Trusight oncology 500 reference guide

DNA libraries were prepared using the hybrid capture based TruSight™ Oncology 500 High Throughput (TSO500‐HT) Library Preparation Kit (Illumina) following Illumina® TruSight™ Oncology 500 Reference Guide (document # 1000000094853 v02). In brief, the genomic DNA was fragmented using the Covaris® ME220 focused‐ultrasonicator (Covaris) for 10 seconds at 50 W. After end repair, A‐tailing, and adapter ligation, the adapter‐ligated fragments were amplified using primers to add index sequences for sample multiplexing. Libraries were enriched through two hybridization/capture steps using specific probes: a pool of oligos specific to 523 genes targeted by TSO500‐HT was hybridized to the DNA libraries overnight. Then, streptavidin magnetic beads were employed to capture probes hybridized to the targeted regions. PCR amplification, cleanup, and quantification of the enriched DNA using Qubit™ dsDNA HS Assay Kit (Invitrogen) were the final steps. Following pooling and denaturation, libraries were diluted to the appropriate loading concentration and finally sequenced on Illumina® NovaSeq™ 6000 Sequencer (Illumina) (read length of 200 bp paired end).

DNA was isolated from archival tumor formalin-fixed, paraffin-embedded samples and sequenced using the hybridization-capture based TruSight Oncology 500 panel (TSO500; Illumina). Sequencing libraries were prepared from 100 ng of extracted tumor DNA according to the Illumina TruSight Oncology 500 Reference Guide (Illumina). Quantification and quality control of the sequencing library were performed using the Qubit DNA HS Assay Kit and Qubit 2.0 fluorometer (Invitrogen). Libraries were pooled and sequenced on the NextSeq 550DX System (Illumina) using the NextSeq High Output v2.5 reagent kit (Illumina). The sequencing data were analyzed using the TruSight Pipeline v2.2 (Illumina) and reviewed using the Clinical Genomics Workspace (PierianDx). Manual review of the BAM files was also performed for detailed analysis of the TERT promoter region.

DNA libraries were prepared using the hybrid capture-based ˋTruSight Oncology 500 Library Preparation Kit´ (Illumina, San Diego, CA, USA) following ˋIllumina’s TruSight Oncology 500 Reference Guide´ (document #1000000067621 v00, Illumina Cambridge, UK) and sequenced on an ˋIllumina NextSeq 500´ instrument. FastQ files were analyzed using ‘CLC Genomics Workbench’ (Qiagen, Hilden, Germany). The reads were mapped on hg19 followed by an initial variant calling.