An improved molecular inversion probe based targeted sequencing approach for low variant allele frequency

Authors:
Open Access
Publisher website
Google Scholar
Abstract
Deep targeted sequencing technologies are still not widely used in clinical practice due to the complexity of the methods and their cost. The Molecular Inversion Probes (MIP) technology is cost effective and scalable in the number of targets, however, suffers from low overall performance especially in GC rich regions. In order to improve the MIP performance, we sequenced a large cohort of healthy individuals (n = 4417), with a panel of 616 MIPs, at high depth in duplicates. To improve the previous state-of-the-art statistical model for low variant allele frequency, we selected 4635 potentially positive variants and validated them using amplicon sequencing. Using machine learning prediction tools, we significantly improved precision of 10–56.25% (P < 0.0004) to detect variants with VAF > 0.005. We further developed biochemically modified MIP protocol and improved its turn-around-time to ∼4 h. Our new biochemistry significantly improved uniformity, GC-Rich regions coverage, and enabled 95% on target reads in a large MIP panel of 8349 genomic targets. Overall, we demonstrate an enhancement of the MIP targeted sequencing approach in both detection of low frequency variants and in other key parameters, paving its way to become an ultrafast cost-effective research and clinical diagnostic tool.
Funding Information
  • EU Horizon 2020 (714731)
  • LLS and rising tide foundation (RTF6005-19)
  • ISF-NSFC (2427/18)
  • ISF-IPMP-Israel Precision Medicine Program (3165/19, BIRAX 713023)
  • Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine
  • Sagol Institute for Longevity Research
  • Barry and Eleanore Reznik Family Cancer Research Fund
  • Steven B. Rubenstein Research Fund
  • Rising Tide Foundation
  • Applebaum Foundation
  • Princess Margaret Cancer Foundation

This publication has 33 references indexed in Scilit: