WO2023030479A1 - 高通量单细胞靶向测序的试剂和方法 - Google Patents

高通量单细胞靶向测序的试剂和方法 Download PDF

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WO2023030479A1
WO2023030479A1 PCT/CN2022/116674 CN2022116674W WO2023030479A1 WO 2023030479 A1 WO2023030479 A1 WO 2023030479A1 CN 2022116674 W CN2022116674 W CN 2022116674W WO 2023030479 A1 WO2023030479 A1 WO 2023030479A1
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cdna
cell
pcr
barcode
enrichment
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朱文奇
魏星
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新格元(南京)生物科技有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C40B20/04—Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes

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  • the present application generally relates to the field of molecular biology, and specifically provides methods, components, kits and systems including for high-throughput single-cell target sequencing.
  • Single-cell transcriptome sequencing is the most popular technology in the field of biology in recent years. Its ultra-high resolution can realize accurate analysis of sample information, and has great application potential in many fields of biology. For example, studying the important influence of tumor heterogeneity on disease development and drug intervention is used to dissect the diversity of immune receptors and translate into vaccination, cancer and autoimmune diseases.
  • RNA splicing events [1,2]
  • single-cell transcriptome sequencing is limited by the current short-read mRNA library sequencing technology, there is obvious selectivity for the 3' end or 5' end of the transcript, and it cannot effectively capture gene sequences far from the 3' end or 5' end, making it more difficult to detect complex gene rearrangements and RNA splicing .
  • each newly differentiated T or B lymphocyte in the immune system carries a different antigen receptor, and DNA rearrangement changes 450 nucleotides at the 5' end of T cell or B cell antigen receptor mRNA [3] .
  • B lymphocytes they use additional DNA rearrangements to "isotype switch" between nine alternative constant region sequences comprising 1000-1500 nucleotides at the 3' end of the heavy chain mRNA [4] and use alternative mRNA splicing changes the nucleotides at the 3' end of IGH mRNA to secrete the encoded receptor as an antibody [5] .
  • complex gene rearrangements and alternative splicing events generate pathological cellular diversity in cancer cells. Therefore, methods that can capture these sequence changes that occur across the entire length of an mRNA molecule at single-cell resolution and combine this information with gene expression signatures are urgently needed.
  • this solution is based on the 3' mRNA capture strategy, which can only quantify the expression of the target gene, but cannot obtain the variation information of the target gene.
  • immune receptor sequences there are several methods and reagents for detection of single cell immune receptors.
  • the immune receptor sequence is captured based on SMART-Seq2, but the above-mentioned method relies on flow cytometry for cell sorting and the detection throughput is small, usually 10-100 cells [6] , and the detection cost of a single cell is relatively high , and cannot simultaneously detect BCR or transcripts. Therefore, it is necessary to develop a method for high-throughput detection of single cell target genes (including but not limited to immune receptor nucleic acid sequences) to achieve the enrichment of massive single cell target sequences.
  • the present application provides methods, compositions, kits and systems for high-throughput single-cell target sequencing.
  • the present application provides a method for analyzing cellular gene expression levels and target gene sequences at the single-cell level, wherein the method includes:
  • each barcode oligonucleotide of the plurality of barcode oligonucleotides includes a cell barcode and a unique molecular identifier ( UMI), wherein the first barcode oligonucleotide of the plurality of barcode oligonucleotides each comprises a polyT sequence capable of binding a polyA tail of a first messenger ribonucleic acid (mRNA) target;
  • mRNA messenger ribonucleic acid
  • Circular cDNA is used for enrichment of target genes.
  • the plurality of barcode oligonucleotide sequences cover two different sequencing primer binding sites; wherein, the binding sites can be used as PCR primer binding sequences to amplify cDNA sequences.
  • the multiple barcode oligonucleotide sequences cover two different sequencing primer binding sites; wherein, the binding sites can be used as sequencing primer binding sites for library sequencing.
  • said cDNA circularization is performed by an enzymatic reaction.
  • the cDNA circularizing enzyme is DNA ligase.
  • the cDNA circularizing enzyme is DNA polymerase.
  • the enrichment of the target gene is carried out by means of inverse PCR using circular cDNA as a template.
  • the way of obtaining the information is gene sequencing.
  • the method of the matching analysis is performed by pairing after Cell barcode identification.
  • said nucleic acid target comprises ribonucleic acid (RNA), messenger RNA (mRNA) and deoxyribonucleic acid (DNA), and/or wherein said nucleic acid target comprises a cell from, in a cell and/or on a cell surface nucleic acid target.
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • DNA deoxyribonucleic acid
  • the source of the enriched target gene can be any vertebrate.
  • Figure 1 is a schematic diagram of a high-throughput single-cell targeted sequencing experiment.
  • FIG. 2 is a quality inspection map of the full-length TCR cDNA in Example 1.
  • Example 3 is a quality inspection map of the full-length TCR enrichment library in Example 1.
  • Figure 4 is a quality inspection map of the full-length BCR cDNA in Example 2.
  • Example 5 is a quality inspection image of the full-length BCR enrichment library in Example 2.
  • Figure 6 is a Perturb-seq cDNA quality inspection map in Example 3.
  • Figure 7 is the quality inspection map of the Perturb-seq transcriptome library in Example 3.
  • Fig. 8 is the quality inspection map of three rounds of enrichment products of Perturb-seq target sequences in Example 3.
  • Single-cell transcriptome sequencing combined with single-cell targeted sequencing technology can simultaneously analyze the cell type and target gene sequence information of the transcriptome, including cell mutation information, and is a powerful tool for studying the relationship between tumor cell development, targeted drugs, and gene hotspot mutations. tool.
  • Single-cell transcriptome combined with targeted mutation can accurately identify the mutated cell type and provide reference for clinical medicine. At the same time, it can dynamically monitor the changes in the type and frequency of mutations during the course of medication. This technology is to circularize a part of the double-stranded cDNA and design strand-specific primers to enrich the target gene far from the 3' end by reverse PCR technology.
  • the single-cell transcriptome can also obtain the target gene of interest and immune receptor sequence.
  • the method disclosed in this paper can not only obtain high-quality single-cell transcriptome data, but also obtain interesting hotspot mutation information at a much lower sequencing depth than transcriptome according to customer needs.
  • the technology has the following characteristics: (1) High-throughput: it can detect mutations in thousands of cells at the same time; (2) Deep customization: corresponding enrichment primers can be designed according to different needs of customers; (3) Cost-effective : Experimental procedures are highly compatible with single-cell transcriptome workflows. It only needs to customize the primer sequence and construct the corresponding enrichment library to achieve the capture of the target region.
  • mice spleen cells mouse spleen cells, cell concentration: 2.5*10 5 cells/mL, cell viability: 95%.
  • the Oligo sequence of FL Barcoding Beads contains illumina Read1 sequencing primer sequence, cell label (Cell Barcode), molecular label (UMI) and PolyT nucleoside Acid sequences are used to capture multiple mRNAs through polyT.
  • the full-length transcriptome is amplified through the PCR handle sequence at the 5' end of FL Barcoding Beads (adapted to the sequencing primers of the illumina next-generation sequencing platform) and the TSO sequence added during the reverse transcription process enrichment to obtain cDNA products.
  • the magnetic beads (purified magnetic beads) were taken out from 4°C 30 minutes in advance and returned to room temperature.
  • the magnetic beads are relatively viscous, pipette the corresponding volume accurately, otherwise the length of the sorted fragments may be inconsistent with the expectation.
  • step 3 for a total of two rinses.
  • the product was purified with 0.6x magnetic beads, and the specific steps were as described above.
  • Step 3.3 product Take 20ng of the Step 3.3 product, put the PCR tube on ice to prepare the third round of enrichment PCR mix according to the following table, vortex to mix and centrifuge briefly.
  • the product was purified with 0.6x magnetic beads, and the specific steps were as described above.
  • the product was sorted using 0.5x and 0.15x sorting coefficients as described above.
  • the sequence and expression information of VDJ (TCR) were obtained by library sequencing.
  • Figure 3 is the quality inspection map of the full-length TCR cDNA
  • Figure 4 is the quality inspection map of the full-length TCR enriched library.
  • Table 1 below is a list of TOP10 clonotypes enriched in full-length TCR
  • Table 2 is a list of important indicators for enrichment of full-length TCR.
  • mice PBMC cells mouse PBMC cells, cell concentration: 2.3*10 5 cells/mL, cell viability: 92%.
  • the PCR machine should be set and run according to the following table.
  • the thermal lid of the PCR machine should be set at 105°C:
  • the product was purified using a 0.6x purification factor in the same manner as described above.
  • the product was purified using a 0.6x purification factor in the same manner as described above.
  • Step 4.3 product 1) Take 20ng of the Step 4.3 product, put the PCR tube on ice to prepare the third round of enrichment PCR mix according to the table below, vortex to mix and centrifuge briefly.
  • the product was purified using a 0.6x purification factor in the same manner as described above.
  • Figure 5 is the quality inspection map of the full-length BCR cDNA
  • Figure 6 is the quality inspection map of the full-length BCR enriched library.
  • Table 3 below is a list of TOP10 clonotypes enriched in full-length BCR
  • Table 4 is a list of important indicators for enrichment of full-length BCR.
  • mice cell line mouse cell line
  • cell concentration 5.28*10 6 cells/mL
  • cell viability 95%.
  • the present invention can also be used for the targeted capture of sgRNA.
  • the difference between the captured magnetic beads used and the FL Barcoding beads in the above-mentioned implementation cases is that the magnetic beads are based on polyT probes, and a targeted probe sequence is added in a certain proportion , to achieve simultaneous capture of mRNA and target sequences;
  • the perturb-seq target sequence enrichment method is also enriched by circularizing the cDNA template, and the primers enriched by PCR are bound to the constant region of the sgRNA;
  • the product was purified using a 1x purification factor as described above.
  • Step 3.4.2.1 1) Take 18 ⁇ L of the product of Step 3.4.2.1, put the PCR tube on ice to prepare the second round of enrichment PCR mix according to the table below, vortex to mix and centrifuge briefly.
  • the product was purified using a 1x purification factor as described above.
  • Step 3.4.2.2 1) Take 20ng of the product of Step 3.4.2.2, put the PCR tube on ice to prepare the third round of enrichment PCR mix according to the table below, vortex to mix and centrifuge briefly.
  • the product was purified using a 0.8x purification factor as described above.
  • Figure 7 shows the quality inspection map of Perturb-seq cDNA
  • Figure 8 shows the quality inspection map of Perturb-seq transcriptome library
  • Figure 9 shows the quality inspection map of three rounds of enrichment products of Perturb-seq target sequences.

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Abstract

本发明提供了用于单细胞分析的方法，包括将细胞和附着有多个条形码寡核苷酸的珠子划分成一个分区。多个条形码寡核苷酸中的每个条形码寡核苷酸可包含细胞条形码和唯一分子标识符（UMI），通过寡核苷酸末尾的polyT序列，与mRNA的polyA序列进行结合，完成mRNA的捕获。通过反转录和PCR扩增，分别完成mRNA的一链和二链合成。将一部分cDNA用于构建转录组测序文库，另一部分cDNA通过环化，形成环状双链cDNA，以此为模板，进行靶基因的特异性富集。由于两部分的cDNA携带的cell barcode信息是匹配的，通过测序之后的Barcode序列匹配，就能获得细胞的表达谱信息和配对的靶基因序列信息。

Description

高通量单细胞靶向测序的试剂和方法 技术领域
本申请总体上涉及分子生物学领域，具体提供包括用于高通量单细胞靶标测序的方法、成分、试剂盒和系统。
背景技术
单细胞转录组测序是近年来生物学领域最流行的技术。其超高分辨率可实现对样品信息的准确分析，在生物学的诸多领域具有巨大的应用潜力。例如，研究肿瘤的异质性对疾病发展和药物干预的重要影响，用于解析免疫受体多样性，并转化应用于疫苗接种、癌症和自身免疫疾病。人类和其他脊椎动物的细胞表型多样性可能源于复杂的基因重排和替代RNA剪接事件 [1,2]，然而，单细胞转录组测序，受限于目前的短读长mRNA建库测序技术，对于转录本的3’端或者5’端存在明显的选择性，不能有效捕获距离3’端或者5’端较远的基因序列，对于检测复杂的基因重排和RNA剪切就更加困难。
例如，免疫系统中每个新分化的T或B淋巴细胞都携带不同的抗原受体，DNA重排改变了T细胞或B细胞抗原受体mRNA 5’端的450个核苷酸 [3]。在B淋巴细胞中，它们使用额外DNA重排在重链mRNA的3’端包含1000-1500个核苷酸的九个替代恒定区序列之间“同种型转换” [4]，并使用替代mRNA剪接来改变IGH mRNA 3’末端的核苷酸以分泌作为抗体的编码受体 [5]。同样，复杂的基因重排和选择性剪接事件在癌细胞中产生病理细胞多样性。因此，迫切需要能够以单细胞分辨率捕获在整个mRNA分子长度上发生的这些序列变化的方法，并将该信息与基因表达特征相结合。
目前，已有的单细胞靶向测序的技术方案，如10X Genomics的单细胞靶向测序的产品，基于探针杂交捕获的方法，在原有的单细胞3’端或者5’端转录组文库的基础上，通过探针杂交捕获感兴趣的基因片段。虽然实现了靶基因的富集，但是未能解决对于末端存在偏好的问题，仍然不能捕获距离3’端或者5’端较远的基因。BD Biosciences的靶向测序产品，基于不同基因设计了富集Panel，采用多重PCR的方式对于感兴趣的基因位点，进行PCR扩增从而获取多个基因的表达信息。但是，这一方案基于3’端的mRNA捕获策略，只能对于靶基因的 表达进行定量，不能获得靶基因的变异信息。对于基因重排后的检测，例如，免疫受体序列，已有一些用于检测单细胞免疫受体的方法和试剂。例如，基于SMART-Seq2捕获免疫受体序列，但是上述的方法依赖于流式细胞仪进行细胞分选检测通量小，通常是10-100个细胞 [6]，单个细胞的检测成本均比较高昂，且不能同时检测BCR或转录本。因此，需要开发一款高通量检测单个细胞靶基因(包括但不限于免疫受体核酸序列)的方法来实现对海量单细胞靶向序列的富集。
发明内容
本申请提供了用于高通量单细胞靶标测序的方法、成分、试剂盒和系统。
本申请提供了一种在单细胞水平分析细胞基因表达水平以及靶基因序列的方法，其中，所述方法包括：
a.将细胞和附着有多个条形码寡核苷酸的珠子共同落入同一个微孔，其中，多个条形码寡核苷酸的每个条形码寡核苷酸包括细胞条形码和唯一分子标识符(UMI)，其中多个条形码寡核苷酸的第一条形码寡核苷酸每个包含能够结合第一信使核糖核酸(mRNA)靶标的polyA尾的polyT序列；
b.在二链合成之后，一部分cDNA会进行环化，形成环状双链cDNA；
c.环状cDNA被用于进行靶基因的富集；以及
d.匹配分析转录组信息与靶基因信息。
优选地，所述多个条码寡核苷酸序列上涵盖两种不同的测序引物结合位点；其中，所述结合位点可用作PCR引物结合序列以扩增cDNA的序列。
优选地，所述多个条码寡核苷酸序列上涵盖两种不同的测序引物结合位点；其中，所述结合位点可用作测序引物结合位点，进行文库测序。
优选地，所述cDNA环化是通过酶促反应进行的。
优选地，所述cDNA环化的酶是DNA连接酶。
优选地，所述cDNA环化的酶是DNA聚合酶。
优选地，所述靶基因的富集是以环状cDNA为模板，用反向PCR的方式进行富集。
优选地，所述信息获取的方式是基因测序。
优选地，所述匹配分析的方法是通过Cell barcode识别后的配对进行。
优选地，所述核酸靶标包括核糖核酸(RNA)、信使RNA(mRNA)和脱氧核糖核酸(DNA)，和/或其中所述核酸靶标包括来自细胞、细胞中和/或细胞表面上的细胞的核酸靶标。
优选地，所述被富集的靶基因来源可以是任意脊椎动物。
附图说明
图1为高通量单细胞靶向测序实验原理图。
图2为实施例1中全长TCR cDNA的质检图。
图3为实施例1中全长TCR富集文库质检图。
图4为实施例2中全长BCR cDNA质检图。
图5为实施例2中全长BCR富集文库质检图。
图6为实施例3中Perturb-seq cDNA质检图。
图7为实施例3中Perturb-seq转录组文库质检图。
图8为实施例3中Perturb-seq靶序列三轮富集产物质检图。
具体实施方式
单细胞转录组测序结合单细胞靶向测序技术，可以同时分析转录组的细胞类型和靶基因序列信息，包括细胞突变信息，是研究肿瘤细胞发育、靶向药物和基因热点突变之间关系的有力工具。单细胞转录组结合靶向突变可以准确识别发生突变的细胞类型，为临床用药提供参考。同时，它可以动态监测用药过程中突变类型和频率的变化。该技术是通过将一部分双链cDNA进行环化，通过反向PCR技术设计链特异性引物富集距离3’端较远的靶基因，并基于Singleron独特的单细胞微流控系统，不仅检测普通的单细胞转录组，还可以获得感兴趣的靶基因以及免疫受体序列。本文所公开的方法不仅可以获得高质量的单细胞转录组数据，还可以根据客户的需求，在比转录组低得多的测序深度上获得感兴趣的热点突变信息。该技术具有以下特点：(1)高通量：可同时检测数千个细胞感兴趣区域的突变；(2)深度定制：可根据客户的不同需求设计相应的富集引物；(3)性价比高：实验程序与单细胞转录组工作流程高度兼容。只需要定制引物序列，构建相应的富集文库即可实现目标区域的捕获。
在以下实施例中更详细地公开了上述实施例的一些方面，其不以任何方式旨在限制本公开的范围。
实施例1：全长TCR序列检测
实验材料：小鼠脾脏细胞，细胞浓度：2.5*10 5个/mL，细胞活性：95％。
实验流程：
1.单细胞分选、mRNA捕获、反转录及PCR富集
(1)将小鼠脾脏细胞分别Loading进Singleron微流控芯片，根据“泊松分布”的原理完成单个细胞的分离；
(2)Loading FL Barcoding Beads进入芯片中，确保90％的孔中均有beads；FL Barcoding Beads的Oligo序列包含illumina Read1测序引物序列，细胞标签(Cell Barcode)，分子标签(UMI)和PolyT核苷酸序列，通过ployT完成多mRNA的捕获。
(3)loading 100μL细胞裂解液至芯片中，室温裂解15min，完成单个细胞的mRNA的捕获和标记；
(4)裂解完成后，用1.5mL离心管回收芯片中的磁珠，并清洗去除上清，加入200μL反转录试剂，42℃，1300rpm，反应90min，完成反转录过程；
(5)反转录完成后，通过FL Barcoding Beads 5’端的PCR handle序列(适配illumina二代测序平台的测序引物)及反转录过程中添加上的TSO序列，扩增完成全长转录组的富集，获得cDNA产物。
(6)获得cDNA产物，一部分用于转录组文库的构建，一部份用于后续全长TCR和BCR的富集。
该部分的具体实验操作可参考GEXSCOPE单细胞RNAseq文库构建试剂盒(Singleron Biotechnologies)的使用说明进行。该部分实验原理可参考附图1。
2.全长TCR富集及文库构建(该部分实验原理可参考附图2。)
2.1环化
1)取250ng cDNA产物，将PCR管置于冰上按照如下表格配制环化mix，涡旋混匀并短暂离心。
Figure PCTCN2022116674-appb-000001
2)使用移液器轻柔充分混匀，短暂离心后将反应管置于PCR仪中，PCR仪根据下表设置并运行反应，PCR仪热盖85℃：
Figure PCTCN2022116674-appb-000002
3)环化后产物不纯化，加入Cyclicase进行酶切。将PCR管置于冰上按照如下表格配制酶切体系。
Figure PCTCN2022116674-appb-000003
4)使用移液器轻柔充分混匀，短暂离心后将反应管置于PCR仪中，PCR仪根据下表设置并运行反应，PCR仪热盖关闭：
Figure PCTCN2022116674-appb-000004
5)产物纯化
①磁珠(纯化磁珠)提前30min从4℃中取出，恢复室温。
注：磁珠使用前需混匀。
②将PCR管中的液体，瞬离，计算体积。加入1.3×产物体积的磁珠，吹打混匀后，室温孵育5min，短暂离心，置于磁力架上静置5min；至液体透明澄清，小心移除上清至新的PCR管中，暂留。
注：磁珠比较粘稠，准确移取相应的体积，否则可能导致分选的片段长度与预期不一致。
③保持PCR管始终处于磁力架上，加入200μL新配制的80％乙醇漂洗磁珠。室温孵育30s，小心移除上清。
④重复步骤③，共计漂洗两次。
⑤取下PCR管，短暂离心，再次置于磁力架上，吸去多余酒精，晾干。
⑥取下PCR管，加入20μL Nuclease-free Water，吹吸混匀磁珠，室温孵育5min，短暂离心后静置于磁力架上，至液体透明澄清。
⑦吸取上清并转移至新的EP管中，即为纯化产物。
2.2全长免疫受体(TCR)第一轮富集
1)取20ng环化产物，将PCR管置于冰上按照如下表格配制第一轮富集PCR mix，涡旋混匀并短暂离心。
Figure PCTCN2022116674-appb-000005
2)使用移液器轻柔充分混匀，短暂离心后将反应管置于PCR仪中，PCR仪根据下表设置并运行反应，PCR仪热盖105℃：
Figure PCTCN2022116674-appb-000006
3)产物纯化
该产物用0.6x磁珠进行纯化，具体步骤同上所述。
2.3全长免疫受体第二轮富集
1)取20ng第一轮富集产物，将PCR管置于冰上按照如下表格配制第一轮富集PCR mix，涡旋混匀并短暂离心。
Figure PCTCN2022116674-appb-000007
2)使用移液器轻柔充分混匀，短暂离心后将反应管置于PCR仪中，PCR仪根据下表设置并运行反应，PCR仪热盖105℃：
Figure PCTCN2022116674-appb-000008
3)产物分选
①提前30min取出纯化磁珠，恢复室温，备用。将PCR管瞬离，量取PCR产物体积，加入25μL磁珠(0.5×产物体积)，涡旋振荡或使用移液器吹打10次充分混匀，室温孵育5min。
②将PCR管短暂离心后置于磁力架上，使磁珠与液体分离，待溶液澄清后(约5min)，小心转移上清至一个新有无菌PCR管中。
③吸取7.5μL(0.15x产物体积)磁珠至步骤2)收集的上清中，涡旋振荡或使用移液器吹打10次充分混匀，室温孵育5min。
④将PCR管短暂离心后置于磁力架上，使磁珠与液体分离，待溶液澄清后(约5min)，小心移除上清。
⑤保持PCR管始终处于磁力架上，加入200μL新鲜配制的80％乙醇漂洗磁珠。室温孵育30s，小心移除上清。
⑥重复步骤⑤，总计漂洗2次。
⑦从磁力架上取下PCR管，短暂离心，再次置于磁力架上，吸去多余酒精，开盖使磁珠暴露于空气中干燥2min。
⑧将PCR管从磁力架上取出，加入20μL Nuclease-free Water洗脱。涡旋振荡使磁珠和液体充分混匀，室温孵育5min。
⑨将PCR管短暂离心并置于磁力架上，使磁珠与液体分离，待溶液澄清后(约5min)小心吸取18μL上清至新的灭菌PCR管中。
4)扩增纯化产物质检
取1μL样品进行Qubit浓度检测。
3.4全长免疫受体(TCR)第三轮富集
(1)取20ng Step 3.3产物，将PCR管置于冰上按照如下表格配制第三轮富集PCR mix，涡旋混匀并短暂离心。
Figure PCTCN2022116674-appb-000009
(2)将反应管置于PCR仪中，PCR仪根据下表设置并运行反应，PCR仪 热盖105℃：
Figure PCTCN2022116674-appb-000010
该产物用0.6x磁珠进行纯化，具体步骤同上所述。
3.5全长免疫受体富集文库构建
3.5.1片段化
(1)提前按照下表设置PCR程序，反应体积35μL，并使PCR仪热盖保持在75℃。
Figure PCTCN2022116674-appb-000011
(2)室温解冻FR Buffer V2和1×TE，确保完全融化，涡旋10s混匀并短暂离心后冰上备用；使用前将FR Mix V2涡旋振荡10s后，置于冰上备用。
(3)将灭菌的PCR管置于冰上，配置如下反应体系：
Figure PCTCN2022116674-appb-000012
(4)用移液器轻柔吹打充分混匀，瞬离后将PCR管置于PCR仪中，跳过PCR程序第一步。
(5)完成反应后立即进行下一步接头连接。
3.5.2接头连接
(1)提前按照下表设置PCR程序，反应体系70μL，并关闭PCR仪热盖加热功能。
Figure PCTCN2022116674-appb-000013
(2)提前室温解冻AFI，涡旋10s混匀后冰上备用。使用前将LA Mix和LA IB涡旋10s混匀，短暂离心后置于冰上备用。LA Mix较粘稠，吸取时注意量取准确体积(多反应体系时建议AFI单独加入)。
(3)将上一步反应的PCR管置于冰上，配置如下反应体系：
Figure PCTCN2022116674-appb-000014
3.5.3接头连接后产物纯化
①该接头连接产物用0.5x纯化系数进行纯化，方法同上所述；不同点在于产物重悬时加入17μL 0.1×TE(使用Nuclease-free Water按1:9稀释1×TE)，涡旋振荡15s混匀磁珠，室温孵育5min。
②将纯化磁珠所在PCR管短暂离心后置于磁力架上，使磁珠与液体分离，待溶液澄清后(约5min)，小心吸取15μL上清至新的灭菌PCR管中用于PCR富集。
3.5.4 PCR富集
(1)将PCR管置于冰上，配置如下反应体系，使用移液器轻柔吹打5次(45μL量程)混匀并短暂离心，将反应管置于PCR仪中。
Figure PCTCN2022116674-appb-000015
Figure PCTCN2022116674-appb-000016
(2)提前按照下表设置PCR程序，并使PCR仪热盖保持在105℃，反应体积50μL。
Figure PCTCN2022116674-appb-000017
3.5.5产物分选
该产物用0.5x和0.15x分选系数进行分选，方法同上所述。通过文库测序获取VDJ(TCR)的序列和表达量信息。
实验结果如图3为全长TCR cDNA的质检图；图4为全长TCR富集文库质检图。如下表1为全长TCR富集TOP10克隆型列表；表2为全长TCR富集重要指标列表。
表1
Figure PCTCN2022116674-appb-000018
表2
Figure PCTCN2022116674-appb-000019
实施例2：全长BCR序列检测
实验材料：小鼠PBMC细胞，细胞浓度：2.3*10 5个/mL，细胞活性：92％。
实验方法：实验方法中除BCR富集方式部分和上述TCR富集方式有差异，其他操作包括单细胞分选、mRNA捕获、反转录及PCR富集、cDNA环化及富集产物文库构建均和全长TCR序列检测一致，全长BCR序列检测富集方式部分具体步骤如下：
1.全长免疫受体(BCR)第一轮富集
1)取20ng环化产物，将PCR管置于冰上按照如下表格配制第一轮富集PCR mix，涡旋混匀并短暂离心。
Figure PCTCN2022116674-appb-000020
2)使用移液器轻柔充分混匀，短暂离心后将反应管置于PCR仪中，PCR仪根 据下表设置并运行反应，PCR仪热盖105℃：
Figure PCTCN2022116674-appb-000021
3)产物纯化
该产物用0.6x纯化系数进行纯化，纯化方式同上所述。
2.全长免疫受体(BCR)第二轮富集
1)取20ng第一轮富集产物，将PCR管置于冰上按照如下表格配制第一轮富集PCR mix，涡旋混匀并短暂离心。
Figure PCTCN2022116674-appb-000022
2)使用移液器轻柔充分混匀，短暂离心后将反应管置于PCR仪中，PCR仪根据下表设置并运行反应，PCR仪热盖105℃：
Figure PCTCN2022116674-appb-000023
3)产物纯化
该产物用0.6x纯化系数进行纯化，纯化方式同上所述。
3.全长免疫受体(BCR)第三轮富集
1)取20ng Step 4.3产物，将PCR管置于冰上按照如下表格配制第三轮富集PCR mix，涡旋混匀并短暂离心。
Figure PCTCN2022116674-appb-000024
2)将反应管置于PCR仪中，PCR仪根据下表设置并运行反应，PCR仪热盖105℃：
Figure PCTCN2022116674-appb-000025
3.产物纯化
该产物用0.6x纯化系数进行纯化，纯化方式同上所述。
实验结果如图5为全长BCR cDNA质检图；图6为全长BCR富集文库质检图。如下表3为全长BCR富集TOP10克隆型列表；表4为全长BCR富集重要指标列表。
表3
Figure PCTCN2022116674-appb-000026
表4
分析指标 M_0427PBMC1_N_B3Nlib
Estimated_Number_of_Cells 15，099
Cells_Match_with_ScRNA-seq_Analysis 6，001
Reads_Mapped_To_Any_V(D)J_Gene 79.09％
Cells_With_Productive_V-J_Spanning_Pair 97.67％
Cells_With_Productive_V-J_Spanning_(IGH，_IGL)_Pair 10.61％
Cells_With_Productive_V-J_Spanning_(IGH，_IGK)_Pair 90.13％
Cells_With_IGH_Contig 99.55％
Cells_With_Productive_IGH_Contig 98.20％
Cells_With_IGL_Contig 43.33％
Cells_With_Productive_IGL_Contig 10.86％
Cells_With_IGK_Contig 99.48％
Cells_With_Productive_IGK_Contig 91.78％
实施例3：perturb-seq靶序列富集
实验材料：小鼠细胞系，细胞浓度：5.28*10 6个/mL，细胞活性：95％。
实验方法：
●本发明亦可用于sgRNA的靶向捕获，所用捕获磁珠与上述实施案例中的 FL Barcoding beads的区别在于，该磁珠在polyT探针的基础上，按一定比例添加了靶向探针序列，实现mRNA和靶向序列的同时捕获；
●perturb-seq靶序列富集方式部分也是通过将cDNA模板环化再在进行富集，PCR富集的引物binding在sgRNA的恒定区域；
●单细胞分选、mRNA捕获、反转录及PCR富集、cDNA环化均和上述全长TCR序列检测一致，perturb-seq靶序列富集方式部分具体步骤如下：
1.Perturb-seq靶序列第一轮富集
1)取10μL环化产物，将PCR管置于冰上按照如下表格配制第一轮富集PCR mix，涡旋混匀并短暂离心。
Figure PCTCN2022116674-appb-000027
2)将反应管置于PCR仪中，PCR仪根据下表设置并运行反应，PCR仪热盖105℃：
Figure PCTCN2022116674-appb-000028
3)产物纯化
该产物用1x纯化系数进行纯化，纯化方法同上所述。
2.Perturb-seq靶序列第二轮富集
1)取18μL Step 3.4.2.1产物，将PCR管置于冰上按照如下表格配制第二轮富 集PCR mix，涡旋混匀并短暂离心。
Figure PCTCN2022116674-appb-000029
2)将反应管置于PCR仪中，PCR仪根据下表设置并运行反应，PCR仪热盖105℃：
Figure PCTCN2022116674-appb-000030
3)产物纯化
该产物用1x纯化系数进行纯化，纯化方法同上所述。
3.Perturb-seq靶序列第三轮富集
1)取20ng Step 3.4.2.2产物，将PCR管置于冰上按照如下表格配制第三轮富集PCR mix，涡旋混匀并短暂离心。
Figure PCTCN2022116674-appb-000031
Figure PCTCN2022116674-appb-000032
2)将反应管置于PCR仪中，PCR仪根据下表设置并运行反应，PCR仪热盖105℃：
Figure PCTCN2022116674-appb-000033
3)产物纯化
该产物用0.8x纯化系数进行纯化，纯化方法同上所述。
实验结果如图7为Perturb-seq cDNA质检图；图8为Perturb-seq转录组文库质检图；以及图9为Perturb-seq靶序列三轮富集产物质检图。
参考文献：
[1]V(D)J Recombination and the Evolution of the Adaptive Immune System[J].PLoS Biology,2003,1(1):e16-.
[2]Eric T,Wang,Rickard,et al.Alternative isoform regulation in human tissue transcriptomes.[J].Nature,2008.
[3]Bassing C H,Swat W,Alt F W.The Mechanism and Regulation of Chromosomal V(D)J Recombination[J].Cell,2002,109(2):S45-S55.
[4]Chaudhuri J,Alt F W.Class-switch recombination:interplay of transcription,DNA deamination and DNA repair[J].Nature Reviews Immunology.
[5]Frederick,W,Alt,et al.Synthesis of secreted and membrane-bound immunoglobulin mu heavy chains is directed by mRNAs that differ at their 3′ends[J].Cell,1980,20(2):293-301.
[6]Picelli,S.et al.Smart-seq2 for sensitive full-length transcriptome profiling in single cells.Nat.Methods 10,1096–1098,(2013).

Claims (11)

  1. 一种在单细胞水平分析细胞基因表达水平以及靶基因序列的方法，其包括：
    a.将细胞和附着有多个条形码寡核苷酸的珠子共同落入同一个微孔，其中，多个条形码寡核苷酸的每个条形码寡核苷酸包括细胞条形码和唯一分子标识符(UMI)，其中多个条形码寡核苷酸的第一条形码寡核苷酸每个包含能够结合第一信使核糖核酸mRNA靶标的polyA尾的polyT序列；
    b.在二链合成之后，一部分cDNA会进行环化，形成环状双链cDNA；
    c.环状cDNA被用于进行靶基因的富集；以及
    d.匹配分析转录组信息与靶基因信息。
  2. 根据权利要求1所述的方法，其中，所述多个条码寡核苷酸序列上涵盖两种不同的测序引物结合位点；其中，所述结合位点可用作PCR引物结合序列以扩增cDNA的序列。
  3. 根据权利要求1所述的方法，其中，所述多个条码寡核苷酸序列上涵盖两种不同的测序引物结合位点；其中，所述结合位点可用作测序引物结合位点，进行文库测序。
  4. 根据权利要求1所述的方法，其中，所述cDNA环化是通过酶促反应进行。
  5. 根据权利要求4所述的方法，其中，所述cDNA环化的酶是DNA连接酶。
  6. 根据权利要求4所述的方法，其中，所述cDNA环化的酶是DNA聚合酶。
  7. 根据权利要求1所述的方法，所述靶基因的富集是以环状cDNA为模板，用反向PCR的方式进行富集。
  8. 根据权利要求1所述的方法，其中，所述信息获取的方式是基因测序。
  9. 根据权利要求1所述的方法，其中，所述匹配分析的方法是通过Cell barcode识别后的配对进行。
  10. 根据权利要求1所述的方法，其中，所述核酸靶标包括核糖核酸RNA、信使核糖核酸mRNA和脱氧核糖核酸DNA，和/或其中所述核酸靶标包括来自细胞、细胞中和/或细胞表面上的细胞的核酸靶标。
  11. 根据权利要求1所述的方法，所述被富集的靶基因来源可以是任意脊椎动物。
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