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

• Published in: Cancer genetics 2015-09, Vol.208 (9), p.415-427
• Main Authors: Serizawa, Masakuni, Yokota, Tomoya, Hosokawa, Ayumu, Kusafuka, Kimihide, Sugiyama, Toshiro, Tsubosa, Yasuhiro, Yasui, Hirofumi, Nakajima, Takashi, Koh, Yasuhiro
• Format: Article
• Language: English
• Subjects: Amplicon-based massively parallel sequencing; Cytosine deamination; DNA - analysis; Esophageal Neoplasms - genetics; Formalin-fixed paraffin-embedded samples; Hematology, Oncology and Palliative Medicine; High-Throughput Nucleotide Sequencing - methods; Humans; Medical Education; Molecular cancer diagnostics; Mutation; Paraffin Embedding; Polymerase Chain Reaction - methods; Sequence Analysis, DNA - methods; Tissue Fixation; Uracil DNA glycosylase; Uracil-DNA Glycosidase - metabolism
• ISSN: 2210-7762; 2210-7770
• DOI: 10.1016/j.cancergen.2015.05.001

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.