Introduction

Gene panels enable the simultaneous screening of a number of genes. Panels are typically customized for specific screening purposes; thus, the genes (and/or specific gene regions) on such panels are unique to the screening goals. In recent years, with technological sequencing advances, panel-based screening has become extremely efficient and cost-effective. These advancements involve the targeted enrichment of selected genes followed by massively parallel sequencing, which is also known as next-generation sequencing (NGS) [1, 2].

NGS gene panels have been implemented into clinical practice to assess inherited risk of cancer [1–3]. These panels include clinically valid genes for which clinical management guidelines have been established, such as genetic risk assessment criteria and mutation-positive management strategies. In the U.S., the National Comprehensive Cancer Network (NCCN) provides such guidelines to maximize clinical utility [4]. The American College of Medical Genetics and Genomics (ACMG) has established clinical laboratory standards for NGS gene panels but, ultimately, these panels are regulated by the Clinical Laboratory Improvement Amendments (CLIA), a federal program that certifies and oversees clinical laboratory testing [5, 6]. CLIA primarily assesses analytical validity–the accuracy of mutation detection–in order to maintain quality standards and ensure the effectiveness of each laboratory test [6].

CLIA does not regulate research-based genetic testing, but similar analytical assessments can be carried out to ensure accurate mutation detection in a research setting. We have developed a research-based gene panel, B.O.P. (Breast, Ovarian, and Prostate), to assess inherited risk of hereditary breast cancer (BC) and associated cancers. B.O.P. is an exploratory gene panel. In addition to targeting clinically valid genes that have NCCN management strategies, it also targets genes that have been suggested to be associated with an increased risk but currently lack sufficient evidence, as well as candidate genes, such as those in DNA repair pathways. Therefore, the ultimate goal in utilizing this panel is to better elucidate risk. Regarding hereditary BC, NCCN clinically valid genes only account for a minority of the associated genes reported in the literature [1, 7, 8]. Furthermore, NCCN risk management strategies have primarily been developed for overtly pathogenic, truncation mutations in clinically valid genes–resulting in the detection of many variants of unknown significance (VUS), and clinically valid mutations explain less than 30% of hereditary BC cases. Additional exploration is critical to fill these knowledge gaps, and B.O.P. can aid in this investigation. However, prior to using B.O.P. as a way to increase knowledge in these areas, it must be evaluated for its ability to accurately detect variants. The purpose of this manuscript is to introduce B.O.P., present the analytical assessment of 10 NCCN regulated genes (in order to ensure the accurate detection of clinically relevant variants), and discuss the future potential of the panel.

Materials and methods

Panel design

B.O.P. targets ~500 kilobases (Kb) of DNA including 87 genes that are suggested, predicted, or clinically proven to be associated with BC, OvC, and/or prostate cancer (PC) risk (S1 Table). Agilent Technologies Haloplex probes were designed using Agilent Technologies SureDesign software (https://earray.chem.agilent.com/suredesign/). The “Advanced HaloPlex” design allowed for the selection of the desired genes of which the targeted regions included both coding and non-coding exons as well as 10 intronic base pairs flanking the intron-exon boundaries. The probe set was designed for 100 base pair reads on an Illumina platform. Overall, probes were predicted to cover 98.93% of the targeted genes/regions (Table 1).

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Table 1. Ten clinically relevant genes on the B.O.P. panel assessed for analytical validity.

https://doi.org/10.1371/journal.pone.0220929.t001

Capture and sequencing

The genomic DNA of 43 cancer–affected individuals (23 African American [AA] and 20 European American [EA]) from the AHCC [9] was selected to undergo the first B.O.P. screening (Figs 1 and 2). Two study participants (1CAD-a and 1CAD-f) were knowingly related (first cousins). The HaloPlex HS Target Enrichment System For Illumina Sequencing Protocol (Version C0, December 2015) was followed for the targeted-capture, allowing each of the 43 samples to be uniquely barcoded/indexed, individually captured, and pooled in equimolar amounts for Illumina paired-end sequencing. One pooled sample with a final concentration of 24.13 nanomoles/liter (and DNA fragments ranging from 175 to 625 base pairs) was sent for sequencing on one lane of a flow cell on an Illumina HiSeq 2500 at the Genomic Services Laboratory (GSL) at HudsonAlpha Institute for Biotechnology. The final DNA quality/quantity of the pooled sample was assessed using the High Sensitivity DNA kit using the ABI 2100 Bioanalyzer.

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Fig 1. Screening process for individuals in the AHCC.

*In situations where blood is unattainable, another set of three distinct biological samples (i.e. saliva) is collected.

https://doi.org/10.1371/journal.pone.0220929.g001

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Fig 2. Pipeline for B.O.P. panel screening: Targeted capture, next-generation sequencing, bioinformatics pipeline, and variant filtering.

https://doi.org/10.1371/journal.pone.0220929.g002

Results

The number of reads that passed QC assessment per individual averaged 11.3 million (M), with 98.6% of those reads mapping to the human genome. On average, 50.9% of the reads mapped to B.O.P. targeted regions (S2 Table). The average sequencing depth for all targeted base pairs was 809X; however, sequencing depth was not uniform with a large interquartile range (Table 1). A probe design report provided by Agilent Technologies predicted that 98.9% of the targeted base pairs would be covered at least 1X, which was similar to the actual coverage of 98.2%; thus, 1.8% of the targeted base pairs were not covered at all (Table 1). The 10 assessed genes had average sequencing depths that ranged from 505X-1017X (Table 1). Furthermore, assessment of the 225 different regions that targeted those 10 genes revealed that the majority had average sequencing depths between 800-899X but ranged from 68X-2053X (Table 2; S3 Table). Although rare, regions with average sequencing depths less than 100X missed on average 24.3% of the targeted base pairs and only covered 52.2%, 34.6%, and 21.3% of targeted base pairs at or greater than 20X, 50X, and 100X, respectively (Table 2; S3 Table). Regions with the highest average sequencing depth, 1500X or greater, had over 99% and 96.5% of the targeted base pairs covered at least 50X and 100X, respectively. However, 28.0% of the 225 regions-of-focus did not, on average, obtain 100% coverage at 1X, which included regions with average sequencing depths ranging from 68X-1354X (Table 2; S3 Table).

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Table 2. Average sequencing depth analyses of the 225 regions targeting the ten B.O.P. genes being assessed.

https://doi.org/10.1371/journal.pone.0220929.t002

Upon variant annotation (Fig 2), a total of 24,915 variants (2,858 unique) were called. After filtering for variants in the 10 genes (Table 1), a total of 1960 (287 unique) remained, 74 (56 unique) of which had MAFs less than 2% in both ethnicities (Table 3; Fig 2). A total of 61 of the 74 variants were validated and classified as TPs, averaging a sequencing depth of 659X and an allele balance of 0.51; this included 100% of the variants in seven out of the 10 genes (ATM, BRCA2, CHEK2, NBN, PALB2, STK11, and TP53), resulting in FDRs of 0 (Table 3). BRCA1 and CDH1 each had one FP, and PTEN had the highest FDR with 11 FPs, all in intron7/exon8 (Table 3). Despite that the average FP sequencing depth was 34X (ranging from 12X-63X), sequencing depths of the three regions harboring FPs revealed that all achieved an average greater than 427X (Table 3 and S3 Table). The average FP allele balance was 0.33, ranging from 0.13–0.68.

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Table 3. Summary of called variants after bioinformatics pipeline and variant filtering.

https://doi.org/10.1371/journal.pone.0220929.t003

Though not optimal, low sequencing depth was not always indicative of a FP. Of the 20 variants covered less than 100X, 13 were FPs, and 7 were TPs with average sequencing depth of 60X and allele balance of 0.48, ranging from 0.33–0.58 (Table 3). In contrast, higher coverage, such as sequencing depth greater than 100X, was an indicator of a TP; all 54 variants covered over 100X were determined to be TPs. This included two homozygous TPs, which were each covered over 1000X with the alternate allele being the only one detected (Table 3), and 52 heterozygous TPs that had an average sequencing depth of 724X and allele balance of 0.50. To note the importance of allele balance, 95% of the TPs had an allele balance above 0.40 compared to only 23% of the FPs. Eighty five percent of the TPs had over 100X coverage and an allele balance of 0.40 (Table 3). No variants with an allele balance less than 0.20 were TPs, and additional filtering to exclude such variants improved FDRs (Table 3).

Prior to B.O.P. screening, positive and negative BRCA1 and BRCA2 mutation status was known for eight of the 43 study participants; thus, sensitivity and specificity could be calculated for those genes. Seven study participants had previously undergone clinical BRCA1/2 screening; six reported negative results with no pathogenic variants identified. One individual, 1CB-a, received a positive report indicating a pathogenic BRCA2 frame-shifting mutation (c.5611_5615delAGTAA [p.S1871fs] also known as c.5616_5620delAGTAA [p.K1872Nfs), which was confirmed using PCR and Sanger sequencing prior to B.O.P. screening (Fig 3 and Table 3). The eighth individual, 1CAD-a, had not personally obtained clinical gene screening; however, a deceased family member had undergone clinical BRCA1/2 screening and received a positive report indicating a pathogenic missense mutation (BRCA1 c.5387T>G [p.M1796R]). Thus, this individual was screened for the familial mutation using PCR and Sanger sequencing prior to B.O.P. screening and tested positive (Fig 3 and Table 3). Noteworthy, another BC-affected family member, 1CAD-f, tested negative for BRCA1 p.M1796R in the research laboratory prior to B.O.P. screening but could not be considered a TN for the specificity calculation since full gene screening had not been carried out (Table 3). B.O.P. variant calling reported 12 and 14 variants in BRCA1 and BRCA2, respectively (Table 3). Upon Sanger sequencing confirmation, this included 11 TPs, zero FNs, six TNs and one FP in BRCA1, and 14 TPs, six TNs, and zero FNs and FPs in BRCA2 (Table 3), which corroborated the previously reported BRCA1 and BRCA2 mutation statuses. Therefore, B.O.P. screening of BRCA1/2 resulted in 100% sensitivity and 92.3% specificity. However, specificity became 100% with the elimination of variants with an allele balance of 0.20 or less.

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Fig 3. B.O.P. positive controls: Mutations that were previously reported through clinical gene screening.

The Sanger sequence electropherogram (above) and the Integrative Genomics Viewer (v.2.4.5) image (below) are depicted for two B.O.P. screened positive controls: 1CB-a (Panel A) and 1CAD-a (Panel B). ($) This deletion is within a short tandem repeat and is commonly referred to as BRCA2 c.5616_5620del5 (p.K1872Nfs) since HGVS nomenclature rules arbitrarily assign the deletion to the most 3’ nucleotide.

https://doi.org/10.1371/journal.pone.0220929.g003

Of the 61 TPs, 45 were detected in AAs; this included 34 unique variants, eight of which were detected in multiple individuals (Table 3). According to ClinVar [13, 14], the 34 variants were categorized as pathogenic/risk factor (n = 4), VUSs (n = 11), or benign/likely benign (n = 19). A total of five variants were predicted to be deleterious in Polyphen, two of which have been defined as pathogenic non-synonymous variants in ClinVar; the other three are currently classified as VUSs (Table 3). Of the eight variants detected in more than one individual, BRCA2 c.5020A>G; p.S1674G, was identified in two first cousins. The remaining seven were in seemingly unrelated individuals. This includes STK11 c.369G>A;p.Q123Q, a seemingly benign variant, which is reported to have a MAF of 1.5% in the general AA population but was detected in five of the 23 AAs in this study, indicating a MAF of 10.8% (P value 8.50 X 10⁻⁴; Odds ratio 7.79 CI₉₅[2.32–20.70]). Furthermore, the 45 AA TPs were detected in 96% of the AAs screened, and multiple variants were detected in 70% of the cases. In contrast, 16 TPs were validated in 55% of the EAs, and only 20% had multiple variants. The difference in the number of individuals from each ethnicity with at least one TP was significant (P value 2.71 X 10⁻³; Odds ratio 16.83 CI₉₅[1.93–819.72]) as well as the number of cases from each ethnicity with multiple TPs (P value 1.95 X 10⁻³; Odds ratio 8.60 CI₉₅[1.89–49.30]). No overtly pathogenic variants were validated in EAs, but 50% of the EA TPs (8/16) were listed as a VUS, three of which were predicted to be deleterious in Polyphen [15].

Discussion

Our group has developed B.O.P., a research-based NGS gene panel, which targets 87 genes that have been suggested, predicted, or clinically proven to be associated with risk of BC, OvC, and/or PC. The overall purpose of this new panel is to gain additional insights toward the genetic risk of and overlap between those three cancers. This manuscript served to introduce B.O.P. by reporting its initial screening, which involved 43 cancer-affected individuals from the AHCC [16]. Targeting ~500 Kb of DNA, 98.9% of the base pairs were covered at least 1X, and an average sequencing depth of 809X was obtained. We took a closer look at 10 NCCN regulated genes in order to begin the analytical assessment of the panel and ensure the accurate detection of clinically relevant variants; upon variant filtering and validation, 100% of the variants in seven of the 10 genes were TPs. TPs had an average sequencing depth of 659X and allele balance of 0.51, whereas the average FP sequencing depth and allele balance was 34X and 0.33, respectively. Although FPs had a much lower average sequencing depth compared to TPs, a low sequencing depth was not always indicative of a FP. Contrarily, all variants called with high sequencing depths (>100X) signified a TPs. Furthermore, sensitivity and specificity of BRCA1/2 were calculated to be 100% and 92.3%, respectively.

There are a number of different targeted enrichment options to choose from when designing gene panels, all of which can have different affects on sequencing outcomes [10]. When comparing methods, Samorodnitsky et al. concluded that Haloplex had the highest on-target read alignment and normalized sequencing depth but the least uniformity [10]. Noteworthy, despite reports of Haloplex resulting in a high percentage (>90%) of on-target read alignments [10, 17], 50.9% of our QC passed reads mapped to the B.O.P. targeted regions. With other Haloplex gene panel studies not reporting such data [18–20], it is difficult to make general conclusions about Haloplex on-target read alignment specificity. However, similar on-target read alignment percentages have been reported; Castera et al. used SureSelect baits in order to target hereditary BC and OvC susceptibility genes, and reported an average of 42% of reads on-target [21]. Ultimately, the percentage of off-target reads is likely dependent on a number of factors, including the specific genes/regions being targeted [22]. Of the reads that mapped on-target, B.O.P.’s overall sequencing depth averaged 809X, and each individually assessed gene obtained average sequencing depths from 505X-1017X. Nevertheless, large interquartile ranges indicated that depth was not uniform. This was expected since no current enrichment and sequencing approach provides complete uniformity primarily because of complex genomic regions that are very difficult to capture/sequence and result in low sequencing depths or even no coverage at all [10, 22].

By focusing on a select set of genes/regions, NGS gene panels target a smaller number of base pairs compared to more broad applications such as exome and whole genome sequencing. The smaller target-capture provides the option to achieve a high average sequencing depth, which aids in variant identification [10, 23]. Therefore, the overall goal is to obtain 100% coverage as well as the appropriate/desired sequencing depths at all targeted base pairs. Since this goal is not generally achieved, complementary assays can be used to fill in gaps, which is commonly implemented for clinical applications. In such cases, regions of low/no coverage are normally Sanger sequenced [22, 23]. B.O.P. was able to cover, on average, 98.2% of its targeted base pairs at 1X. Being a research panel, no gap-filling assays were carried out; however, region-specific coverage analyses provided insight towards the feasibility of gap-filling. Gap-filling criteria has been described in a number of BC NGS gene panel publications, specifically, those that highlighted panel performance and analytical validity [21, 24–26]. Being clinical panels, regions covered less than 20X [21] or 50X [24–26] were checked by conventional methods. Interestingly, only two B.O.P. regions had average sequencing depths less than 100X (68X and 82X), which would not have required complementary assays to fill in gaps according to the criteria set in the referenced studies [21, 24–26]. This is despite that, on average, those two B.O.P. regions missed 24.3% of the targeted base pairs and only covered 52.2%, 34.6%, and 21.3% of targeted base pairs at or greater than 20X, 50X, and 100X, respectively. Furthermore, 63 of the 225 B.O.P. regions-of-focus did not, on average, obtain 100% coverage at 1X; these regions had average sequencing depths ranging from 68X-1354X. Thus, regions with high sequencing depths still had base pairs with no/low coverage, which happened to be where FPs were detected in this study. Therefore, only gap-filling regions with ‘low’ (20X or 50X) sequencing depths, will not guarantee 100% coverage. Establishing mapping criteria to ensure all base pairs are covered at a desired depth is ideal but would likely reveal gaps in too many regions, making gap-filling infeasible. Overall, gaps in B.O.P. as well as other panels, even with gap-filling criteria, can provide less than definitive negative results [23]; however, in noting that, zero FNs were identified in BRCA1 and BRCA2, resulting in 100% sensitivity.

In addition to gap-filling, conventional approaches are also used to validate called variants. A total of 1960 variants were detected in the 10 B.O.P. assessed genes and, to reduce the number of variants to validate for this analytical assessment, only variants with MAFs less than 2% in both ethnicities were Sanger sequenced. This included 74 variants, 61 of which were confirmed and defined as TPs revealing 13 FPs. The validation process ultimately provided insight regarding the likelihood of confirmation based on variant quality, such as sequencing depth since all 54 variants covered over 100X were TPs. These results corroborated the criteria established by Mu et al., which set high confidence calls as having a minimum sequencing depth of 100X and allele balance of 40%. Additionally, Mu et al. indicated that such calls did not require confirmation. Although, all B.O.P. variants covered at or above 100X were TPs despite allele balance, the criteria from Mu et al. will be implemented in the future in order to be thorough. This will limit validation efforts to low confidence calls, reducing the cost and time of validation.

In this study, 22 variants had low confidence calls. This included nine TPs, seven of which were covered less than 100X and two that failed to meet the required allele balance. The remaining 13 were FPs with an average sequencing depth of 34X and allele balance of 0.33, reiterating that low sequencing depths are susceptible to sequencing artifacts [10, 23]. Interestingly, as mentioned in the previous paragraph, the regions harboring the FPs did not have low sequencing depths, stressing the potential lack of uniformity within a targeted region. On another note, 11 of the 13 FPs were in PTEN. Considering PTEN has a processed pseudogene, PTENP1 on chromosome 9, their homology could have contributed to probe mis-priming as well as read mis-alignments. Overall, encountering problematic regions, such as regions with high homology or GC rich content, is common and referred to in many studies [18, 22, 23]. Overall, for each assessed gene, FDRs ranged from 0 to 0.92, the latter being PTEN. Of course, FDRs improved as additional filtering was implemented. Initial B.O.P. specificity, which could only be calculated for BRCA1/BRCA2, was 92.3%. Upon filtering out variants with an allele balance equal to or less than 0.20, specificity was 100%. Ultimately, Sanger sequencing all low confidence calls will eliminate FPs and provide 100% specificity; therefore, it is common to complement NGS gene panels with Sanger sequencing validation in order to consider the test complete and optimize specificity [21, 24–28].

In addition to enabling B.O.P.’s initial analytical assessment, the first B.O.P. screening, which involved 23 AAs and 20 EAs from the AHCC [9], has provided insight regarding variant contributions and ethnic differences. Overall, compared to EAs, AAs had a significantly higher number of individuals with at least one TP (P value 2.71 X 10⁻³) as well as individuals with multiple TPs (P value 1.95 X 10⁻³). Of course, comparisons to ethnic-specific controls will determine if these differences contribute to an inherited cancer risk. Interestingly, according to ClinVar [13, 14], none of the variants identified in EAs were considered pathogenic/risk variants, whereas 17.4% (4/23) of the AAs had a variant with that classification. The majority of the detected variants were classified as VUSs or benign/likely benign; ultimately, elucidating how VUSs and, even, synonymous variants contribute towards risk is very important. Synonymous variants, though normally ignored and considered benign, can affect splicing, gene expression or translation dynamics, all of which can contribute to a disease phenotype [29]. To further stress their importance, they have been reported to act as driver mutations in human cancers [30] and, through this initial B.O.P. screening, STK11 c.369G>A;p.Q123Q was detected in significantly more AA cases than controls (P value 8.50 X 10⁻⁴). Additionally, despite recognizing that hereditary BC risk is polygenic [31], little effort has been put forth to thoroughly investigate all variants in clinically relevant BC susceptibility genes, not to mention variants in genes currently lacking clinical significance, and determine if different variant combinations increase risk. Altogether, seemingly benign variant combinations could, in fact, be pathogenic, and paired with the striking difference between ethnicities regarding the number of cases with multiple variants, further investigation is warranted. Of course, a larger number of cases will be required for this effort.

In summary, this effort assesses the analytical validity of the B.O.P. panel and demonstrates the panel’s ability to accurately detect mutations in 10 NCCN clinically actionable genes [8]. Despite the potential biases of the B.O.P. capture and NGS, the high depth of coverage, low FDR, and great sensitivity and specificity strongly support the use of this research gene panel to further elucidate hereditary BC/OvC/PC genetics. Although the cohort for this initial assessment is small, B.O.P. has begun to determine the mutation contributions of clinically valid genes in different ethnicities as well as permit the investigation of VUSs and other variant types and their effect towards polygenic risk. Furthermore, continued B.O.P. screening can provide additional evidence to confirm or refute previously suggested susceptibility genes, lessening the number of genes that lack clinical validity on commercially available panels [1]. Additionally, with the incorporation of candidate genes on B.O.P., it has the potential to identify novel genetic risk factors that are contributing towards BC, OvC, and PC. Lastly, as new susceptibility genes are discovered that are not currently on the B.O.P. panel, it is important to stress the ability to edit the targeted genes in order to best reflect clinical screening. As described herein, there are many potential benefits of B.O.P. screening, and we aim to make advances in cancer genetics research through its implementation in our research efforts.

Supporting information

Acknowledgments

First, and most importantly, we would like to thank our study participants. The establishment of the Alabama Hereditary Cancer Cohort could not have been accomplished without their involvement and contribution. Furthermore, this effort also acknowledges and appreciates the continued support and commitment from the study’s community partners and patient advocates. Additionally, we thank The Cancer Center at East Alabama Medical Center in Opelika, Alabama for their essential partnership and efforts towards hospital recruitment. This research was supported by the American Association of Colleges of Pharmacy (AACP) New Investigator Award, AU Research Initiative in Cancer (AURIC) Seed Grant, the Joy to Life Foundation Grant, the AU Innovative Research grant through the Internal Grant Program with matching funds provided by AURIC and the Joy to Life Foundation, and the National Science Foundation (NSF) Research Excellence for Undergraduates (REU; Award number: 1560115). This research was also supported by the AURIC Graduate Fellowship Program (to M.R.B. and A.L.W.H.), the AU Cellular and Molecular Biosciences (CMB) Peaks of Excellence Research Fellowship (to M.R.B. and A.L.W.H.; NSF-EPS-1158862, grant G00006750), the NSF REU Fellowship (to J.H.), the Department of Drug Discovery and Development in the AU Harrison School of Pharmacy, the Department of Pathobiology in the AU College of Veterinary Medicine, and the AU College of Sciences and Mathematics. Lastly, we would like to acknowledge the Office of Information Technology at Auburn University for support and compute time on the Hopper High-Performance Computing Cluster.