Cas9 targeted enrichment of mobile elements using nanopore sequencing

Abstract

Mobile element insertions (MEIs) are highly repetitive genomic sequences that contribute to inter- and intra-individual genetic variation and can lead to genetic disorders. Targeted and whole-genome approaches using short-read sequencing have been developed to identify reference and non-reference MEIs; however, the read length hampers detection of these elements in complex genomic regions. Here, we pair Cas9 targeted nanopore sequencing with computational methodologies to capture active MEIs in human genomes. We demonstrate parallel enrichment for distinct classes of MEIs, averaging 44% of reads on targeted signals. We show an individual flow cell can recover a remarkable fraction of MEIs (97% L1Hs, 93% AluYb, 51% AluYa, 99% SVA_F, and 65% SVA_E). We identify twenty-one non-reference MEIs in GM12878 overlooked by modern, long-read analysis pipelines, primarily in repetitive genomic regions. This work introduces the utility of nanopore sequencing for MEI enrichment and lays the foundation for rapid discovery of elusive, repetitive genetic elements.

CRISPR-LRS for mapping transgenes in the mouse genome

“…We propose a more targeted approach to map a transgene by combining (i) the RNA programmable CRISPR-Cas9 system (10) and (ii) long-read length coverage of the ONT sequencing platform (11,12). Previous studies have combined CRISPR-Cas9 with LRS to enrich for genomic elements (13)(14)(15)(16)(17)(18)(19)(20); however, no studies until now have combined CRISPR and LRS to map transgenes in an animal model. CRISPR-LRS can be designed (i) to target a genomic section (Targeted-CRISPR-LRS) or (ii) to enrich genomic sections (Enrichment-CRISPR-LRS).…”

An efficient CRISPR-Cas9 enrichment sequencing strategy for characterizing complex and highly duplicated genomic regions. A case study in the Prunus salicina LG3-MYB10 genes cluster

“…Long-read approaches have improved the contiguity of genome assemblies, while special effort is currently being made to resolve particularly complicated regions of relatively small sizes in selected genotypes. In humans, several authors have used a strategy based on enrichment and sequencing using targeted cleavage of chromosomal DNA with Cas9 combined with Nanopore sequencing to resolve genomic SVs as well as mobile elements (23, 36-38). In plants, this strategy has been validated to fine map short regions (24) and to characterize the transposon insertion landscape (39).…”

Emerging roles for endogenous retroviruses in immune epigenetic regulation*

“…Polymorphic elements will also often be absent from the species reference genome, requiring specialized software to analyze 160 . Detecting transposon‐derived structural variants can be technologically challenging, but the adoption of long‐read sequencing technologies will significantly facilitate their identification 161,162 . However, to truly incorporate newly identified transposon polymorphisms into genomic studies, adoption of graph‐based “pan‐genome” reference genomes that include structural variation will be necessary 163 …”