Performance comparison of three DNA extraction kits on human whole-exome data from formalin-fixed paraffin-embedded normal and tumor samples

Abstract

Next-generation sequencing (NGS) studies are becoming routinely used for the detection of novel and clinically actionable DNA variants at a pangenomic scale. Such analyses are now used in the clinical practice to enable precision medicine. Formalin-fixed paraffin-embedded (FFPE) tissues are still one of the most abundant source of cancer clinical specimen, unfortunately this method of preparation is known to degrade DNA and therefore compromise subsequent analysis. Some studies have reported that variant detection can be performed on FFPE samples sequenced with NGS techniques, but few or none have done an in-depth coverage analysis and compared the influence of different state-of-the-art FFPE DNA extraction kits on the quality of the variant calling. Here, we generated 42 human whole-exome sequencing data sets from fresh-frozen (FF) and FFPE samples. These samples include normal and tumor tissues from two different organs (liver and colon), that we extracted with three different FFPE extraction kits (QIAamp DNA FFPE Tissue kit and GeneRead DNA FFPE kit from Qiagen, Maxwell™ RSC DNA FFPE Kit from Promega). We determined the rate of concordance of called variants between matched FF and FFPE samples on all common variants (representing at least 86% of the total number of variants for SNVs). The concordance rate is very high between all matched FF / FFPE pairs, with equivalent values for the three kits we analyzed. On the other hand, when looking at the difference between the total number of variants in FF and FFPE, we find a significant variation for the three different FFPE DNA extraction kits. Coverage analysis shows that FFPE samples have less good indicators than FF samples, yet the coverage quality remains above accepted thresholds. We detect limited but statistically significant variations in coverage indicator values between the three FFPE extraction kits. Globally, the GeneRead and QIAamp kits have better variant calling and coverage indicators than the Maxwell kit on the samples used in this study, although this kit performs better on some indicators and has advantages in terms of practical usage. Taken together, our results confirm the potential of FFPE samples analysis for clinical genomic studies, but also indicate that the choice of a FFPE DNA extraction kit should be done with careful testing and analysis beforehand in order to maximize the accuracy of the results.

Suitability of melanoma FFPE samples for NGS libraries: time and quality thresholds for downstream molecular tests

“…There are several studies in the literature that have evaluated QC and DIN/RIN values prior to NGS [11]. Yakovleva etal .…”

“…Among others, DNA integrity number (DIN) and RNA integrity number (RIN) from Agilent Technologies, CA, USA, in a range of 1 to 10, give information regarding the fragmentation of the DNA or RNA based on electrophoresis [10,11]. Also, the quality control (QC) value from Illumina, CA, USA, enables the researcher to test the quality of the DNA based on a qPCR, giving values closer to zero when the quality is the highest [12,13].…”

Why Formalin-fixed, Paraffin-embedded Biospecimens Must Be Used in Genomic Medicine: An Evidence-based Review and Conclusion

“…9,17 However, the value of routinely applying UDG treatment to correct artifactual C deamination is open to question: Studies consistently show a limited effect of UDG treatment. Bonnet et al 14 compared C:G > T:A substitution rates in 25 matched FFPE and FF tumor samples using three different FFPE extraction kits, one including and the other two not including UDG treatment. They found that, overall, FFPE samples had 1% higher C:G > T:A substitution rates than FF (presumably the effect of formalin-induced C deamination), but for the FFPE samples, the extraction kit that included the UDG treatment only reduced the substitutions by 0.01%.…”

Bioinformatics and DNA-extraction strategies to reliably detect genetic variants from FFPE breast tissue samples

“…Others have reported use of FFPE DNA in NGS applications [3,[11][12][13][14][15]. Spencer et al compared clinical NGS sequencing data from unpaired FFPE and fresh frozen DNA samples using a capture based approach [3].…”