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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 longread sequencing technologies. These methodological pitfalls need to be avoided if the opportunities provided by long-read sequencers are to be fully exploited.
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https://www.hal.inserm.fr/inserm-02457710
Contributor : Sylvie Le Bihan <>
Submitted on : Tuesday, January 28, 2020 - 11:49:55 AM
Last modification on : Wednesday, January 29, 2020 - 1:28:34 AM

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Thomas Laver, Richard Caswell, Karen Moore, Jérémie Poschmann, Matthew Johnson, et al.. Pitfalls of haplotype phasing from amplicon-based long-read sequencing. Scientific Reports, Nature Publishing Group, 2016, 6 (1), ⟨10.1038/srep21746⟩. ⟨inserm-02457710⟩

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