Targeted Enrichment of Clinically Relevant Genes

The FFPE and Non-FFPE samples were pooled separately using 500 ng of library for each sample. These two pools of libraries were then hybridized in solution to the HGSC VCRome 2.1 design (Bainbridge et al, 2011 (link)) (42 Mb [mega base]; NimbleGen) according to the manufacturer’s protocol NimbleGen SeqCap EZ Exome Library SR User’s Guide (Version 2.2) with minor revisions. For ∼3,500 clinically relevant genes that had low coverage (<20× coverage at ∼2.72 Mb sequencing data) probes were supplemented with PKv1 and PKv2 reagent spiked into the VCRome 2.1. Human COT1 DNA and xGen Universal Blocking oligonucleotides (Integrated DNA Technologies) were added into the hybridization to block repetitive genomic sequences and the adaptor sequences and hybridization was carried out at 42°C for 72 h. Post-capture LM-PCR amplification was performed using the Library Amplification Readymix containing KAPA HiFi DNA Polymerase (Cat no. KK2612; Kapa Biosystems, Inc.) with 12 cycles of amplification. After the final AMPure XP bead purification, quantity and size of the capture library was analyzed using the Agilent Bioanalyzer 2100 DNA Chip 7500.