Figure 5.

Tumour mutational signature (TMS) assessment for biallelic MUTYH-carrier CRCs. A representative CRC from a biallelic MUTYH-carrier (M05) was selected to demonstrate the changes in the SBS18 and SBS36 TMS profile related to changes in experimental conditions namely, (a), changes in variant allele fraction (VAF) filtering (at a minimum read depth (DP) fixed at 50bp), and (b) changes in minimum DP (at a VAF fixed at 0.1). The SBS18 and SBS36 TMS were robust and dominant with VAF thresholds between 0.075 and 0.25 (a), and with DP thresholds between 25–100bp (b) across all 12 biallelic MUTYH carrier CRCs (images not shown), with the upper thresholds of VAF and DP corresponding to limits where the number of somatic variants started to decline and the TMS error increased. The sum of SBS18 and SBS36 separated CRCs from biallelic MUTYH carriers (light green) from CRCs from the MMRp and MMRd controls (brown) from the discovery group when the VAF was between 0.075 and 0.175 (c) and when DP was >25 (d). The calculated 95% (blue line), and 5% (red line) probabilities reflect the likelihood that a CRC is from a biallelic MUTYH pathogenic variant carrier. When applied to the validation set of CRCs, the sum of SBS18 and SBS36 similarly provided the best separation of CRCs from biallelic MUTYH carriers from the MMRp and MMRd control CRCs at VAF thresholds between 0.075 and 0.15 (e) and at DP thresholds between 25bp and 100bp (f).