High coverage sequencing of a single sample can account for the problem of intratumor heterogeneity

Tumor heterogeneity is a consequence of clonal evolution, resulting in a fractal-like architecture with spatially separated main clones, sub-clones and single-cells. Here, we tested the effect of sample size, pooling strategy as well as sequencing depth using next-generation sequencing of ovarian tumor samples. Our results show that sequencing from spatially neighboring regions show similar genetic compositions, with few private mutations. Pooling samples from multiple distinct regions of the primary tumor did not substantially increase the overall number of identified mutations but may increase the robustness of detecting clonal mutations. Hypermutating tumors are a special case, since increasing sample size can easily dilute sub-clonal private mutations below detection thresholds. In view of the limitations of present tools and technologies, single sequencing run per sample combined with high coverage (100-300x) sequencing is affordable and practical, regardless of the number of samples taken from the same patient.EGA study EGAS00001004200