FFPE Tissue DNA Extraction and Exome Sequencing

Genomic DNA was extracted from microdissected formalin-fixed paraffin-embedded (FFPE) tissue using a QIAamp DNA FFPE Tissue Kit (Qiagen). At least 250 ng of genomic DNA was used for each sample. DNA quality was assessed on a 4200 TapeStation (Agilent). Genomic DNA libraries were generated using the SureSelectXT Kit (Agilent Technologies), followed by exon enrichment using the SureSelectXT Human All Exon V7 bait set (Agilent Technologies). The resulting exon-enriched libraries were subjected to paired-end, 100-cycle sequencing performed on a HiSeq 4000 (Illumina). Sequencing results were analysed using an institutionally established pipeline for alignment and calling of single nucleotide variants (SNVs) and insertions or deletions (indels), which were annotated and ranked by putative pathogenicity using a workflow named “medal ceremony” and subsequently manually reviewed [18 (link)–20 (link)].