Deamination of nucleotides causes C:G>T:A changes in formalin-fixed, paraffin-embedded (FFPE) tissue samples and produces false positives during next-generation sequencing (NGS). Uracil DNA glycosylase (UDG) helps eliminate this issue, but the effect of UDG in different tissue preparation conditions has not been rigorously studied. To investigate whether UDG can reduce false-positive single-nucleotide variant (SNV) calls, we used tumor and normal tissues from gastric adenocarcinoma patients prepared using different fixation times and pH conditions. FFPE tumor blocks >10 years were also evaluated for the comparison. We performed semiconductor-based NGS to evaluate nucleotide changes and used UDG to test deamination-related effects. Sequencing quality parameters mildly worsened with prolonged fixation time, acidic pH, and delayed fixation. SNV calls and C:G>T:A changes increased after >48 hours of fixation. In both recently prepared and old FFPE tissue blocks, UDG treatment reduced deamination-induced nucleotide changes. In the recently prepared samples, both high-quality SNVs and mean target coverage were remarkably increased on treatment with UDG. However, the quality of NGS results from old-age samples varied irrespective of UDG treatment. In conclusion, based on our findings, we believe that when performing NGS on recently embedded blocks, it is important to consider that certain poorly fixed samples may be at the risk of being deaminated, which can be corrected with UDG treatment.
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.