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DNA was extracted using the Qiagen AllPrep DNA/RNA Mini Kit. Sequencing of exome samples was performed using Illumina’s next-generation sequencing methodology [32 (link)]. In detail, total DNA was quality checked using Agilent 4200 TapeStation System (Agilent Genomic DNA ScreenTape) and quantified using Quant-iT™ PicoGreen™. Libraries were prepared from 3 µg of input material using SureSelect Human All Exon V6 (manufacturer’s instructions) and subsequently quantified and quality checked using Agilent 4200 TapeStation System (D1000 ScreenTape). Libraries were pooled and sequenced on NextSeq 500 System (High Output Flow Cell) running in 150 cycle (2 × 75 bp paired-end) mode. Sequence information was converted to FASTQ format using bcl2fastq v2.20.0.422. The WES data were used to search for HLA-presented individual neoepitopes in the complete OPSCC HLA ligandome dataset of each patient with available whole exome sequencing data (38/40 patients). Database search and spectral annotation were performed against the combination of the human proteome as comprised in the UniProtKB/Swiss-Prot database and the mutated protein sequences as defined for the respective patients.

Samples for DNA extraction and quality control assessments were collected in sterile Eppendorf tubes (Eppendorf, Germany) and immediately transferred to − 20 °C for short-term storage and submitted to Integrated Genomics Operation (IGO) core. DNA from the frozen tissue was isolated with DNeasy Blood & Tissue Kit (QIAGEN catalog #69504) with 1-h of incubation at 55 °C for digestion. DNA was eluted in 0.5X Buffer AE. The quantity and quality of DNA were estimated using Quant-it PicoGreen and Agilent TapeStation D1000. For MSK-IMPACT, 100 ng DNA was used to prepare libraries using KAPA Hyper Prep Kit (Kapa Biosystems KK8504) with 8 PCR-cycles. 100 ng of each barcoded library were captured by hybridization in equimolar pools using the Integrated Mutation Profiling of Actionable Cancer Targets (IMPACT) assay (Nimblegen SeqCap), designed to capture all protein-coding exons and select introns of 505 commonly implicated oncogenes, tumor suppressor genes, and members of pathways deemed actionable by targeted therapies. Captured pools were sequenced on a HiSeq 4000 in a PE100 run using the HiSeq 3000/4000 SBS Kit (Illumina) producing an average of 500X coverage per tumor. The IMPACT data was analyzed on cBioportal^{28 (link),29 (link)}.