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Purpose The percentage of a maternal cell-free DNA (cfDNA) sample that is fetal-derived (the fetal fraction; FF) is a key driver of the sensitivity and specificity of noninvasive prenatal screening (NIPS). On certain NIPS platforms, >20% of women with high body mass index (and >5% overall) receive a test failure due to low FF (

Purpose Carrier status associates strongly with genetic ancestry, yet current carrier screening guidelines recommend testing for a limited set of conditions based on a patient’s self-reported ethnicity. Ethnicity, which can reflect both genetic ancestry and cultural factors (e.g., religion), may be imperfectly known or communicated by patients. We sought to quantitatively assess the efficacy and equity with which ethnicity-based carrier screening captures recessive disease risk. Methods For 93,419 individuals undergoing a 96-gene expanded carrier screen (ECS), correspondence was assessed among carrier status, self-reported ethnicity, and a dual-component genetic ancestry (e.g., 75% African/25% European) calculated from sequencing data. Results Self-reported ethnicity was an imperfect indicator of genetic ancestry, with 9% of individuals having >50% genetic ancestry from a lineage inconsistent with self-reported ethnicity. Limitations of self-reported ethnicity led to missed carriers in at-risk populations: for 10 ECS conditions, patients with intermediate genetic ancestry backgrounds—who did not self-report the associated ethnicity—had significantly elevated carrier risk. Finally, for 7 of the 16 conditions included in current screening guidelines, most carriers were not from the population the guideline aimed to serve. Conclusion Substantial and disproportionate risk for recessive disease is not detected when carrier screening is based on ethnicity, leading to inequitable reproductive care.

Background: Noninvasive prenatal screening (NIPS) utilization has grown dramatically and is increasingly offered to the general population by nongenetic specialists. Web-based technologies and telegenetic services offer potential solutions for efficient results delivery and genetic counseling. Introduction: All major guidelines recommend patients with both negative and positive results be counseled. The main objective of this study was to quantify patient utilization, motivation for posttest counseling, and satisfaction of a technology platform designed for large-scale dissemination of NIPS results. Methods: The technology platform provided general education videos to patients, results delivery through a secure portal, and access to telegenetic counseling through phone. Automatic results delivery to patients was sent only to patients with screen-negative results. For patients with screen-positive results, either the ordering provider or a board-certified genetic counselor contacted the patient directly through phone to communicate the test results and provide counseling. Results: Over a 39-month period, 67,122 NIPS results were issued through the platform, and 4,673 patients elected genetic counseling consultations; 95.2% (n = 4,450) of consultations were for patients receiving negative results. More than 70% (n = 3,370) of consultations were on-demand rather than scheduled. A positive screen, advanced maternal age, family history, previous history of a pregnancy with a chromosomal abnormality, and other high-risk pregnancy were associated with the greatest odds of electing genetic counseling. By combining web education, automated notifications, and telegenetic counseling, we implemented a service that facilitates results disclosure for ordering providers. Discussion: This automated results delivery platform illustrates the use of technology in managing large-scale disclosure of NIPS results. Further studies should address effectiveness and satisfaction among patients and providers in greater detail. Conclusions: These data demonstrate the capability to deliver NIPS results, education, and counseling—congruent with professional society management guidelines—to a large population.

We evaluated whether there is an association between β-globin (HBB) pathogenic variants and fetal fraction (FF), and whether the association has a clinically relevant impact on non-invasive prenatal screening (NIPS).A whole-genome sequencing NIPS laboratory database was retrospectively queried for women who underwent NIPS and carrier screening of both HBB and the α-globin genes (HBA1/HBA2). Women affected with either condition were excluded from the study, yielding a cohort size of 15,853. A "corrected FF" was obtained via multivariable linear regression adjusted for the systematic impacts of maternal age, gestational age and BMI. Corrected FF distributions of HBB and HBA1/HBA2 carriers were each compared to non-carriers using the Kolmogorov-Smirnov test.In this cohort, 291 women were carriers for HBB alone, and 1016 were carriers for HBA1/HBA2 alone. The HBB carriers had a lower corrected FF when compared to non-carriers (p 0.0001). There was no difference in corrected FF among carriers and non-carriers of HBA1/HBA2.Carriers of pathogenic variants in the HBB gene, but not the HBA1/HBA2 genes, are more likely to have lower FF when compared to women with structurally normal hemoglobin. This decrease in FF could result in an elevated test-failure rate if FF thresholds were used.

Objective Women with high body mass index (BMI) tend to have reduced fetal fraction (FF) during cell‐free DNA‐based noninvasive prenatal screening (NIPS), causing test failure rates up to 24.3% and prompting guidelines that recommend aneuploidy screening other than NIPS for patients with significant obesity. Because alternatives to NIPS are only preferable if they perform better, we compared the respective sensitivities at different BMI levels of traditional aneuploidy screening and a customized whole‐genome sequencing NIPS. Method The relationship between FF, aneuploidy, and BMI was quantified from 58 105 patients screened with a customized NIPS that does not fail samples because of low FF alone. Expected analytical sensitivity as a function of aneuploidy and BMI (eg, trisomy 18 sensitivity when BMI = 35) was determined by scaling the BMI‐ and aneuploidy‐specific FF distribution by the FF‐ and aneuploidy‐specific sensitivity calculated from empirically informed simulations. Results Across all classes of obesity and assuming zero FF‐related test failures, analytical sensitivity for the investigated NIPS exceeded that of traditional aneuploidy screening for trisomies 13, 18, and 21. Conclusion Relative to traditional aneuploidy screening, a customized NIPS with high accuracy at low FF and a low test‐failure rate is a superior screening option for women with high BMI., What's already known about this topic? Women with high body mass index (BMI) often receive a test failure on noninvasive prenatal screening (NIPS) because of low fetal fraction (FF).The American College of Medical Genetics and Genomics recommends offering traditional aneuploidy screening to patients with “significant obesity.”NIPS offerings differ in their efficacy at low FF. What does this study add? Irrespective of BMI and without FF‐based test failures, it is possible for a customized NIPS to provide all women with accurate prenatal screening.

Expanded carrier screening (ECS) panels that use next‐generation sequencing aim to identify pathogenic variants in coding and clinically relevant non‐coding regions of hundreds of genes, each associated with a serious recessive condition. ECS has established analytical validity and clinical utility, meaning that variants are accurately identified and pathogenic variants tend to alter patients' clinical management, respectively. However, the clinical validity of ECS, that is, correct discernment of whether an identified variant is indeed pathogenic, has only been shown for single conditions, not for panels. Here, we evaluate the clinical validity of a >170‐condition ECS panel by assessing concordance between >12 000 variant interpretations classified with guideline‐based criteria to their corresponding per‐variant combined classifications in ClinVar. We observe 99% concordance at the level of unique variants. A more clinically relevant frequency‐weighted analysis reveals that fewer than 1 in 500 patients are expected to receive a report with a variant that has a discordant classification. Importantly, gene‐level concordance is not diminished for rare ECS conditions, suggesting that large panels do not balloon the panel‐wide false‐positive rate. Finally, because ECS is intended to serve all reproductive‐age couples, we show that classification of novel variants is feasible and scales predictably for a large population.

Purpose: Expanded 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. Methods: Couples 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. Results: Three 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. Conclusions: ECS results impacted couples’ reproductive decision-making and led to altered pregnancy management that effectively eliminates the risk of having affected offspring.

Clinical genomic tests increasingly use a next-generation sequencing (NGS) platform due in part to the high fidelity of variant calls, yet rare errors are still possible. In germline DNA screening, 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 by Sanger sequencing is required to verify NGS results, especially low-confidence positives with depressed allele fraction ( 15,000 samples. Licensed reviewers manually inspected both raw and processed data at the batch, sample, and variant levels, including raw NGS read pileups. Of ambiguous variant calls with 99% (n = 1701) as true positives (enriched for long insertions or deletions and homopolymers) or true negatives (often conspicuous NGS artifacts), with the remaining

Purpose The American College of Obstetricians and Gynecologists (ACOG) and the American College of Medical Genetics and Genomics (ACMG) suggest carrier screening panel design criteria intended to ensure meaningful results. This study used a data-driven approach to interpret the criteria to identify guidelines-consistent panels. Methods Carrier frequencies in >460,000 individuals across 11 races/ethnicities were used to assess carrier frequency. Other criteria were interpreted on the basis of published data. A total of 176 conditions were then evaluated. Stringency thresholds were set as suggested by ACOG and/or ACMG or by evaluating conditions already recommended by ACOG and ACMG. Results Forty and 75 conditions had carrier frequencies of ≥1 in 100 and ≥1 in 200, respectively; 175 had a well-defined phenotype; and 165 met at least 1 severity criterion and had an onset early in life. Thirty-seven conditions met conservative thresholds, including a carrier frequency of ≥1 in 100, and 74 conditions met permissive thresholds, including a carrier frequency of ≥1 in 200; thus, both were identified as guidelines-consistent panels. Conclusion Clear panel design criteria are needed to ensure quality and consistency among carrier screening panels. Evidence-based analyses of criteria resulted in the identification of guidelines-consistent panels of 37 and 74 conditions.

PurposeThe percentage of a maternal cell-free DNA (cfDNA) sample that is fetal-derived (the fetal fraction; FF) is a key driver of the sensitivity and specificity of noninvasive prenatal screening (NIPS). On certain NIPS platforms, >20% of women with high body-mass index (and >5% overall) receive a test failure due to low FF (MethodsA scalable fetal-fraction amplification (FFA) technology was analytically validated on 1,264 samples undergoing whole-genome sequencing (WGS)-based NIPS. All samples were tested with and without FFA.ResultsZero samples had FFConclusionsFFA transforms low-FF samples into high-FF samples. By combining FFA with WGS-based NIPS, a single round of NIPS can provide nearly all women with confident results about the broad range of potential fetal chromosomal abnormalities across the genome.

Background Noninvasive prenatal screening (NIPS) of common aneuploidies using cell-free DNA from maternal plasma is part of routine prenatal care and is widely used in both high-risk and low-risk patient populations. High specificity is needed for clinically acceptable positive predictive values. Maternal copy-number variants (mCNVs) have been reported as a source of false-positive aneuploidy results that compromises specificity. Methods We surveyed the mCNV landscape in 87,255 patients undergoing NIPS. We evaluated both previously reported and novel algorithmic strategies for mitigating the effects of mCNVs on the screen’s specificity. Further, we analyzed the frequency, length, and positional distribution of CNVs in our large dataset to investigate the curation of novel fetal microdeletions, which can be identified by NIPS but are challenging to interpret clinically. Results mCNVs are common, with 65% of expecting mothers harboring an autosomal CNV spanning more than 200 kb, underscoring the need for robust NIPS analysis strategies. By analyzing empirical and simulated data, we found that general, outlier-robust strategies reduce the rate of mCNV-caused false positives but not as appreciably as algorithms specifically designed to account for mCNVs. We demonstrate that large-scale tabulation of CNVs identified via routine NIPS could be clinically useful: together with the gene density of a putative microdeletion region, we show that the region’s relative tolerance to duplications versus deletions may aid the interpretation of microdeletion pathogenicity. Conclusions Our study thoroughly investigates a common source of NIPS false positives and demonstrates how to bypass its corrupting effects. Our findings offer insight into the interpretation of NIPS results and inform the design of NIPS algorithms suitable for use in screening in the general obstetric population. Electronic supplementary material The online version of this article (10.1186/s12920-018-0410-6) contains supplementary material, which is available to authorized users.

Objective 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. Methods Computational models were parameterized with up-to-date published data and used to compare the performance of the two methods at calling common fetal trisomies (T21, T18, T13) at low fetal fractions. Furthermore, clinical experience data were reviewed to determine aneuploidy detection rates based on compliance with recent invasive screening recommendations. Results The SNP method's performance is dependent on the origin of the trisomy, and is lowest for the most common trisomies (maternal M1 nondisjunction). Consequently, the SNP method cannot maintain acceptable performance at fetal fractions below ~3%. In contrast, the WGS method maintains high specificity independent of fetal fraction and has >80% sensitivity for trisomies in low fetal fraction samples. Conclusion The WGS method will detect more aneuploidies below the fetal fraction threshold at which many labs issue a no-call result, avoiding unnecessary invasive procedures. © 2017 Counsyl Inc. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

PurposeDespite strong association between genetic ancestry and carrier status, current carrier-screening guidelines recommend testing for a limited set of conditions based on a patient’s self-reported ethnicity, which conflates genetic and cultural factors.Materials and MethodsFor 93,419 individuals undergoing a 96-gene expanded carrier screen (ECS), correspondence was assessed among carrier status, self-reported ethnicity, and a dual-component genetic ancestry (e.g., 75% African/25% European) calculated from sequencing data.ResultsSelf-reported ethnicity was an imperfect indicator of genetic ancestry, with 9% of individuals having >50% genetic ancestry from a lineage inconsistent with self-reported ethnicity. Self-reported ethnicity-based carrier-screening guidelines are incomplete, as several conditions not included in guidelines had similarly strong correlation between carrier rate and genetic ancestry as conditions included in screening guidelines. Limitations of self-reported ethnicity led to missed carriers in at-risk populations: for 10 ECS conditions, patients with intermediate genetic ancestry backgrounds—who did not self-report the associated ethnicity—had significantly elevated carrier risk. Finally, for seven of the 16 conditions included in current screening guidelines, most carriers were not from the population the guideline aimed to serve.ConclusionTo provide equitable reproductive care, guidelines should discontinue the use of ethnicity as a basis for determining which patients are appropriate for carrier screening and instead recommend pan-ethnic ECS.

Objective To describe our clinical experience across the entire range of fetal‐fraction (FF) measurements of a non‐invasive prenatal screen (NIPS) that uses whole‐ genome sequencing (WGS). Methods We analyzed retrospectively results from 58 105 singleton pregnancies that underwent NIPS on a customized WGS platform during an 8‐month period and assessed clinical test performance for trisomy 21, trisomy 18 and trisomy 13. Pregnancy outcomes were sought for all screen‐positive patients and for 18% of screen‐negative patients. As differences in outcome‐collection response rates could artificially impact test‐performance calculations, we computed inferred sensitivity, specificity, positive predictive values (PPV) and negative predictive values adjusted for ascertainment bias. Results The screening test yielded a result for 99.9% (n = 58 048) of patients, meaning that approximately 1 in 1000 patients received a test failure (i.e. test failure rate = 0.1%). Of pregnancies with a test result, 572 (1%) screened positive for one of the common aneuploidies (362 for trisomy 21, 142 for trisomy 18 and 68 for trisomy 13). Informative outcomes were received for 237 (41.4%) patients with a screen‐positive result and 3258 (5.7%) of those with a screen‐negative result. In the full cohort, inferred sensitivities for trisomy 21, trisomy 18 and trisomy 13 were 99.7%, 96.8% and 94.3%, respectively, and PPVs were 93.1%, 85.2% and 48.4%, respectively. If a FF threshold of 4% had been employed to guard against false negatives, calculated sensitivities for the three aneuploidies would not have changed significantly, yet, importantly, the overall test‐failure rate would have increased to 6.6% (n = 3829), impacting 1 in 15 women. Conclusions Our clinical experience demonstrates that a customized WGS‐based NIPS without a FF threshold achieves high accuracy while maintaining a low test‐failure rate of 0.1%. As such, alternative strategies to ensure high accuracy of detection of common aneuploidies in samples with low FF (such as redraw after test failure, redrawing at a later gestational age, risk scoring based on FF) are not necessary for this screening approach. © 2019 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology., Linked article: There is a comment on this article by Jelsema et al. Click here to view the Correspondence.

Background Pathogenic variants in HEXA that impair β‐hexosaminidase A (Hex A) enzyme activity cause Tay‐Sachs Disease (TSD), a severe autosomal‐recessive neurodegenerative disorder. Hex A enzyme analysis demonstrates near‐zero activity in patients affected with TSD and can also identify carriers, whose single functional copy of HEXA results in reduced enzyme activity relative to noncarriers. Although enzyme testing has been optimized and widely used for carrier screening in Ashkenazi Jewish (AJ) individuals, it has unproven sensitivity and specificity in a pan‐ethnic population. The ability to detect HEXA variants via DNA analysis has evolved from limited targeting of a few ethnicity‐specific variants to next‐generation sequencing (NGS) of the entire coding region coupled with interpretation of any discovered novel variants. Methods We combined results of enzyme testing, retrospective computational analysis, and variant reclassification to estimate the respective clinical performance of TSD screening via enzyme analysis and NGS. We maximized NGS accuracy by reclassifying variants of uncertain significance and compared to the maximum performance of enzyme analysis estimated by calculating ethnicity‐specific frequencies of variants known to yield false‐positive or false‐negative enzyme results (e.g., pseudodeficiency and B1 alleles). Results In both AJ and non‐AJ populations, the estimated clinical sensitivity, specificity, and positive predictive value were higher by NGS than by enzyme testing. The differences were significant for all comparisons except for AJ clinical sensitivity, where NGS exceeded enzyme testing, but not significantly. Conclusions Our results suggest that performance of an NGS‐based TSD carrier screen that interrogates the entire coding region and employs novel variant interpretation exceeds that of Hex A enzyme testing, warranting a reconsideration of existing guidelines.

Purpose Medical society guidelines recommend offering genotyping-based cystic fibrosis (CF) carrier screening to pregnant women or women considering pregnancy. We assessed the performance of sequencing-based CF screening relative to genotyping, in terms of analytical validity, clinical validity, clinical impact, and clinical utility. Methods Analytical validity was assessed using orthogonal confirmation and reference samples. Clinical validity was evaluated using the CFTR2 database. Clinical impact was assessed using ~100,000 screened patients. Three screening strategies were compared: genotyping 23 guideline-recommended variants (“CF23”), sequencing all coding bases in CFTR (“NGS”), and sequencing with large copy-number variant (CNV) identification (“NGS + CNV”). Clinical utility was determined via self-reported actions of at-risk couples (ARCs). Results Analytical accuracy of NGS + CNV was 100% for SNVs, indels, and CNVs; interpretive clinical specificity relative to CFTR2 was 99.5%. NGS + CNV detected 58 ARCs, 18 of whom would have gone undetected with CF23 alone. Most ARCs (89% screened preconceptionally, 56% prenatally) altered pregnancy management, and no significant differences were observed between ARCs with or without at least one non-CF23 variant. Conclusion Modern NGS and variant interpretation enable accurate sequencing-based CF screening. Limiting screening to 23 variants does not improve analytical validity, clinical validity, or clinical utility, but does fail to detect approximately 30% (18/58) of ARCs.

PurposeThe 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.MethodsCarrier 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.ResultsDifferent 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.ConclusionsThe 1-in-100 criterion limits at-risk couple detection and should be reconsidered.

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.

Background: As noninvasive prenatal screening usage grows in the general obstetrics setting, proper patient education on the screen’s benefits and limitations is needed. Objective: Describe the use of a technology platform designed for large-scale dissemination of noninvasive prenatal screening information and results. Study Design: The technology platform functioned as follows: Patients were emailed a link to an noninvasive prenatal screening general-education video upon laboratory receipt of a test requisition. Providers were then notified upon availability of patients’ results. If noninvasive prenatal screening results were negative, the patient was sent an automated email with instructions to access results through a secure portal where she could watch tailored informational videos, request “on-demand” or scheduled genetic counseling, or decline any further services. If genetic counseling was elected, a summary of the session was sent to the ordering provider and patient upon completion. If noninvasive prenatal screening results were positive, either the ordering provider or a board-certified genetic counselor contacted the patient directly to communicate test results and provide counseling. The number and type of results issued through the platform, the number and type of genetic counseling consultations completed, and factors associated with requesting laboratory-delivered genetic counseling were tracked and analyzed for a 39-month period. Results: Over the study period, 67,122 noninvasive prenatal screening results were issued through the platform, and 4,673 patients elected genetic counseling consultations; 95.2% (n=4,450) of consultations were for patients receiving negative results. Over 70% (n= 3,370) of consultations were on-demand rather than scheduled. Median consultation time was 14 minutes for positive results and six minutes for negative results. A positive screen, advanced maternal age, family history, previous history of a pregnancy with a chromosomal abnormality, and other high-risk pregnancy were associated with the greatest odds of electing laboratory-delivered genetic counseling.Conclusions: By combining web education, automated notifications, and genetic counseling, we implemented a service that effectively facilitates results disclosure for ordering providers. These data demonstrate the capability to deliver noninvasive prenatal screening results, education, and counseling—congruent with management guidelines—to a large population, which is imperative to quality care as uptake increases.

Clinical genomic tests increasingly use a next-generation sequencing (NGS) platform due in part to the high fidelity of variant calls, yet rare errors are still possible. In germline DNA screening, 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 by Sanger sequencing is required to verify NGS results, especially low-confidence positives with depressed allele fraction (30% of alternate allele). We evaluated whether an alternative method of confirmation-software-assisted manual call review-performed comparably with Sanger confirmation in15,000 samples. Licensed reviewers manually inspected both raw and processed data at the batch, sample, and variant levels, including raw NGS read pileups. Of ambiguous variant calls with30% allele fraction (1707 total calls at 38 unique sites), manual call review classified99% (n = 1701) as true positives (enriched for long insertions or deletions and homopolymers) or true negatives (often conspicuous NGS artifacts), with the remaining1% (n = 6) being mosaic. Critically, results from software-assisted 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 with routine Sanger confirmation.

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, 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 fraction (15,000 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 99% (1,701) as true positives (enriched for long insertions or deletions (“indels”) and homopolymers) or true negatives (often conspicuous NGS artifacts), with the remaining

BACKGROUND By identifying pathogenic variants across hundreds of genes, expanded carrier screening (ECS) enables prospective parents to assess the risk of transmitting an autosomal recessive or X-linked condition. Detection of at-risk couples depends on the number of conditions tested, the prevalence of the respective diseases, and the screen's analytical sensitivity for identifying disease-causing variants. Disease-level analytical sensitivity is often METHODS We present an analytical validation and preliminary clinical characterization of a 235-gene sequencing-based ECS with full coverage across coding regions, targeted assessment of pathogenic noncoding variants, panel-wide CNV calling, and specialized assays for technically challenging genes. Next-generation sequencing, customized bioinformatics, and expert manual call review were used to identify single-nucleotide variants, short insertions and deletions, and CNVs for all genes except FMR1 and those whose low disease incidence or high technical complexity precluded novel variant identification or interpretation. RESULTS Screening of 36859 patients' blood or saliva samples revealed the substantial impact on fetal disease-risk detection attributable to novel CNVs (9.19% of risk) and technically challenging conditions (20.2% of risk), such as congenital adrenal hyperplasia. Of the 7498 couples screened, 335 were identified as at risk for an affected pregnancy, underscoring the clinical importance of the test. Validation of our ECS demonstrated >99% analytical sensitivity and >99% analytical specificity. CONCLUSIONS Validated high-fidelity identification of different variant types—especially for diseases with complicated molecular genetics—maximizes at-risk couple detection.

The core goal of cell-free DNA-based prenatal testing (at its introduction called “NIPT”) is to provide minimally invasive, clinically accurate, and financially accessible screening for fetal chromosomal aneuploidies in the early stages of pregnancy. This goal poses certain important constraints: minimal invasiveness means the test must operate from analytes in a maternal blood sample; clinical accuracy requires that even small enrichments in placenta-derived cell-free DNA (cfDNA) must be detectable and attributable to specific genomic regions; and financial accessibility is possible only with modern, scalable genomic technologies. Next-generation sequencing satisfies each of these constraints and consequently is the chosen platform for the majority of cfDNA-based prenatal testing offerings. This chapter provides a general overview of how NGS technology works and focuses particularly on its application to cfDNA prenatal testing.

The primary challenge of cell-free DNA (“cfDNA”) based prenatal testing (commonly called noninvasive prenatal testing or NIPT) is to identify fetal chromosomal anomalies from maternal plasma samples, where maternal cfDNA molecules far outnumber their fetal counterparts. Further complicating this task is the fact that the relative amounts of fetal and maternal fragments (i.e., the fetal fraction) varies across pregnancies and gestational ages, reaching as high as 40% and as low as 1%. Therefore, to achieve maximal sensitivity and specificity, a cfDNA test's analysis pipeline must determine both the fetal fraction (“FF”) of a sample and the likelihood of an aneuploid fetus. Several elegant molecular and bioinformatic strategies have emerged to infer FF and ploidy status reliably and at low cost, yielding a range of cfDNA test offerings that hold in common a remarkably high clinical sensitivity that has driven the rapid adoption of this screening modality. In this chapter, the analysis fundamentals are described for three cfDNA test platforms: whole-genome sequencing (WGS), single-nucleotide polymorphism (SNP), and microarray. The methodologies of aneuploidy identification, FF inference, and microdeletion detection are discussed in turn. These topics provide a basic understanding of how cell-free DNA based prenatal testing works in routine pregnancies and lay the groundwork for a discussion at the end of the chapter about edge cases that will themselves become common as use of the screening grows.

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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.

PurposeBy identifying pathogenic variants across hundreds of genes, expanded carrier screening (ECS) enables prospective parents to assess risk of transmitting an autosomal recessive or X-linked condition. Detection of at-risk couples depends on the number of conditions tested, the diseases’ respective prevalences, and the screen’s sensitivity for identifying disease-causing variants. Here we present an analytical validation of a 235-gene sequencing-based ECS with full coverage across coding regions, targeted assessment of pathogenic noncoding variants, panel-wide copy-number-variant (CNV) calling, and customized assays for technically challenging genes.MethodsNext-generation sequencing, a customized bioinformatics pipeline, and expert manual call review were used to identify single-nucleotide variants, short insertions and deletions, and CNVs for all genes except FMR1 and those whose low disease incidence or high technical complexity precludes novel variant identification or interpretation. Variant calls were compared to reference and orthogonal data.ResultsValidation of our ECS data demonstrated >99% analytical sensitivity and >99% specificity. A preliminary assessment of 15,177 patient samples reveals the substantial impact on fetal disease-risk detection attributable to novel CNV calling (13.9% of risk) and technically challenging conditions (15.5% of risk), such as congenital adrenal hyperplasia.ConclusionValidated, high-fidelity identification of different variant types—especially in diseases with complicated molecular genetics—maximizes at-risk couple detection.