Additional data quality criteria were as follows: read mapping percentage, >95% reference genome-wide coverage 10×, >90% reference genome-wide coverage 20×, >70% tumor genome-wide 30× coverage, >80% and tumor genome-wide 60× coverage, >65%. No minimal threshold was applied regarding the mean coverage but instead the Gbase sequencing output for the tumor and blood samples had to be >300 and >100 Gb, respectively, to be eligible for downstream diagnostic analysis. The Illumina HiSeqX and NovaSeq6000 platforms were used for sequencing tumor (approximately 90×) and blood (approximately 30×) genomes. All procedures were automated on the Beckman Coulter Biomek 4000 and Biomek i7 liquid handling robots (Beckman Coulter, Brea, CA). Next, 50 to 200 ng DNA was fragmented by sonication on the Covaris LE220 Focused ultrasonicator (Covaris, Brighton, UK median fragment size, 450 bp) for TruSeq Nano DNA Library (Illumina, San Diego, CA) preparation, including PCR amplification (eight cycles). The QIAsymphony DSP DNA Mini kit was used for tissue DNA isolation. DNA extraction is performed on the QIAsymphony (Qiagen, Hilden, Germany) following standard reagents and protocols: 1 mL of blood was used for DNA isolation using the QIAsymphony DSP DNA Midi kit (Qiagen). The WGS test used DNA extracted from fresh-frozen or frozen archived tumor tissue (primary or metastatic) and from matching blood samples (reference). Whole genome sequencing (WGS) was performed under ISO-17025 accreditation at the Hartwig Medical Foundation laboratory (Amsterdam, the Netherlands). In conclusion, whole genome sequencing has a >95% sensitivity and precision compared with routinely used DNA techniques in diagnostics, and all relevant mutation types can be detected reliably in a single assay. Microsatellite (in)stability assessment showed a sensitivity of 100% with a precision of 94%, and virus detection (human papillomavirus), an accuracy of 100% compared with standard testing. Gene fusion analysis showed a concordance of 91.3% between DNA-based WGS and an orthogonal RNA-based gene fusion assay. Independent validation of identified biomarkers against commonly used diagnostic assays showed a high sensitivity (recall 98.5%) and precision (positive predictive value 97.8%) for detection of somatic single-nucleotide variants and insertions and deletions (across 22 genes), and high concordance for detection of gene amplification (97.0% EGFR and MET) as well as somatic complete loss (100% CDKN2A/p16). To meet high-quality diagnostics standards, we optimized and clinically validated WGS sample and data processing procedures, resulting in a technical success rate of 95.6% for fresh-frozen samples with sufficient (≥20%) tumor content. With the increasing availability of small biopsies and the need to screen more number of biomarkers, the use of a single all-inclusive test is preferable over multiple consecutive assays. Whole genome sequencing (WGS) using fresh-frozen tissue and matched blood samples from cancer patients may become the most complete genetic tumor test.
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