Journal article
A long-read sequencing strategy with overlapping linkers on adjacent fragments (OLAF-Seq) for targeted resequencing and enrichment
Scientific reports, v 14(1), pp 5583-5583
07 Mar 2024
PMID: 38448490
Featured in Collection : UN Sustainable Development Goals @ Drexel
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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Details
- Title
- A long-read sequencing strategy with overlapping linkers on adjacent fragments (OLAF-Seq) for targeted resequencing and enrichment
- Creators
- Lahari Uppuluri - Drexel UniversityChristina Huan Shi - Discovery InstituteDharma Varapula - Drexel UniversityEleanor Young - Drexel UniversityRachel L Ehrlich - Drexel UniversityYilin Wang - Drexel UniversityDanielle Piazza - Drexel UniversityJoshua Chang Mell - Drexel UniversityKevin Y Yip - Discovery InstituteMing Xiao - Drexel University
- Publication Details
- Scientific reports, v 14(1), pp 5583-5583
- Publisher
- Springer Nature
- Grant note
- HG005946 / NIH HHS R56 HG005946 / NHGRI NIH HHS R01 HG005946 / NHGRI NIH HHS
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Microbiology and Immunology; School of Biomedical Engineering, Science, and Health Systems
- Web of Science ID
- WOS:001180918400002
- Scopus ID
- 2-s2.0-85186932857
- Other Identifier
- 991021861707104721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Genetics & Heredity