Figure 4.

Effects of prolonged formalin fixation on base substitution. Amplicon sequencing using the TruSeq Amplicon Cancer Panel (Illumina) was performed using colorectal cancer specimens from two cases. The samples were fixed for 1, 2, or 3 days. Refer to Figure 3 for the sample preparation details. When the DNA quality was determined using ΔC _t values, samples fixed for 7 or 14 days did not meet the quality standard for use with this panel, and the libraries could not be prepared. Therefore, only samples fixed for 1, 2, or 3 days were used in this analysis. An analysis of the sequencing data revealed no large deviations in the total number or read depth among these three samples. (a) An analysis of the numbers of each type of base change in the sequenced regions of this gene panel (35 kb) revealed that the number of base changes, including the C > T, A > G, and A > T substitutions, increased significantly on Day 3 of formalin fixation. (b) In the additional analyses focusing on an allele frequency of ≤0.1%, the number of base changes in (b) was similar to the total number of changes in (a), suggesting that the change was likely to be an artifact generated by fixation