Transposase-Assisted Tagmentation: An Economical and Scalable Strategy for Single-Worm Whole-Genome Sequencing (elegans)

AlphaMissense identifies 23 million human missense variants as likely pathogenic, but only 0.1% have been clinically classified. To experimentally validate these predictions, chemical mutagenesis presents a rapid, cost-effective method to produce billions of mutations in model organisms. However, the prohibitive costs and limitations in the throughput of whole-genome sequencing (WGS) technologies, crucial for variant identification, constrain its widespread application. Here, we introduce a Tn5 transposase-assisted tagmentation technique for conducting WGS in C. elegans, E. coli, S. cervisiae, and C. reinhardtii. This method, demands merely 20 minutes of hands-on time for a single worm or single-cell clones and incurs a cost below 10 US dollars. It effectively pinpoints causal mutations in mutants defective in cilia or neurotransmitter secretion and in mutants synthetically sterile with a variant analogous to the oncogenic BRAF(V600E) mutation. Integrated with chemical mutagenesis, our approach can generate and identify missense variants economically and efficiently, facilitating experimental investigations of missense variants in diverse species.

AlphaMissense 可鉴定出2300万个人类错义变异为可能致病性变异，但其中仅有0.1%得到了临床分类。为实验验证这些预测结果，化学诱变是一种快速且经济高效的手段，可在模式生物中产生数十亿个突变。然而，作为变异鉴定核心技术的全基因组测序（Whole-Genome Sequencing, WGS），其高昂的成本与有限的通量限制了该诱变方法的广泛应用。在此研究中，我们提出了一种基于Tn5转座酶辅助标记片段化（tagmentation）的技术，可用于秀丽隐杆线虫（C. elegans）、大肠杆菌（E. coli）、酿酒酵母（S. cervisiae）以及莱茵衣藻（C. reinhardtii）的全基因组测序。该方法仅需20分钟的手工操作时间即可完成单条线虫或单细胞克隆的建库流程，单次实验成本低于10美元。其可有效定位纤毛缺陷、神经递质分泌缺陷突变体，以及携带类似致癌性BRAF(V600E)变异的合成不育突变体中的致病突变。将该技术与化学诱变手段整合后，我们的方案可经济高效地产生并鉴定错义变异，从而推动跨物种错义变异的实验研究。