WO2019061198A1 - 一种扩增tcr全长序列的试剂盒及其应用 - Google Patents

一种扩增tcr全长序列的试剂盒及其应用 Download PDF

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WO2019061198A1
WO2019061198A1 PCT/CN2017/104109 CN2017104109W WO2019061198A1 WO 2019061198 A1 WO2019061198 A1 WO 2019061198A1 CN 2017104109 W CN2017104109 W CN 2017104109W WO 2019061198 A1 WO2019061198 A1 WO 2019061198A1
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primer
tcr
sequence
cycles
sequencing
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French (fr)
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周清
王飞
赵小莹
尹悦露
李贵波
赵正琦
杨乃波
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深圳华大生命科学研究院
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Priority to PCT/CN2017/104109 priority Critical patent/WO2019061198A1/zh
Priority to CN201780095486.3A priority patent/CN111344418A/zh
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  • the invention belongs to the field of molecular biology, and relates to a kit for amplifying a full-length TCR sequence and an application thereof, in particular to a kit for amplifying a full-length TCR sequence, a method for amplifying a full-length TCR sequence and an application thereof.
  • T lymphocyte precursor enters the thymus from the bone marrow and undergoes a positive and negative screening process to develop into mature T cells. During this period, T cells acquire a functional T cell receptor (TCR), express surface molecules of mature T cells (CD4/CD8, etc.) and obtain MHC restriction and autotolerance.
  • T cell type associated with the complexity and diversity of the TCR rearrangements in the thymus, TRB (TCR beta) gene via DJ, VDJ, VDJC region rearrangement eventually produce about 108 different gene rearrangement TRB; TRA (TCR ⁇ ) gene via VJ, VJC region rearrangement, eventually produce about 104 different gene rearrangement TRA.
  • TCR diversity is up to 10 18 due to the rearrangement of TRB and TRA, which determines how the human immune system adapts to environmental changes.
  • Previous TCR amplification methods include: 1. Multiplex PCR amplification combined with high-throughput sequencing technology, by designing multiple primers covering all TRA, TRB typing, capturing the most complete TRA, TRB subtypes in the population, high throughput Sequencing reads the TRA and TRB sequences of each subtype; 2.
  • TCR ⁇ / ⁇ pairing analysis based on single cell level is more and more widely used in immunology research.
  • the TCR receptor is a heterodimer formed by the TCR ⁇ and TCR ⁇ chains. Since the TCR gene undergoes rearrangement of DNA sequences during T cell formation, T cell diversity is produced, and T cell diversity is effective. The immunity has played a huge role. Recently, a variety of TCR-based high-throughput technologies have emerged, such as:
  • PairSEQ high-throughput TCR sequencing technology This technique adopts the characteristics of T cell diversity.
  • the TCR sequences corresponding to T cells are different among different subgroups, and the number of subpopulations in the sample, the number of T cells in the subgroup and corresponding
  • the TCR ⁇ / ⁇ pairing is correlated.
  • the strategy of this technique is to perform mRNA isolation and first-strand cDNA amplification of cells in each well by randomized distinct subsets of T cells in a 96-well plate, followed by addition of TCR V and C-region primers. The area of the TCR CDR3.
  • a sample-specific barcode is ligated to the amplification primer so that a specific DNA barcode is combined with the amplification product at the time of amplification, and each sample can be accurately calibrated.
  • High-throughput sequencing was performed after pooling amplification products from all samples of 96-well plates. By simultaneously reading the TCR sequence information and the DNA barcode information, the source of the hole corresponding to the TCR can be determined by the DNA barcode information between the different wells.
  • the TCR ⁇ / ⁇ pairing in each well T cell subset needs to be analyzed by subsequent construction models. Single-cell paired TCR ⁇ / ⁇ CDR3 region information was obtained by computer model.
  • the technology is characterized by the initial amplification and analysis of high-throughput single-cell TCR pairing sequences.
  • the previous sample processing process is complicated. It is necessary to first group the T cells, and then spread them into a 96-well plate combined with random DNA barcode for sequencing. The data simulation trains the TCR ⁇ / ⁇ pairing information of different T cell subtypes, and the sequenced assembled TRA. And TRB has a greater limit on the accuracy of subsequent ⁇ / ⁇ pairing.
  • the technology is characterized by the initial amplification and analysis of high-throughput single-cell TCR pairing sequences.
  • the shortcomings are very obvious.
  • the throughput of the technology is relatively low. Due to the 96-well plate, the flux and application are limited.
  • the technique is based on amplification of the CDR3 region and does not achieve amplification of the full-length TCR sequence, and full-length sequences are critical for their use, particularly in immunotherapy.
  • TraCeR This technology reconstructs the full-length TCR ⁇ / ⁇ pairing sequence by computer-captured TCR transcriptome information in single cells by T cell single-cell transcriptome data, and distinguishes functional T cell clones based on TCR sequence differences.
  • Single-cell transcriptome data can reveal the transcriptional heterogeneity between cells and the evolutionary process between different cells, thereby deducing a distinct subset of T cells.
  • the technology combines the single-cell transcriptome data, and obtains the TCR ⁇ / ⁇ complete pairing sequence information at the same time when obtaining the expression amount, which has high advantages and innovations.
  • this technique also implements TCR ⁇ / ⁇ pairing at the single cell level based on the multiplex PCR method, only the CDR3 region of the TCR can be obtained, and the full-length TCR sequence cannot be obtained. Since the technique relies on computer reconstruction software, the diversity and high variability of the TCR sequence itself determines the difficulty of aligning the rearrangement sequences. Therefore, the development of appropriate computer software for evaluating the integrity of the reconstructed TCR sequence and Accuracy is the bottleneck of the technology, and accuracy is worth considering.
  • the existing TCR amplification technology stays on the 96-well plate scale, and there is no mature high-throughput single-cell amplification method.
  • low flux is difficult to achieve detailed analysis of T cell heterogeneity and different cell subpopulations.
  • TCR ⁇ / ⁇ pairing information It is also impossible to understand the TCR ⁇ / ⁇ pairing information, and there is no suitable method for full-length TCR expansion. Increase, which limits its application to some extent.
  • recent advances in full-length amplification of TCR in single-cell aspects although to some extent solve the problem of TCR ⁇ / ⁇ pairing, most of the obtained sequence information of the single-cell TCR CDR3 region.
  • CN 105543064 A discloses a digital PCR chip and a method of using the same: the chip comprises a silicon micropore chip, a microfluidic channel filled with a sample, and an aluminum thermally conductive susceptor having 20,000 processed on the silicon microporous chip
  • the inner wall is a hydrophilic, surface-hydrophobic via that is used to disperse micronucleic acids or cell solutions, but it is not suitable for high-throughput TCR amplification.
  • the present invention provides a kit for amplifying a full-length TCR sequence and the use thereof, which can realize full-length amplification of TCR ⁇ / ⁇ pairing sequences at a single cell level, and can cover All TRA, TRB subtypes are known.
  • the invention provides a kit for amplifying a full length TCR sequence, the kit comprising a nanoscale microplate.
  • the invention adopts a nano-upgraded microporous chip as a reaction container, and can perform up to 5124 single T cell TCR ⁇ / ⁇ pair amplification at one time, which can improve the flux and can efficiently acquire thousands of TCR ⁇ chains of a single T cell layer at a time. Pair the full-length sequences and construct a TCR immune pool.
  • the nano-upgraded micro-hole chip is a micro-hole chip with a volume-upgrading, and the number of micro-holes on the nano-upgraded micro-hole chip does not affect the present invention, and those skilled in the art can select according to needs. It is not specifically limited here.
  • the kit further comprises a primer pair that amplifies the full length sequence of the TCR.
  • the primer pair that amplifies the full length sequence of the TCR comprises an upstream primer and a downstream primer, the upstream primer being a complementary sequence designed according to a sequencing adaptor sequence, and the downstream primer being a complementary sequence designed according to the C region of the TCR. .
  • the present invention designs the downstream primer according to the C region, and can be carried out at any position from the 5' end to the 3' end of the C region. Design, and then by adding a sequencing linker sequence (TSO) at the 3' end of the TCR, and designing the upstream primer according to its design, the complete V(D)J sequence can be obtained by such upstream and downstream primers, thereby obtaining the full length of the TCR. .
  • TSO sequencing linker sequence
  • the designed primers follow general primer design principles, such as GC content 40-60%, no secondary hairpin structure, no primer dimer, etc., and primer design should be designed as far as possible without base polymorphism. .
  • the sequencing linker sequence is introduced by a transformation template method, wherein the linker primer can be any fixed length sequence, and the skilled person can design the nucleotide sequence of the linker primer of the present invention as needed.
  • the nucleotide sequence shown in SEQ ID NO. 1 is as follows: 5'-AAGCAGTGGTATCAACGCAGAGT-3';
  • the sequencing linker sequence is 18-35 nt in length, for example 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt or 28 nt, preferably 28 nt.
  • nucleotide sequence of the upstream primer is shown in SEQ ID NO. 2, and the nucleotide sequence shown in SEQ ID NO. 2 is as follows: 5'-AAGCAGTGGTATCAACGCAGAGT-3'.
  • the downstream primer is a nested primer
  • the inventors have found that the nested primer can not only target a small starting sample in a single cell of the present application, but also has a very complex mechanism for the TCR, and the result after amplification is very Accurately, the downstream primer comprises an external primer and/or an internal primer.
  • the full length of the TCR is composed of the full length of the TCR ⁇ gene and the full length of the TCR ⁇ gene or the full length of the TCR ⁇ gene and the full length of the TCR ⁇ gene;
  • nucleotide sequences of the external primer and the internal primer of the downstream primer which amplifies the full length of the TCR ⁇ gene are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively, and the nucleotide sequence of the primer is as follows:
  • External primer (SEQ ID NO. 3): GCAGACAGACTTGTCACTGG;
  • nucleotide sequences of the external primer and the internal primer of the downstream primer which amplifies the full length of the TCR ⁇ gene are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively;
  • the nucleotide sequence of the primer is as follows:
  • External primer (SEQ ID NO. 5): TGGTCGGGGAAGAAGCCTGTG;
  • the invention provides a method of amplifying a full length TCR sequence, using the kit of the first aspect.
  • the method of amplifying a full length TCR sequence comprises the steps of:
  • a step of single cell separation and lysis is further included before the step (1), the single cell separation is performed by flow cytometry to sort single cells, and the sorted single cells are dispensed into each using a sample micrometer.
  • the single cells are derived from peripheral blood mononuclear cells of peripheral blood.
  • TCR full-length sequencing and TCR ⁇ / ⁇ matching can be completely solved from a high-throughput single-cell level.
  • the problem can not only distinguish the paired ⁇ sequence information, but also distinguish different single cell samples, so that the single tube TCR pairing full-length amplification technology can be effectively applied to high-throughput platforms to achieve high throughput.
  • the system for reverse transcription of mRNA described in the step (1) comprises sequencing a linker sequence, and after the system for preparing the reverse transcription of the mRNA, the system is dispensed into each micro by a sample micro-dispenser. In the micropores of the hole chip.
  • the sequencing linker sequence has a final concentration of 0.8 to 3 ⁇ M, and may be, for example, 0.8 ⁇ M, 0.9 ⁇ M, 1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.8 ⁇ M, 2 ⁇ M, 2.2 ⁇ M, 2.4 ⁇ M. , 2.5 ⁇ M, 2.6 ⁇ M, 2.8 ⁇ M or 3 ⁇ M, preferably 2 ⁇ M;
  • the reverse transcription conditions of the mRNA described in the step (1) are: 38-45 ° C 85-95 min 1-3 cycles; 48-53 ° C 1-5 min, 38-45 ° C 1-5 min, 8-15 cycles ; 68-73 ° C 10-20 min 1-3 cycles; stored at 4 ° C;
  • the reverse transcription conditions of the mRNA described in the step (1) are: 42 ° C for 90 min 1 cycle; 50 ° C for 2 min, 42 ° C for 2 min, 10 cycles; 70 ° C for 15 min 1 cycle; stored at 4 ° C.
  • the PCR amplification is nested PCR
  • the number of times of nested PCR is 1-3 times, preferably 2 times;
  • the nested PCR specifically includes:
  • the first round of nested PCR conditions described in the step (1') are: 92-98 ° C 1-6 min, 1-3 cycles; 95-100 ° C 15-25 s, 53-58 ° C 10-20 s, 70-75 ° C 35-45 s, 35-45 cycles; 70-75 ° C 3-8 min, 1-3 cycles; stored at 4 ° C;
  • the first round of nested PCR conditions described in step (1') are: 95 ° C for 3 min, 1 cycle; 98 ° C for 20 s, 55 ° C for 15 s, 72 ° C for 40 s, 40 cycles; 72 ° C for 5 min, 1 cycle ; stored at 4 ° C;
  • the first round nested PCR conditions described in the step (2') are: 92-98 ° C 1-6 min, 1-3 cycles; 95-100 ° C 15-25 s, 53-58 ° C 10-20 s, 70-75 ° C 35-45 s, 35-45 cycles; 70-75 ° C 3-8 min, 1-3 cycles; stored at 4 ° C;
  • the first round nested PCR conditions described in the step (2') are: 95 ° C for 3 min, 1 cycle; 98 ° C for 20 s, 55 ° C for 15 s, 72 ° C for 40 s, 40 cycles; 72 ° C for 5 min, 1 cycle. ; stored at 4 ° C;
  • the sequencing described in step (3) is sanger sequencing and/or Miseq sequencing.
  • the present invention provides the use of the kit of the first aspect for the construction of a TCR.
  • the present invention provides the use of a kit according to the first aspect for the preparation of a medicament for immunological diagnosis and/or prognosis monitoring of a disease.
  • the present invention has the following beneficial effects:
  • the present invention can adopt a nano-upgraded microporous chip as a reaction container, and can perform up to 5124 single T-cell TCR ⁇ / ⁇ pair-amplification at a time, which can increase flux and efficiently acquire thousands of individual T cell layers at one time.
  • the TCR ⁇ chain is paired with the full-length sequence to construct a TCR immune group library, and the reagent cost per cell is reduced by about 70%, and the labor cost is also greatly reduced;
  • the invention can simultaneously acquire thousands of TCR ⁇ VDJ region sequences of cell samples with a starting amount of picogram (1-100 pg) and nanograms, which is simple in operation and short in time, and the whole process can be within 10-12 hours. Finish Into, subtype coverage is complete and amplification preference is low;
  • the method of the invention is suitable for high-throughput cell heterogeneity and T cell subtype grouping, exploring a new immunological mechanism, constructing a large-scale full-length TCR immune group library, facilitating disease diagnosis and health management, and combining tumor new antigen
  • Figure 1 is a flow chart of microwell high-flux single-cell TCR ⁇ full-length sequence amplification technology
  • Fig. 2(a) is the result of the qPCR amplification real-time monitoring curve of the product of the 7th row and the 45th column of the microporous chip as a function of the number of cycles
  • Fig. 2(b) is the qPCR of the product of the 45th row of the microporous chip. Amplifying the results of the real-time monitoring curve as a function of temperature;
  • Figure 3 shows the results of the micropore amplification product 2100 taken by the microstep instrument
  • Electrophoresis results of high-throughput T-cell TCR amplification products of microwell microchips wherein 1-11 indicates electrophoretic detection results of single-cell amplification products in different chip wells, marker is 100 bp marker, and bottom-up is 100 bp, 200 bp, 300 bp, 400 bp, 500 bp (brightest band), 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, and 1500 bp.
  • the upstream primer is designed according to the adaptor sequence, which is shown in SEQ ID NO. 2, and the nucleotide sequence shown in SEQ ID NO. 2 is as follows:
  • the downstream primer is designed according to the C region of the TCR gene, and the complete sequence of the C region of the T cell ⁇ and ⁇ chain is as follows:
  • TCR ⁇ C region (SEQ ID NO. 7):
  • TCR ⁇ C region (SEQ ID NO. 8):
  • the primers for the Miseq sequencing platform are as follows:
  • the sequencing strategy of PE300 requires that the length of the library Insert be no more than 600bp, and the sequence of the 5' UTR sequence +V+D+J of the TCR ⁇ chain is just within 600bp, so we will
  • the downstream primer is placed 20 to 30 bp near the 5' end of the C region as follows:
  • the method for amplifying the full length TCR sequence is shown in FIG. 1 and includes the following steps:
  • the prepared lysate was mixed and mixed into a clean 384-well plate, and 24 wells were dispensed into 14 ⁇ L per well. After sealing, 2600 rcf, centrifuge at 1 ° C for 1 min (drop the droplets to the bottom of the tube and remove air bubbles). Place the 384-well plate on the MSND sample micro-dispenser, select the 35nL 72 sample spray mode, spray 35nL per micropore on the chip, seal it, 2600rcf, centrifuge at 5°C for 5min, store at 4°C, wait Cell addition;
  • the above chip was placed in a Biorad chip-specific PCR instrument, incubated at 72 ° C for 5 min, the hot lid temperature was 75 ° C, and immediately placed on ice for 1 min after the completion of the lysis; 2600 rcf was centrifuged at 4 ° C for 5 min, and immediately transferred to ice; this step Afterwards, all mRNAs are released from single cells, and Oligo-dT primers have also been bound to mRNAs;
  • the reverse transcription buffer was prepared in a 1.5 mL nuclease-free PCR tube, mixed by pipetting, and dispensed.
  • dispense 24 wells, 14 ⁇ L per well seal the membrane, 2600rcf, centrifuge at 1 °C for 1 min (drop the droplets to the bottom of the tube and remove air bubbles), and place the 384-well plate on the MSND sample micro-dispenser
  • select 35nL72 sample spray mode 35nl of each micropore spray on the chip, after sealing, 2600rcf, centrifuge at 5 °C for 5min;
  • the first strand cDNA of all mRNAs was synthesized, and after reverse transcription was completed, it was centrifuged at 2600 rcf for 5 min at 4 ° C;
  • the cell buffer was prepared in a 1.5 mL PCR tube, mixed by pipetting, and dispensed into a clean 384-well plate, and 24 wells were dispensed, 14 ⁇ L per well, and the membrane was sealed. Place the 384-well plate on the MSND sample micro-dispenser, select the 35nL 72 sample spray mode, spray 35nl per micropore on the chip, seal the membrane, 2600rcf, centrifuge at 5 °C for 5min;
  • the microstep instrument was used to aspirate the amplified product in the microwell of the chip into a 96-well plate, and the PCR system was prepared according to the following table:
  • pre-amplification is carried out according to the following conditions:
  • PCR 26th At the beginning of the cycle, the amplification products are significantly increased, and the melting curve of the lower left is revealed. The melting curve shows a single peak, indicating that the amplification product is relatively simple. As can be seen from Figure 3, the fragment distribution is detected by agilent 2100, indicating that the pores are indeed There are only a few relatively specific spikes, and the size is the size of the target segment;
  • the remaining samples were detected by PCR electrophoresis. Using 2% agarose gel, 25 ⁇ L of the product was added, and 3 ⁇ l of the addition buffer was added and mixed, and electrophoresed at 130 V for 45 min. The target strip was recovered by gelation. The results are shown in Fig. 4.
  • the microporous chip pore amplification product (the fifth sample) was further amplified by TCR-internal primers, and the product specificity was good, and it was about 600 bp. Consistent with the expected product size, it was again confirmed that the TCR amplification results in the wells of the microwell chip were good.
  • sequencing can directly obtain the TCR ⁇ VDJ region sequence, and can also achieve TCR ⁇ / ⁇ pairing.
  • the present invention can adopt a nano-upgraded micro-hole chip as a reaction container, and can perform up to 5124 single T-cell TCR ⁇ / ⁇ pair-amplification at a time, which can improve the throughput and can efficiently acquire thousands of individual Ts at a time.
  • the TCR ⁇ chain at the cell level was paired with the full-length sequence to construct a TCR immunological pool.

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Abstract

本发明提供了一种扩增TCR全长序列的试剂盒及其应用，所述试剂盒包含纳升级微孔芯片。本发明通过采用纳升级微孔芯片作为反应容器，一次最多可进行5124个单T细胞的TCRα/β配对扩增。

Description

一种扩增TCR全长序列的试剂盒及其应用 技术领域
本发明属于分子生物学领域，涉及一种扩增TCR全长序列的试剂盒及其应用，具体涉及一种扩增TCR全长序列的试剂盒，扩增TCR全长序列的方法及其应用。
背景技术
T淋巴细胞前体从骨髓进入胸腺，经历阳性和阴性筛选过程，发育为成熟的T细胞。期间，T细胞获得功能性T细胞受体(T cell receptor，TCR)，表达成熟T细胞的表面分子(CD4/CD8等)且获得MHC限制性和自身耐受性。T细胞种类复杂多样性与TCR重排相关，在胸腺中，TRB(TCRβ)基因经过D-J，V-D-J，V-D-J-C区域重排，最终产生约108不同TRB重排基因；TRA(TCRα)基因经过V-J，V-J-C区域重排，最终产生约104不同TRA重排基因。在人体外周血中，TCR多样性由于TRB、TRA的重排多达1018，决定着人的免疫系统如何适应环境的变化。
目前，对TCR的主要鉴别方法之一是对α和β链进行测序。传统免疫学研究中，基于群体细胞测序获得的数据很难实现对细胞异质性和不同细胞亚群的特性分析。以往的TCR扩增方法有：1.多重PCR扩增配合高通量测序技术，通过设计覆盖所有TRA、TRB分型的多重引物，在群体中捕获尽量完整的TRA、TRB亚型，高通量测序读取每一个亚型的TRA、TRB序列；2.RACE扩增配合高通量测序技术，通过TCR序列中恒定已知序列设计引物，在群体中扩增TCR基因序列，高通量测序技术读取每个亚型的TRA、TRB序列；3.T细胞群体转录组测序，生物信息组装TCR技术等。这些技术无法通过TCR测序准确区分T 细胞亚群及TCRα/β配对关系，而要更进一步理解T细胞免疫反应机理，需要对不同T细胞亚群内部和相互问的表型差异进行更精确的识别。因此，基于单细胞层面的TCRα/β配对分析越来越广泛的被应用于免疫学研究中。
TCR受体是由TCRα与TCRβ链形成的异二聚体，由于TCR基因在T细胞形成过程中，会发生DNA序列的重排，因此产生了T细胞的多样性，T细胞的多样性对于有效的免疫起了巨大作用。近期，多种基于TCR的高通量技术层出不穷，如：
(1)PairSEQ高通量TCR测序技术：该技术采用T细胞多样性特点，不同亚群之间T细胞对应的TCR序列不同，而样本中的亚群数目，亚群中T细胞的数目与对应的TCRα/β配对有相关性。该技术的策略是通过在96孔板中随机的明显亚群的T细胞，对每个孔中的细胞进行mRNA分离和第一链cDNA扩增，之后添加TCR V区和C区引物，扩增TCR CDR3的区域。在扩增引物上连接sample特异性的barcode，这样，在扩增时特异性的DNA barcode与扩增产物结合，可以准确标定每一个样本。将96孔板所有样本的扩增产物pooling后，进行的高通量测序。通过同时读取TCR序列信息和DNA barcode信息，可以通过不同孔之间的DNA barcode信息确定TCR对应的孔来源。每个孔T细胞亚群中的TCRα/β配对需要通过后续构建模型进行分析。通过计算机模型，获得单细胞配对的TCRα/β CDR3区域信息。
该技术的特点为，可以初步实现高通量单细胞TCR配对序列的扩增及分析。不过缺点十分明显，前期样本处理流程复杂，需要先对T细胞进行分群，铺到96孔板结合随机DNA barcode进行测序，数据模拟训练不同T细胞亚型的TCRα/β配对信息，测序组装的TRA和TRB对后续α/β配对的准确性限制较大。 该技术的特点为，可以初步实现高通量单细胞TCR配对序列的扩增及分析。不过缺点十分明显，首先，该技术通量比较低，由于在96孔板进行，限制了其通量及应用。其次，该技术还是基于CDR3区域的扩增，没有实现TCR全长配对序列的扩增，而全长序列对于其应用，尤其是免疫治疗方面的应用十分关键。
(2)TraCeR：该技术是通过T细胞单细胞转录组数据，通过计算机捕获单细胞中的TCR转录组信息，重构全长TCRα/β配对序列，根据TCR序列差异从而区分功能性T细胞克隆，单细胞转录组数据可以揭示细胞之间的转录异质性和不同细胞之间的演化进程，从而推算出明显的T细胞亚群。该技术是结合单细胞转录组数据，在获得表达量时，同时组装获得TCRα/β完整配对序列信息，具有较高的优势和创新。但由于该技术同样基于多重PCR方法在单细胞层面实现TCRα/β配对，但只能获得TCR的CDR3区域，不能获得TCR全长序列。由于该技术依赖于计算机重构软件，TCR序列本身的多样性和高度可变性，决定了比对重排序列是的难度，因此，开发合适的计算机软件，对于评估重构TCR序列的完整性和准确是该技术的瓶颈，准确率值得考虑。
(3)Single cell V(D)J：该技术是2017年4月份10Xgenomics公司推出的基于液滴微流控技术，一次性获取100-10000个单个免疫细胞TCR序列信息的技术。然而，基于液滴微流控的技术获取高通量的TCR序列是一个很好的切入点，但是基于液滴微流控方法实现单细胞层面TCRα/β配对，但是有液滴双包裹单细胞之间交叉污染、包裹效率低及成本高等缺陷。
目前现有TCR扩增技术，多停留在96孔板规模，未有成熟的高通量单细胞扩增方法。一方面，通量低较难实现T细胞异质性和不同细胞亚群的细致分析。对于TCRα/β配对信息也无法了解，同时无合适方法进行TCR全长序列扩 增，这一定程度上限制了其应用。另一方面，最近单细胞方面TCR全长扩增测序的进展，虽然可以一定程度上解决TCRα/β配对的问题，但大多数获得的都是单细胞TCR CDR3区域的序列信息。
CN 105543064 A公开了一种数字PCR芯片及其使用方法：该芯片包括硅质微孔芯片、加注样品的微流体通道和铝质导热基座，所述硅质微孔芯片上加工有20,000个内壁亲水、表面疏水的通孔，其用于分散微量核酸或细胞溶液，但是其并不适用于高通量TCR扩增。
然而，目前尚没有理想的技术可以在单细胞层面实现TCRα/β配对序列全长扩增，且具有覆盖已知的所有TRA、TRB亚型。
发明内容
针对现有技术中的缺陷，本发明提供了一种扩增TCR全长序列的试剂盒及其应用，所述试剂盒可以在单细胞层面实现TCRα/β配对序列全长扩增，且能够覆盖已知的所有TRA、TRB亚型。
一方面，本发明提供了一种扩增TCR全长序列的试剂盒，所述试剂盒包含纳升级微孔芯片。
本发明通过采用纳升级微孔芯片作为反应容器，一次最多可进行5124个单T细胞的TCRα/β配对扩增，可以提高通量，能够一次高效地获取数千个单个T细胞层面的TCRαβ链配对全长序列，构建TCR免疫组库。
本发明中，所述纳升级微孔芯片为容积为纳升级别的微孔芯片，所述纳升级微孔芯片上微孔的数量不会对本发明造成影响，本领域技术人员可以根据需要进行选择，在此不作特殊限定。
根据本发明，所述试剂盒还包括扩增TCR全长序列的引物对。
根据本发明，所述扩增TCR全长序列的引物对包括上游引物和下游引物，所述上游引物为根据测序接头序列设计的互补序列，所述下游引物为根据TCR的C区设计的互补序列。
本发明中，尽管TCR具有种类多样性，其C区序列同源性较好，所以本发明将下游引物根据C区进行设计，可以在C区的5’端到3’端中的任意位置进行设计，再通过在TCR的3’端加入一段测序接头序列(TSO)，同时根据其设计上游引物，通过这样的上游和下游引物就可以获得完整的V(D)J序列，从而得到TCR全长。
本发明中，所述设计引物遵循一般引物设计原则，例如GC含量40-60％、无二级发夹结构、无引物二聚体等，引物设计应该尽量设计在没有碱基多态性的位置。
根据本发明，所述测序接头序列是通过转换模板法引入，其中所述接头引物可以是任意一段固定长度的序列，本领域技术人员可以根据需要进行设计，本发明的接头引物的核苷酸序列如SEQ ID NO.1所示，所述SEQ ID NO.1所示的核苷酸序列如下：5’-AAGCAGTGGTATCAACGCAGAGT-3’；
根据本发明，所述测序接头序列的长度为18-35nt，例如可以是18nt、19nt、20nt、21nt、22nt、23nt、24nt、25nt、26nt、27nt或28nt，优选为28nt。
根据本发明，所述上游引物的核苷酸序列如SEQ ID NO.2所示，所述SEQ ID NO.2所示的核苷酸序列如下：5’-AAGCAGTGGTATCAACGCAGAGT-3’.
根据本发明，所述下游引物为巢式引物，发明人发现巢式引物不仅能够针对本申请单细胞中的起始量小的样本，且其针对TCR如此复杂的机构，扩增后的结果非常准确，所述下游引物包括外部引物和/或内部引物。
根据本发明，所述TCR全长由TCRα基因全长和TCRβ基因全长组成或由TCRγ基因全长和TCRδ基因全长组成；
根据本发明，扩增TCRα基因全长的下游引物的外部引物和内部引物的核苷酸序列分别如SEQ ID NO.3和SEQ ID NO.4所示，所述引物的核苷酸序列如下：
外部引物(SEQ ID NO.3)：GCAGACAGACTTGTCACTGG；
内部引物(SEQ ID NO.4)：GGTACACGGCAGGGTCAGGGTTC；
根据本发明，扩增TCRβ基因全长的下游引物的外部引物和内部引物的核苷酸序列分别如SEQ ID NO.5和SEQ ID NO.6所示；
所述引物的核苷酸序列如下：
外部引物(SEQ ID NO.5)：TGGTCGGGGAAGAAGCCTGTG；
内部引物(SEQ ID NO.6)：TTCTGATGGCTCAAACACAGCGA；
第二方面，本发明提供一种扩增TCR全长序列的方法，采用如第一方面所述的试剂盒。
根据本发明，所述扩增TCR全长序列的方法包括如下步骤：
(1)在所述纳升级微孔芯片中进行mRNA逆转录，得到第一链cDNA；
(2)以步骤(1)得到的第一链cDNA为模板，进行PCR扩增；
(3)测序验证，得到所述TCR全长扩增产物。
根据本发明，在步骤(1)之前还包括单细胞分离和裂解的步骤，所述单细胞分离采用流式细胞仪分选单细胞，采用样品微型分液仪将分选的单细胞分装到每个微孔芯片的微孔中，所述单细胞来源于外周血的外周血单个核细胞。
本发明中，可以从高通量单细胞层面彻底解决TCR全长测序和TCRα/β配 对的问题，既可以区分配对的αβ序列信息，同时又可以区分不同单细胞样本，从而将单管的TCR配对全长扩增技术有效的应用于高通量平台，实现高通量。
根据本发明，步骤(1)所述的mRNA反转录的体系包括测序接头序列，所述mRNA反转录的体系通过配制好体系后，采用样品微型分液仪将体系分装到每个微孔芯片的微孔中。
根据本发明，所述测序接头序列的终浓度为0.8-3μM，例如可以是0.8μM、0.9μM、1μM、1.2μM、1.3μM、1.5μM、1.6μM、1.8μM、2μM、2.2μM、2.4μM、2.5μM、2.6μM、2.8μM或3μM，优选为2μM；
根据本发明，步骤(1)所述的mRNA反转录的条件为：38-45℃85-95min1-3循环；48-53℃1-5min，38-45℃1-5min，8-15循环；68-73℃10-20min 1-3循环；保存在4℃；
根据本发明，步骤(1)所述的mRNA反转录的条件为：42℃90min 1循环；50℃2min，42℃2min，10循环；70℃15min 1循环；保存在4℃。
根据本发明，所述下游引物为巢式引物时，所述PCR扩增为巢式PCR；
根据本发明，所述巢式PCR的次数为1-3次，优选为2次；
根据本发明，所述巢式PCR具体包括：
(1’)以所述上游引物和所述外部引物为引物，以第一链cDNA为模板，进行第一轮巢式PCR，得到第一轮扩增产物；
(2’)以所述上游引物和所述内部引物为引物，以步骤(1’)得到的第一轮扩增产物为模板，进行第二轮巢式PCR，得到第二轮扩增产物；
根据本发明，步骤(1’)所述的第一轮巢式PCR条件为：92-98℃1-6min，1-3循环；95-100℃15-25s，53-58℃10-20s，70-75℃35-45s，35-45个循环； 70-75℃3-8min，1-3循环；保存在4℃；
根据本发明，步骤(1’)所述的第一轮巢式PCR条件为：95℃3min，1循环；98℃20s，55℃15s，72℃40s，40个循环；72℃5min，1循环；保存在4℃；
根据本发明，步骤(2’)所述的第一轮巢式PCR条件为：92-98℃1-6min，1-3循环；95-100℃15-25s，53-58℃10-20s，70-75℃35-45s，35-45个循环；70-75℃3-8min，1-3循环；保存在4℃；
根据本发明，步骤(2’)所述的第一轮巢式PCR条件为：95℃3min，1循环；98℃20s，55℃15s，72℃40s，40个循环；72℃5min，1循环；保存在4℃；
根据本发明，步骤(3)所述的测序为sanger测序和/或Miseq测序。
第三方面，本发明提供一种如第一方面所述的试剂盒用于TCR的建库的用途。
第四方面，本发明提供一种如第一方面所述的试剂盒用于制备疾病的免疫学诊断治疗和/或预后监控的药物的用途。
与现有技术相比，本发明具有如下有益效果：
(1)本发明通过采用纳升级微孔芯片作为反应容器，一次最多可进行5124个单T细胞的TCRα/β配对扩增，可以提高通量，能够一次高效地获取数千个单个T细胞层面的TCRαβ链配对全长序列，构建TCR免疫组库，同时每个细胞的试剂成本减少约70％，人力成本也大大降低；
(2)本发明能够同时获取数千个起始量为皮克级(1-100pg)和纳克级的细胞样品的TCRαβVDJ区序列，操作简单，耗时短，整个过程可在10-12h内完 成，亚型覆盖完整和扩增偏好性低；
(3)本发明方法适用于高通量的细胞异质性和T细胞亚型分群，探索免疫学新机制，构建大规模全长TCR免疫组库，方便疾病诊断及健康管理，结合肿瘤新抗原开发neo TCR-T肿瘤免疫细胞疗法，进行肿瘤免疫治疗，癌症或自体免疫疾病预后监测，有助于指导医生用药和科学研究等。
附图说明
图1微孔高通量单细胞TCRαβ全长序列扩增技术流程图；
图2(a)为微孔芯片第7行第45列产物的qPCR扩增实时监测曲线随着循环数变化的结果图，图2(b)为微孔芯片第7行第45列产物的qPCR扩增实时监测曲线随着温度变化的结果图；
图3采用microstep仪器吸出的芯片微孔扩增产物2100检测结果；
图4微孔芯片高通量T细胞TCR扩增产物电泳结果，其中，1-11表示不同芯片孔内单细胞扩增产物电泳检测结果，marker为100bp marker，从下到上为100bp、200bp、300bp、400bp、500bp(最亮条带)、600bp、700bp、800bp、900bp、1000bp和1500bp。
具体实施方式
下面结合附图和实施例对本发明作进一步的详细说明。可以理解的是，此处所描述的具体实施例仅仅用于解释本发明，而非对本发明的限定。另外还需要说明的是，为了便于描述，附图中仅示出了与本发明相关的部分而非全部结构。
实施例1试剂盒的组装
(1)引物设计：利用测序接头序列通过转换模板法在cDNA第一链的3’ 端引入测序接头序列，所述测序接头序列如SEQ ID NO.1所示，所述SEQ ID NO.1所示的核苷酸序列如下：
Figure PCTCN2017104109-appb-000001
根据接头序列设计上游引物，所述上游引物如SEQ ID NO.2所示，所述SEQ ID NO.2所示的核苷酸序列如下：
Figure PCTCN2017104109-appb-000002
所述下游引物根据TCR基因C区进行设计，所述T细胞α和β链的C区完整序列如下：
TCRαC区(SEQ ID NO.7)：
Figure PCTCN2017104109-appb-000003
TCRβC区(SEQ ID NO.8)：
Figure PCTCN2017104109-appb-000004
Figure PCTCN2017104109-appb-000005
采用Miseq测序平台，所述Miseq测序平台的引物如下：
因Miseq目前最长的读长为300bp/reads，采用PE300的测序策略，要求文库Insert长度不超过600bp，TCRαβ链5’端UTR序列+V+D+J区序列正好在600bp以内，因此我们将下游引物设置在靠近C区的5’端20～30bp的位置，如下所示：
表1人T细胞C区引物(下游)序列信息
Figure PCTCN2017104109-appb-000006
(2)将设计好的引物与WaferGen的微孔芯片组装后装入试剂盒中。
实施例2 TCR在Miseq测序平台测序
所述扩增TCR全长序列的方法，流程图如图1所示，包括如下步骤：
(1)第一链cDNA合成：
a)配制裂解液，所述裂解液配方如下表所示：
Figure PCTCN2017104109-appb-000007
配制好的裂解液吹打混匀后分装到洁净384孔板中，分装24孔，每孔14μL，封膜后，2600rcf，4℃离心1min(将液滴离心到管底并去除气泡)，将384孔板置于MSND样品微型分液仪上，选择35nL 72样品喷液模式，芯片上每个微孔喷液35nL，封膜后，2600rcf，4℃离心5min，置于4℃保存，等待细胞加入；
b)单细胞分离
按下表配制细胞缓冲液：
Figure PCTCN2017104109-appb-000008
在0.2mL的无核酸酶PCR管中配制该细胞缓冲液，吹打混匀后，采用FACS (流式细胞仪)分选5800个目的单细胞分选至该缓冲液中，轻轻吹打混匀，分装到洁净384孔板中，分装24孔，每孔14μL，封膜，将384孔板置于MSND样品微型分液仪上，选择35nL72样品喷液模式，芯片上每个微孔喷液35nl，封膜后，2600rcf，4℃离心5min，细胞按照泊松分布规则分布在芯片的纳升级微孔中；
c)细胞裂解
将上述芯片置于Biorad芯片专用PCR仪内，72℃，5min孵育，热盖温度为75℃，裂解完成后立即置于冰上1min；2600rcf 4℃离心5min，后立即转至冰上；此步后，所有mRNAs都从单细胞中释放，并且Oligo-dT引物也已与mRNAs结合；
d)mRNA反转录
配制反转录体系如下：
Figure PCTCN2017104109-appb-000009
在1.5mL的无核酸酶PCR管中配制该逆转录缓冲液，吹打混匀后，分装 到洁净384孔板中，分装24孔，每孔14μL，封膜，2600rcf，4℃离心1min(将液滴离心到管底并去除气泡)，将384孔板置于MSND样品微型分液仪上，选择35nL72样本喷液模式，芯片上每个微孔喷液35nl，封膜后，2600rcf，4℃离心5min；
将上述芯片置于Biorad芯片专用PCR仪内，按如下条件进行反转录反应(75℃热盖)：
Figure PCTCN2017104109-appb-000010
所有mRNAs的第一链cDNA合成完毕，逆转录完成后，2600rcf，4℃离心5min；
(2)特异性TCR序列扩增，采用实施例1中的引物：
a)第一轮PCR
以所述上游引物和所述外部引物为引物，以第一链cDNA为模板，进行第一轮巢式PCR，得到第一轮扩增产物；
按下表配制PCR体系：
Figure PCTCN2017104109-appb-000011
Figure PCTCN2017104109-appb-000012
在1.5mL PCR管中配制该细胞缓冲液，吹打混匀后，分装到洁净384孔板中，分装24孔，每孔14μL，封膜。将384孔板置于MSND样品微型分液仪上，选择35nL 72样品喷液模式，芯片上每个微孔喷液35nl，封膜后，2600rcf，4℃离心5min；
在BIORAD专用的芯片PCR仪上，按下述条件预扩增：
Figure PCTCN2017104109-appb-000013
b)第二轮PCR
以所述上游引物和所述中间引物为引物，以步骤(a)得到的第一轮扩增产物为模板，进行第二轮巢式PCR，得到第二轮扩增产物；
采用microstep仪器将芯片微孔中扩增产物吸出到96孔板中，按下表配制PCR体系：
Figure PCTCN2017104109-appb-000014
Figure PCTCN2017104109-appb-000015
吹打混匀、瞬时离心后，按下述条件预扩增：
Figure PCTCN2017104109-appb-000016
(3)电泳检测
巢式PCR完成后取1μl进行agilent 2100检测，检测结果如图2(a)、2(b)和图3所示，从图2(a)-2(b)可以看出，在PCR第26个循环开始，扩增产物明显递增，且通过左下图熔解曲线可发现，熔解曲线呈现单峰，说明扩增产物较为单一；从图3可以看出，通过agilent 2100检测片段分布，显示孔内确实只有几条相对特异的尖峰，且大小符合目的片段大小；
剩余样品进行PCR电泳检测，采用2％琼脂糖凝胶，取25μL产物，加3μl加样缓冲液混匀，130V电泳45min。目的条带切胶回收，结果如图4所示，采用微孔芯片孔扩增产物(第5个样品)采用TCR-内部引物进行进一步扩增发现，产物特异性较好，且在600bp左右，与预期产物大小一致，再次佐证微孔芯片孔内TCR扩增结果良好。
(4)将回收的产物进行TA克隆和Sanger测序，测序结果如下：
α链序列
α1：V12-2+J45+C
Figure PCTCN2017104109-appb-000017
β链序列(V27+J2)
Figure PCTCN2017104109-appb-000018
将上述结果整理如下表3所示：
表3
Figure PCTCN2017104109-appb-000019
由表3可知，样品的α、B型别分别是：
α：V12-2和J45
β：V9J1-6。
可见，采用Sanger测序平台，测序可以直接得到TCRαβVDJ区序列，还可实现TCRα/β配对。
综上所述，本发明通过采用纳升级微孔芯片作为反应容器，一次最多可进行5124个单T细胞的TCRα/β配对扩增，可以提高通量，能够一次高效地获取数千个单个T细胞层面的TCRαβ链配对全长序列，构建TCR免疫组库。
上述仅为本发明的较佳实施例及所运用技术原理。本领域技术人员会理解，本发明不限于这里所述的特定实施例，对本领域技术人员来说能够进行各种明显的变化、重新调整和替代而不会脱离本发明的保护范围。因此，虽然通过以上实施例对本发明进行了较为详细的说明，但是本发明不仅仅限于以上实施例，在不脱离本发明构思的情况下，还可以包括更多其他等效实施例，而本发明的范围由所附的权利要求范围决定。

Claims (14)

  1. 一种扩增TCR全长序列的试剂盒，其包含纳升级微孔芯片和扩增TCR全长序列的引物对。
  2. 根据权利要求1所述的试剂盒，其特征在于，所述扩增TCR全长序列的引物对包括上游引物和下游引物，所述上游引物为根据测序接头序列设计的互补序列，所述下游引物为根据TCR的C区设计的互补序列。
  3. 根据权利要求2所述的试剂盒，其特征在于，所述测序接头序列是通过转换模板法引入。
  4. 根据权利要求2所述的试剂盒，其特征在于，所述测序接头序列的长度为18-35nt，优选为28nt；
    优选地，所述测序接头序列的核苷酸序列如SEQ ID NO.1所示；
    优选地，所述上游引物的核苷酸序列如SEQ ID NO.2所示；
    优选地，所述下游引物为巢式引物；
    优选地，所述下游引物包括外部引物和/或内部引物。
  5. 根据权利要求1-4中任一项所述的试剂盒，其特征在于，所述TCR全长由TCRα基因全长和TCRβ基因全长组成或由TCRγ基因全长和TCRδ基因全长组成；
    优选地，扩增TCRα基因全长的下游引物的外部引物和内部引物的核苷酸序列分别如SEQ ID NO.3和SEQ ID NO.4所示；
    优选地，扩增TCRβ基因全长的下游引物的外部引物和内部引物的核苷酸序列分别如SEQ ID NO.5和SEQ ID NO.6所示。
  6. 一种扩增TCR全长序列的方法，其特征在于，采用如权利要求1-5中任一项所述的试剂盒。
  7. 根据权利要求6所述的方法，其特征在于，包括如下步骤：
    (1)在所述纳升级微孔芯片中进行mRNA逆转录，得到第一链cDNA；
    (2)以步骤(1)得到的第一链cDNA为模板，进行PCR扩增；
    (3)测序验证，得到所述TCR全长扩增产物。
  8. 根据权利要求7所述的方法，其特征在于，步骤(1)所述的mRNA逆转录的体系包括测序接头序列。
  9. 根据权利要求8所述的方法，其特征在于，所述测序接头序列的终浓度为0.8-3μM，优选为2μM。
  10. 根据权利要求7所述的方法，其特征在于，步骤(1)所述的mRNA逆转录的条件为：38-45℃ 85-95min 1-3循环；48-53℃ 1-5min，38-45℃ 1-5min，8-15循环；68-73℃ 10-20min 1-3循环；保存在4℃；
    优选地，步骤(1)所述的mRNA逆转录的条件为：42℃ 90min 1循环；50℃ 2min，42℃ 2min，10循环；70℃ 15min 1循环；保存在4℃。
  11. 根据权利要求6-10中任一项所述的方法，其特征在于，所述下游引物为巢式引物，所述PCR扩增为巢式PCR；
    优选地，所述巢式PCR的次数为1-3次，优选为2次；
    优选地，所述巢式PCR具体包括：
    (1’)以所述上游引物和所述外部引物为引物，以第一链cDNA为模板，进行第一轮巣式PCR，得到第一轮扩增产物；
    (2’)以所述上游引物和所述内部引物为引物，以步骤(1’)得到的第一轮扩增产物为模板，进行第二轮巣式PCR，得到第二轮扩增产物；
    优选地，步骤(1’)所述的第一轮巣式PCR条件为：92-98℃ 1-6min，1-3 循环；95-100℃ 15-25s，53-58℃ 10-20s，70-75℃ 35-45s，35-45个循环；70-75℃ 3-8min，1-3循环；保存在4℃；
    优选地，步骤(1’)所述的第一轮巣式PCR条件为：95℃ 3min，1循环；98℃ 20s，55℃ 15s，72℃ 40s，40个循环；72℃ 5min，1循环；保存在4℃；
    优选地，步骤(2’)所述的第一轮巣式PCR条件为：92-98℃ 1-6min，1-3循环；95-100℃ 15-25s，53-58℃ 10-20s，70-75℃ 35-45s，35-45个循环；70-75℃ 3-8min，1-3循环；保存在4℃；
    优选地，步骤(2’)所述的第一轮巣式PCR条件为：95℃ 3min，1循环；98℃ 20s，55℃ 15s，72℃ 40s，40个循环；72℃ 5min，1循环；保存在4℃；
    优选地，步骤(3)所述的测序为sanger测序和/或Miseq测序。
  12. 根据权利要求7-11中任一项所述的方法，其特征在于，在步骤(1)之前还包括单细胞分离和裂解的步骤；
    优选地，所述单细胞来源于外周血的外周血单个核细胞。
  13. 一种如权利要求1-5中任一项所述的试剂盒用于TCR的建库的用途。
  14. 一种如权利要求1-5中任一项所述的试剂盒用于制备疾病的免疫学诊断治疗和/或预后监控的药物的用途。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1856571A (zh) * 2003-09-18 2006-11-01 西福根有限公司 连接目标序列的方法
CN103627808A (zh) * 2013-12-10 2014-03-12 江南大学 一种t-发卡结构介导的测定dna侧翼未知序列的方法
WO2016015349A1 (zh) * 2014-08-01 2016-02-04 深圳华大基因科技有限公司 确定容器中核酸是否来源于单个细胞的方法及其装置和用途
CN105506746A (zh) * 2014-09-22 2016-04-20 深圳华大基因科技有限公司 构建可变区测序文库的方法及确定可变区核酸序列的方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9012148B2 (en) * 2008-04-16 2015-04-21 Jian Han Method for evaluating and comparing immunorepertoires
CN108138365B (zh) * 2015-11-17 2022-08-02 深圳华大智造科技股份有限公司 一种高通量的单细胞转录组建库方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1856571A (zh) * 2003-09-18 2006-11-01 西福根有限公司 连接目标序列的方法
CN103627808A (zh) * 2013-12-10 2014-03-12 江南大学 一种t-发卡结构介导的测定dna侧翼未知序列的方法
WO2016015349A1 (zh) * 2014-08-01 2016-02-04 深圳华大基因科技有限公司 确定容器中核酸是否来源于单个细胞的方法及其装置和用途
CN105506746A (zh) * 2014-09-22 2016-04-20 深圳华大基因科技有限公司 构建可变区测序文库的方法及确定可变区核酸序列的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"SMARTer Human TCR a/b Profiling Kit User Manual TECH NOTE", TAKARA BIO U.S.A. INC, 31 December 2016 (2016-12-31), pages 1 - 23, XP055587891, Retrieved from the Internet <URL:http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/NGS_Learning_Resources/Technical-Notes/Human-TCR-Profiling> *

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