CN114107287A

CN114107287A - Preparation method for comprehensively amplifying humann TCR beta chain library by adopting a small amount of degenerate primers

Info

Publication number: CN114107287A
Application number: CN202111522460.7A
Authority: CN
Inventors: 张翼冠; 赵海洋; 魏平
Original assignee: Yunce Intelligent Technology Co ltd
Current assignee: Sichuan Yunshixin Medical Laboratory Co.,Ltd.
Priority date: 2021-12-13
Filing date: 2021-12-13
Publication date: 2022-03-01

Abstract

The invention provides a preparation method for comprehensively amplifying a humanTCR beta chain library by adopting a small amount of degenerate primers, and relates to the technical field of amplification preparation of an immune repertoire sequencing library. Lymphocyte lysis and total RNA extraction: step one, pretreating a sample, and then extracting RNA; step two, reverse transcription: carrying out reverse transcription on the obtained total RNA of the lymphocyte to obtain cDNA; step three, multiplex PCR: performing specific amplification on a TCR beta chain gene of the cDNA by performing a first round of PCR and a second round of PCR on the cDNA; and step four, adding a sequencing joint sequence and an index to construct a library which is shorter but contains a complete CDR3 segment, so that higher flux and lower price can be obtained, and the addition amount of each specific primer and an amplification system are determined after a large amount of experimental optimization.

Description

Preparation method for comprehensively amplifying humann TCR beta chain library by adopting a small amount of degenerate primers

Technical Field

The invention relates to the technical field of amplification preparation of an immune repertoire sequencing library, in particular to a preparation method for comprehensively amplifying a humanTCR beta chain library by adopting a small amount of degenerate primers.

Background

The Immune Repertoire (IR) is the sum of all functional diversity of B cells and T cells in the circulatory system of an individual at any given time, the diversity of the Immune system is crucial to the health of the organism, the more diverse the Immune system is, the healthier the Immune system of the organism is, the stronger the ability to recognize and eliminate antigens is, the acquired immunity of the organism can recognize and respond to countless antigen molecules based on the specificity recognition antigen segments of T cell surface receptors and B cell surface receptors (TCR and BCR), the Immune response is activated, the T cell Repertoire and the B cell Repertoire in the Immune system respectively contain all T cell clones and B cell grams with different specificities, the diversity of the recognition receptors is generated in the process of gene rearrangement, the diversity is embodied by the recombination of receptor genes V (D) J fragments, and the variable region of the beta gene group comprises V beta, T cell clones and T cells, Three types of gene segments, namely D beta and J beta, the TCR alpha gene group comprises two types of gene segments, namely V alpha and J alpha, and 1016 possibilities can be generated by the combination of different gene segments and different chains, so that the analysis of diversity is mainly to analyze the sequence characteristics of the V (D) J segment.

At present, the technology for preparing NGS of immune repertoire includes multiplex PCR and 5' RACE (Rapid Amplification of cDNA Ends), wherein in the process of enzymatic reaction of reverse transcription, TdT tailing and PCR Amplification, failure of any step leads to the final completion of previous work, even if the reaction is smooth, the result usually shows some non-specific products or non-full-length products, and the cost is higher. Moreover, the more the primer pairs, the higher the experimental cost and the greater the difficulty, at present, there are no two preparation methods related to immune repertoire at home and abroad, one is full primer amplification, while there are 152 variable region V region genes of TCR, adopt the traditional multiplex PCR amplification technical route, need hundreds of pairs of primers, this undoubtedly add many difficulties and costs to the amplification preparation of immune repertoire, the latter is local amplification, adopt a part of V region primers to amplify, utilize the whole of local detection, and this method is very easy to cause deviation to the result, and the randomness is too strong, the repeatability is bad, the experimental result is unstable, therefore the invention utilizes a unique computer algorithm to design 43 pairs of TCR primers for 152V genes, through a series of experimental condition optimization, has realized utilizing a small amount of primers to fully cover the amplified humann beta chain sequencing library.

Disclosure of Invention

The invention aims to provide a preparation method for comprehensively amplifying a humanTCR beta chain library by adopting a small amount of degenerate primers.

43 pairs of degenerate primers are designed aiming at 152V genes through a unique computer algorithm, and experimental conditions are optimized, so that the aim of amplifying the humann TCR beta chain sequencing library in a full-coverage manner by using a small amount of primers is realized.

To achieve low cost, comprehensive amplification of the humanTCR β chain CDR3 library.

The invention provides the following technical scheme: a preparation method for comprehensively amplifying a humanTCR beta chain library by adopting a small amount of degenerate primers comprises the following steps:

s1, lymphocyte lysis and total RNA extraction: pretreating a sample, and then extracting RNA;

s2, reverse transcription: carrying out reverse transcription on the obtained total RNA of the lymphocyte to obtain cDNA;

s3, multiplex PCR: performing specific amplification on a TCR beta chain gene of the cDNA by performing a first round of PCR and a second round of PCR on the cDNA;

s4, adding a sequencing adaptor sequence and an index;

s5, sequencing result analysis: and after the high-throughput sequencing is completed, performing data analysis by using a fastq file, comparing a sequencing result to an V, D, J, C gene reference sequence of a T cell receptor by using a unique analysis tool taking MIXCR as a core, splicing clonotypes by using the comparison result obtained in the previous step, outputting the comparison result, and finally generating the immune repertoire diversity map.

Further, according to the operation step in S1, the method includes the steps of:

s101, transferring 250 mu L of blood to a 1.5mL RNase-free centrifuge tube, adding 750 mu L of lysate, repeatedly blowing, violently shaking, uniformly mixing, and standing at room temperature for 5 min;

s102, adding 200 mu L of chloroform, violently oscillating for 15sec, uniformly mixing to obtain a sample, and standing for 2min at room temperature;

s103, placing the sample in a centrifuge, layering the mixed solution, transferring the upper aqueous phase to a 1.5mL RNase-free centrifuge tube, adding 0.5 volume of absolute ethyl alcohol, and reversing and uniformly mixing to obtain a pretreatment mixed solution;

s104, sleeving the RNA adsorption column into a 2mL collection tube, adding the pretreated mixed solution into the RNA adsorption column, centrifuging, and discarding waste liquid;

s105, adding 500 mu L of deproteinized liquid, centrifuging, and discarding waste liquid;

s106, putting the RNA adsorption column back to the collecting pipe, adding 500 mu L of rinsing liquid, centrifuging at room temperature for 30sec, discarding the waste liquid, and repeatedly circulating once;

s107, putting the RNA adsorption column back to the collection tube, centrifuging the empty column at room temperature for 2min, and removing residual rinsing liquid;

s108, putting the RNA adsorption column into a new 1.5mLRNase-free centrifuge tube, and putting RNase-free₂And O, eluting, standing at room temperature for 2min, centrifuging for 1min, collecting filtrate to obtain RNA solution, centrifuging for 1min, and collecting filtrate to obtain RNA solution.

Further, the centrifuge is set to be at 4 ℃ and rotate at 12000rpm for 10 min.

Further, according to the operation step in S2, the method includes the steps of:

s201, preparing a mixed solution in an RNase-free centrifugal tube, heating the prepared mixed solution for 5min at the temperature of 65 ℃, and rapidly placing the mixed solution on ice for quenching;

s202, preparing a first strand cDNA synthesis reaction solution, gently blowing and uniformly mixing the first strand cDNA synthesis reaction solution by using a pipette, and then carrying out a first strand cDNA synthesis reaction;

further, the mixed solution is RNase-freeddH₂O, Gene-Specific-Primers and Total-RNA were mixed to prepare a mixture.

Further, the first strand cDNA synthesis reaction solution is prepared by mixing a mixed solution, 2 XRT Mix and Enzyme Mix.

Further, according to the operation step in S3, the method includes the steps of:

s301, first round PCR 25.25. mu.L Master mix, 5. mu. L V-Primer mix (SEQ ID NO:1-SEQ ID NO:43), 5. mu.LC region Primer (SEQ ID NO:44), cDNA template and ddH₂O, mixing to prepare a multiple PCR reaction solution;

s302, performing electrophoresis detection and magnetic bead purification on the prepared multiplex PCR reaction solution, and purifying a PCR product by using DNA Clean Beads magnetic Beads;

s303, performing a second round of PCR by mixing 25. mu.L of Polymerase mix, 1. mu.L of LV-PrimerADmix (SEQ ID NO:47-SEQ ID NO:90), 1. mu.L of LC-PrimerAD, the first round of PCR-purified product, and 50. mu.L of ddH₂Mixing O to prepare a PCR reaction solution;

s304, preparing a PCR reaction solution, recovering and purifying glue, and verifying through an electrophoresis band.

Further, according to the operation step in S302, the method includes the following steps:

s3021, taking out the magnetic beads from the refrigerator, balancing at room temperature for at least 30min, preparing 80% ethanol, and oscillating or fully inverting the magnetic beads through vortex;

s3022, sucking 1.0 XDNA Clean Beads into the DNA solution, and incubating for 5min at room temperature;

(not less than 20 mu L), vortexing and shaking or gently blowing and beating by using a pipette until the mixture is fully mixed, and standing for 5min at room temperature.

Further, according to the operation step in S304, the method includes the following steps:

s3041, transferring the gel block with the target fragment into a 1.5mL centrifuge tube, weighing to obtain the weight of the gel block, determining the volume of the gel block approximately, adding a Binding Buffer with the same volume, carrying out warm bath in a water bath at 50-60 ℃ until the gel is completely melted, and oscillating or vortexing the mixture every 2-3 min;

s3042, putting a DNA binding column in a 2mL collecting tube, transferring all the obtained DNA glue solution to the DNA binding column, centrifuging at room temperature at 10000xg for 1min, discarding the filtrate in the collecting tube, and sleeving the column back to the collecting tube in the 2mL collecting tube;

s3043, collecting the filtrate in the tube, sleeving the DNA Binding column back to the collection tube in the 2mL collection tube, transferring 300 mu L Binding Buffer to the column, centrifuging at the maximum speed (not less than 13000) for 1min at room temperature, and discarding the filtrate;

s3044, sleeving the DNA binding column back to a collection tube in a 2mL collection tube, transferring 700 μ l of elution Buffer to the DNA binding column, centrifuging at room temperature at 10000xg for 1min, discarding the filtrate, and repeating the steps once;

s3045, sleeving the DNA binding column back to a collection tube in a 2mL collection tube, and centrifuging for 2min at room temperature at the speed of more than or equal to 13000Xg to spin-dry residual liquid of the DNA binding column matrix;

s3046, the DNA binding column is placed in a clean 1.5mL centrifuge tube, 15-30 μ L of Elution Buffer is added to the matrix of the DNA binding column, the mixture is placed at room temperature for 1min, and 13000Xg is centrifuged for 1min to elute the DNA.

Further, according to the operation step in S4, the method includes the steps of:

s401, 25. mu.L of Polymerase, 1. mu.L of Primer-P5, 1. mu.L of Primer-P7, second round of PCR purified product cDNA, ddH₂Mixing O to prepare PCR reaction solution;

s402, after the glue is recovered and purified, the library building final product is subjected to electrophoresis band verification.

The invention provides a preparation method for comprehensively amplifying a humanTCR beta chain library by adopting a small amount of degenerate primers, which has the following beneficial effects: the constructed library is shorter, but contains a complete CDR3 segment, higher flux and lower price can be obtained, the addition amount of each specific primer and an amplification system are determined after a large amount of experiments are optimized, and the method can greatly reduce the number of primer dimers and obtain a high-quality library.

Drawings

FIG. 1 is a first round of PCR electrophoresis of a library of humanTCR β chains of the invention fully amplified using a small number of degenerate primers;

FIG. 2 is a second round of PCR products of a comprehensive amplification of a humanTCR β chain library using a small number of degenerate primers according to the invention;

FIG. 3 is a schematic diagram showing an electrophoretic band of a library-constructed end product of a humanTCR β chain library comprehensively amplified using a small number of degenerate primers according to the present invention;

FIG. 4 is an immune diversity map of a comprehensive amplification of a humanTCR β chain library using a small number of degenerate primers according to the present invention;

FIG. 5 is a comparison of clonotype spectra of a comprehensive amplification of a library of humanTCR β chains using a small number of degenerate primers according to the invention.

Detailed Description

Referring to fig. 1-5, the present invention provides a technical solution: a preparation method for comprehensively amplifying a humanTCR beta chain library by adopting a small amount of degenerate primers comprises the following steps:

s4, adding a sequencing adaptor sequence and an index;

Specifically, according to the operation step in S1, the method includes the following steps:

s108, putting the RNA adsorption column into a new 1.5mL RNase-free centrifuge tube, and putting the RNase-free H₂And O, eluting, standing at room temperature for 2min, centrifuging for 1min, collecting filtrate to obtain RNA solution, centrifuging for 1min, and collecting filtrate to obtain RNA solution.

Specifically, the centrifuge is set at 4 ℃ and at 12000rpm for 10 min.

Specifically, according to the operation step in S2, the method includes the following steps:

specifically, the mixed solution is RNase-freeddH₂O, Gene-Specific-Primers and TotalR NA were mixed to prepare a mixture.

Specifically, the first strand cDNA synthesis reaction solution is a mixed solution, and is prepared by mixing 2 XRT Mix and Enzyme Mix.

Specifically, according to the operation step in S3, the method includes the following steps:

s301, first round PCR 25.25. mu.L Mastermix, 5. mu. L V-Primer mix (SEQ ID NO:1-SEQ ID NO:43), 5. mu. L C region Primer (SEQ ID NO:44), cDNA template and ddH₂O, mixing to prepare a multiple PCR reaction solution;

s303, performing a second round of PCR by mixing 25. mu.L of Polymerase mix, 1. mu. L V-PrimerADmix (SEQ ID NO:47-SEQ ID NO:90), 1. mu. L C-PrimerAD, the first round of PCR-purified product, and 50. mu.L ddH₂Mixing O to prepare a PCR reaction solution;

Specifically, according to the operation steps in S302, the method includes the following steps:

Specifically, according to the operation steps in S304, the method includes the following steps:

s3043, collecting the filtrate in the tube, sleeving the DNA Binding column back to the 2mL tube in the tube, transferring 300 mu L Binding Buffer into the column, centrifuging at the maximum speed (not less than 13000) for 1min at room temperature, and discarding the filtrate;

s3044, sleeving the DNA binding column back to a collection tube in a 2mL collection tube, transferring 700 mu L of elution Buffer to the DNA binding column, centrifuging at room temperature at 10000xg for 1min, discarding the filtrate, and repeating the steps once;

Specifically, according to the operation step in S4, the method includes the following steps:

The method of the examples was performed for detection analysis and compared to the prior art to yield the following data:

	library quality	Price
			Examples	Is higher than	Is lower than
Prior Art	Is lower than	Is higher than

According to the above table data, it can be concluded that when the embodiment is used, the library is constructed to be shorter, but the complete CDR3 segment is contained, higher flux can be obtained, the price is lower, and the PCR amplification system provided by the invention can greatly reduce the generation of primer dimer, and high-quality library can be obtained.

The invention provides a preparation method for comprehensively amplifying a humanTCR beta chain library by adopting a small amount of degenerate primers, which comprises the following steps:

s4, adding a sequencing adaptor sequence and an index;

s5, sequencing result analysis: after the high-throughput sequencing is completed, performing data analysis by using a fastq file, comparing a sequencing result to an V, D, J, C gene reference sequence of a T cell receptor by using a unique analysis tool taking MIXCR as a core, splicing clonotpes by using the comparison result obtained in the previous step, outputting the comparison result, and finally generating an immune repertoire diversity map, as shown in FIG. 4, wherein each color block in the map represents a clonotype, the size of the color block represents the number of each clonotype, the more the types of the color blocks are, the closer the sizes are, the more the diversity of the immune repertoire of a subject is reflected, the healthier the immunity is, in order to verify the stability and reliability of the method, the blood sample of the same tester is divided into 5 parts, each blood sample is subjected to a parallel experiment, the obtained 5 parts of repertoire are respectively added with different indexes to be sequenced on a computer together, the sequencing result is analyzed, and the clone type spectrums of the 5 parts of the blood sample are highly consistent, the types and the quantities of clonotypes are very close, which indicates that the method is used for amplifying the humanTCR beta chain sequencing library and has high stability and reliability, and the comparison of the clonotype frequency spectrum is shown in figure 5.

According to the operation step in S1, comprising the steps of:

s108, putting the RNA adsorption column into a new 1.5mL RNase-free centrifuge tube, and putting the RNase-free H₂And (3) eluting, standing at room temperature for 2min, centrifuging for 1min, collecting filtrate to obtain RNA solution, and storing the RNA solution at-80 deg.C for a long time.

The centrifuge is set at 4 ℃ and the rotating speed is 12000rpm, and the centrifuge is centrifuged for 10 min.

According to the operation step in S2, comprising the steps of:

s202, preparing a first strand cDNA synthesis reaction solution, mixing 8 mu L of mixed solution, 10 mu LRTMix and 2 mu LENzyme Mix, gently blowing and beating the mixed solution by a pipette, uniformly mixing the mixed solution, reacting the mixed solution at 50 ℃ for 45min, reacting the mixed solution at 85 ℃ for 2min, and carrying out a first strand cDNA synthesis reaction, wherein the product can be immediately used for PCR reaction or stored at-20 ℃ and used within half a year, long-term storage is recommended to be subpackaged and stored at-70 ℃, and repeated freeze thawing of cDNA is avoided;

the mixed solution is RNase-freeddH₂O, Gene-Specific-Primers and TotalRNA were mixed to prepare a mixture.

The first chain cDNA synthesis reaction solution is prepared by mixing a mixed solution, RTmix and Enzyme Mix.

According to the operation step in S3, comprising the steps of:

s301, first round PCR 25. mu.L of Polymerase mix, 5. mu. L V-Primer mix (SEQ ID NO:1-SEQ ID NO:43), 5. mu. L C region Primer (SEQ ID NO:44), cDNA template and ddH₂O, mixing, preparing a multiple PCR reaction solution, reacting at 94 ℃ for 1min at (1), reacting at 94 ℃ for 30s at (2), reacting at 94 ℃, reacting at 60 ℃ for 1min at (4), reacting at 72 ℃ for 10min, and repeating the steps (2) and (3)30 times, wherein V-Primer mix is a 5' end multiple PCR Primer, and the name and sequence of each Primer are shown in the following table;

s302, performing electrophoresis detection and magnetic bead purification on the prepared multiplex PCR reaction solution, purifying a PCR product by using DNA Clean Beads magnetic Beads, and detecting an amplification result by electrophoresis, wherein a band is between 100 and 200 bp;

s303, performing a second round of PCR, mixing 25 μ L of Polymerase mix, 1 μ L V-PrimerADmix (SEQ ID NO:47-SEQ I)DNO 90), 1. mu. L C-primeraD, first round PCR purified product and 50. mu.L ddH₂O, mixing to prepare a PCR reaction solution, reacting the PCR reaction solution at the temperature of 94 ℃ for 1min (5) for 30s at the temperature of 94 ℃ for (7) at the temperature of 60 ℃ for 1min, reacting at the temperature of 72 ℃ for 10min (8), and repeating the step (6) and the step (7) for 10 times, wherein V-PrimerADmix is a 5' end multiple PCR library building primer, the primers are mixed according to the volume ratio of 1:1, the final concentration is 10 mu M, and the name and the sequence of each primer are shown in the following table;

According to the operation steps in S302, the method includes the following steps:

According to the operation steps in S304, the method includes the following steps:

According to the operation step in S4, comprising the steps of:

s401, 25 μ L of Polymerase, 1 μ L of Primer-P5, 1 μ L of Primer-P7, second round PCR purified product cDNA, ddH₂Mixing O to prepare PCR reaction liquid, reacting the PCR reaction liquid at the temperature of 98 ℃ for 1min (9), reacting at the temperature of 98 ℃ for 10s (11), reacting at the temperature of 60 ℃ for 5s (12), reacting at the temperature of 72 ℃ for 5s (13), reacting at the temperature of 72 ℃ for 3min, repeating the steps of (10), (11) and (12) for 15 times, wherein in a primer sequence, an i5 sequence is embedded with a P5 sequence, an i7 sequence is embedded with a P7 sequence, and the specific sequence is as follows;

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

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<211> 53

<212> DNA

<213> ADhTRBV-9

<400> 54

gtgactggag ttcagacgtg tgctcttccg atcctcagaa cccagggact cag 53

<210> 55

<211> 59

<212> DNA

<213> ADhTRBV-10

<400> 55

gtgactggag ttcagacgtg tgctcttccg atccagagaa gtctgaaata ttcgatgat 59

<210> 56

<211> 53

<212> DNA

<213> ADhTRBV-11

<400> 56

gtgactggag ttcagacgtg tgctcttccg atcccttgag atccaggcta cga 53

<210> 57

<211> 58

<212> DNA

<213> ADhTRBV-12

<400> 57

gtgactggag ttcagacgtg tgctcttccg atctggattt gtcattgaca agtttccc 58

<210> 58

<211> 52

<212> DNA

<213> ADhTRBV-13

<400> 58

gtgactggag ttcagacgtg tgctcttccg atcgcgtctc tcgggagaag aa 52

<210> 59

<211> 56

<212> DNA

<213> ADhTRBV-14

<400> 59

gtgactggag ttcagacgtg tgctcttccg atcctctaga tcaaacacag aggacc 56

<210> 60

<211> 54

<212> DNA

<213> ADhTRBV-15

<400> 60

gtgactggag ttcagacgtg tgctcttccg atcttgaacg attctccgca caac 54

<210> 61

<211> 51

<212> DNA

<213> ADhTRBV-16

<400> 61

gtgactggag ttcagacgtg tgctcttccg atccaggagg ccgaacactt c 51

<210> 62

<211> 54

<212> DNA

<213> ADhTRBV-18

<400> 62

gtgactggag ttcagacgtg tgctcttccg atcgactcgg ccatgtatct ctgt 54

<210> 63

<211> 52

<212> DNA

<213> ADhTRBV-19

<400> 63

gtgactggag ttcagacgtg tgctcttccg atcgagaagt ccccgatggc ta 52

<210> 64

<211> 55

<212> DNA

<213> ADhTRBV-21

<400> 64

gtgactggag ttcagacgtg tgctcttccg atcgcccaat gctccaaaaa ctcat 55

<210> 65

<211> 57

<212> DNA

<213> ADhTRBV-22

<400> 65

gtgactggag ttcagacgtg tgctcttccg atccaccagt tccctaacta tagctct 57

<210> 66

<211> 52

<212> DNA

<213> ADhTRBV-23

<400> 66

gtgactggag ttcagacgtg tgctcttccg atcctgaaga tccagcgcac ag 52

<210> 67

<211> 53

<212> DNA

<213> ADhTRBV-24

<400> 67

gtgactggag ttcagacgtg tgctcttccg atcgaacatg agctccttgg agc 53

<210> 68

<211> 51

<212> DNA

<213> ADhTRBV-25

<400> 68

gtgactggag ttcagacgtg tgctcttccg atctgccatc cccaaccaga c 51

<210> 69

<211> 55

<212> DNA

<213> ADhTRBV-26

<400> 69

gtgactggag ttcagacgtg tgctcttccg atcctgaaag acctaacgga acgtc 55

<210> 70

<211> 52

<212> DNA

<213> ADhTRBV-27

<400> 70

gtgactggag ttcagacgtg tgctcttccg atcagcacca gccagaccta tc 52

<210> 71

<211> 59

<212> DNA

<213> ADhTRBV-28

<400> 71

gtgactggag ttcagacgtg tgctcttccg atcctggagg attctggagt ttatttctg 59

<210> 72

<211> 58

<212> DNA

<213> ADhTRBV-29

<400> 72

gtgactggag ttcagacgtg tgctcttccg atctaactct cgctctgaga tgaatgtg 58

<210> 73

<211> 52

<212> DNA

<213> ADhTRBV-30

<400> 73

gtgactggag ttcagacgtg tgctcttccg atcccagcac caaccagaca tc 52

<210> 74

<211> 52

<212> DNA

<213> ADhTRBV-31

<400> 74

gtgactggag ttcagacgtg tgctcttccg atcgctacgt gtctgccaag ag 52

<210> 75

<211> 55

<212> DNA

<213> ADhTRBV-32

<400> 75

gtgactggag ttcagacgtg tgctcttccg atcgagaggc ctgatggatc attct 55

<210> 76

<211> 55

<212> DNA

<213> ADhTRBV-33

<400> 76

gtgactggag ttcagacgtg tgctcttccg atcgtctcca gaataaggac ggagc 55

<210> 77

<211> 52

<212> DNA

<213> ADhTRBV-34

<400> 77

gtgactggag ttcagacgtg tgctcttccg atccacacct gaatgccctg ac 52

<210> 78

<211> 53

<212> DNA

<213> ADhTRBV-35

<400> 78

gtgactggag ttcagacgtg tgctcttccg atcacagagg tctgagggat cca 53

<210> 79

<211> 50

<212> DNA

<213> ADhTRBV-36

<400> 79

gtgactggag ttcagacgtg tgctcttccg atcgctgctc cctcccagac 50

<210> 80

<211> 54

<212> DNA

<213> ADhTRBV-37

<400> 80

gtgactggag ttcagacgtg tgctcttccg atcagcgatt ctcatctcaa tgcc 54

<210> 81

<211> 52

<212> DNA

<213> ADhTRBV-38

<400> 81

gtgactggag ttcagacgtg tgctcttccg atctagtgcg aggagattcg gc 52

<210> 82

<211> 55

<212> DNA

<213> ADhTRBV-39

<400> 82

gtgactggag ttcagacgtg tgctcttccg atcgcacaga tgcctgatgt atcat 55

<210> 83

<211> 55

<212> DNA

<213> ADhTRBV-40

<400> 83

gtgactggag ttcagacgtg tgctcttccg atcggagatg ttcctgaagg gtaca 55

<210> 84

<211> 53

<212> DNA

<213> ADhTRBV-41

<400> 84

gtgactggag ttcagacgtg tgctcttccg atcctactag caccagccag acc 53

<210> 85

<211> 56

<212> DNA

<213> ADUny-1#

<400> 85

gtgactggag ttcagacgtg tgctcttccg atcctcccag acatctgtgt acttct 56

<210> 86

<211> 50

<212> DNA

<213> ADUny-2#

<400> 86

gtgactggag ttcagacgtg tgctcttccg atcgttggag tcggctgctc 50

<210> 87

<211> 57

<212> DNA

<213> ADUny-3#

<400> 87

gtgactggag ttcagacgtg tgctcttccg atctccagag caaacacaga tgatttc 57

<210> 88

<211> 57

<212> DNA

<213> ADUny-4#

<400> 88

gtgactggag ttcagacgtg tgctcttccg atcctctctt aaaccttcac ctacacg 57

<210> 89

<211> 56

<212> DNA

<213> ADUny-5#

<400> 89

gtgactggag ttcagacgtg tgctcttccg atccaatggc tacaatgtct ccagat 56

<210> 90

<211> 52

<212> DNA

<213> ADhTRBC1

<400> 90

acactctttc cctacacgac gctcttccga tctgggtggg aacaccttgt tc 52

<210> 91

<211> 55

<212> DNA

<213> P7-i7-1

<400> 91

caagcagaag acggcatacg agataatcgt tagtgactgg agttcagacg tgtgc 55

<210> 92

<211> 55

<212> DNA

<213> P7-i7-2

<400> 92

caagcagaag acggcatacg agatgtctac atgtgactgg agttcagacg tgtgc 55

<210> 93

<211> 55

<212> DNA

<213> P7-i7-3

<400> 93

caagcagaag acggcatacg agatcgctgc tcgtgactgg agttcagacg tgtgc 55

<210> 94

<211> 55

<212> DNA

<213> P7-i7-4

<400> 94

caagcagaag acggcatacg agatgatcaa cagtgactgg agttcagacg tgtgc 55

<210> 95

<211> 55

<212> DNA

<213> P7-i7-5

<400> 95

caagcagaag acggcatacg agatcgaagg acgtgactgg agttcagacg tgtgc 55

<210> 96

<211> 55

<212> DNA

<213> P7-i7-6

<400> 96

caagcagaag acggcatacg agatgatgcc gggtgactgg agttcagacg tgtgc 55

<210> 97

<211> 55

<212> DNA

<213> P7-i7-7

<400> 97

caagcagaag acggcatacg agatctacga aggtgactgg agttcagacg tgtgc 55

<210> 98

<211> 55

<212> DNA

<213> P7-i7-8

<400> 98

caagcagaag acggcatacg agatgatgcg tcgtgactgg agttcagacg tgtgc 55

<210> 99

<211> 59

<212> DNA

<213> P5-i5-1

<400> 99

aatgatacgg cgaccaccga gatctacaca ataacgtaca ctctttccct acacgacgc 59

<210> 100

<211> 59

<212> DNA

<213> P5-i5-2

<400> 100

aatgatacgg cgaccaccga gatctacact tcttgaaaca ctctttccct acacgacgc 59

Claims

1. A preparation method for comprehensively amplifying a humanTCR beta chain library by adopting a small amount of degenerate primers is characterized by comprising the following steps:

s4, adding a sequencing adaptor sequence and an index;

2. The method of claim 1, wherein the method comprises the following steps according to the procedure of S1:

s108, putting the RNA adsorption column into the new 1.5mL RNase-free centrifuge tube, RNase-free H₂And O, eluting, standing at room temperature for 2min, centrifuging for 1min, collecting filtrate to obtain RNA solution, centrifuging for 1min, and collecting filtrate to obtain RNA solution.

3. The method of claim 2, wherein the centrifuge is set at 4 ℃ and 12000rpm for 10 min.

4. The method of claim 1, wherein the method comprises the following steps according to the procedure of S2:

s202, preparing a first strand cDNA synthesis reaction solution, gently and uniformly blowing and beating the first strand cDNA synthesis reaction solution by using a pipette, and then carrying out a first strand cDNA synthesis reaction.

5. The method of claim 4, wherein the mixture of RNase-freeddH is the same as RNase-freeddH in the method for fully amplifying the library of humann TCR beta chains using a small amount of degenerate primers₂O, Gene-Specific-Primers and TotalRNA were mixed to prepare a mixture.

6. The method of claim 4, wherein the first chain cDNA synthesis reaction solution is a mixture of 2 XTMix and EnzymeMix.

7. The method of claim 1, wherein the method comprises the following steps according to the procedure of S3:

s301, first round PCR 25. mu.L of Polymerase mix, 5. mu.L of LV-Primer mix (SEQ ID NO: 1)ID NO:1-SEQ ID NO:43), 5. mu.LC region primer (SEQ ID NO:44), cDNA template and ddH₂O, mixing to prepare a multiple PCR reaction solution;

s303. mu.L of Polymerase mix, 1. mu.L of LV-PrimerADmix (SEQ ID NO:47-SEQ ID NO:90), 1. mu. L C-PrimerAD, the first PCR purified product and 50. mu.L of ddH were subjected to the second round of PCR₂Mixing O to prepare a PCR reaction solution;

8. The method according to claim 7, wherein the method comprises the following steps according to the procedure in S302:

9. The method according to claim 7, wherein the method comprises the following steps according to the procedure in S304:

s3042, placing a DNA binding column in a 2mL collecting tube, transferring all the obtained DNA glue solution to the DNA binding column, centrifuging at room temperature at 10000xg for 1min, discarding the filtrate in the collecting tube, and sleeving the column back to the collecting tube in the 2mL collecting tube;

10. The method of claim 1, wherein the method comprises the following steps according to the procedure of S4: