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.

• Authors: Masakuni Serizawa, Tomoya Yokota, Ayumu Hosokawa, Kimihide Kusafuka, Toshiro Sugiyama, Yasuhiro Tsubosa, Hirofumi Yasui, Takashi Nakajima, Yasuhiro Koh
• Journal: Cancer genetics (Cancer Genet) Vol. 208 Issue 9 Pg. 415-27 (Sep 2015) ISSN: 2210-7762 [Print] United States
• PMID: 26194062 (Publication Type: Journal Article)
• Copyright: Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Chemical References

• DNA
• Uracil-DNA Glycosidase

Topics

• DNA (analysis)
• Esophageal Neoplasms (genetics)
• High-Throughput Nucleotide Sequencing (methods)
• Humans
• Mutation
• Paraffin Embedding
• Polymerase Chain Reaction (methods)
• Sequence Analysis, DNA (methods)
• Tissue Fixation
• Uracil-DNA Glycosidase (metabolism)