Fragmentation assessment of FFPE DNA helps in evaluating NGS library complexity and interpretation of NGS results

Formalin-fixed paraffin-embedded (FFPE) tissue remains the most common source for DNA extraction from human tissue both in research and routine clinical practice. FFPE DNA can be considerably fragmented, and the amount of DNA measured in nanograms may not represent the amount of amplifiable DNA available for next-generation sequencing (NGS). Two samples with similar input DNA amounts in nanograms can yield NGS analyses of considerably different quality. Nevertheless, many protocols for NGS library preparation from FFPE DNA describe input DNA in nanograms without indication of a minimum requirement of amplifiable genome equivalent DNA. An important NGS quality metric is the library complexity, reflecting the number of DNA fragments from the original specimen represented in the final library. Aiming to illustrate the relationship between DNA fragmentation degree and library complexity, we assessed the fragmentation degree of 116 lung cancer FFPE DNA samples to calculate the amount of amplifiable input DNA used for library preparation. Mean unique coverage, coverage uniformity, and mean number of PCR duplicates with the same unique molecular identifier were used to evaluate library complexity. We showed that the amount of amplifiable input DNA predicted library complexity better than the input measured in nanograms. The frequent discrepancy between DNA amount in nanograms and the amount of amplifiable DNA indicate that the fragmentation degree should be considered when performing NGS of FFPE DNA. Importantly, the fragmentation assessment may help when interpreting NGS data and be a useful tool for evaluating library complexity in the absence of unique molecular identifiers.