CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing

作者：Amelia D. Wallace, Thomas A. Sasani, Jordan Swanier, Brooke L. Gates, Jeff Greenland, Brent S. Pedersen, Katherine E. Varley, Aaron R. Quinlan · 发表于：PLoS ONE · 年份：2021 · DOI：10.1371/journal.pone.0241253 · 被引用次数：33 · 研究领域：CRISPR and Genetic Engineering、Chromosomal and Genetic Variations、Genomics and Phylogenetic Studies

A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore's MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore's MinION long-read sequencing technology. Enrichment with CaBagE resulted in a median of 116X coverage (range 39-416) of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical e...