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

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), whilst 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. ispartof: The Journal of Molecular Diagnostics vol:20 issue:2 pages:195-202 ispartof: location:United States status: published