Establishing reference samples for detection of somatic mutations and germline variants with NGS technologies

We characterized two reference samples for NGS technologies: a human triple-negative breast cancercell line and a matched normal cell line. Leveraging several whole-genome sequencing(WGS) platforms, multiple sequencingreplicates, and orthogonal mutation detection bioinformatics pipelines, we minimized the potential biases from sequencing technologies, assays, and informatics. Thus, our "truth sets" were defined using evidence from 21 repeats of WGS runs with coverages ranging from 50X to 100X (a total of 140 billion reads). These "truth sets" present many relevant variants/mutations including 193 COSMIC mutations and 9,016 germline variants from the ClinVar database, nonsense mutationsin BRCA1/2 and missense mutationsin TP53 and FGFR1. Independent validation in three orthogonal experiments demonstrated a successful stress test of the truth set. We expect these reference materials and "truth sets" to facilitate assay development, qualification, validation, and proficiency testing. In addition, our methods can be extended to establish new fully characterized reference samples for the community.