WGS and Panel Sequencing
WGS was performed on samples from study participants as part of the NIHR BioResource Rare Diseases study.5 Blood DNA samples were fragmented (mean size 450 bp) with the Covaris LE220 kit and further processed with an Illumina TruSeq DNA PCR-Free Library Prep Kit. Libraries were sequenced with an Illumina HiSeq 2500 sequencer with one library per two lanes. FASTQ files were generated by HiSeq Analysis Software v.2.0 (Illumina). Alignment (GRCh37) and variant calling (including structural variants [SVs])9, 10 was performed with Isaac (Illumina).
For 411 samples, the Illumina TruSight Cancer Panel (TCP) was also used (gene list in Table S2), and libraries were sequenced with an Illumina MiSeq. BCL files resulting from the sequencing were converted to FASTQ files with Illumina’s bcl2fastq. FASTQ files were checked for coverage and other quality-control parameters with fastqc software. FASTQ files were aligned to the UCSC Genome Browser (hg19) with the Burrows-Wheeler Aligner (BWA-MEM) with default parameters and SAMtools for the generation of a binary compressed sequence alignment map (BAM) files.11, 12 Variants were called from BAM files with the Genome Analysis Toolkit Unified Genotyper algorithm.13, 14 All data were annotated with Variant Effect Predictor (VEP) v.87 on the basis of canonical transcripts.15