Figure 5

Low template copies are associated with higher probability of sequencing artefacts post-PCR amplification. In good quality DNA from sources such as blood and fresh frozen tissue, fragmentation and uracil lesions are present at very low levels. In this circumstance, high amounts of amplifiable template increase the likelihood of accurately identifying mutations due to high sequencing coverage with little or no stochastic enrichment of sequencing artefacts. In FFPE DNA with moderate fragmentation, the number of amplifiable templates is reduced, with some formalin-induced uracil lesions being present in template DNA. Subsequently PCR amplification results in lower coverage due to less amplifiable template numbers. Uracil lesions are also amplified, and due to the lower copy numbers, can appear as non-reproducible sequencing artefacts (C>T/G>A changes). These artefacts will be low in frequency. In the case of FFPE with high amounts of fragmentation, the numbers of amplifiable template are severely limited. An artefact in one of these templates can then appear as a moderate to high frequency sequencing variant. These can subsequently be interpreted as real mutations.