Targeted DNA sequencing of Homo sapiens: adult recurrent glioblastoma tumor tissue at trial enrollment surgery
Genomic DNA was extracted from formalin-fixed, paraffin-embedded tumor tissue of the recurrent glioblastoma tumor specimens using the QIAamp DNA FFPE Tissue Kit (Qiagen). Tumor tissue was selectively scraped from unstained slides or punched from formalin-fixed, paraffin-embedded blocks using biopsy punches (Integra Miltex Instruments) to enrich for maximal tumor content (ideally >25% tumor fraction). For most patients, a constitutional DNA sample was extracted from a buccal swab or peripheral blood specimen using the QIAamp DNA Blood Midi Kit (Qiagen) and simultaneously sequenced to enable accurate discrimination of germline versus somatic origin of variants. Capture-based next-generation DNA sequencing was performed on a clinical basis in a CLIA-certified laboratory using the UCSF500 NGS panel as previously described that targets all coding exons of 529 cancer-related genes, select introns and upstream regulatory regions of 73 genes to enable detection of structural variants including gene fusions, and DNA segments at regular intervals along each chromosome to enable genome-wide copy number and zygosity analysis, with a total sequencing footprint of 2.8 Mb. Multiplex library preparation was performed using the KAPA Hyper Prep Kit (Roche) according to the manufacturers specifications using 200 ng of sample DNA. Hybrid capture of pooled libraries was performed using a custom oligonucleotide library (Integrated DNA Technologies). Captured libraries were sequenced as paired-end reads on an Illumina NovaSeq 6000 instrument. Sequence reads were mapped to the reference human genome build GRCh37 (hg19) using the Burrows-Wheeler aligner (BWA). Recalibration and deduplication of reads was performed using the Genome Analysis Toolkit (GATK).