FFPE DNA Extraction and Targeted Sequencing

DNA from bioptic or surgical FFPE specimens was extracted using Maxwell nucleic acid extractor (Promega) and quantified using Qubit dsDNA HS assay kit (ThermoFisher) as previously described [38 (link),39 (link)]. Truseq custom amplicon low input kit (Illumina) was used for library preparation. The genes included in the panel were summarized in Table S6. Sequencing was performed on Illumina Nextseq 500 Mid output reagent cartridge v2 300 cycles (2 × 151). Primary bioinformatic analysis was performed on Illumina Basespace environment using Amplicon DS software. Variant studio software (Illumina) was used to visualize .vcf files, investigate mutations occurred in each sample, annotate them and apply selection filters. Mutations were considered reliable if the locus presented a minimum coverage of 1000×.