High-Quality Library Preparation for NGS-Based Immunoglobulin Germline Gene Inference and Repertoire Expression Analysis
High-Quality Library Preparation for NGS-Based Immunoglobulin Germline Gene Inference and Repertoire Expression Analysis
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DOI：
10.3389/fimmu.2019.00660
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发表时间：
2019-04-05
影响因子：
7.3
通讯作者：
Hedestam, Gunilla B. Karlsson
中科院分区：
文献类型：
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作者：
Bernat, Nestor Vazquez;Corcoran, Martin;Hedestam, Gunilla B. Karlsson
免疫球蛋白（IG）库（Rep-seq）的下一代测序（NGS）使得能够在前所未有的水平上检查适应性免疫系统。应用包括研究表达谱、基因使用、体细胞超突变水平、IG谱系追踪和通过推断方法鉴定IG基因座内的遗传变异。所有这些应用都需要启动文库，其允许产生具有低错误率和表达库的最佳表示的序列数据。在这里，我们提供了详细的协议，适用于人类IG种系基因推断和IG库研究的图书馆的生产。测试了该过程中使用的各种参数，以证明获得高质量文库的关键因素。我们展示了一种改进的5 'RACE技术，该技术减少了基于Illumina MiSeq的Rep-seq分析的长度限制，但允许获得IG V基因上游的序列，这对引物设计有用。然后，我们描述了一种5'多重文库制备方法，该方法产生了适合于基因型鉴定和新基因推断的全长V（D）J序列。我们提供针对IGHV、IGKV和IGLV基因的全面引物组。使用优化的方案，我们产生了IgM、IgG、IgK和IgL文库，并使用种系推断工具IgDiscover对其进行分析以鉴定表达的种系V等位基因。该过程还在单个个体中发现了三个IGHV、一个IGKV和六个IGLV新等位基因，这些等位基因在IMGT参考数据库中不存在，这突出了进一步研究IG遗传变异的必要性。本文提出的文库生成方案能够实现分析表达的IG库、鉴定新等位基因和从人类产生个体化生殖系基因数据库的稳健手段。
Next generation sequencing (NGS) of immunoglobulin (Ig) repertoires (Rep-seq) enables examination of the adaptive immune system at an unprecedented level. Applications include studies of expressed repertoires, gene usage, somatic hypermutation levels, Ig lineage tracing and identification of genetic variation within the Ig loci through inference methods. All these applications require starting libraries that allow the generation of sequence data with low error rate and optimal representation of the expressed repertoire. Here, we provide detailed protocols for the production of libraries suitable for human Ig germline gene inference and Ig repertoire studies. Various parameters used in the process were tested in order to demonstrate factors that are critical to obtain high quality libraries. We demonstrate an improved 5'RACE technique that reduces the length constraints of Illumina MiSeq based Rep-seq analysis but allows for the acquisition of sequences upstream of Ig V genes, useful for primer design. We then describe a 5' multiplex method for library preparation, which yields full length V(D)J sequences suitable for genotype identification and novel gene inference. We provide comprehensive sets of primers targeting IGHV, IGKV, and IGLV genes. Using the optimized protocol, we produced IgM, IgG, IgK, and IgL libraries and analyzed them using the germline inference tool IgDiscover to identify expressed germline V alleles. This process additionally uncovered three IGHV, one IGKV, and six IGLV novel alleles in a single individual, which are absent from the IMGT reference database, highlighting the need for further study of Ig genetic variation. The library generation protocols presented here enable a robust means of analyzing expressed Ig repertoires, identifying novel alleles and producing individualized germline gene databases from humans.