Abstract 54: Marked reduction of sequence artifacts in massively parallel sequencing of formalin-fixed paraffin-embedded DNA by depletion of uracil containing templates.

Abstract

Abstract Formalin-fixed and paraffin-embedded (FFPE) tissues are commonly used for the detection of mutational biomarkers in cancer patients. One of the challenges associated with FFPE DNA in genetic testing is its high number of sequence artefacts, thus increasing the risk of false positive results particularly for low level mutations. We have previously shown that uracil lesions in FFPE DNA due to deamination of cytosines are the major cause of C:G>T:A sequence artefacts and pretreatment of damaged FFPE DNA with uracil-DNA glycosylase (UDG) prior to PCR amplification markedly reduces the C:G>T:A sequence artefacts using methodologies suitable for the testing of limited numbers of genomic loci. As the number of cancer associated mutations increases, massively parallel sequencing (MPS) approaches are increasingly necessary. In particular, the use of targeted amplicon based strategies is being implemented in many laboratories. We set out to test whether sequence artifacts were a serious problem and whether the UDG pretreatment strategy is also applicable to a amplicon based targeted MPS approach (Truseq Cancer Panel). Samples not treated with UDG showed multiple sequence artefacts with a significant portion of changes greater than background sequencing error, with C:G>T:A changes forming the vast majority of artefacts. Treatment of the samples with UDG prior to sequencing dramatically reduced the majority of C:G>T:A changes without affecting the detection of known SNPs and somatic mutations. Elimination of artefactual changes by UDG treatment also improved the accurate detection of low-level sequence variants. Interestingly, most C:G>T:A changes that occurred in the context of CpG dinucleotides were resistant to the UDG treatment as a likely consequence of 5-methyl cytosine being deaminated to thymine. This approach will greatly facilitate accurate detection of mutations in FFPE samples using amplicon-based approaches which is imperative for personalised cancer medicine, especially when working with samples of low tumour purity. This work also has implications for choice of tissue and protocols for the assessment of heterogeneity in tumours. Citation Format: Hongdo Do, Stephen Q. Wong, Jason Li, Alexander Dobrovic. Marked reduction of sequence artifacts in massively parallel sequencing of formalin-fixed paraffin-embedded DNA by depletion of uracil containing templates. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 54. doi:10.1158/1538-7445.AM2013-54 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.