Figure 11.

Chemically induced base substitution due to prolonged duration of formalin‐fixed paraffin‐embedded (FFPE) block storage and the effect of uracil DNA N‐glycosylase (UNG)‐treatment on base repair. Amplicon sequencing using the TruSeq Amplicon Cancer Panel (Illumina) was performed on surgical specimens obtained from two colorectal cancer patients, fixed for 1 and 3 days, respectively. The analysis was performed twice in 2014 (immediately after FFPE block preparation) and again in 2017 (3 years post‐preparation). A comparative analysis was performed in 2017 to examine the effects of UNG treatment on samples fixed for up to 3 days using two nucleic acid extraction kits, the QIAamp DNA FFPE Tissue Kit (without treatment), and the GeneRead DNA FFPE Tissue Kit (with treatment) (Qiagen). (a) In both samples (1 and 3 days fixation), the base substitution increased due to long‐term storage [A–D]. In particular, the C > T substitution increased significantly (lower insets in A and B are magnifications of upper diagrams). The number of base changes resulting from long‐term storage was proportional to the number at the beginning of storage. (b) A lower number of C > T base substitutions were observed in the UNG‐treated samples (UNG+) compared to the untreated (UNG‐) samples, while the number of some types of base substitutions, such as T > C substitutions, increased. The total number of reads and the mean read depth were ≥eightfold higher in the UNG+ samples. As previously reported, DNA quality improved with UNG treatment; however, the effect was not uniform. Thus, the effects in preliminary experiments should be checked to decide whether UNG treatment is required