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Pitfalls of haplotype phasing from amplicon-based long-read sequencing.

ABSTRACT: The long-read sequencers from Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT) offer the opportunity to phase mutations multiple kilobases apart directly from sequencing reads. In this study, we used long-range PCR with ONT and PacBio sequencing to phase two variants 9 kb apart in the RET gene. We also re-analysed data from a recent paper which had apparently successfully used ONT to phase clinically important haplotypes at the CYP2D6 and HLA loci. From these analyses, we demonstrate PCR-chimera formation during PCR amplification and reference alignment bias are pitfalls that need to be considered when attempting to phase variants using amplicon-based long-read sequencing technologies. These methodological pitfalls need to be avoided if the opportunities provided by long-read sequencers are to be fully exploited.

SUBMITTER: Laver TW

PROVIDER: S-EPMC4756330 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Pitfalls of haplotype phasing from amplicon-based long-read sequencing.

Laver Thomas W TW Caswell Richard C RC Moore Karen A KA Poschmann Jeremie J Johnson Matthew B MB Owens Martina M MM Ellard Sian S Paszkiewicz Konrad H KH Weedon Michael N MN

Scientific reports 20160217

The long-read sequencers from Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT) offer the opportunity to phase mutations multiple kilobases apart directly from sequencing reads. In this study, we used long-range PCR with ONT and PacBio sequencing to phase two variants 9 kb apart in the RET gene. We also re-analysed data from a recent paper which had apparently successfully used ONT to phase clinically important haplotypes at the CYP2D6 and HLA loci. From these analyses, we demon ...[more]

PMID: 26883533

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