Figure 2

Sequencing throughput and strategy for variant calling. (A) Uniformity of coverage in the exonic and intergenic targeted regions. The mean coverage in each sample is highlighted in red. More than 50% of targeted regions were sequenced at least at 300× coverage in all four tumors. (B) Pipeline for variant calling. First, filters for quality scores and for propensity to accumulate errors were applied. Second, statistical tests were applied to account for the coverage and quality score of the variant site (Bernoulli distribution and Chernoff bound) and for the error accumulation of the surrounding region (Binomial distribution). Each test was performed on forward and reverse reads independently, and the resulting four Ps were adjusted using Bonferroni correction. Candidate variants were retained if they passed all filters on mismatches and all statistical tests of the variant calling. The resulting ensemble of all somatic mutations at various frequencies constituted the adenoma mutation profile. (C) Variation of the quality score at different positions along the read. In sample A1 mismatches were evenly distributed along the read, with a slight decrease towards the end. In the other samples there was higher occurrence of mismatches at the beginning and at the end of the read, indicating that these positions were prone to accumulate errors. (D) Cumulative percentage of mismatches at each read position for base calls with quality score ≥30. In each sample, we only considered the portion of the reads where a linear correlation was observed. This corresponded to positions (20 to 76) for sample A1 and to positions (20 to 60) for samples and A2, A3, A4.