Next-Generation Sequencing Using S1 Nuclease for Poor-Quality Formalin-Fixed, Paraffin-Embedded Tumor Specimens

Next-generation sequencing (NGS) testing of formalin-fixed, paraffin-embedded (FFPE) tissues is widely used in clinical diagnosis. However, the failure of high-quality DNA library construction, a critical step for NGS, often limits NGS application for FFPE tissues, particularly for old FFPE tissues. The aim was to develop a high-quality DNA library construction method optimized for NGS of FFPE specimens. DNA library construction was developed for FFPE specimens by using S1 nuclease and compared with the Covaris method-the widely accepted DNA library construction protocol. The Covaris method includes a DNA shearing step with sonication to generate shortened DNA fragments. DNA shearing was found to be unnecessary, and S1 nuclease treatment only during genomic DNA extraction significantly improved the success rate of library construction from FFPE tissues. Moreover, poor-quality FFPE tissues that failed the Covaris method were rescued with the S1 method. Higher complexity was observed in DNA libraries constructed by the S1 method. Among 223 FFPE specimens, DNA libraries were successfully constructed for 134 cases with the Covaris method (60.0% success rate), whereas the success rate was 86.5% (193 of 223) with the S1 method (P = 5.255e^-10). Furthermore, we were able to rescue 59 samples, including 25 primary lung cancers, from the 89 failed Covaris method cases. In conclusion, the S1 method is optimal for NGS testing of poor-quality FFPE specimens.