RT Journal Article
SR Electronic
T1 Cas12a-Capture: a novel, low-cost, and scalable method for targeted sequencing
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.11.18.388876
DO 10.1101/2020.11.18.388876
A1 Taylor L. Mighell
A1 Andrew Nishida
A1 Brendan L. O’Connell
A1 Caitlin V. Miller
A1 Sally Grindstaff
A1 Casey A. Thornton
A1 Andrew C. Adey
A1 Daniel Doherty
A1 Brian J. O’Roak
YR 2020
UL http://biorxiv.org/content/early/2020/11/20/2020.11.18.388876.abstract
AB Targeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive 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 preprogramed 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 7,176 guides targeting 3.5 megabases 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 megabases. 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 SNVs, 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.Competing Interest StatementOregon Health & Science University, TLM, CAT, AA, and BJO have submitted a patent application for the Cas12a-Capture method, PCT/US2020/049966.