Pitfalls of haplotype phasing from amplicon-based long-read sequencing.
Author
Laver, T. W.
Caswell, Richard C.
Moore, K. A.
Poschmann, J
Johnson, M B
Owens, Martina
Ellard, Sian
Paszkiewicz, K H
Weedon, M. N.
Date
2016-02-17Journal
Scientific reportsType
Journal ArticleReport
Publisher
NatureDOI
10.1038/srep21746Rights
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Show full item recordAbstract
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.