12-042-N1
Genomic DNA was extracted from tumor and matched normal bladder tissues using a QIA DNA tissue kit (Qiagen, Germantown, MD) according to the manufacturers instruction. Targeted sequencing library preparation was performed with KAPA HyperChoice Custom Target Enrichment system (Roche, Indianapolis, IN). Briefly, genomic DNA was enzymatically fragmented to 150300 bp using the KAPA HyperPrep Kit. Custom biotinylated DNA probes targeting all exons and selected introns of 29 genes within the chromosome 9p21.3 region were designed using HyperDesign (Roche) and synthesized for hybridization-based capture. Target enrichment was performed via hybridization to the custom probe set, followed by streptavidin bead capture. Sequencing libraries were then constructed using the KAPA HyperPrep kit. Captured libraries were pooled and sequenced on Illumina NovaSeq6000 platform to achieve high, uniform coverage (>500 median coverage). Genomic alterations including single nucleotide variants (SNVs), insertions/deletions (indels), copy number alterations, and structural rearrangements were called relative to the patients matched normal DNA.