Unbiased quantification of immunoglobulin diversity at the DNA level with VDJ-seq
Unbiased quantification of immunoglobulin diversity at the DNA level with VDJ-seq
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DOI：
10.1038/nprot.2018.021
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发表时间：
2018-06-01
期刊：
影响因子：
14.8
通讯作者：
Corcoran, Anne E.
中科院分区：
文献类型：
--
作者：
Chovanec, Peter;Bolland, Daniel J.;Corcoran, Anne E.
对于免疫球蛋白库的高通量测序和定量，大多数方法使用RNA。然而，由于不同的启动子强度和淋巴细胞亚群的差异激活，重组基因之间的输出变化很大，排除了每个细胞基础上的重组体的定量。迄今为止，基于DNA的方法使用V基因引物鸡尾酒，具有实质性的固有偏差。在这里，我们描述了VDJ测序（VDJ-seq），它以无偏的方式在DNA水平上准确地定量免疫球蛋白的多样性。这通过使用生物素化的J基因引物的单一引物延伸步骤来完成。通过在引物延伸之前添加独特的分子标识符（UMI），我们可靠地去除重复序列并校正测序和PCR错误。此外，VDJ-seq在每个细胞的基础上捕获生产性和非生产性VDJ和DJ重组事件。文库制备需要3天，2天测序，1天数据处理和分析。
For high-throughput sequencing and quantification of immunoglobulin repertoires, most methodologies use RNA. However, output varies enormously between recombined genes due to different promoter strengths and differential activation of lymphocyte subsets, precluding quantitation of recombinants on a per-cell basis. To date, DNA-based approaches have used V gene primer cocktails, with substantial inherent biases. Here, we describe VDJ sequencing (VDJ-seq), which accurately quantitates immunoglobulin diversity at the DNA level in an unbiased manner. This is accomplished with a single primer-extension step using biotinylated J gene primers. By addition of unique molecular identifiers (UMIs) before primer extension, we reliably remove duplicate sequences and correct for sequencing and PCR errors. Furthermore, VDJ-seq captures productive and nonproductive VDJ and DJ recombination events on a per-cell basis. Library preparation takes 3 d, with 2 d of sequencing and 1 d of data processing and analysis.