Sureselect human all exon v5 capture library

A minimum of 500 ng of gDNA was prepared for whole exome sequencing (WES) using the Agilent SureSelect Human All Exon v5 capture library, according to the manufacturers’ protocol. The resulting libraries were sequenced to a mean depth of 100× using paired-end 100 reads on an Illumina HiSeq 2500. High-quality reads were aligned to the National Center for Biotechnology information (NCBI) reference genome (hg19) using BWA (v0.7.12) and SAMtools (v0.1.19) to remove duplicates. Tumor content was estimated based on the CNVkit (v0.8.1) copy number profile.

Next generation sequencing libraries were built according to established laboratory protocols at the Science for Life Laboratory Stockholm and the Institute for Molecular Medicine Finland (FIMM). The exome libraries were processed according to the Agilent SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, CA, USA) for Illumina Paired-End Sequencing Libraries (Illumina, San Diego, CA, USA) using the SureSelect Human All Exon V5 capture library (Agilent Technologies, Santa Clara, CA, USA). Libraries were sequenced with 101 bp read length (HiSeq1500 sequencing platform, Illumina, San Diego, CA, USA). The read mapping, variant calling and genome annotation were performed as described previously (28 (link)). We focused the search on the haplotype that segregated with the phenotype, prioritizing rare, heterozygous coding variants that negatively affect conserved residues and shared by both whole genome sequenced individuals. Only one such variant was identified, localized within the TMEM173 gene. The G207E variant was absent from all major public [The Exome Aggregation Consortium (ExAC), 1,000 Genomes, NHLBI Exome variant server and UK TWIN and ALSPAC study cohorts (2–4)], as well as in-house databases.