Targeted Exome Sequencing of SCN9A, SCN10A, and SCN11A

The custom capture oligo set was designed by the SureDesign platform from Agilent Technologies (SureSelectXT Custom Capture Oligo) against SCN9A, SCN10A, and SCN11A. This set was specifically designed to capture all exons, the proximal promoter sequence, as well as limited intron sequences and untranslated regions near exons. The target-captured sequencing libraries were constructed using the KAPA LTP Library Preparation Kit (Kapa Biosystems, Inc., Wilmington, MA) combined with a SureSelectXT Reagent Kit (Agilent Technologies), and sequenced on an Illumina Hiseq 2500 sequencer (Illumina, Inc, San Diego, CA) at read length of paired-end 2 × 100 bp. Targeted exome sequencing was performed on an initial cohort of 46 IBD patients that included a mixture of individuals with and without inflammation and abdominal pain. Generated reads were aligned with the GRCh37 human reference genome using the Burrows-Wheeler alignment (20 (link)). Variant detection and analysis were performed using the GATK Best Practice for germline SNP/indel finding workflow (Broad Institute). ANNOVAR software (21 (link)) was used to annotate the variants and identify synonymous, non-synonymous, and deleterious variants for further analysis.