Germline mutation detection by whole-exome sequencing (WES)
SENSITIVITY
Peripheral blood was collected from all patients before ICI treatment initiation (baseline). Mononucleated cells were isolated by Ficoll-Biocoll density centrifugation, and genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen) following the manufacturer’s instructions. DNA was quantified with Qubit 2.0 using the dsDNA High Sensitivity Kit (Thermo Fisher Scientific) and qualified with TapeStation 4150 using the Genomic DNA ScreenTape Assay (Agilent Technologies) according to the manufacturer’s instructions. WES libraries were prepared using the SureSelect XT Human All Exon V7 Enrichment Kit (Agilent Technologies). Equimolar multiplexed libraries were then sequenced using 100 bp paired-end runs on Illumina NovaSeq 6000 S1 platform (Illumina) at the DKFZ NGS Core Facility according to the manufacturer’s instructions. The quality scores Q30 were between 91.4% and 92.5%, total read count was 103 129 510x mean coverage per library (range: 88 189 108x – 115 652 520x). Raw sequencing reads were processed and aligned using the automated pipeline OTP