Illumina TruSight Oncology 500 (TSO) analyses

DNA libraries were prepared using the hybrid capture-based TSO Library Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer’s instructions (#1000000067621 v00). Library concentrations and peak heights were evaluated on a Tape Station (Agilent, Santa Clara, USA). Equal amounts of up to eight library samples were pooled and diluted to 4 nM. 10 µl of the library pool was mixed in 0.1 M NaOH and incubated for 5 min at RT. The library was neutralized and diluted to 20 pMwith 990 µl HT1, mixed and kept on ice. To generate 200,000 clusters/mm² the pool was diluted to 0.6 pM by the addition of 1261 µl HT1, 39 µl library (20 pM) and 1 µl PhiX (20 pM). Libraries were sequenced on an Illumina NextSeq 500 instrument. The FastQ files were analyzed in CLC Biomedical Workbench (Qiagen). Reads were mapped to hg19 followed by initial variant calling. Then local realignments, primer clipping, and low-frequency variant calling were performed. False-positives were removed based on read quality and forward/reverse balance. All variants were checked manually for sequencing artefacts. The average coverage was > 500 in all samples; the mutations had at least 50 variant reads. In total, 11 samples were analyzed (2 adenocarcinoma, 2 carcinoma NOS, 3 rhabdomyosarcoma, 1 angiosarcoma, 2 sarcoma).