Detailed analysis of a 1.1 Mb genomic region surrounding the <i>VKORC1</i> gene locus in East Asia.

<p>The boundaries of the region displayed (chr16:30,271,572-31,391,123; UCSC human genome build hg18) were chosen so as to include the three clusters of significant scores detected in East Asia by the selection tests in the 2 Mb region centered on <i>VKORC1</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053049#pone-0053049-g003" target="_blank">Figure 3</a>). (<b>A</b>) <b>Name and location of genes.</b> Exons are displayed as blue boxes and the transcribed strand is indicated with an arrow. Genes located in the block of strong LD encompassing <i>VKORC1</i> and including the SNPs in the red box shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053049#pone-0053049-g004" target="_blank">Figure 4C</a>, are highlighted in the grey area. (<b>B</b>) <b>XP-EHH results in East Asia.</b> The significance of the XP-EHH scores (−log₁₀ empirical <i>p</i>-value) are shown for individual SNPs with a MAF ≥0.01 in East Asia. Horizontal dashed lines indicate 0.05 and 0.01 chromosome-wide significance levels. Recombination hotspots detected in HapMap Phase II data are indicated by red vertical dotted lines. The data and methods used to derive these hotspots are available from the HapMap website (<a href="http://www.hapmap.org/" target="_blank">http://www.hapmap.org/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053049#pone.0053049-McVean1" target="_blank">[83]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053049#pone.0053049-Winckler1" target="_blank">[84]</a>. (<b>C</b>) <b>LD plot.</b> Pairwise LD values, depicted as <i>D</i>’, are shown for SNPs with a MAF ≥0.01 in East Asia. <i>D</i>’ values are displayed in different colors from yellow to red for <i>D</i>’ = 0 to <i>D</i>’ = 1, respectively. The red box highlights SNPs included in the LD block encompassing <i>VKORC1.</i> The plot was produced using the snp.plotter R package <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053049#pone.0053049-Luna1" target="_blank">[74]</a>.</p

Distribution of –log₁₀ (<i>p-</i>values) for four selection tests across a 2 Mb region centered on <i>VKORC1</i>.

<p>A black vertical line indicates the physical position of <i>VKORC1</i> on chromosome 16. Horizontal red dotted and dashed lines show 0.05 and 0.01 chromosome-wide significance levels, respectively. The selection tests (inter-regional <i>F_ST</i>, XP-CLR, XP-EHH and iHS, respectively) were separately applied in each of the seven geographic regions.</p

Atypical patterns of genetic differentiation observed for <i>VKORC1</i> SNPs.

<p>Genome-wide empirical distributions of <i>F_ST</i> values were constructed from 644,143 SNPs having a MAF ≥0.001 at the global level. Individual values of <i>F_ST</i> calculated for each of the seven <i>VKORC1</i> SNPs are plotted against their global MAF. The functional rs9923231 SNP is shown in red. The 50^th, 95^th and 99^th percentiles are indicated as dotted, dashed and full red lines, respectively.</p

Results of the inter-regional <i>F_ST</i>, intra-regional <i>F_ST</i>, XP-EHH and iHS tests in the seven geographic regions.

a<p>Derived allele frequency estimated at the global level.</p>b<p><i>F_ST</i> estimated at the inter-regional level, <i>i.e.</i> between a given geographic region and the remaining ones.</p>c<p><i>P</i>-values are derived from the genome-wide empirical distribution of <i>F_ST</i> values.</p>d<p><i>F_ST</i> estimated at the intra-regional level, <i>i.e.</i> among populations within a region.</p>e<p><i>P</i>-values are derived from the empirical distribution of the iHS and XP-EHH scores along the chromosome 16.</p>*<p><i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.005.</p><p>NA: Not Applicable (for iHS: when a gap>200 kb between successive SNPs is found in the region in the region delimited by the SNPs where the EHH value drops below 0.05 around the core SNP).</p

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

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

Percentage of reads mapping outside target regions.

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

Percentage of duplicated reads for FF and FFPE samples.

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

Difference in number of variants between FF and FFPE samples for all matched FF/FFPE pairs.

<p>A: SNVs. B: INDELs.</p