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

TL;DR

OLAF-Seq is a new sequencing method that uses CRISPR to fragment DNA and link adjacent pieces, enabling efficient and detailed analysis of targeted genomic regions.

Contribution

OLAF-Seq introduces a CRISPR-based strategy for targeted long-read sequencing with physical linkage information between fragments.

Findings

OLAF-Seq enables targeted enrichment of genomic regions with long-read sequencing platforms like PacBio and Oxford Nanopore.

The method successfully sequences cancer-related genes, including exons and introns, with improved haplotype assembly.

Linker regions in OLAF-Seq provide physical linkage information, aiding genome assembly and phasing.

Abstract

In this report, we present OLAF-Seq, a novel strategy to construct a long-read sequencing library such that adjacent fragments are linked with end-terminal duplications. We use the CRISPR-Cas9 nickase enzyme and a pool of multiple sgRNAs to perform non-random fragmentation of targeted long DNA molecules (> 300kb) into smaller library-sized fragments (about 20 kbp) in a manner so as to retain physical linkage information (up to 1000 bp) between adjacent fragments. DNA molecules targeted for fragmentation are preferentially ligated with adaptors for sequencing, so this method can enrich targeted regions while taking advantage of the long-read sequencing platforms. This enables the sequencing of target regions with significantly lower total coverage, and the genome sequence within linker regions provides information for assembly and phasing. We demonstrated the validity and efficacy of the method first using phage and then by sequencing a panel of 100 full-length cancer-related genes (including both exons and introns) in the human genome. When the designed linkers contained heterozygous genetic variants, long haplotypes could be established. This sequencing strategy can be readily applied in both PacBio and Oxford Nanopore platforms for both long and short genes with an easy protocol. This economically viable approach is useful for targeted enrichment of hundreds of target genomic regions and where long no-gap contigs need deep sequencing.

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