适配器(计算)
计算生物学
清脆的
结扎测序
DNA测序
计算机科学
DNA
生物
基因组文库
遗传学
基序列
计算机硬件
基因
作者
Taylor L. Mighell,Andrew Nishida,Brendan O’Connell,Caitlin V. Miller,Sally Grindstaff,Casey Thornton,Andrew Adey,Daniel Doherty,Brian J. O’Roak
出处
期刊:The CRISPR journal
[Mary Ann Liebert]
日期:2022-08-01
卷期号:5 (4): 548-557
标识
DOI:10.1089/crispr.2021.0140
摘要
Targeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive guanine-cytosine (GC) sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogrammed guide RNAs to direct CRISPR-Cas12a cleavage of double-stranded DNA in vitro and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7176 guides targeting 3.5 Mb of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 Mb. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for single nucleotide variants, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double-stranded DNA template or genome.
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