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Phasing of de novo mutations using a scaled-up multiple amplicon long-read sequencing approach
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  • G.S. Holt,
  • L. Batty,
  • B. Alobaidi,
  • H. Smith,
  • Manon Oud,
  • Liliana Ramos,
  • M.J. Xavier,
  • Joris Veltman
G.S. Holt
Newcastle University

Corresponding Author:[email protected]

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L. Batty
Newcastle University
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B. Alobaidi
Newcastle University
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H. Smith
Newcastle University
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Manon Oud
Radboud Universiteit Donders Institute for Brain Cognition and Behaviour
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Liliana Ramos
Radboud Universiteit Donders Institute for Brain Cognition and Behaviour
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M.J. Xavier
Newcastle University
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Joris Veltman
Newcastle University
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Abstract

De novo mutations (DNMs) play an important role in severe genetic disorders that reduce fitness. To better understand the role of DNMs in disease, it is important to determine the parent-of-origin and timing of the mutational events that give rise to the mutations, especially in sex-specific developmental disorders such as male infertility. However, currently available short-read sequencing approaches are not ideally suited for phasing as this requires long continuous DNA strands that span both the DNM and one or more informative SNPs. To overcome these challenges, we optimised and implemented a multiplexed long-read sequencing approach using the Oxford Nanopore technologies MinION platform. We specifically focused on improving target amplification, integrating long-read sequenced data with high-quality short-read sequence data, and developing an anchored phasing computational method. This approach was able to handle the inherent phasing challenges that arise from long-range target amplification and the normal accumulation of sequencing error associated with long-read sequencing. In total, 77 out of 109 DNMs (71%) were successfully phased and parent-of-origin identified. The majority of phased DNMs were prezygotic (90%), the accuracy of which is highlighted by the average mutant allele frequency of 49.6% and a standard error margin of 0.84%. This study demonstrates the benefits of using an integrated short-read and long-read sequencing approach for large-scale DNM phasing.
05 May 2022Submitted to Human Mutation
09 May 2022Submission Checks Completed
09 May 2022Assigned to Editor
22 May 2022Reviewer(s) Assigned
28 Jun 2022Review(s) Completed, Editorial Evaluation Pending
06 Jul 2022Editorial Decision: Revise Major
31 Jul 20221st Revision Received
12 Aug 2022Submission Checks Completed
12 Aug 2022Assigned to Editor
12 Aug 2022Review(s) Completed, Editorial Evaluation Pending
18 Aug 2022Editorial Decision: Accept
Nov 2022Published in Human Mutation volume 43 issue 11 on pages 1545-1556. 10.1002/humu.24450