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

<div><p>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.</p></div

Alpha diversity indices for TcGP63I amplicon diversity derived from pairs of congenital Chagas disease cases.

<p>Diversity indices were derived from STs defined at 99% sequence similarity. Bar plot and associated <i>x</i>-axis on the right hand side shows the Shannon diversity index calculated in Mothur [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003458#pntd.0003458.ref034" target="_blank">34</a>], with error bars defining upper and lower 95% confidence intervals.</p

Principal coordinates analysis of sequence diversity between chronic Chagas Disease patient TcGP63I antigenic repertoires.

<p>Genetic distances are based on a weighted unifrac metric. Plot A shows diversity comparisons among Go-as asymptomatic (asympt) and symptomatic (sympt) clinical cases, as well as one acute case. Plot B shows Goias cases with symptoms categorised as acute, card (cardiopathy), card + mega (cardiopathy as well as megacolon and / or megaesophagous), mega (megacolon and / or megaesophagous) or asympt (asymptomatic). Plot C shows comparisons among Cochabamba clinical cases (not including congenital cases) classified as either asymptomatic (asympt) and symptomatic (sympt). The dashed circle on plot C indicates samples unambiguously defined as TcI at the ND5 locus. Pairs of sequential isolates from the same patient are labelled x and y respectively.</p

Percentage of reads mapping outside target regions.

<p>A: FF and FFPE samples. B: FFPE samples grouped by extraction method.</p

Yang and Neilson estimates for positive selection within and among abundant 97% STs identified in this study.

<p>^a Numbers in brackets represent the number of 99% STs define within each cluster from which estimates were generated.</p><p>^b P values are give for Fisher’s exact tests for deviation from the neutral expectation of Ka/Ks = 0.</p><p>Yang and Neilson estimates for positive selection within and among abundant 97% STs identified in this study.</p

Bar plot showing sequence type identity and abundance defined at 97% similarity for the ND5 locus across all samples.

<p>A—Goias cohort chronic/intermediate cases; B—Cochabamba chronic/intermediate cases; C—Cochabamba congenital cases. Y axes show log transformed abundance (read counts). X axes show clustered bars for individual samples. Sequence type identities are given in the legend. Stars denote congenital pair from Goias. Labels x (6416 / 6452), y (6401 / 6536) and z (6379 / 6445) sample pairs from the same patient at different time points (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003458#pntd.0003458.t001" target="_blank">Table 1</a>).</p

Rate of SNVs C.G > T.A substitution in FF and FFPE samples.

<p>A: FF and FFPE samples. B: FFPE samples grouped by extraction method.</p

Samples provenance and symptoms.

<p>^a Samples from Goias congenital case</p><p>^x Samples from the same patient taken >12 months apart</p><p>^y Samples from the same patient taken < 6 months apart</p><p>^z Samples taken from the same patient >12 months apart</p><p>Samples provenance and symptoms.</p

Percentage of positions having a coverage greater then or equal to 30X.

<p>A: FF and FFPE samples. B: FFPE samples grouped by extraction method.</p

Single nucleotide variants (SNVs) analysis between FF and FFPE sample pairs.

<p>NFF: Number of SNV in FF samples, NFFPE: number of SNV in FFPE samples, NPos: number of common positions between FF and FFPE samples, Nco: number of concordant positions, Ndi: number of discordant positions, P: concordance rate (Nco/NPos * 100).</p