2015 ASHG: Effect of Formalin Fixation on Targeted DNA Sequencing and SNV Identification

Tumor biopsies are often Formalin-Fixed and Paraffin-Embedded (FFPE) for histological staining, genetic testing and archival purposes. Formalin treatment preserves tissue by crosslinking proteins, but also leads to mutation of the nucleic acid bases and poses a challenge to identication of true variants in the tumor using next-generation sequencing (NGS) methods. Studies have shown that it is possible to isolate high quality nucleic acids from good FFPE samples and to profile small variants using NGS. These studies used tissue fixed with 10% neutral buered formalin for 24 hours, a standard protocol in the pathology field. In our initial handling of FFPE samples we found that the quality of the isolated DNA and subsequent sequencing results vary widely. We hypothesized that this may be due to deviations from the standard protocol, such as inaccurate logging of the fixation protocol, variation in the fixation time, and varied storage conditions of the samples. To understand the role of formalin fixation on the quality of variants called, we performed an augmented target enrichment and sequencing assay on fresh frozen (FF) and formalin treated reference cell line NA12878. We assessed raw DNA quality, library quality, sequencing metrics (alignment rate, duplication rate and on-target eciency) and variant concordance profiles between FF and formalin treated cells. Using the results of this study, we intend to provide guidance to labs preparing FFPE samples about conditions that minimize formalin- induced variations. We also demonstrate how a deeper understanding of the effects of formalin can improve sequencing analysis results from formalin fixed tissues, especially at lower allele frequencies where formalin-related errors have the greatest impact.