ena-STUDY-KU Leuven-09-07-2017-13:35:55:887-756
Illumina and PacBio targeted sequencing of IKBKG, IRAK4 & MYD88
The primary immunodeficiency associated gene IKBKG can be difficult to sequence due to the presence of the IKBKGP1 pseudogene.  Here we used long-range PCRs to bypass the pseudogene and isolate only the IKBKG and associated primary immunodeficiency genes IRAK4 and MYD88.  Following PCR, the amplicons were divided in half and sequenced on both an Illumina short-read sequencing platform and PacBio long-read single-molecule sequencer. Targeted bioinformatic analyses were then evaluated to determine robust variant calling methods and compare the two sequencing platforms. Supplemented with traditional sequencing of IKBKG exons 1 and 2 (Frans et al. 2017. J Allergy Clin Immunol. PMID: 28479328), these methods present targeted techniques to sequence the full coding regions of IKBKG, IRAK4, and MYD88, whilst bypassing the IKBKGP1 pseudogene.PacBio barcodes 57, 65, 73, 81, and 89 from PacBio’s Barcoded Adapter Complete Prep kit correspond to samples S1, S2, S3, S4, and S5, respectively.
The primary immunodeficiency associated gene IKBKG can be difficult to sequence due to the presence of the IKBKGP1 pseudogene.  Here we used long-range PCRs to bypass the pseudogene and isolate only the IKBKG and associated primary immunodeficiency genes IRAK4 and MYD88.  Following PCR, the amplicons were divided in half and sequenced on both an Illumina short-read sequencing platform and PacBio long-read single-molecule sequencer. Targeted bioinformatic analyses were then evaluated to determine robust variant calling methods and compare the two sequencing platforms. Supplemented with traditional sequencing of IKBKG exons 1 and 2 (Frans et al. 2017. J Allergy Clin Immunol. PMID: 28479328), these methods present targeted techniques to sequence the full coding regions of IKBKG, IRAK4, and MYD88, whilst bypassing the IKBKGP1 pseudogene.PacBio barcodes 57, 65, 73, 81, and 89 from PacBio’s Barcoded Adapter Complete Prep kit correspond to samples S1, S2, S3, S4, and S5, respectively.