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

Abstract

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 novel 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 up to 416X coverage 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 enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the ‘hidden genome’ underlying human disease.

Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing

“…From a technical perspective, we achieved > 500× enrichment on CNBP using the Cas9 protocol, which is robust and comparable to similar assays for the assessment of microsatellite length(Giesselmann et al 2019; Mizuguchi et al 2021; Sone et al 2019; Wallace et al 2021). We also compared Cas9-mediated singleplex vs multiplex enrichment protocols for the first time on clinical samples and observed a consistently lower performance (10-fold lower enrichment, with 70% unclassified reads) in the multiplex environment, as reported by other ONT users in the ONT for other type of samples (Oxford Nanopore Community 2022).…”

“…Reads derived from the expanded alleles (41 on average per sample, IQR = 11; Figure 1E ) ranged from 344 bp to as much as 46.6 kbp ( Figure 2D ), confirming the presence of extremely large expansions in these patients. To our knowledge, the latter is the longest repeat expansion analyzed thus far at single-nucleotide resolution(Mizuguchi et al 2021; Sone et al 2019; Giesselmann et al 2019; Wallace et al 2021) and is one of the longest DNA fragments captured by Cas9-mediated enrichment with no specific adjustment(Gilpatrick et al 2020; Iyer et al 2020). Considering average values per sample, the number of repetitive quadruplets varied from 1,371 to 4,421, corresponding to expansion lengths of 5,485–17,685 bp.…”

“…Targeted enrichment approaches coupled to long-read sequencing have already been used for the in-depth characterization of repeat expansions in fragile X syndrome ( FMR1 ), Huntington’s disease ( HTT ) and neuronal intranuclear inclusion disease ( NOTCH2NLC ), although these expansions are significantly shorter than the (CCTG) n repeats in DM2 patients(Dejesus-Hernandez et al 2021; Ebbert et al 2018; Giesselmann et al 2019; Grosso et al 2021; Hafford-Tear et al 2019; Höijer et al 2018; Mizuguchi et al 2021; Sone et al 2019; Tsai et al 2017; Wallace et al 2021; Wieben et al 2019; Mitsuhashi and Matsumoto 2020). The longest allele thus far characterized by third-generation sequencing is a 21-kbp allele of the C9orf72 gene that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (Dejesus-Hernandez et al 2021).…”

ACME: an Affinity-based Cas9 Mediated Enrichment method for targeted nanopore sequencing

“…Our results therefore specifically highlight the advantage offered by ACME’s ability to reduce competing background reads. Two other Cas9-based targeting approaches that involve background reduction - CaBagE 14 and Negative Enrichment 15 - use exonuclease digestion to eliminate non-target DNA. Both studies have only successfully achieved target read lengths of <35kb in size and pose the additional risk of digesting nicked target fragments that are not protected by Cas9 binding.…”

“…Two groups have adapted the principle behind nCATS to perform exonuclease digestion of non-target DNA. Cas9-based Background Elimination (CaBagE) 14 and Negative Enrichment 15 focus on protecting target DNA ends with Cas9, followed by exonuclease digestion of non-target reads. However, both methods have only achieved target read lengths of <35kb.…”