Human Health Therapeutics Research Centre, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada, K1A 0R6.
Biotechniques. 2018 Dec;65(6):351-356. doi: 10.2144/btn-2018-0123.
Antibody (Ab) repertoire sequencing using high-throughput massively parallel technologies has contributed substantially to the understanding of Ab responses following infection, vaccination and autoimmunity. Because individual B-cell receptors are recombined and diversified somatically, genomic comparisons are limited, and distinguishing rare variants from sequencing errors is a major challenge. Oxford Nanopore Technologies' MinION is a highly portable and cost-effective third-generation sequencing instrument, but has not been used for Ab repertoire sequencing due to its high error rate (approximately 1/10 bases). Here, we applied nanopore sequencing to single-domain Ab (sdAb) repertoires and phage-displayed sdAb libraries. We show that despite low overall data fidelity, sdAb sequences could be reconstructed above a frequency threshold (∼100 copies); however, distinguishing clonal sdAb variants was not always possible. The data quality was sufficient to enable rapid identification of antigen-specific sdAb sequences enriched during panning of phage display libraries, obviating the need for screening single clones.
利用高通量大规模平行技术进行抗体（Ab）库测序，极大地促进了对感染、疫苗接种和自身免疫后 Ab 反应的理解。由于个体 B 细胞受体通过体细胞重组和多样化，基因组比较受到限制，因此区分稀有变体和测序错误是一个主要挑战。牛津纳米孔技术的 MinION 是一种高度便携且具有成本效益的第三代测序仪器，但由于其错误率较高（约为 1/10 个碱基），尚未用于 Ab 库测序。在这里，我们将纳米孔测序应用于单域抗体（sdAb）库和噬菌体展示 sdAb 文库。我们表明，尽管整体数据保真度较低，但可以在频率阈值（~100 个拷贝）以上重建 sdAb 序列；然而，并不总是能够区分克隆 sdAb 变体。数据质量足以快速识别在噬菌体展示文库筛选过程中富集的抗原特异性 sdAb 序列，从而无需筛选单个克隆。
