The hotspot analysis aims to identify small focal regions with high mutation rates. We first considered all mutated 21 bp regions by taking 10 bp flanks on each side of each mutation. Then we calculated the mutation recurrence scores for all 21 bp regions with three or more mutated samples (two or more for indels). The P value of mutation recurrence of each hotspot was calculated using the Poisson binomial model described in the previous section. The total number of hypothesis tested is equal to the number of bases in the masked non-coding genome. We used the Bonferroni correction to adjust for multiple testing of 2,533,374,732 hypotheses, to maintain the overall α at 0.01.