The efficacy of uracil DNA glycosylase pretreatment in amplicon-based massively parallel sequencing with DNA extracted from archived formalin-fixed paraffin-embedded esophageal cancer tissues

Abstract

Advances in mutation testing for molecular-targeted cancer therapies have led to the increased use of archived formalin-fixed paraffin-embedded (FFPE) tumors. However, DNA extracted from FFPE tumors (FFPE DNA) is problematic for mutation testing, especially for amplicon-based massively parallel sequencing (MPS), owing to DNA fragmentation and artificial C:G > T:A single nucleotide variants (SNVs) caused by deamination of cytosine to uracil. Therefore, to reduce artificial C:G > T:A SNVs in amplicon-based MPS using FFPE DNA, we evaluated the efficacy of uracil DNA glycosylase (UDG) pretreatment, which can eliminate uracil-containing DNA molecules, with 126 archived FFPE esophageal cancer specimens. We also examined the association between the frequency of C:G > T:A SNVs and DNA quality, as assessed by a quantitative PCR (qPCR)-based assay. UDG pretreatment significantly lowered the frequency of C:G > T:A SNVs in highly fragmented DNA (by approximately 60%). This effect was not observed for good- to moderate-quality DNA, suggesting that a predictive assay (i.e., DNA quality assessment) needs to be performed prior to UDG pretreatment. These results suggest that UDG pretreatment is efficacious for mutation testing by amplicon-based MPS with fragmented DNA from FFPE samples.

Keywords: Amplicon-based massively parallel sequencing; Cytosine deamination; Formalin-fixed paraffin-embedded samples; Molecular cancer diagnostics; Uracil DNA glycosylase.