stroma or immune cell infiltration, all of which are commonly seen in clinical samples. Importantly, our data suggest that in order to increase the reliability and identify mutations present at less than 5% prevalence, the accuracy of the next generation sequencing technology needs to increase, with improvements of both chemistry, instrument and bioinformatics analysis. Increasing sequence depth coverage only is unlikely to solve the systematic bias observed that limits the ability to accurately measure the abundance of alleles present at less than 5% prevalence. This is exemplified by the notable improvement in the substitution rate observed on the MiSeq instrument, where the samples were sequenced at lower depth.
