Criteria for the enrollment of high-risk <i>BRCA1</i>- and <i>BRCA2</i>-negative individuals in this study and number of identified mutations in each group.

<p>BC—breast cancer; HBOC—hereditary breast and ovarian cancer; HOC—hereditary ovarian cancer; OC—ovarian cancer; y/o—years old. Number of rare missense variants are in brackets.</p><p>Criteria for the enrollment of high-risk <i>BRCA1</i>- and <i>BRCA2</i>-negative individuals in this study and number of identified mutations in each group.</p

cDNA analysis of patient No.1273 uncovered with an intronic variant c.1026+5_1026+7delGTA causing RAD51C out-of-frame exon 8 skipping.

<p>The electrophoresis (left) of PCR products amplified with primers located in exon 5 and 3’ UTR sequence (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127711#pone.0127711.s001" target="_blank">S1 Table</a>) shows two products in a patient No.1273 compared to a wild-type control (C) sample. Sequencing chromatogram of the patient’s PCR product shows the presence of aberrantly spliced mRNA with exon 8 skipping.</p

Validation of CZECANCA (CZEch CAncer paNel for Clinical Application) for targeted NGS-based analysis of hereditary cancer syndromes

<div><p>Background</p><p>Carriers of mutations in hereditary cancer predisposition genes represent a small but clinically important subgroup of oncology patients. The identification of causal germline mutations determines follow-up management, treatment options and genetic counselling in patients’ families. Targeted next-generation sequencing-based analyses using cancer-specific panels in high-risk individuals have been rapidly adopted by diagnostic laboratories. While the use of diagnosis-specific panels is straightforward in typical cases, individuals with unusual phenotypes from families with overlapping criteria require multiple panel testing. Moreover, narrow gene panels are limited by our currently incomplete knowledge about possible genetic dispositions.</p><p>Methods</p><p>We have designed a multi-gene panel called CZECANCA (CZEch CAncer paNel for Clinical Application) for a sequencing analysis of 219 cancer-susceptibility and candidate predisposition genes associated with frequent hereditary cancers.</p><p>Results</p><p>The bioanalytical and bioinformatics pipeline was validated on a set of internal and commercially available DNA controls showing high coverage uniformity, sensitivity, specificity and accuracy. The panel demonstrates a reliable detection of both single nucleotide and copy number variants. Inter-laboratory, intra- and inter-run replicates confirmed the robustness of our approach.</p><p>Conclusion</p><p>The objective of CZECANCA is a nationwide consolidation of cancer-predisposition genetic testing across various clinical indications with savings in costs, human labor and turnaround time. Moreover, the unified diagnostics will enable the integration and analysis of genotypes with associated phenotypes in a national database improving the clinical interpretation of variants.</p></div

Validation of CZECANCA (CZEch CAncer paNel for Clinical Application) for targeted NGS-based analysis of hereditary cancer syndromes - Fig 7

<p><b>Comparison of variant detection (shown as values of variant allelic fraction; AF) in DNA reference standards</b> (NA12878, NA24149, NA24385, NA24631 and NA24143) obtained from CZECANCA analysis (x-axis) and AF from VCF files for these standards downloaded from <a href="http://jimb.stanford.edu/giab/" target="_blank">http://jimb.stanford.edu/giab/</a> (y-axis). The graph shows all variants with GATK quality >100 reached in CZECANCA analysis (including FP variants) and undetected (FN) variants. Heterozygote variants clustered in the center, while homozygote variants in right upper corner. Variant distribution was partially influenced by the differences in mean sequencing coverage targeting 100X and 300X in CZECANCA and DNA reference standards VCFs, respectively. The number of TP, TN, FP, FN, and total number of variant (= CZECANCA target) was used to calculate of sensitivity, specificity, and accuracy of CZECANCA analysis.</p

Identification of c.661_662delinsTA double substitution (p.Val221*) in <i>PALB2</i> (NM_024675).

<p>The BAM file displayed in IGV shows the <i>cis</i>-position of both substitutions in approximately 50% of forward (pink bars) and reverse (blue bars) reads, respectively.</p

Validation of CZECANCA (CZEch CAncer paNel for Clinical Application) for targeted NGS-based analysis of hereditary cancer syndromes - Fig 4

<p><b>Analysis of intra-run (A), inter-run (B), and inter-laboratory (C) replicates.</b> The panels show sequencing coverages (y-axis) of the identified variants arranged according to chromosomal localizations (x-axis). We used moving average curves (average of 3 values) to compare trends in coverages. Panel (A) describes the results of an analysis of three independently processed intra-run replicates from an identical DNA sample pooled in 33 ng (considered as 100%), 24.75 ng (75%), and 16.5 ng (50%), respectively. Panel (B) demonstrates variant coverages identified in two independent inter-run (run 8 and 14) replicates. All coverage values of sample #3647 in run 14 were corrected by a factor of 1.3880 to normalize coverages between samples (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195761#pone.0195761.s004" target="_blank">S4 Table</a>). Panel (C) shows coverages of variants identified in an inter-laboratory control sequenced in four laboratories (Lab) participating in panel validation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195761#pone.0195761.s005" target="_blank">S5 Table</a>). The coverages of variants identified in Lab 2, 3, and 4 were normalized to the average coverage of Lab 1 for better comparisons of coverages.</p

Coverage (y-axis) of coding sequences (x-axis) of 219 CZECANCA target genes from a routine, randomly selected run targeting 100X coverage.

<p>Note: Fully covered genes are depicted in green letters, genes with coverage <20X in a single exon are in orange letters, and genes with uncovered regions exceeding single exon or >10% of coding sequence are in red letters. Green horizontal bars (below individual graphs constructed using “Boudalyzer” script) indicate coverage ≥ 20X; red horizontal bars indicate regions covered <20X and uncovered regions.</p

CNV detection is influenced by a DNA preparation method.

<p>Panels show analyses of remaining ACMG genes (not shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195761#pone.0195761.g005" target="_blank">Fig 5B and 5C</a>) from four runs performed in laboratory 1 (116 DNA samples fragmented by ultrasound) and laboratory 3 (125 DNA samples fragmented enzymatically). The numbers in parentheses express number of samples with possible CNVs from all analyzed samples in contributing laboratories. *indicate samples analyzed by MLPA negatively (FP–black) or positively (TP–red). Bin set covering exon 1 in <i>RET</i> was excluded from the analysis due to the large coverage variability.</p

Validation of CZECANCA (CZEch CAncer paNel for Clinical Application) for targeted NGS-based analysis of hereditary cancer syndromes - Fig 3

<p><b>Coverage of selected genes from the CZECANCA (A-E) and TruSight Cancer sequencing (F) panels.</b> The pictures show coverage (at y-axis) alongside the coding sequences of <i>BRCA1</i> (NM_007294), <i>BRCA2</i> (NM_000059), <i>PALB2</i> (NM_024675), and <i>TP53</i> (NM_000546), the vertical lines represent exon boundaries. Panels A–D show results obtained from a CZECANCA NGS analysis of various samples performed in four participating laboratories using the ultrasound (A, B) or enzymatic (C, D) DNA fragmentation protocol. Examples of the identified CNV aberrations in the depicted genes (deletions in <i>BRCA1</i>, <i>BRCA2</i> and <i>TP53</i> and duplication in <i>PALB2</i>) are shown in panel E. For comparison, panel F demonstrates the uneven coverage of the depicted genes by sequencing using the TruSight Cancer panel (Illumina).</p