Recent advances in next generation sequencing have led to the increased use of formalin-fixed and paraffin-embedded (FFPE) tissues for medical samples in disease and scientific research. Single Molecule Real-Time (SMRT®) sequencing offers a unique advantage in that it allows direct analysis of FFPE samples without amplification. However, obtaining ample long read information from FFPE samples has been a challenge due to the quality and quantity of the extracted DNA. DNA samples extracted from FFPE often contain damaged sites, including breaks in the backbone and missing or altered nucleotide bases, which directly impact sequencing and amplification. Additionally, the quality and quantity of the recovered DNA also vary depending on the extraction methods used.

We have evaluated the Adaptive Focused Acoustics (AFA™) system by Covaris® as a method for obtaining high molecular weight DNA suitable for SMRTbell template preparation and subsequent single molecule sequencing. Using this method, genomic DNA was extracted from normal kidney FFPE scrolls acquired from Cooperative Human Tissue Network (CHTN), University of Pennsylvania. Damaged sites present in the extracted DNA were repaired using a DNA Damage Repair step, and the treated DNA was constructed into SMRTbell libraries suitable for sequencing on the RSII System. Using the same repaired DNA, we also tested PCR efficiency of target gene regions of up to 5 kb. The resulting amplicons were constructed into SMRTbell templates for full-length sequencing on the RS II System.

We found the Adaptive Focused Acoustics (AFA™) system by Covaris® to be effective and efficient. This system is easy and simple to use, and the resulting DNA is compatible with SMRTbell™ library preparation for targeted and whole genome SMRT sequencing. The data presented here demonstrates single molecule sequencing of DNA samples extracted from tissues embedded in FFPE.

Citation Format: Primo Baybayan, Michael Weiand, Kevin Eng, Guillaume Durin, Steve Kujawa. SMRT® sequencing of DNA samples extracted from formalin-fixed and paraffin embedded tissues. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3611.