than 3 to the Hamming distance of paired sequencing sequence, have same index but from the object of the sequencing sequence of different DNA profiling to reach to distinguish;
3) each the order-checking base for the sequencing sequence in the serial repeated fragment of each DNA profiling amplification formation carries out mutual comparison, if the concordance rate of certain base type in sequencing sequence reaches 80%, then remember this base base type for this reason of new sequencing sequence, otherwise be designated as N, so just, obtain the new sequencing sequence representing original DNA template sequence, correct by this method, effectively can remove the random mistake introduced in order-checking and PCR, improve the accuracy detected;
4) data filter is carried out to new sequencing sequence, the sequencing sequence that removal joint sequence and comparison quality are less than 30;
5) according to 4) in the sequencing sequence that obtains and IMGT database the embryonal system reference sequences of V, D and J district gene fragment compare and annotate, simultaneously according to the uniquely tagged of unique tags to each template, statistics is carried out quantitatively to each immune subclone;
6) according to 5) result carry out other correlation analyses.
The invention provides said detecting system and detect the application in blood plasma cfDNA in BCR and TCR immune group storehouse.
The invention provides aforesaid method and the application of said detecting system in preparation disorder in screening test kit.
Further, described disease is tumour, autoimmune disorder, infectious diseases.
The invention provides aforesaid method and the application of said detecting system in the test kit of preparation evaluation function of immune system such as stem cell and/or organ transplantation.
Present invention also offers a kind of combination of primers for detecting BCR and TCR immune group storehouse in blood plasma cfDNA, containing 3 groups of primers, the primer sequence that first group of primer contains is respectively as shown in SEQIDNO.1-28; The primer sequence that second group of primer contains is respectively as shown in SEQIDNO.29-77; The 3rd group of primer sequence that primer contains is respectively as shown in SEQIDNO.78-79.
Above-mentioned first group, second group primer is referred to as PCR1 primer system in an embodiment of the present invention, 3rd group of primer is called PCR2 primer system, PCR1 primer system designs respectively based on BCR and TCR, and object is all amplified the BCRH chain of total length and TCRBeta chain CDR3 district.The immune polymorphism of BCR arranges based on the restructuring of V (D) J district fragment; TCRCDR3 district just covers TRBV-Junction-TRBD-Junction-TRBJ region, maximum owing to making a variation, thus directly determines the antigen-specific of TCR.
PCR2 primer system is the universal primer of BCR/TCR second when taking turns PCR, and its PCR-based 1 product is template, introduces order-checking label and sequencing sequence, complete the structure in library by primer.
Primer construction for the PCR1 of BCR and TCR: the upstream and downstream primer comprising joint sequence, specific sequence label and BCR/TCR amplification from 5 ' end to 3 ' end all successively.
The upstream and downstream design of primers of BCR amplification: according to the homology analysis to BCRV gene 7 great Lei family ORF (being respectively FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4), the upstream primer designed with the leader district of V gene (22 altogether, as shown in SEQIDNO.1-22) and at least one downstream primer (6 altogether, as shown in SEQIDNO.23-28) that designs respectively according to the feature of the C district gene order of BCRmIgG, mIgA, mIgM, mIgE or mIgD.Details are in table 1.
TCR amplification upstream and downstream design of primers: people TCR β chain V gene family comprises 45 functioning gene families and 7 ORF, altogether designs 45 upstream primers (as shown in SEQIDNO.29-73) according to the homology of 52 genes; And the C gene very high homology of people TCR β chain, therefore the general downstream primer (as shown in SEQIDNO.74-77) that design 4 is different, random and 46 upstream primers match, and to avoid because blood plasma fragment random fracture reason causes increasing unsuccessfully, improve the success ratio of fragment amplification.Details are in table 2.
The feature that PCR1 upstream and downstream primer all has is: (1) joint sequence is with the Illumina platform of existing maturation for reference design, and upstream primer is 34bp, and downstream primer is 33bp; (2) specific sequence label: the sequence being set to 7 randomized bases compositions according to data empirical analysis display, obtains higher data user rate by being more conducive to.4 of its generation
7plant tag combination, meet the mark to each DNA profiling molecule completely.(3) based on the feature of the average 170bp of blood plasma fragment, for more effectively realizing the amplification to blood plasma fragment, upstream and downstream primer length controls at about 16bp as far as possible.
The primer construction of BCR and TCR-PCR2: in PCR2 primer system, the basic primer sequence of upstream and downstream primer is complementary with the joint sequence at the product two ends of PCR1 primer system respectively, its upstream primer only comprises sequencing sequence simultaneously, downstream primer then comprises sequencing sequence and order-checking label---8 known fixed bases (index), for carrying out the differentiation mixing order-checking sample room.Details are in table 3.
The invention provides the application of above-mentioned combination of primers in preparation disorder in screening test kit or diagnostic reagent.
The invention provides a kind of test kit detecting BCR and TCR immune group storehouse in blood plasma cfDNA, it contains combination of primers of the present invention.
In detection blood plasma cfDNA provided by the invention, BCR and TCR immune group storehouse method has comprehensively, efficiently, precisely, feature easily, relative to prior art, the present invention introduces label coding technology and is optimized, based on the unique tags that 7 randomized bases are formed, to set in each tuftlet any two simultaneously and 3 are no more than to the Hamming distance of paired sequencing sequence, meet and each template is marked, realize higher data separate efficiency, not only can correct the mistake introduced in order-checking and PCR, and according to the uniquely tagged to each template, can maximizedly realize without the data analysis of Preference, realize the precisely quantitative of each subclone in immune group storehouse.The inventive method is discharged into the gene order of BCR and TCR of DNA fragmentation in blood by detecting different subcloned cells apoptosis in blood plasma, analyzes its polymorphism, for the early screening of disease of immune system provides technical support with rear direction of medication usage.On the other hand, the primer of the present invention's design almost enumerates the sequence for the immune polymorphism of all BCRH chain VDJ and the immune polymorphism in TCRBeta chain CDR3 district, thus the assessment of immunological status can be carried out more comprehensively accurately, can be used for preparing the test kit detecting malignant tumour, autoimmune pathologies, also can be used for stem cell transplantation and filed of organ transplantation carries out thoroughly evaluating to the immunologic function of body, have broad application prospects in Non-invasive detection field.
Accompanying drawing explanation
Fig. 1 is the schema of detection method.
Fig. 2 adopts detection method to the TCR immunity polymorphic detection result of patients with lung adenocarcinoma blood plasma cfDNA.
Fig. 3 adopts detection method to the BCR cluster analysis detected result of patients with lung adenocarcinoma blood plasma cfDNA.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, chemical reagent used in embodiment is conventional commercial reagent, the conventional means that technique means used in embodiment is well known to those skilled in the art.The sequencing device adopted in the embodiment of the present invention is IlluminaHiSeq2500, in sequencing steps of the present invention, is not limited to this sequencing device.
Embodiment 1 detects the design of the primer of BCR and TCR in blood plasma cfDNA
The immune polymorphism of BCR arranges based on the restructuring of V (D) J district fragment.TCRCDR3 district just covers TRBV-Junction-TRBD-Junction-TRBJ region, maximum owing to making a variation, thus directly determines the antigen-specific of TCR.Carry out design of primers respectively for BCR and tcr gene sequence, object is all amplified the BCRH chain of total length and TCRBeta chain CDR3 district.
The primer construction of BCR/TCR-PCR1: the upstream and downstream primer comprising joint sequence, specific sequence label and BCR/TCR amplification from 5 ' end to 3 ' end all successively.
The upstream and downstream design of primers of BCR amplification: according to the homology analysis to BCRV gene 7 great Lei family ORF (being respectively FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4), the upstream primer designed with the leader district of V gene (22 altogether, in the B-PCR1 upstream primer of table 1, or as shown in SEQIDNO.1-22) and at least one downstream primer (6 altogether that designs respectively according to the feature of the C district gene order of BCRmIgG, mIgA, mIgM, mIgE or mIgD, in the B-PCR1 downstream primer of table 1, or as shown in SEQIDNO.23-28).
TCR amplification upstream and downstream design of primers: people TCR β chain V gene family comprises 45 functioning gene families and 7 ORF, 45 upstream primers (see the T-PCR1 upstream primer of table 2, or as shown in SEQIDNO.29-73) are altogether designed according to the homology of 52 genes; And the C gene very high homology of people TCR β chain, therefore the general downstream primer that design 4 is different is (see the T-PCR1 downstream primer of table 2, or as shown in SEQIDNO.74-77), random and 46 upstream primers match, to avoid because blood plasma fragment random fracture reason causes increasing unsuccessfully, improve the success ratio of fragment amplification.
Table 1BCR-PCR1 primer system
Table 2TCR-PCR1 primer system
The primer construction of BCR/TCR-PCR2: the basic primer sequence of PCR2 upstream and downstream primer is complementary with the joint sequence at the product two ends of PCR1 respectively, its upstream primer only comprises sequencing sequence simultaneously, downstream primer then comprises sequencing sequence and order-checking label---8 known fixed bases (index), for carrying out the differentiation mixing order-checking sample room.Details are in table 3.
Table 3BCR/TCR-PCR2 primer system
| Primer | Primer sequence (5 ' → 3 ') |
| PCR2 upstream primer | aatgatacggcgaccaccgagatctacactctttccctacacgacgctcttccgatct |
| PCR2 downstream primer | caagcagaagacggcatacgagatxxxxxxxxgtgactggagttcagacgtgtgctcttccgatct |
Embodiment 2 detects the foundation of BCR and TCR immune group storehouse method in blood plasma cfDNA
1, blood plasma cfDNA extracts:
1.1 extract blood plasma extracts person under inspection peripheral bloods 2 and manages (5mL/ pipe) in EDTA anticoagulant tube, softly turn upside down (preventing cell rupture), fully mixes for 6-8 time, carries out following process taking a blood sample in the 4-6 hour same day; Under 4 DEG C of conditions, centrifugal 10 minutes of 1600g, is dispensed into supernatant (blood plasma) in multiple 1.5mL/2mL centrifuge tube after centrifugal, can not be drawn onto middle layer white corpuscle in suction process; Centrifugal 10 minutes of 16000g under 4 DEG C of conditions, removes residual cells, is transferred in new 1.5mL/2mL centrifuge tube by supernatant (blood plasma), can not be drawn onto white corpuscle at the bottom of pipe, namely obtains being separated rear required blood plasma.
After 1.2 plasma samples process, the blood plasma that separation obtains and residue hemocyte are all saved in-80 DEG C of refrigerators, avoid multigelation.
The extraction of 1.3 blood plasma cfDNA/ctDNA is with quantitative: get isolated blood plasma and be about 2-3ml, extract reagent specification sheets, carry out the extraction of blood plasma cfDNA according to QIAampCirculatingNucleicAcidKit (Qiagen).The DNA that Qubit (Invitrogen, theQuant-iTTMdsDNAHSAssayKit) is quantitative extracted, total amount is about 30 ~ 50ng.
2, carry out BCR/TCR multiplex PCR 1 respectively to increase
Table 1 in embodiment 1 and the upstream and downstream primer in table 2 mix by 2.1 respectively, form BCR and TCR upstream and downstream primer MIX, and the single primer concentration in downstream are 20 times of the single primer concentration in upstream.
2.2BCRH chain total length multiplex PCR 1 reaction amplification, operates based on QIAGEN company MultiplexPCR test kit:
In above-mentioned PCR reaction system, B-PCR1 upstream primer sequence is respectively as shown in SEQIDNO.1-22, and B-PCR1 downstream primer sequence is respectively as shown in SEQIDNO.23-28, and wherein in B-PCR1 upstream and downstream primer Mix, each bar primer is the mixing of equal-volume isoconcentration.
BCRH chain total length multiplexed PCR amplification condition is: 98 DEG C of denaturation 45s; 98 DEG C of sex change 15s, 60 DEG C of annealing 30s, 72 DEG C extend 30s, totally 8 circulations; 72 DEG C of ends extend 60s; 4 DEG C of maintenances.
2.3TCR β chain CDR3 district multiplex PCR 1 reacts amplification, operates based on QIAGEN company MultiplexPCR test kit:
In above-mentioned PCR reaction system, TCRCDR3PCR1 upstream primer sequence is respectively as shown in SEQIDNO.29-74, TCRCDR3PCR1 downstream primer sequence is respectively as shown in SEQIDNO.75-78, and wherein in upstream and downstream primer Mix, each bar primer is the mixing of equal-volume isoconcentration.
TCR β chain CDR3 multiplexed PCR amplification condition is: 98 DEG C of denaturation 45s; 98 DEG C of sex change 15s, 54 DEG C of annealing 30s, 72 DEG C extend 30s, totally 8 circulations; 72 DEG C of ends extend 60s; 4 DEG C of maintenances.
2.4 PCR primer using the above-mentioned amplification of TBE-PAGE gel electrophoresis to obtain, obtain the fragment of clip size at about 300bp or 190bp respectively, output recovery is carried out according to QIAquickGelExtractionKit (Qiagen) reagent specification sheets, obtain the amplified production of BCR/TCRPCR1, Qubit (Invitrogen, theQuant-iTdsDNABRAssayKit) is adopted to carry out quantitatively afterwards.
3, amplified production equal proportion is mixed into performing PCR 2 and increases: for same sample, equal proportion respectively gets the PCR1 purified product of 50ngBCR and TCR, and carry out PCR2 amplification, reaction system is as follows:
PCR response procedures is: 98 DEG C of denaturation 45s; 98 DEG C of sex change 15s, 65 DEG C of annealing 30s, 72 DEG C extend 30s, totally 15 circulations; 72 DEG C of ends extend 60s; 4 DEG C of maintenances.
PCR2 upstream and downstream primer sequence is respectively as shown in SEQIDNO.79,80.
4, upper machine order-checking: adopt IlluminaHiSeq2500PE101+8+101 program to carry out upper machine order-checking, the process specifications (announcing cBot see Illumina/Solexa official) that order-checking experiment provides according to manufacturers carries out upper machine sequencing procedures.
5, immune group storehouse accurate information is analyzed:
1) determine the position of 7 randomized bases N based on known joint sequence, 7 randomized bases can form 4
7plant unique tags, the sequencing sequence 1 of paired sequencing sequence and the unique tags sequence of sequencing sequence 2 are joined end to end, form an index of 14bp, and using this 14bp as paired sequencing sequence index carry out external sort, to reach the object be brought together by all sequencing sequences deriving from same DNA profiling;
2) central cluster is carried out to the sequencing sequence having same index gathered together, according to the Hamming distance between insertion sequence, large bunch of same index is had to be gathered into several tuftlets by each, in each tuftlet, any two are no more than 3 to the Hamming distance of paired sequencing sequence, have same index but from the object of the sequencing sequence of different DNA profiling to reach to distinguish;
3) each the order-checking base for the sequencing sequence in the serial repeated fragment of each DNA profiling amplification formation carries out mutual comparison, if the concordance rate of certain base type in sequencing sequence reaches 80%, then remember this base base type for this reason of new sequencing sequence, otherwise be designated as N, so just, obtain the new sequencing sequence representing original DNA template sequence, correct by this method, effectively can remove the random mistake introduced in order-checking and PCR, improve the accuracy detected;
4) data filter is carried out to new sequencing sequence, the sequencing sequence that removal joint sequence and comparison quality are less than 30;
5) according to 4) in the sequencing sequence that obtains and IMGT database the embryonal system reference sequences of V, D and J district gene fragment compare and annotate, simultaneously according to the uniquely tagged of unique tags to each template, statistics is carried out quantitatively to each immune subclone;
6) according to 5) result carry out Sequence structure analysis, immune group storehouse expression pattern analysis, Biomarker analyze.
9. sequencing result analysis
By the plasma sample of 1 routine adenocarcinoma of lung postoperative patient, detect based on above method steps:
9.1 patient TCR immunity polymorphisms: see Fig. 2.Result illustrates, the distribution of patient T lymphocyte each V, J gene hypotype is more homogeneous, and the highest clone's accounting 0.60%---T lymphocyte group storehouse diversity is good.
9.2 patient B CR cluster analysis results (Lineage analysis): see Fig. 3.Result illustrates, Lineage is substantially in being dispersed in distribution, and indication B cell group storehouse diversity is good.
Comprehensive analysis shows: after operation in patients, overall immune is in good condition.
Above embodiment is only be described the preferred embodiment of the present invention; not scope of the present invention is limited; under not departing from the present invention and designing the prerequisite of spirit; the various modification that the common engineering technical personnel in this area make technical scheme of the present invention and improvement, all should fall in protection domain that claims of the present invention determine.
Claims (20)
1. detect the method in BCR and TCR immune group storehouse in blood plasma cfDNA, comprise the following steps:
(1) blood plasma cfDNA extracts;
(2) BCRH chain total length multiplexed PCR amplification and TCR β chain CDR3 multiplexed PCR amplification;
(3) pcr amplification is carried out again after two kinds of amplified production mixing in step (2);
(4) high-flux sequence;
(5) immune group storehouse accurate information is analyzed.
2. method according to claim 1, it is characterized in that, the upstream primer totally 22 that in step (2), BCRH chain total length multiplexed PCR amplification adopts, sequence is respectively as shown in SEQIDNO.1-22, downstream primer totally 6, sequence is respectively as shown in SEQIDNO.23-28;
The upstream primer totally 45 that TCR β chain CDR3 multiplexed PCR amplification adopts, sequence is respectively as shown in SEQIDNO.29-73, and downstream primer totally 4, sequence is respectively as shown in SEQIDNO.74-77.
3. method according to claim 2, is characterized in that, in step (2), the concentration of each bar upstream primer is identical, and the concentration of each bar downstream primer is identical, and the single primer concentration in downstream is 20 times of the single primer concentration in upstream.
4. method according to claim 3, is characterized in that, in step (2), BCRH chain total length multiplexed PCR amplification condition is: 98 DEG C of denaturation 45s; 98 DEG C of sex change 15s, 60 DEG C of annealing 30s, 72 DEG C extend 30s, totally 8 circulations; 72 DEG C of ends extend 60s; 4 DEG C of maintenances.
5. method according to claim 3, is characterized in that, in step (2), TCR β chain CDR3 multiplexed PCR amplification condition is: 98 DEG C of denaturation 45s; 98 DEG C of sex change 15s, 54 DEG C of annealing 30s, 72 DEG C extend 30s, totally 8 circulations; 72 DEG C of ends extend 60s; 4 DEG C of maintenances.
6. method according to claim 1, it is characterized in that, two kinds of amplified productions described in step (3) refer to the product of clip size after the fragment of 200-250bp reclaims purifying in product after the fragment of 300-350bp reclaims purifying of clip size in BCRH chain total length multiplexed PCR amplification product and TCR β chain CDR3 multiplexed PCR amplification product respectively.
7. method according to claim 6, is characterized in that, carries out a pcr amplification again in step (3) by after two kinds of amplified production equal-volume mixing of step (2); The primer sequence adopted is as shown in SEQIDNO.78-79.
8., according to the arbitrary described method of claim 1-7, it is characterized in that, in step (3), PCR response procedures is: 98 DEG C of denaturation 45s; 98 DEG C of sex change 15s, 65 DEG C of annealing 30s, 72 DEG C extend 30s, totally 15 circulations; 72 DEG C of ends extend 60s; 4 DEG C of maintenances.
9., according to the arbitrary described method of claim 1-7, it is characterized in that, the immune group storehouse accurate information analysis of step (5) comprises the following steps:
1) determine the position of 7 randomized bases N based on known joint sequence, 7 randomized bases can form 4
7plant unique tags, the sequencing sequence 1 of paired sequencing sequence and the unique tags sequence of sequencing sequence 2 are joined end to end, form an index of 14bp, and using this 14bp as paired sequencing sequence index carry out external sort, to reach the object be brought together by all sequencing sequences deriving from same DNA profiling;
2) central cluster is carried out to the sequencing sequence having same index gathered together, according to the Hamming distance between insertion sequence, large bunch of same index is had to be gathered into several tuftlets by each, in each tuftlet, any two are no more than 3 to the Hamming distance of paired sequencing sequence, have same index but from the object of the sequencing sequence of different DNA profiling to reach to distinguish;
3) each the order-checking base for the sequencing sequence in the serial repeated fragment of each DNA profiling amplification formation carries out mutual comparison, if the concordance rate of certain base type in sequencing sequence reaches 80%, then remember this base base type for this reason of new sequencing sequence, otherwise be designated as N, so just, obtain the new sequencing sequence representing original DNA template sequence, correct by this method, effectively can remove the random mistake introduced in order-checking and PCR, improve the accuracy detected;
4) data filter is carried out to new sequencing sequence, the sequencing sequence that removal joint sequence and comparison quality are less than 30;
5) according to 4) in the sequencing sequence that obtains and IMGT database the embryonal system reference sequences of V, D and J district gene fragment compare and annotate, simultaneously according to the uniquely tagged of unique tags to each template, statistics is carried out quantitatively to each immune subclone;
6) according to 5) result carry out Sequence structure analysis, immune group storehouse expression pattern analysis, Biomarker analyze.
10. detect the system in BCR and TCR immune group storehouse in blood plasma cfDNA, comprise following operating unit:
(1) blood plasma cfDNA extraction unit;
(2) BCRH chain total length multiplexed PCR amplification and TCR β chain CDR3 multiplexed PCR amplification unit;
(3) a pcr amplification unit is carried out again after two kinds of amplified production mixing in step (2);
(4) high-flux sequence unit;
(5) immune group storehouse accurate information analytical unit.
11. systems as claimed in claim 10, it is characterized in that, the upstream primer totally 22 that in described unit (2), BCRH chain total length multiplexed PCR amplification adopts, sequence is respectively as shown in SEQIDNO.1-22, downstream primer totally 6, sequence is respectively as shown in SEQIDNO.23-28;
The upstream primer totally 45 that TCR β chain CDR3 multiplexed PCR amplification adopts, sequence is respectively as shown in SEQIDNO.29-73, and downstream primer totally 4, sequence is respectively as shown in SEQIDNO.74-77.
12. systems as claimed in claim 10, is characterized in that, are to carry out a pcr amplification again after two kinds of amplified production equal-volume mixing by unit (2) in unit (3); The primer sequence adopted is as shown in SEQIDNO.78-79.
13. systems as claimed in claim 10, is characterized in that, the immune group storehouse accurate information analysis of unit (5) comprises the following steps:
1) determine the position of 7 randomized bases N based on known joint sequence, 7 randomized bases can form 4
7plant unique tags, the sequencing sequence 1 of paired sequencing sequence and the unique tags sequence of sequencing sequence 2 are joined end to end, form an index of 14bp, and using this 14bp as paired sequencing sequence index carry out external sort, to reach the object be brought together by all sequencing sequences deriving from same DNA profiling;
2) central cluster is carried out to the sequencing sequence having same index gathered together, according to the Hamming distance between insertion sequence, large bunch of same index is had to be gathered into several tuftlets by each, in each tuftlet, any two are no more than 3 to the Hamming distance of paired sequencing sequence, have same index but from the object of the sequencing sequence of different DNA profiling to reach to distinguish;
3) each the order-checking base for the sequencing sequence in the serial repeated fragment of each DNA profiling amplification formation carries out mutual comparison, if the concordance rate of certain base type in sequencing sequence reaches 80%, then remember this base base type for this reason of new sequencing sequence, otherwise be designated as N, so just, obtain the new sequencing sequence representing original DNA template sequence, correct by this method, effectively can remove the random mistake introduced in order-checking and PCR, improve the accuracy detected;
4) data filter is carried out to new sequencing sequence, the sequencing sequence that removal joint sequence and comparison quality are less than 30;
5) according to 4) in the sequencing sequence that obtains and IMGT database the embryonal system reference sequences of V, D and J district gene fragment compare and annotate, simultaneously according to the uniquely tagged of unique tags to each template, statistics is carried out quantitatively to each immune subclone;
6) according to 5) result carry out other correlation analyses.
The arbitrary described system of 14. claim 10-13 is in the application detecting BCR and TCR immune group storehouse in blood plasma cfDNA.
The arbitrary described method of 15. claim 1-9 or the application of the arbitrary described system of claim 10-13 in preparation disorder in screening test kit.
16. apply as claimed in claim 16, it is characterized in that, described disease is tumour, autoimmune disorder, infectious diseases.
The arbitrary described method of 17. claim 1-9 or the arbitrary described system of claim 10-13 evaluate the application in function of immune system test kit in preparation.
18. 1 kinds for detecting the combination of primers in BCR and TCR immune group storehouse in blood plasma cfDNA, containing 3 groups of primers, the primer sequence that first group of primer contains is respectively as shown in SEQIDNO.1-28; The primer sequence that second group of primer contains is respectively as shown in SEQIDNO.29-77; The 3rd group of primer sequence that primer contains is respectively as shown in SEQIDNO.78-79.
The application of 19. combination of primers according to claim 18 in preparation disorder in screening test kit or diagnostic reagent.
20. 1 kinds of test kits detecting BCR and TCR immune group storehouse in blood plasma cfDNA, it contains combination of primers according to claim 18.
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