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

Sci Rep. 2016 Feb 17:6:21746. doi: 10.1038/srep21746.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cytochrome P-450 CYP2D6 / genetics
  • Genome, Human
  • Haplotypes*
  • High-Throughput Nucleotide Sequencing / methods*
  • High-Throughput Nucleotide Sequencing / standards
  • Humans
  • Mutation
  • Polymerase Chain Reaction / standards*
  • Proto-Oncogene Proteins c-ret / genetics
  • Repetitive Sequences, Nucleic Acid
  • Sequence Analysis, DNA / methods*
  • Sequence Analysis, DNA / standards

Substances

  • Cytochrome P-450 CYP2D6
  • Proto-Oncogene Proteins c-ret
  • RET protein, human