Multiplex structural variant detection by whole-genome mapping and nanopore sequencing

Abstract

Identification of structural variants (SVs) breakpoints is important in studying mutations, mutagenic causes, and functional impacts. Next-generation sequencing and whole-genome optical mapping are extensively used in SV discovery and characterization. However, multiple platforms and computational approaches are needed for comprehensive analysis, making it resource-intensive and expensive. Here, we propose a strategy combining optical mapping and cas9-assisted targeted nanopore sequencing to analyze SVs. Optical mapping can economically and quickly detect SVs across a whole genome but does not provide sequence-level information or precisely resolve breakpoints. Furthermore, since only a subset of all SVs is known to affect biology, we attempted to type a subset of all SVs using targeted nanopore sequencing. Using our approach, we resolved the breakpoints of five deletions, five insertions, and an inversion, in a single experiment.

A long-read sequencing strategy with overlapping linkers on adjacent fragments (OLAF-Seq) for targeted resequencing and enrichment

“…For this, additional alternative sgRNAs may be designed. For example, to capture a large heterozygous insertion or deletion, two or more pairs of sgRNAs, for targeting both haplotype with SV and without SV may be designed such that the SV falls within the linkers 23 . Even if one of the linkers is missed due to the presence of an SV, the fragments can still be captured by the adjacent linkers.…”