title: 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.
authors.affiliation: University of Exeter Medical School, RILD Building, Barrack Road, Exeter EX2 5DW, UK.
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Laver
authors.affiliation: University of Exeter Medical School, RILD Building, Barrack Road, Exeter EX2 5DW, UK.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Caswell
authors.affiliation: Wellcome Trust Biomedical Informatics Hub, Geoffrey Pope Building, Stocker Road, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: Karen A
authors.initials: KA
authors.lastname: Moore
authors.affiliation: Wellcome Trust Biomedical Informatics Hub, Geoffrey Pope Building, Stocker Road, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: Jeremie
authors.initials: J
authors.lastname: Poschmann
authors.affiliation: University of Exeter Medical School, RILD Building, Barrack Road, Exeter EX2 5DW, UK.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Johnson
authors.affiliation: Department of Molecular Genetics, RILD Level 3, Royal Devon and Exeter NHS Foundation Trust, Barrack Road, Exeter, UK.
authors.forename: Martina M
authors.initials: MM
authors.lastname: Owens
authors.affiliation: University of Exeter Medical School, RILD Building, Barrack Road, Exeter EX2 5DW, UK.
authors.forename: Sian
authors.initials: S
authors.lastname: Ellard
authors.affiliation: Wellcome Trust Biomedical Informatics Hub, Geoffrey Pope Building, Stocker Road, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: Konrad H
authors.initials: KH
authors.lastname: Paszkiewicz
authors.affiliation: University of Exeter Medical School, RILD Building, Barrack Road, Exeter EX2 5DW, UK.
authors.forename: Michael N
authors.initials: MN
authors.lastname: Weedon
mesh_terms: D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics; D015894:Genome, Human; D006239:Haplotypes; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000592:standards*; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards
chemical_list: D019389:Cytochrome P-450 CYP2D6; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human