Cas9-Assisted Targeting of CHromosome segments (CATCH) for targeted nanopore sequencing and optical genome mapping

Abstract

Variations in the genetic code, from single point mutations to large structural or copy number alterations, influence susceptibility, onset, and progression of genetic diseases and tumor transformation. Next-generation sequencing analysis is unable to reliably capture aberrations larger than the typical sequencing read length of several hundred bases. Long-read, singlemolecule sequencing methods such as SMRT and nanopore sequencing can address larger variations, but require costly whole genome analysis. Here we describe a method for isolation and enrichment of a large genomic region of interest for targeted analysis based on Cas9 excision of two sites flanking the target region and isolation of the excised DNA segment by pulsed field gel electrophoresis. The isolated target remains intact and is ideally suited for optical genome mapping and long-read sequencing at high coverage. In addition, analysis is performed directly on native genomic DNA that retains genetic and epigenetic composition without amplification bias. This method enables detection of mutations and structural variants as well as detailed analysis by generation of hybrid scaffolds composed of optical maps and sequencing data at a fraction of the cost of whole genome sequencing.

Xdrop: Targeted sequencing of long DNA molecules from low input samples using droplet sorting

“…The level of enrichment by Xdrop technology is comparable to other long-read targeted enrichment strategies (Gabrieli et al, 2017;Gilpatrick et al, 2019;Tsai et al, 2017) interest, and they require micrograms of input DNA (Gilpatrick et al, 2019;Hoijer et al, 2018;Stangl et al, 2019;Tsai et al, 2017).…”

Detecting genetic variation and base modifications together in the same single molecules of DNA and RNA at base pair resolution using a magnetic tweezer platform

“…Our MT platform is well suited to this dual analysis in the same single molecules of DNA. To analyze specific gene loci without DNA amplification, we developed the CRISPR/Cas9 enrichment method that achieved target specificities that are far greater than we have seen reported elsewhere in the published literature [10][11][12] . From human genomic DNA, we enriched fragments containing FMR1 and observed that the allele frequency of expanded repeats to normal was in the expected 1:1 ratio for a heterozygous sample.…”

“…Compared to full genome sequencing, these protocols dramatically reduce per-sample costs as multiple samples can be pooled and multiplexed; however, because these methods involve sample amplification, this benefit comes at the penalty of loss of epigenetic information and the introduction of bias into the results 9 . Recently amplification-free Cas9-based enrichment strategies have been attempted by several groups [10][11][12] but typically only achieve an enrichment of 20-to 60-fold (compared to 10,000-fold for PCR) 13 .…”

Harnessing “A Billion Years of Experimentation”: The Ongoing Exploration and Exploitation of CRISPR–Cas Immune Systems

“…250 Cas9 may also be useful to directly enrich for desired sequences, either through dCas9-based affinity purification or through the excision and subsequent isolation of genomic regions of interest. 251 Cas9 has even proven useful for physical mapping of genomic DNA. In one study, a Cas9 mutant was used to nick specific genomic sequences, followed by DNA polymerase-mediated local incorporation of fluorescent nucleotides; the resulting sites could then be directly visualized within nanochannel arrays using fluorescence microscopy to identify structural variants.…”