Maternal SARS-CoV-2 exposure alters infant DNA methylation.

Background: Infection during pregnancy can increase the risk of neurodevelopmental disorders in offspring. The impact of maternal SARS-CoV-2 infection on infant neurodevelopment is poorly understood. The maternal immune response to infection may be mimicked in rodent models of maternal immune activation which recapitulate altered neurodevelopment and behavioural disturbances in the offspring. In these models, epigenetic mechanisms, in particular DNA methylation, are one pathway through which this risk is conferred in utero to offspring. We hypothesised that in utero exposure to SARS-CoV-2 in humans may alter infant DNA methylation, particularly in genes associated with neurodevelopment. We aimed to test this hypothesis in a pilot sample of children in Victoria, Australia, who were exposed in utero to SARS-CoV-2. Methods: DNA was extracted from buccal swab specimens from (n = 4) SARS-CoV-2 in utero exposed and (n = 4) non-exposed infants and methylation status assessed across 850,000 methylation sites using an Illumina EPIC BeadChip. We also conducted an exploratory enrichment analysis using Gene Ontology annotations. Results: 1962 hypermethylated CpG sites were identified with an unadjusted p-value of 0.05, where 1133 CpGs mapped to 959 unique protein coding genes, and 716 hypomethylated CpG sites mapped to 559 unique protein coding genes in SARS-CoV-2 exposed infants compared to non-exposed. One differentially methylated position (cg06758191), located in the gene body of AFAP1 that was hypomethylated in the SARS-CoV-2 exposed cohort was significant after correction for multiple testing (FDR-adjusted p-value <0.00083). Two significant differentially methylated regions were identified; a hypomethylated intergenic region located in chromosome 6p proximal to the genes ZP57 and HLA-F (fwer <0.004), and a hypomethylated region in the promoter and body of the gene GAREM2 (fwer <0.036). Gene network enrichment analysis revealed differential methylation in genes corresponding to pathways relevant to neurodevelopment, including the ERBB pathway. Conclusion: These pilot data suggest that exposure to SARS-CoV-2 in utero differentially alters methylation of genes in pathways that play a role in human neurodevelopment.

Population-based estimates of breast cancer risk for carriers of pathogenic variants identified by gene-panel testing.

Population-based estimates of breast cancer risk for carriers of pathogenic variants identified by gene-panel testing are urgently required. Most prior research has been based on women selected for high-risk features and more data is needed to make inference about breast cancer risk for women unselected for family history, an important consideration of population screening. We tested 1464 women diagnosed with breast cancer and 862 age-matched controls participating in the Australian Breast Cancer Family Study (ABCFS), and 6549 healthy, older Australian women enroled in the ASPirin in Reducing Events in the Elderly (ASPREE) study for rare germline variants using a 24-gene-panel. Odds ratios (ORs) were estimated using unconditional logistic regression adjusted for age and other potential confounders. We identified pathogenic variants in 11.1% of the ABCFS cases, 3.7% of the ABCFS controls and 2.2% of the ASPREE (control) participants. The estimated breast cancer OR [95% confidence interval] was 5.3 [2.1-16.2] for BRCA1, 4.0 [1.9-9.1] for BRCA2, 3.4 [1.4-8.4] for ATM and 4.3 [1.0-17.0] for PALB2. Our findings provide a population-based perspective to gene-panel testing for breast cancer predisposition and opportunities to improve predictors for identifying women who carry pathogenic variants in breast cancer predisposition genes.

Repeatability of methylation measures using a QIAseq targeted methyl panel and comparison with the Illumina HumanMethylation450 assay.

OBJECTIVE: In previous studies using Illumina Infinium methylation arrays, we have identified DNA methylation marks associated with cancer predisposition and progression. In the present study, we have sought to find appropriate technology to both technically validate our data and expand our understanding of DNA methylation in these genomic regions. Here, we aimed to assess the repeatability of methylation measures made using QIAseq targeted methyl panel and to compare them with those obtained from the Illumina HumanMethylation450 (HM450K) assay. We included in the analysis high molecular weight DNA extracted from whole blood (WB) and DNA extracted from formalin-fixed paraffin-embedded tissues (FFPE). RESULTS: The repeatability of QIAseq-methylation measures was assessed at 40 CpGs, using the Intraclass Correlation Coefficient (ICC). The mean ICCs and 95% confidence intervals (CI) were 0.72 (0.62-0.81), 0.59 (0.47-0.71) and 0.80 (0.73-0.88) for WB, FFPE and both sample types combined, respectively. For technical replicates measured using QIAseq and HM450K, the mean ICCs (95% CI) were 0.53 (0.39-0.68), 0.43 (0.31-0.56) and 0.70 (0.59-0.80), respectively. Bland-Altman plots indicated good agreement between QIAseq and HM450K measurements. These results demonstrate that the QIAseq targeted methyl panel produces reliable and reproducible methylation measurements across the 40 CpGs that were examined.

VTRNA2-1: Genetic Variation, Heritable Methylation and Disease Association.

VTRNA2-1 is a metastable epiallele with accumulating evidence that methylation at this region is heritable, modifiable and associated with disease including risk and progression of cancer. This study investigated the influence of genetic variation and other factors such as age and adult lifestyle on blood DNA methylation in this region. We first sequenced the VTRNA2-1 gene region in multiple-case breast cancer families in which VTRNA2-1 methylation was identified as heritable and associated with breast cancer risk. Methylation quantitative trait loci (mQTL) were investigated using a prospective cohort study (4500 participants with genotyping and methylation data). The cis-mQTL analysis (334 variants ± 50 kb of the most heritable CpG site) identified 43 variants associated with VTRNA2-1 methylation (p < 1.5 × 10-4); however, these explained little of the methylation variation (R2 < 0.5% for each of these variants). No genetic variants elsewhere in the genome were found to strongly influence VTRNA2-1 methylation. SNP-based heritability estimates were consistent with the mQTL findings (h2 = 0, 95%CI: -0.14 to 0.14). We found no evidence that age, sex, country of birth, smoking, body mass index, alcohol consumption or diet influenced blood DNA methylation at VTRNA2-1. Genetic factors and adult lifestyle play a minimal role in explaining methylation variability at the heritable VTRNA2-1 cluster.

Rare Germline Pathogenic Variants Identified by Multigene Panel Testing and the Risk of Aggressive Prostate Cancer.

While gene panel sequencing is becoming widely used for cancer risk prediction, its clinical utility with respect to predicting aggressive prostate cancer (PrCa) is limited by our current understanding of the genetic risk factors associated with predisposition to this potentially lethal disease phenotype. This study included 837 men diagnosed with aggressive PrCa and 7261 controls (unaffected men and men who did not meet criteria for aggressive PrCa). Rare germline pathogenic variants (including likely pathogenic variants) were identified by targeted sequencing of 26 known or putative cancer predisposition genes. We found that 85 (10%) men with aggressive PrCa and 265 (4%) controls carried a pathogenic variant (p < 0.0001). Aggressive PrCa odds ratios (ORs) were estimated using unconditional logistic regression. Increased risk of aggressive PrCa (OR (95% confidence interval)) was identified for pathogenic variants in BRCA2 (5.8 (2.7-12.4)), BRCA1 (5.5 (1.8-16.6)), and ATM (3.8 (1.6-9.1)). Our study provides further evidence that rare germline pathogenic variants in these genes are associated with increased risk of this aggressive, clinically relevant subset of PrCa. These rare genetic variants could be incorporated into risk prediction models to improve their precision to identify men at highest risk of aggressive prostate cancer and be used to identify men with newly diagnosed prostate cancer who require urgent treatment.

Population-Based Estimates of the Age-Specific Cumulative Risk of Breast Cancer for Pathogenic Variants in CHEK2: Findings from the Australian Breast Cancer Family Registry.

Case-control studies of breast cancer have consistently shown that pathogenic variants in CHEK2 are associated with about a 3-fold increased risk of breast cancer. Information about the recurrent protein-truncating variant CHEK2 c.1100delC dominates this estimate. There have been no formal estimates of age-specific cumulative risk of breast cancer for all CHEK2 pathogenic (including likely pathogenic) variants combined. We conducted a population-based case-control-family study of pathogenic CHEK2 variants (26 families, 1071 relatives) and estimated the age-specific cumulative risk of breast cancer using segregation analysis. The estimated hazard ratio for carriers of pathogenic CHEK2 variants (combined) was 4.9 (95% CI 2.5-9.5) relative to non-carriers. The HR for carriers of the CHEK2 c.1100delC variant was estimated to be 3.5 (95% CI 1.02-11.6) and the HR for carriers of all other CHEK2 variants combined was estimated to be 5.7 (95% CI 2.5-12.9). The age-specific cumulative risk of breast cancer was estimated to be 18% (95% CI 11-30%) and 33% (95% CI 21-48%) to age 60 and 80 years, respectively. These findings provide important information for the clinical management of breast cancer risk for women carrying pathogenic variants in CHEK2.

Genetic testing in Poland and Ukraine: should comprehensive germline testing of BRCA1 and BRCA2 be recommended for women with breast and ovarian cancer?

PURPOSE: To characterize the spectrum of BRCA1 and BRCA2 pathogenic germline variants in women from south-west Poland and west Ukraine affected with breast or ovarian cancer. Testing in women at high risk of breast and ovarian cancer in these regions is currently mainly limited to founder mutations. METHODS: Unrelated women affected with breast and/or ovarian cancer from Poland (n = 337) and Ukraine (n = 123) were screened by targeted sequencing. Excluded from targeted sequencing were 34 Polish women who had previously been identified as carrying a founder mutation in BRCA1. No prior testing had been conducted among the Ukrainian women. Thus, this study screened BRCA1 and BRCA2 in the germline DNA of 426 women in total. RESULTS: We identified 31 and 18 women as carriers of pathogenic/likely pathogenic (P/LP) genetic variants in BRCA1 and BRCA2, respectively. We observed five BRCA1 and eight BRCA2 P/LP variants (13/337, 3.9%) in the Polish women. Combined with the 34/337 (10.1%) founder variants identified prior to this study, the overall P/LP variant frequency in the Polish women was thus 14% (47/337). Among the Ukrainian women, 16/123 (13%) women were identified as carrying a founder mutation and 20/123 (16.3%) were found to carry non-founder P/LP variants (10 in BRCA1 and 10 in BRCA2). CONCLUSIONS: These results indicate that genetic testing in women at high risk of breast and ovarian cancer in Poland and Ukraine should not be limited to founder mutations. Extended testing will enhance risk stratification and management for these women and their families.

Rare germline genetic variants and risk of aggressive prostate cancer.

Few genetic risk factors have been demonstrated to be specifically associated with aggressive prostate cancer (PrCa). Here, we report a case-case study of PrCa comparing the prevalence of germline pathogenic/likely pathogenic (P/LP) genetic variants in 787 men with aggressive disease and 769 with nonaggressive disease. Overall, we observed P/LP variants in 11.4% of men with aggressive PrCa and 9.8% of men with nonaggressive PrCa (two-tailed Fisher's exact tests, P = .28). The proportion of BRCA2 and ATM P/LP variant carriers in men with aggressive PrCa exceeded that observed in men with nonaggressive PrCa; 18/787 carriers (2.3%) and 4/769 carriers (0.5%), P = .004, and 14/787 carriers (0.02%) and 5/769 carriers (0.01%), P = .06, respectively. Our findings contribute to the extensive international effort to interpret the genetic variation identified in genes included on gene-panel tests, for which there is currently an insufficient evidence-base for clinical translation in the context of PrCa risk.

Mismatch repair gene pathogenic germline variants in a population-based cohort of breast cancer.

The advent of gene panel testing is challenging the previous practice of using clinically defined cancer family syndromes to inform single-gene genetic screening. Individual and family cancer histories that would have previously indicated testing of a single gene or a small number of related genes are now, increasingly, leading to screening across gene panels that contain larger numbers of genes. We have applied a gene panel test that included four DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2) to an Australian population-based case-control-family study of breast cancer. Altogether, eight pathogenic variants in MMR genes were identified: six in 1421 case-families (0.4%, 4 MSH6 and 2 PMS2) and two in 833 control-families (0.2%, one each of MLH1 and MSH2). This testing highlights the current and future challenges for clinical genetics in the context of anticipated gene panel-based population-based screening that includes the MMR genes. This testing is likely to provide additional opportunities for cancer prevention via cascade testing for Lynch syndrome and precision medicine for breast cancer treatment.

Hi-Plex2: a simple and robust approach to targeted sequencing-based genetic screening.

We have previously reported Hi-Plex, a multiplex PCR methodology for building targeted DNA sequencing libraries that offers a low-cost protocol compatible with high-throughput processing. Here, we detail an improved protocol, Hi-Plex2, that more effectively enables the robust construction of small-to-medium panel-size libraries while maintaining low cost, simplicity and accuracy benefits of the Hi-Plex platform. Hi-Plex2 was applied to three panels, comprising 291, 740 and 1193 amplicons, targeting genes associated with risk for breast and/or colon cancer. We show substantial reduction of off-target amplification to enable library construction for small-to-medium-sized design panels not possible using the previous Hi-Plex chemistry.

Is RNASEL:p.Glu265* a modifier of early-onset breast cancer risk for carriers of high-risk mutations?

BACKGROUND: Breast cancer risk for BRCA1 and BRCA2 pathogenic mutation carriers is modified by risk factors that cluster in families, including genetic modifiers of risk. We considered genetic modifiers of risk for carriers of high-risk mutations in other breast cancer susceptibility genes. METHODS: In a family known to carry the high-risk mutation PALB2:c.3113G>A (p.Trp1038*), whole-exome sequencing was performed on germline DNA from four affected women, three of whom were mutation carriers. RESULTS: RNASEL:p.Glu265* was identified in one of the PALB2 carriers who had two primary invasive breast cancer diagnoses before 50 years. Gene-panel testing of BRCA1, BRCA2, PALB2 and RNASEL in the Australian Breast Cancer Family Registry identified five carriers of RNASEL:p.Glu265* in 591 early onset breast cancer cases. Three of the five women (60%) carrying RNASEL:p.Glu265* also carried a pathogenic mutation in a breast cancer susceptibility gene compared with 30 carriers of pathogenic mutations in the 586 non-carriers of RNASEL:p.Glu265* (5%) (p < 0.002). Taqman genotyping demonstrated that the allele frequency of RNASEL:p.Glu265* was similar in affected and unaffected Australian women, consistent with other populations. CONCLUSION: Our study suggests that RNASEL:p.Glu265* may be a genetic modifier of risk for early-onset breast cancer predisposition in carriers of high-risk mutations. Much larger case-case and case-control studies are warranted to test the association observed in this report.

FANCM and RECQL genetic variants and breast cancer susceptibility: relevance to South Poland and West Ukraine.

BACKGROUND: FANCM and RECQL have recently been reported as breast cancer susceptibility genes and it has been suggested that they should be included on gene panel tests for breast cancer predisposition. However, the clinical value of testing for mutations in RECQL and FANCM remains to be determined. In this study, we have characterised the spectrum of FANCM and RECQL mutations in women affected with breast or ovarian cancer from South-West Poland and West Ukraine. METHODS: We applied Hi-Plex, an amplicon-based enrichment method for targeted massively parallel sequencing, to screen the coding exons and proximal intron-exon junctions of FANCM and RECQL in germline DNA from unrelated women affected with breast cancer (n = 338) and ovarian cancer (n = 89) from Poland (n = 304) and Ukraine (n = 123). These women were at high-risk of carrying a genetic predisposition to breast and/or ovarian cancer due to a family history and/or early-onset disease. RESULTS: Among 427 women screened, we identified one carrier of the FANCM:c.1972C > T nonsense mutation (0.23%), and two carriers of the frameshift insertion FANCM:c.1491dup (0.47%). None of the variants we observed in RECQL were predicted to be loss-of-function mutations by standard variant effect prediction tools. CONCLUSIONS: Our study of the Polish and Ukrainian populations has identified a carrier frequency of truncating mutations in FANCM consistent with previous reports. Although initial reports suggesting that mutations in RECQL could be associated with increased breast cancer risk included women from Poland and identified the RECQL:c.1667_1667 + 3delAGTA mutation in 0.23-0.35% of breast cancer cases, we did not observe any carriers in our study cohort. Continued screening, both in research and diagnostic settings, will enable the accumulation of data that is needed to establish the clinical utility of including RECQL and FANCM on gene panel tests.

Targeted massively parallel sequencing characterises the mutation spectrum of PALB2 in breast and ovarian cancer cases from Poland and Ukraine.

Loss-of-function germline mutations in the PALB2 gene are associated with an increase of breast cancer risk. The purpose of this study was to characterise the spectrum of PALB2 mutations in women affected with breast or ovarian cancer from South-West Poland and West Ukraine. We applied Hi-Plex, an amplicon-based enrichment method for targeted massively parallel sequencing, to screen the coding exons and proximal intron-exon junctions of PALB2 in germline DNA from unrelated women affected with breast cancer (n = 338) and ovarian cancer (n = 89) from Poland (n = 304) and Ukraine (n = 123). These women were at high-risk of carrying a genetic predisposition to breast and/or ovarian cancer due to a family history and/or early-onset disease. Targeted-sequencing identified two frameshift deletions: PALB2:c.509_510del; p.R170Ifs in three women affected with breast cancer and PALB2:c.172_175del;p.Q60Rfs in one woman affected with ovarian cancer. A number of other previously described missense (some predicted to be damaging by PolyPhen-2 and CADD) and synonymous mutations were also identified in this population. This study is consistent with previous reports that PALB2:c.509_510del and PALB2:c.172_175del are recurrent mutations associated with breast cancer predisposition in Polish women with a family history of the disease. Our study contributes to the accumulating evidence indicating that PALB2 should be included in genetic testing for breast cancer susceptibility in these populations to enhance risk assessment and management of women at high-risk of developing breast cancer. This data could also contribute to ongoing work that is assessing the possible association between ovarian cancer risk and PALB2 mutations for which there is currently no evidence.

Hi-Plex for Simple, Accurate, and Cost-Effective Amplicon-based Targeted DNA Sequencing.

Hi-Plex is a suite of methods to enable simple, accurate, and cost-effective highly multiplex PCR-based targeted sequencing (Nguyen-Dumont et al., Biotechniques 58:33-36, 2015). At its core is the principle of using gene-specific primers (GSPs) to "seed" (or target) the reaction and universal primers to "drive" the majority of the reaction. In this manner, effects on amplification efficiencies across the target amplicons can, to a large extent, be restricted to early seeding cycles. Product sizes are defined within a relatively narrow range to enable high-specificity size selection, replication uniformity across target sites (including in the context of fragmented input DNA such as that derived from fixed tumor specimens (Nguyen-Dumont et al., Biotechniques 55:69-74, 2013; Nguyen-Dumont et al., Anal Biochem 470:48-51, 2015), and application of high-specificity genetic variant calling algorithms (Pope et al., Source Code Biol Med 9:3, 2014; Park et al., BMC Bioinformatics 17:165, 2016). Hi-Plex offers a streamlined workflow that is suitable for testing large numbers of specimens without the need for automation.

Mutation screening of ACKR3 and COPS8 in kidney cancer cases from the CONFIRM study.

An apparently balanced t(2;3)(q37.3;q13.2) translocation that appears to segregate with renal cell carcinoma (RCC) has indicated potential areas to search for the elusive genetic basis of clear cell RCC. We applied Hi-Plex targeted sequencing to analyse germline DNA from 479 individuals affected with clear cell RCC for this breakpoint translocation and genetic variants in neighbouring genes on chromosome 2, ACKR3 and COPS8. While only synonymous variants were found in COPS8, one of the missense variants in ACKR3:c.892C>T, observed in 4/479 individuals screened (0.8%), was predicted likely to damage ACKR3 function. Identification of causal genes for RCC has potential clinical utility, where risk assessment and risk management can offer better outcomes, with surveillance for at-risk relatives and nephron sparing surgery through earlier intervention.

Abridged adapter primers increase the target scope of Hi-Plex.

Previously, we reported Hi-Plex, an amplicon-based method for targeted massively parallel sequencing capable of generating 60 amplicons simultaneously. In further experiments, however, we found our approach did not scale to higher amplicon numbers. Here, we report a modification to the original Hi-Plex protocol that includes the use of abridged adapter oligonucleotides as universal primers (bridge primers) in the initial PCR mixture. Full-length adapter primers (indexing primers) are included only during latter stages of thermal cycling with concomitant application of elevated annealing temperatures. Using this approach, we demonstrate the application of Hi-Plex across a broad range of amplicon numbers (16-plex, 62-plex, 250-plex, and 1003-plex) while preserving the low amount (25 ng) of input DNA required.

Mutation screening of PALB2 in clinically ascertained families from the Breast Cancer Family Registry.

Loss-of-function mutations in PALB2 are associated with an increased risk of breast cancer, with recent data showing that female breast cancer risks for PALB2 mutation carriers are comparable in magnitude to those for BRCA2 mutation carriers. This study applied targeted massively parallel sequencing to characterize the mutation spectrum of PALB2 in probands attending breast cancer genetics clinics in the USA. The coding regions and proximal intron-exon junctions of PALB2 were screened in probands not known to carry a mutation in BRCA1 or BCRA2 from 1,250 families enrolled through familial cancer clinics by the Breast Cancer Family Registry. Mutation screening was performed using Hi-Plex, an amplicon-based targeted massively parallel sequencing platform. Screening of PALB2 was successful in 1,240/1,250 probands and identified nine women with protein-truncating mutations (three nonsense mutations and five frameshift mutations). Four of the 33 missense variants were predicted to be deleterious to protein function by in silico analysis using two different programs. Analysis of tumors from carriers of truncating mutations revealed that the majority were high histological grade, invasive ductal carcinomas. Young onset was apparent in most families, with 19 breast cancers under 50 years of age, including eight under the age of 40 years. Our data demonstrate the utility of Hi-Plex in the context of high-throughput testing for rare genetic mutations and provide additional timely information about the nature and prevalence of PALB2 mutations, to enhance risk assessment and risk management of women at high risk of cancer attending clinical genetic services.

Rare mutations in RINT1 predispose carriers to breast and Lynch syndrome-spectrum cancers.

UNLABELLED: Approximately half of the familial aggregation of breast cancer remains unexplained. A multiple-case breast cancer family exome-sequencing study identified three likely pathogenic mutations in RINT1 (NM_021930.4) not present in public sequencing databases: RINT1 c.343C>T (p.Q115X), c.1132_1134del (p.M378del), and c.1207G>T (p.D403Y). On the basis of this finding, a population-based case-control mutation-screening study was conducted that identified 29 carriers of rare (minor allele frequency < 0.5%), likely pathogenic variants: 23 in 1,313 early-onset breast cancer cases and six in 1,123 frequency-matched controls [OR, 3.24; 95% confidence interval (CI), 1.29-8.17; P = 0.013]. RINT1 mutation screening of probands from 798 multiple-case breast cancer families identified four additional carriers of rare genetic variants. Analysis of the incidence of first primary cancers in families of women carrying RINT1 mutations estimated that carriers were at increased risk of Lynch syndrome-spectrum cancers [standardized incidence ratio (SIR), 3.35; 95% CI, 1.7-6.0; P = 0.005], particularly for relatives diagnosed with cancer under the age of 60 years (SIR, 10.9; 95% CI, 4.7-21; P = 0.0003). SIGNIFICANCE: The work described in this study adds RINT1 to the growing list of genes in which rare sequence variants are associated with intermediate levels of breast cancer risk. Given that RINT1 is also associated with a spectrum of cancers with mismatch repair defects, these findings have clinical applications and raise interesting biological questions.

ROVER variant caller: read-pair overlap considerate variant-calling software applied to PCR-based massively parallel sequencing datasets.

BACKGROUND: We recently described Hi-Plex, a highly multiplexed PCR-based target-enrichment system for massively parallel sequencing (MPS), which allows the uniform definition of library size so that subsequent paired-end sequencing can achieve complete overlap of read pairs. Variant calling from Hi-Plex-derived datasets can thus rely on the identification of variants appearing in both reads of read-pairs, permitting stringent filtering of sequencing chemistry-induced errors. These principles underly ROVER software (derived from Read Overlap PCR-MPS variant caller), which we have recently used to report the screening for genetic mutations in the breast cancer predisposition gene PALB2. Here, we describe the algorithms underlying ROVER and its usage. RESULTS: ROVER enables users to quickly and accurately identify genetic variants from PCR-targeted, overlapping paired-end MPS datasets. The open-source availability of the software and threshold tailorability enables broad access for a range of PCR-MPS users. METHODS: ROVER is implemented in Python and runs on all popular POSIX-like operating systems (Linux, OS X). The software accepts a tab-delimited text file listing the coordinates of the target-specific primers used for targeted enrichment based on a specified genome-build. It also accepts aligned sequence files resulting from mapping to the same genome-build. ROVER identifies the amplicon a given read-pair represents and removes the primer sequences by using the mapping co-ordinates and primer co-ordinates. It considers overlapping read-pairs with respect to primer-intervening sequence. Only when a variant is observed in both reads of a read-pair does the signal contribute to a tally of read-pairs containing or not containing the variant. A user-defined threshold informs the minimum number of, and proportion of, read-pairs a variant must be observed in for a 'call' to be made. ROVER also reports the depth of coverage across amplicons to facilitate the identification of any regions that may require further screening. CONCLUSIONS: ROVER can facilitate rapid and accurate genetic variant calling for a broad range of PCR-MPS users.

Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2.

BACKGROUND: Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. METHODS: Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. RESULTS: We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. CONCLUSIONS: These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings.