PDF(Pubmed)
Abstract：
Next-generation sequencing (NGS) is an indispensable tool in antibody discovery projects. However, the limits on NGS read length make it difficult to reconstruct full antibody sequences from the sequencing runs, especially if the six CDRs are randomized. To overcome that, we took advantage of Illumina\'s cluster mapping capabilities to pair non-overlapping reads and reconstruct full Fab sequences with accurate VL:VH pairings. The method relies on in silico cluster coordinate information, and not on extensive in vitro manipulation, making the protocol easily deployable and less prone to PCR-derived errors. This work maintains the throughput necessary for antibody discovery campaigns, and a high degree of fidelity, which potentiates not only phage-display and synthetic library-based discovery methods, but also the NGS-driven analysis of naïve and immune libraries.
摘要：
下一代测序（NGS）是抗体发现项目中不可或缺的工具。然而,NGS读取长度的限制使得很难从测序运行中重建完整的抗体序列，特别是如果六个CDR是随机的。为了克服这一点，我们利用Illumina的簇作图能力,对非重叠读段进行配对,并利用准确的VL:VH配对重建完整的Fab序列。该方法依赖于计算机集群坐标信息，而不是广泛的体外操作，使协议易于部署，不易发生PCR衍生错误。这项工作保持了抗体发现活动所需的吞吐量，和高度的保真度，这不仅加强了噬菌体展示和基于合成文库的发现方法，还有NGS驱动的幼稚和免疫库分析。
