A critical spotlight on the paradigms of FFPE-DNA sequencing

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most widely available material for diagnostics and retrospective clinical studies. Reliable sequencing data obtained from such specimens would have enormous potential for basic and clinical research. However, formaldehyde fixation introduces chemical modifications which ultimately can lead to false interpretations. Here we describe a start-to-end approach to mitigate FFPE-problems comprising preanalytical sample specification and quality, repair treatments, analytical sample preparation, and bioinformatic sub-processes. We have also developed an alternative sequential DNA repair technique (IQBErepair) addressing the cause of formalin-induced false positives: too low and uneven coverage. For low-input approaches this technique increased the unique on-target bases by 53% and 80% in two independent centers, respectively. Furthermore, we present other measures to reduce FFPE artifacts: replicate strategies (up to 98% for 1% VAF detection threshold), bioinformatic filtering (>99.9%). Combined with general recommendations for sample quality and preparation this can create reliable data quality, even from low amounts (50 ng) of low-quality (DIN 2.0/5% Q129 bp/Q41 bp) FFPE-DNA.EGA study EGAS00001005757