Standardization of the preanalytical phase of DNA extraction from fixed tissue for next-generation sequencing analyses

Highlights

• DNA extracted from both FFPE and PFPE tissue is fit-for-purpose for NGS.

• Different tissue fixative/DNA extraction method combinations influence DNA quality.

• Different tissue fixative/DNA extraction method combinations influence NGS results.

• Combinatorial QC including %dsDNA, amplifiable DNA size and WGA DNA yield matters.

Abstract

Next-generation sequencing (NGS) analyses on DNA derived from archived Formalin-Fixed Paraffin-Embedded (FFPE) clinical material can provide a powerful tool in oncology research and clinical diagnostics. Although several studies have established that NGS can be performed using DNA from FFPE tissue, the accuracy and reproducibility of such analyses, as well as their robustness to the biomolecular quality of the samples used, remains a matter of debate. Excellent reviews have recently been published, providing evidence-based best practices for FFPE DNA extraction. Alternative fixatives exist, although their implementation in clinical practice is difficult. In this article, we present (i) a review of fixed tissue DNA preanalytics with a special focus on DNA extraction and fixed tissue sample qualification and (ii) results from comparisons between different methods of DNA extraction from tissue samples that have been fixed or stabilized by different methods, in terms of NGS metrics and different DNA quality metrics.