As next-generation sequencing becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Through the International Cancer Genome Consortium (ICGC), we compared sequencing pipelines at five independent centers (CNAG, DKFZ, OICR, RIKEN and WTSI) using a single tumor-blood DNA pair. Analyses by each center and with one standardized algorithm revealed significant discrepancies. Although most pipelines performed well for coding mutations, library preparation methods and sequencing coverage metrics clearly influenced downstream results. PCR-free methods showed reduced GC-bias and more even coverage. Increasing sequencing depth to ~100x (two- to three-fold higher than current standards) showed a benefit, as long as the tumor:control coverage ratio remained balanced. To become part of routine clinical care, high-throughput sequencing must be globally compatible and comparable. This benchmarking exercise has highlighted several fundamental parameters to consider in this regard, which will allow for better optimization and planning of both basic and translational studies.

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