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Fig. 2 | Genome Medicine

Fig. 2

From: PathOS: a decision support system for reporting high throughput sequencing of cancers in clinical diagnostic laboratories

Fig. 2

Variant allele frequency (VAF) distributions. The variant data for the first six months of 2016 have been aggregated to show the VAF distributions for amplicon and hybrid capture panels. All scatter plots display a bimodal distribution with a peak at 50% allele frequency for heterozygous variants and 100% for homozygous variants. The top left plot shows all variants in the custom myeloid amplicon panel prior to filtering (n = 66,210). It shows a number of peaks that are due to technical panel artefacts. The top right plot shows variants remaining (n = 13,649 20.6%) after removing; variants occurring in one sample replicate only, variants occurring in more than 35% of samples in the myeloid panel (panel artefacts) and variants with less than 100 total reads or less than 20 alternative reads. The resulting distribution is far smoother and free from technical artefacts. Note the large peak at low VAF%. The amplicon panel samples have high read coverage (mean 2297×) which captures low frequency variants from both the wet lab PCR processes and sequencer errors. In contrast, the bottom left plot shows variants from the hybrid capture cancer panel and has no low VAF peak (mean coverage 246×). This is due to multiple factors including lower coverage meaning fewer low VAF variants pass the variant caller threshold (3.0%), more stringent pipeline filtering for hybrid capture and different wet lab processing. The histogram shows all manually reported somatic variants over this period and shows a skew towards low VAF% due to tumour purity (samples of mixed tumour and normal cells) and tumour heterogeneity (variants occurring only within clones in a heterogeneous tumour)

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