Figure 5.

Effects of prolonged formalin fixation on total read number. Amplicon sequencing using two types of small gene panels, the GeneRead Actionable Insight Tumor Panel (12 genes) [AIT12] (Qiagen) and the TruSight Tumor 15 Kit (15 genes) [TST15] (Illumina) was performed on four tumor samples (two colon, one lung, and one gastrointestinal stromal tumor) that had undergone 1‐, 3‐, or 7‐day fixation. For the TST15 panel, the DNA quality was determined using a real‐time PCR assay (quality control [QC] results are shown in Figure 3a, Panel B). For amplicon sequencing using the AIT12 and TST15 panels, libraries were prepared using 40 and 20 ng dsDNA, respectively. After performing a library QC, the samples were analyzed using the GeneReader (Qiagen) and MiSeq systems (Illumina). All the samples were successfully analyzed with both small gene panels. Therefore, it can be inferred that the small gene panels are useful for the analysis of formalin‐fixed paraffin‐embedded (FFPE) samples containing low‐quality nucleic acids. However, the total number of reads decreased as formalin fixation time increased [A, B]; this effect varied depending on the gene panel used for analysis