Relationship between formalin reagent and success rate of targeted sequencing analysis using formalin fixed paraffin embedded tissues

Background: Tumor genetic alterations are determined to aid in selecting therapy and predicting prognosis. In routine clinical practice, targeted sequencing analysis is performed using formalin-fixed paraffin embedded (FFPE) tissues. However, successful genetic analysis remains challenging because FFPE DNA is fragmented during the sample preparation process. Methods: Real-time PCR was performed to assess DNA quality and quantities. Targeted sequencing was performed using FFPE tissues fixed with different types of formalin. Results: DNA was less fragmented from samples fixed in low formalin concentration (10% vs. 20%) and neutral buffered conditions (neutral buffered vs. non-neutral). DNA fragmentation increased over the fixation time. In a preliminary test study, we compared fixation using 10% neutral buffered formalin (n = 180) and 20% formalin (n = 26). The success rate of targeted analysis was higher using 10% neutral formalin (98.3%; 177/180) compared with 20% formalin (34.6%; 9/26). In a validation study with additional formalin-fixed paraffin embedded tissues fixed with 10% neutral buffered formalin (n = 860), we reproduced these results and achieved a high success rate for targeted sequencing analysis (98.4%; 846/860). Conclusion: Our data show that 10% neutral buffered formalin is recommended for fixation of formalin-fixed paraffin embedded samples to achieve high success rate of targeted sequencing analysis.