Complete characterization of the human immune cell transcriptome using accurate full-length cDNA sequencing
Complete characterization of the human immune cell transcriptome using accurate full-length cDNA sequencing
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DOI：
10.1101/gr.257188.119
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发表时间：
2020-04-01
期刊：
影响因子：
7
通讯作者：
Vollmers, Christopher
中科院分区：
文献类型：
--
作者：
Cole, Charles;Byrne, Ashley;Vollmers, Christopher
人类免疫系统依赖于高度复杂和多样的转录本及其编码的蛋白质。这些包括编码人白细胞抗原（HLA）受体以及B细胞和T细胞受体（BCR和TCR）的转录物。确定一个人拥有每个HLA基因的等位基因（高分辨率HLA分型）对于在器官和骨髓移植中建立供体-受体相容性至关重要。反过来，每个个体中数百万个独特的BCR和TCR转录本的库携带了大量的健康相关信息。基于短读段RNA-seq的HLA分型和BCR/TCR库测序（AIRR-seq）目前都依赖于我们对HLA和BCR/TCR基因座遗传多样性的不完全了解。在这里，我们使用我们的基于纳米孔测序的滚环扩增串联共有序列（R2 C2）方案以97.9%的中位准确度生成了超过10，000，000个全长cDNA序列。我们使用该数据集（1）表明深度和准确的全长cDNA测序可用于为9000多个基因座提供亚型水平的转录组分析，（2）生成HLA等位基因的准确序列，以及（3）提取详细的AIRR数据用于分析适应性免疫系统。我们在这里介绍的HLA和AIRR分析方法是非靶向的，因此不需要HLA和BCR/TCR基因座的组成或遗传多样性的先验知识。
The human immune system relies on highly complex and diverse transcripts and the proteins they encode. These include transcripts encoding human leukocyte antigen (HLA) receptors as well as B cell and T cell receptors (BCR and TCR). Determining which alleles an individual possesses for each HLA gene (high-resolution HLA typing) is essential to establish donor-recipient compatibility in organ and bone marrow transplantations. In turn, the repertoires of millions of unique BCR and TCR transcripts in each individual carry a vast amount of health-relevant information. Both short-read RNA-seq-based HLA typing and BCR/TCR repertoire sequencing (AIRR-seq) currently rely on our incomplete knowledge of the genetic diversity at HLA and BCR/TCR loci. Here, we generated over 10,000,000 full-length cDNA sequences at a median accuracy of 97.9% using our nanopore sequencing-based Rolling Circle Amplification to Concatemeric Consensus (R2C2) protocol. We used this data set to (1) show that deep and accurate full-length cDNA sequencing can be used to provide isoform-level transcriptome analysis for more than 9000 loci, (2) generate accurate sequences of HLA alleles, and (3) extract detailed AIRR data for the analysis of the adaptive immune system. The HLA and AIRR analysis approaches we introduce here are untargeted and therefore do not require prior knowledge of the composition or genetic diversity of HLA and BCR/TCR loci.