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Next generation sequencing of all variable loops of synthetic single framework scFv-Application in anti-HDL antibody selections

机译：合成单框架scFv所有可变环的下一代测序-在抗HDL抗体选择中的应用

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Next generation sequencing (NGS) can be applied to monitoring antibody phage display library selection processes to follow the enrichment of each individual antibody clone. Utilising the recent development of the Illumina sequencing platform enabling sequencing up to 2 x 300 bp, we have developed a method to deep sequence all complementarity determining regions (CDRs) in the clones obtained from a synthetic single framework antibody library. This was complemented by an in-house bioinformatics pipeline for efficient analysis of the sequencing results. The method was utilised to study antibody selections against high density lipoprotein (HDL) particles. Sequencing of the output from each selection round enabled extraction of useful information on both the total copy numbers as well as the relative enrichment rates of the clones. Ten antibody clones showing different ranking in terms of frequency were reproduced from synthetic DNA constructs and their capacity to bind HDL was verified by an immunoassay. The method thus facilitates the isolation of clones of interest, and in particular can assist retrieval of less efficiently enriched, yet interesting clones, which are unlikely to be identified by conventional, random colony picking based, screening. (C) 2016 Elsevier B.V. All rights reserved.
机译：下一代测序（NGS）可以应用于监测抗体噬菌体展示文库的选择过程，以追踪每个抗体克隆的富集情况。利用Illumina测序平台的最新发展，该测序平台可进行高达2 x 300 bp的测序，我们开发了一种方法，可对从合成的单框架抗体文库获得的克隆中的所有互补决定区（CDR）进行深度测序。内部生物信息学管道可对测序结果进行有效分析，从而对其进行补充。该方法用于研究针对高密度脂蛋白（HDL）颗粒的抗体选择。通过对每个选择回合的输出进行测序，可以提取出有关总拷贝数以及克隆相对富集率的有用信息。从合成的DNA构建体中复制了十个在频率上显示出不同等级的抗体克隆，并通过免疫测定证实了它们结合HDL的能力。因此，该方法促进了目的克隆的分离，并且特别是可以帮助回收不太有效的富集但有趣的克隆，这不太可能通过常规的基于随机菌落挑选的筛选来鉴定。 （C）2016 Elsevier B.V.保留所有权利。

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