September 22, 2019  |  

Conventional and single-molecule targeted sequencing method for specific variant detection in IKBKG while bypassing the IKBKGP1 pseudogene.

Authors: Frans, Glynis and Meert, Wim and Van der Werff Ten Bosch, Jutte and Meyts, Isabelle and Bossuyt, Xavier and Vermeesch, Joris R and Hestand, Matthew S

In addition to Sanger sequencing, next-generation sequencing of gene panels and exomes has emerged as a standard diagnostic tool in many laboratories. However, these captures can miss regions, have poor efficiency, or capture pseudogenes, which hamper proper diagnoses. One such example is the primary immunodeficiency-associated gene IKBKG. Its pseudogene IKBKGP1 makes traditional capture methods aspecific. We therefore developed a long-range PCR method to efficiently target IKBKG, as well as two associated genes (IRAK4 and MYD88), while bypassing the IKBKGP1 pseudogene. Sequencing accuracy was evaluated using both conventional short-read technology and a newer long-read, single-molecule sequencer. Different mapping and variant calling options were evaluated in their capability to bypass the pseudogene using both sequencing platforms. Based on these evaluations, we determined a robust diagnostic application for unambiguous sequencing and variant calling in IKBKG, IRAK4, and MYD88. This method allows rapid identification of selected primary immunodeficiency diseases in patients suffering from life-threatening invasive pyogenic bacterial infections. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Journal: The Journal of molecular diagnostics
DOI: 10.1016/j.jmoldx.2017.10.005
Year: 2018

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