Finally, we recently demonstrated that intra-tumor heterogeneity can be seen using ultra-deep targeted sequencing (Shah et al., 2009b). The allelic frequencies of SNVs in rare clones in the tumor population will likely result in false negative predictions at conventional sequencing depths (i.e. between 20× and 40×), and confound the estimation of the false negative rates of prediction. Future investigation of all of these problems will be necessary if the goal of sequencing studies is to characterize all mutations present in the heterogeneous mixture of genomes that make up a tumor.
