Dale Muzzey

Understanding the Basics of NGS: From Mechanism to Variant Calling.

Identifying disease-causing mutations in DNA has long been the goal of genetic medicine. In the last decade, the toolkit for discovering DNA variants has undergone rapid evolution: mutations that were historically discovered by analog approaches like Sanger sequencing and multiplex ligation-dependent probe amplification (“MLPA”) can now be decoded from a digital signal with next-generation sequencing (“NGS”). Given the explosive growth of NGS-based tests in the clinic, it is of the utmost importance that medical practitioners have a fundamental understanding of the newest NGS methodologies. To that end, here we provide a very basic overview of how NGS works, with particular emphasis on the close resemblance between the underlying chemistry of Sanger sequencing and NGS. Using a pair of simple analogies, we develop an intuitive framework for understanding how high-confidence detection of single-nucleotide polymorphisms, indels, and large deletions/duplications is possible with NGS alone.

Systematic design and comparison of expanded carrier screening panels

PurposeThe recent growth in pan-ethnic expanded carrier screening (ECS) has raised questions about how such panels might be designed and evaluated systematically. Design principles for ECS panels might improve clinical detection of at-risk couples and facilitate objective discussions of panel choice.MethodsGuided by medical-society statements, we propose a method for the design of ECS panels that aims to maximize the aggregate and per-disease sensitivity and specificity across a range of Mendelian disorders considered serious by a systematic classification scheme. We evaluated this method retrospectively using results from 474,644 de-identified carrier screens. We then constructed several idealized panels to highlight strengths and limitations of different ECS methodologies.ResultsBased on modeled fetal risks for “severe” and “profound” diseases, a commercially available ECS panel (Counsyl) is expected to detect 183 affected conceptuses per 100,000 US births. A screen’s sensitivity is greatly impacted by two factors: (i) the methodology used (e.g., full-exon sequencing finds more affected conceptuses than targeted genotyping) and (ii) the detection rate of the screen for diseases with high prevalence and complex molecular genetics (e.g., fragile X syndrome).ConclusionThe described approaches enable principled, quantitative evaluation of which diseases and methodologies are appropriate for pan-ethnic expanded carrier screening.

Noninvasive prenatal screening at low fetal fraction: comparing whole‐genome sequencing and single‐nucleotide polymorphism methods

Performance of noninvasive prenatal screening (NIPS) methodologies when applied to low fetal fraction samples is not well established. The single‐nucleotide polymorphism (SNP) method fails samples below a predetermined fetal fraction threshold, whereas some laboratories employing the whole‐genome sequencing (WGS) method report aneuploidy calls for all samples. Here, the performance of the two methods was compared to determine which approach actually detects more fetal aneuploidies.

A Data-Driven Evaluation of the Size and Content of Expanded Carrier Screening Panels

Purpose The American College of Obstetricians and Gynecologists (ACOG) proposed seven criteria for expanded carrier screening (ECS) panel design. To ensure that screening for a condition is sufficiently sensitive to identify carriers and reduce residual risk of non-carriers, one criterion requires a per-condition carrier rate greater than 1-in-100. However, it is unestablished whether this threshold corresponds with a loss in clinical detection. The impact of the proposed panel-design criteria on at-risk couple detection warrants data-driven evaluation. Methods Carrier rates and at-risk couple rates were calculated in 56,281 patients who underwent a 176-condition ECS and evaluated for panels satisfying various criteria. Condition-specific clinical detection rate was estimated via simulation. Results Different interpretations of the 1-in-100 criterion have variable impact: a compliant panel would include between 3 and 38 conditions, identify 11%-81% fewer at-risk couples, and detect 36%-79% fewer carriers than a 176-condition panel. If the carrier-rate threshold must be exceeded in all ethnicities, ECS panels would lack prevalent conditions like cystic fibrosis. Simulations suggest that clinical detection rate remains >84% for conditions with carrier rates as low as 1-in-1000. Conclusions The 1-in-100 criterion limits at-risk couple detection and should be reconsidered.

Clinical Impact and Cost-Effectiveness of a 176-Condition Expanded Carrier Screen

Purpose Carrier screening identifies couples at high risk for conceiving offspring affected with serious heritable conditions. Minimal screening guidelines mandate testing for cystic fibrosis and spinal muscular atrophy, but expanded carrier screening (ECS) assesses reproductive risk for hundreds of conditions simultaneously. Although medical societies consider ECS an acceptable practice, the health economics of ECS remain incompletely characterized. Methods The clinical impact and cost-effectiveness of a 176-condition ECS panel were investigated using a decision-tree model comparing minimal screening and ECS in a preconception setting. Carrier rates from >50,000 patients informed disease-incidence estimates, while cost and life-years-lost data were aggregated from the literature and a cost-of-care database. Model robustness was evaluated using one-way and probabilistic sensitivity analyses. Results For every 100,000 pregnancies, 300 are predicted to be affected by ECS-panel conditions, which, on average, individually incur

Software-assisted manual review of clinical NGS data: an alternative to routine Sanger sequencing confirmation with equivalent results in >15,000 hereditary cancer screens

1,300,000 in lifetime costs and increase mortality by 26 undiscounted life-years on average. Relative to minimal screening, ECS reduces the affected-birth rate and is cost-effective (i.e., <

Clinical utility of expanded carrier screening: results-guided actionability and outcomes

50,000 incremental cost per life-year), findings robust to reasonable model-parameter perturbation. Conclusion ECS is predicted to reduce the population burden of Mendelian disease in a cost-effective manner compared to many other common medical interventions.

Detecting clinically actionable variants in the 3′ exons of PMS2 via a reflex workflow based on equivalent hybrid capture of the gene and its pseudogene

Clinical genomic tests increasingly utilize a next generation sequencing (NGS) platform due in part to the high fidelity of variant calls, yet rare errors are still possible. In hereditary cancer screening (HCS), failure to correct such errors could have serious consequences for patients, who may follow an unwarranted screening or surgical-management path. It has been suggested that routine orthogonal confirmation via Sanger sequencing is required to verify NGS results, especially low-confidence positives with depressed allele balance (<30% of the alternate allele). We evaluated whether an alternative method of confirmation—software-assisted manual call review—performed comparably to Sanger confirmation in screening of >15,000 HCS samples. Licensed reviewers manually inspected both raw and processed data at the batch-, sample-, and variant-level, including raw NGS read pileups. Of ambiguous variant calls with <30% allele balance (1,719 total calls at 42 unique sites), manual call review classified >99% (1,711) as true positives (enriched for long indels and homopolymers) or true negatives (often conspicuous NGS artifacts), with the remaining <1% (8) being mosaic. Critically, results from manual review and retrospective Sanger sequencing were concordant for samples selected from all ambiguous sites. We conclude that the confirmation required for high confidence in NGS-based germline testing can manifest in different ways: a trained NGS expert operating platform-tailored review software achieves quality comparable to routine Sanger confirmation.

Development and validation of an expanded carrier screen that optimizes sensitivity via full-exon sequencing and panel-wide copy-number-variant identification

PurposeExpanded carrier screening (ECS) informs couples of their risk of having offspring affected by certain genetic conditions. Limited data exists assessing the actions and reproductive outcomes of at-risk couples (ARCs). We describe the impact of ECS on planned and actual pregnancy management in the largest sample of ARCs studied to date.MethodsCouples who elected ECS and were found to be at high risk of having a pregnancy affected by at least one of 176 genetic conditions were invited to complete a survey about their actions and pregnancy management.ResultsThree hundred ninety-one ARCs completed the survey. Among those screened before becoming pregnant, 77% planned or pursued actions to avoid having affected offspring. Among those screened during pregnancy, 37% elected prenatal diagnostic testing (PNDx) for that pregnancy. In subsequent pregnancies that occurred in both the preconception and prenatal screening groups, PNDx was pursued in 29%. The decision to decline PNDx was most frequently based on the fear of procedure-related miscarriage, as well as the belief that termination would not be pursued in the event of a positive diagnosis.ConclusionECS results impacted couples’ reproductive decision-making and led to altered pregnancy management that effectively eliminates the risk of having affected offspring.

Validation of an Expanded Carrier Screen that Optimizes Sensitivity via Full-Exon Sequencing and Panel-wide Copy Number Variant Identification

BackgroundHereditary cancer screening (HCS) for germline variants in the 3′ exons of PMS2, a mismatch repair gene implicated in Lynch syndrome, is technically challenging due to homology with its pseudogene PMS2CL. Sequences of PMS2 and PMS2CL are so similar that next-generation sequencing (NGS) of short fragments—common practice in multigene HCS panels—may identify the presence of a variant but fail to disambiguate whether its origin is the gene or the pseudogene. Molecular approaches utilizing longer DNA fragments, such as long-range PCR (LR-PCR), can definitively localize variants in PMS2, yet applying such testing to all samples can have logistical and economic drawbacks.MethodsTo address these drawbacks, we propose and characterize a reflex workflow for variant discovery in the 3′ exons of PMS2. We cataloged the natural variation in PMS2 and PMS2CL in 707 samples and designed hybrid-capture probes to enrich the gene and pseudogene with equal efficiency. For PMS2 exon 11, NGS reads were aligned, filtered using gene-specific variants, and subject to standard diploid variant calling. For PMS2 exons 12–15, the NGS reads were permissively aligned to PMS2, and variant calling was performed with the expectation of observing four alleles (i.e., tetraploid calling). In this reflex workflow, short-read NGS identifies potentially reportable variants that are then subject to disambiguation via LR-PCR-based testing.ResultsApplying short-read NGS screening to 299 HCS samples and cell lines demonstrated >99% analytical sensitivity and >99% analytical specificity for single-nucleotide variants (SNVs) and short insertions and deletions (indels), as well as >96% analytical sensitivity and >99% analytical specificity for copy-number variants. Importantly, 92% of samples had resolved genotypes from short-read NGS alone, with the remaining 8% requiring LR-PCR reflex.ConclusionOur reflex workflow mitigates the challenges of screening in PMS2 and serves as a guide for clinical laboratories performing multigene HCS. To facilitate future exploration and testing of PMS2 variants, we share the raw and processed LR-PCR data from commercially available cell lines, as well as variant frequencies from a diverse patient cohort.