Sample processing from tissue or buffy coat, library preparation, hybrid capture, and sequencing were performed at Personal Genome Diagnostics Inc. (Baltimore, MD)15 (link). Briefly, DNA was extracted from FFPE tissue and matched-normal buffy coat cells using the Qiagen FFPE Tissue Kit and DNA Blood Mini Kit, respectively (Qiagen, Hilden, Germany; catalog numbers 56404 and 51104, respectively). Genomic DNA was sheared using a Covaris sonicator (Woburn, MA, USA) to a size range of 150–450 bp, and subsequently used to generate a genomic library using the New England Biolabs (Ipswich, MA, USA) end-repair, A-tailing, and adapter ligation modules (catalog numbers E6050, E6053, and E6056, respectively). Finally, genomic libraries were amplified and captured using the Agilent SureSelect XT in-solution hybrid capture system with a 120 bp RNA panel targeting the pre-defined regions of interest across full exonic regions. Captured libraries were sequenced on the Illumina HiSeq 2000 or 2500 (Illumina, San Diego, CA, USA) with 100-bp paired-end reads.
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