Repair Of Challenging Ffpe Dna Improves Library Success Rate And Sequencing Quality

Formalin-fixed, paraffin-embedded (FFPE) clinical samples are an invaluable source of information about genetic alterations in human disease, especially cancer. Next generation sequencing is a powerful tool to mine that information. Unfortunately, sequencing DNA from FFPE samples is challenging due to limited quantities and poor quality, a result of DNA damage incurred during fixation and storage. In this study, we investigated the effects of DNA repair on library preparation and sequencing from FFPE samples. We evaluated a repair enzyme mix that is designed to work on a broad range of DNA damage including modified bases, nicks, gaps, and blocked 3′ ends. Results from FFPE DNA samples of varying quality show that DNA repair generally increases library yields by 10-170%, with the largest improvement on samples of poor quality. Careful analysis of sequencing data shows that base calling qualities for all 4 bases are improved upon DNA repair. Aberrant G:C to A:T mutation were significantly reduced, consistent with cytosine deamination being induced during fixation and storage at room temperature. Interestingly, sequencing miscalls for base pair changes not typically associated with fixation were also reduced. We are currently investigating the source of these mutations. With DNA repair a noticeably positive impact on read mapping and read pairing was observed, resulting in the generation of more useable data. Pretreatment of FFPE DNA by the repair enzyme mix is easily implemented upstream of library construction, allowing fast turnaround time and easy automation. We expect that these improvements will enable the analysis of many FFPE samples that would otherwise not be accessible. Citation Format: Pingfang Liu, Lixin Chen, Laurence Ettwiller, Christine Sumner, Fiona Stewart, Eileen Dimalanta, Theodore Davis, Thomas Evans. Repair of challenging FFPE DNA improves library success rate and sequencing quality. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4886. doi:10.1158/1538-7445.AM2015-4886