s and 70 °C for 60 s, the reaction was then held at 4 °C until addition of barcoded second-round primers, followed by 19 cycles of 98 °C for 20 s, 62 °C for 15 s and 72 °C for 30 s, then 72 °C 60 s; Group 2, 10 ng DNA input in 5 μl PCR reaction, 95 °C for 2 min, seven cycles of 95 °C for 20 s, 58 °C for 17 min and 70 °C for 60 s, then held at 4 °C until addition of barcoded second-round primers, followed by 23 cycles of 98 °C for 20 s, 62 °C for 15 s and 72 °C for 30 s, then 72 °C 60 s. Each sample was pooled, size selected by SPRI (×0.8) and quantified before being stored at −20 °C until sequencing. Two MiSeq runs (300-bp paired-end) were used for variant confirmation, and reads were mapped with BWA. We analysed positions where the coverage was higher than 100× (159/172). With this approach, we were able to validate 91.2% of the original calls: 14/159 (8.8%) calls had VAF values <0.1 and were deemed false positives. The normal distribution of VAFs around 0.5 is consistent with
