Prenatal Genome-Wide Sequencing analysis (Exome or Genome) in detecting pathogenic Single Nucleotide Variants in fetal Central Nervous System Anomalies: systematic review and meta-analysis.

Prenatal Exome (pES) or Genome (pGS) Sequencing analysis showed a significant incremental diagnostic yield over karyotype and chromosomal microarray analysis (CMA) in fetal structural anomalies. Optimized indications and detection rates in different fetal anomalies are still under investigation. The aim of this study was to assess the incremental diagnostic yield in prenatally diagnosed Central Nervous System (CNS) anomalies. A systematic review on antenatal CNS anomalies was performed according to PRISMA guidelines, including n = 12 paper, accounting for 428 fetuses. Results were pooled in a meta-analysis fitting a logistic random mixed-effect model. The effect of interest was the incremental diagnostic rate of pES over karyotype/CMA in detecting likely pathogenic/pathogenic Single Nucleotide Variants (SNVs). A further meta-analysis adding the available pGS studies (including diagnostic coding SNVs only) and submeta-analysis on three CNS subcategories were also performed. The pooled incremental diagnostic yield estimate of pES studies was 38% (95% C.I.: [29%;47%]) and 36% (95% C.I.: [28%;45%]) when including diagnostic SNVs of pGS studies. The point estimate of the effect resulted 22% (95% C.I.: [15%;31%]) in apparently isolated anomalies, 33% (95% C.I.: [22%;46%]) in CNS-only related anomalies (≥1) and 46% (95% C.I.: [38%;55%]) in non-isolated anomalies (either ≥ 2 anomalies in CNS, or CNS and extra-CNS). Meta-analysis showed a substantial diagnostic improvement in performing Prenatal Genome-Wide Sequencing analysis (Exome or Genome) over karyotype and CMA in CNS anomalies.

A systematic review of the assessment of the clinical utility of genomic sequencing: Implications of the lack of standard definitions and measures of clinical utility.

PURPOSE: Exome sequencing (ES) and genome sequencing (GS) are diagnostic tests for rare genetic diseases. Studies report clinical utility of ES/GS. The goal of this systematic review is to establish how clinical utility is defined and measured in studies evaluating the impacts of ES/GS results for pediatric patients. METHODS: Relevant articles were identified in PubMed, Medline, Embase, and Web of Science. Eligible studies assessed clinical utility of ES/GS for pediatric patients published before 2021. Other relevant articles were added based on articles' references. Articles were coded to assess definitions and measures of clinical utility. RESULTS: Of 1346 articles, 83 articles met eligibility criteria. Clinical utility was not clearly defined in 19% of studies and 92% did not use an explicit measure of clinical utility. When present, definitions of clinical utility diverged from recommended definitions and varied greatly, from narrow (diagnostic yield of ES/GS) to broad (including decisions about withdrawal of care/palliative care and/or impacts on other family members). CONCLUSION: Clinical utility is used to guide policy and practice decisions about test use. The lack of a standard definition of clinical utility of ES/GS may lead to under- or overestimations of clinical utility, complicating policymaking and raising ethical issues.

Diagnostic uplift through the implementation of short tandem repeat analysis using exome sequencing.

To date, approximately 50 short tandem repeat (STR) disorders have been identified; yet, clinical laboratories rarely conduct STR analysis on exomes. To assess its diagnostic value, we analyzed STRs in 6099 exomes from 2510 families with mostly suspected neurogenetic disorders. We employed ExpansionHunter and REViewer to detect pathogenic repeat expansions, confirming them using orthogonal methods. Genotype-phenotype correlations led to the diagnosis of thirteen individuals in seven previously undiagnosed families, identifying three autosomal dominant disorders: dentatorubral-pallidoluysian atrophy (n = 3), spinocerebellar ataxia type 7 (n = 2), and myotonic dystrophy type 1 (n = 2), resulting in a diagnostic gain of 0.28% (7/2510). Additionally, we found expanded ATXN1 alleles (≥39 repeats) with varying patterns of CAT interruptions in twelve individuals, accounting for approximately 0.19% in the Korean population. Our study underscores the importance of integrating STR analysis into exome sequencing pipeline, broadening the application of exome sequencing for STR assessments.

Comprehensive prenatal diagnostics: Exome versus genome sequencing.

OBJECTIVE: This study aimed to assess the diagnostic yield of prenatal genetic testing using trio whole exome sequencing (WES) and trio whole genome sequencing (WGS) in pregnancies with fetal anomalies by comparing the results with conventional chromosomal microarray (CMA) analysis. METHODS: A total of 40 pregnancies with fetal anomalies or increased nuchal translucency (NT ≥ 5 mm) were included between the 12th and 21st week of gestation. Trio WES/WGS and CMA were performed in all cases. RESULTS: The trio WES/WGS analysis increased the diagnostic yield by 25% in cases with negative CMA results. Furthermore, all six chromosomal aberrations identified by CMA were independently detected by WES/WGS analysis. In total, 16 out of 40 cases obtained a genetic sequence variant, copy number variant, or aneuploidy explaining the phenotype, resulting in an overall WES/WGS diagnostic yield of 40%. WES analysis provided a more reliable identification of mosaic sequence variants than WGS because of its higher sequencing depth. CONCLUSIONS: Prenatal WES/WGS proved to be powerful diagnostic tools for fetal anomalies, surpassing the diagnostic yield of CMA. They have the potential to serve as standalone methods for prenatal diagnosis. The study highlighted the limitations of WGS in accurately detecting mosaic variants, which is particularly relevant when analyzing chorionic villus samples.

System analysis of the sequencing quality of human whole exome samples on BGI NGS platform.

Human exome sequencing is a classical method used in most medical genetic applications. The leaders in the field are the manufacturers of enrichment kits based on hybridization of cRNA or cDNA biotinylated probes specific for a genomic region of interest. Recently, the platforms manufactured by the Chinese company MGI Tech have become widespread in Europe and Asia. The reliability and quality of the obtained data are already beyond any doubt. However, only a few kits compatible with these sequencers can be used for such specific tasks as exome sequencing. We developed our own solution for library pre-capture pooling and exome enrichment with Agilent probes. In this work, using a set of the standard benchmark samples from the Platinum Genome collection, we demonstrate that the qualitative and quantitative parameters of our protocol which we called "RSMU_exome" exceed those of the MGI Tech kit. Our protocol allows for identifying more SNV and indels, generates fewer PCR duplicates, enables pooling of more samples in a single enrichment procedure, and requires less raw data to obtain results comparable with the MGI Tech's protocol. The cost of our protocol is also lower than that of MGI Tech's solution.

Challenges in variant interpretation in prenatal exome sequencing.

The use of exome sequencing (ES) in the prenatal setting improves the diagnostic yield of genetic testing for fetuses with ultrasound anomalies. However, while the purpose of ES is to explain the fetal phenotype, secondary or incidental findings unrelated to the observed abnormalities might be detected. Recently, requests for ES in fetuses with no sonographic abnormalities have been increasing, raising serious ethical and medico-legal concerns. Variant interpretation is complex even in the postnatal setting and performing broad genomic data analyses in the prenatal setting presents additional dilemmas. This article discusses challenges and questions related to prenatal ES, including variant interpretation of incidental findings in cases of indicated prenatal ES, as well as in situations where ES is performed in asymptomatic fetuses.

Interest of exome sequencing trio-like strategy based on pooled parental DNA for diagnosis and translational research in rare diseases.

BACKGROUND: Exome sequencing (ES) has become the most powerful and cost-effective molecular tool for deciphering rare diseases with a diagnostic yield approaching 30%-40% in solo-ES and 50% in trio-ES. We applied an innovative parental DNA pooling method to reduce the parental sequencing cost while maintaining the diagnostic yield of trio-ES. METHODS: We pooled six (Agilent-CRE-v2-100X) or five parental DNA (TWIST-HCE-70X) aiming to detect allelic balance around 8-10% for heterozygous status. The strategies were applied as second-tier (74 individuals after negative solo-ES) and first-tier approaches (324 individuals without previous ES). RESULTS: The allelic balance of parental-pool variants was around 8.97%. Sanger sequencing uncovered false positives in 1.5% of sporadic variants. In the second-tier approach, we evaluated than two thirds of the Sanger validations performed after solo-ES (41/59-69%) would have been saved if the parental-pool segregations had been available from the start. The parental-pool strategy identified a causative diagnosis in 18/74 individuals (24%) in the second-tier and in 116/324 individuals (36%) in the first-tier approaches, including 19 genes newly associated with human disorders. CONCLUSIONS: Parental-pooling is an efficient alternative to trio-ES. It provides rapid segregation and extension to translational research while reducing the cost of parental and Sanger sequencing.

Toward best practice in cancer mutation detection with whole-genome and whole-exome sequencing.

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

Benefits of Exome Sequencing in Children with Suspected Isolated Hearing Loss.

PURPOSE: Hearing loss is characterized by an extensive genetic heterogeneity and remains a common disorder in children. Molecular diagnosis is of particular benefit in children, and permits the early identification of clinically-unrecognized hearing loss syndromes, which permits effective clinical management and follow-up, including genetic counselling. METHODS: We performed whole-exome sequencing with the analysis of a panel of 189 genes associated with hearing loss in a prospective cohort of 61 children and 9 adults presenting mainly with isolated hearing loss. RESULTS: The overall diagnostic rate using exome sequencing was 47.2% (52.5% in children; 22% in adults). In children with confirmed molecular results, 17/32 (53.2%) showed autosomal recessive inheritance patterns, 14/32 (43.75%) showed an autosomal dominant condition, and one case had X-linked hearing loss. In adults, the two patients showed an autosomal dominant inheritance pattern. Among the 32 children, 17 (53.1%) had nonsyndromic hearing loss and 15 (46.7%) had syndromic hearing loss. One adult was diagnosed with syndromic hearing loss and one with nonsyndromic hearing loss. The most common causative genes were STRC (5 cases), GJB2 (3 cases), COL11A1 (3 cases), and ACTG1 (3 cases). CONCLUSIONS: Exome sequencing has a high diagnostic yield in children with hearing loss and can reveal a syndromic hearing loss form before other organs/systems become involved, allowing the surveillance of unrecognized present and/or future complications associated with these syndromes.

Utility of fetal whole exome sequencing in the etiological evaluation and outcome of nonimmune hydrops fetalis.

INTRODUCTION: Nonimmune hydrops fetalis (NIHF) has varied etiology. We assessed the etiological spectrum and evaluated the utility of fetal whole exome sequencing (fWES) for the diagnosis of NIHF. METHODS: In this prospective cohort study, we evaluated antenatally diagnosed fetuses with NIHF between July 2018 and December 2019 according to the routine diagnostic algorithm. Fetuses that remained undiagnosed after routine NIHF workup were subjected to fetal chromosomal microarray and/or WES. Pregnancies were followed up for clinical outcomes. RESULTS: Of the 45 fetuses, consanguinity and recurrent hydrops fetalis were observed in 13.3% (6/45) and 28.8% (13/45), respectively. Overall, an etiological diagnosis was possible in 75.5% (34/45) of fetuses, while the cause remained unknown in 24.4% (11/45). A genetic etiology was identified in 46.6% (21/45): aneuploidy and monogenic disorders in 28.8% (13/45) and 17.8% (8/45), respectively. fWES on 19 fetuses detected disease-causing variants in 42.1% (8/19). Nine novel variants were detected in RAPSN, ASCC1, NEB, PKD1L1, GUSB, and PIEZO1. Only 8.8% (4/45) of the cohort survived without morbidity. CONCLUSIONS: This study describes the etiological spectrum and the disease-causing variants in an Indian cohort of hydropic fetuses.

Whole-exome sequencing increases the diagnostic rate for prenatal fetal structural anomalies.

BACKGROUND: Prenatal whole-exome sequencing (WES) is becoming increasingly used when karyotype and microarray tests are not diagnostic of fetal malformations. Although the value of WES clearly emerges in terms of higher diagnostic rates, the limitations of prenatal phenotyping together with the counseling challenges for variants of uncertain significance and incidental results suggest that the routine application of prenatal WES is not yet easy. METHODS: Structurally abnormal fetuses with a mean gestational age of 24 weeks (range 13-38 weeks) were recruited from the Chong Qing Health Center for Women and Children. We performed a retrospective WES investigation in 85 fetuses, using DNA from amniotic fluid (66 samples, 77.6%), umbilical cord blood (10 samples, 11.8%), and fetal tissues (9 samples, 10.6%). Parental DNA was extracted from peripheral blood. RESULTS: Molecular diagnosis was obtained in 16 of the 85 fetuses (18.8%). According to the variant segregation mode and family history, 7 fetuses (43.75%) were affected by an autosomal dominant condition (6 variants were de novo and 1 variant was inherited from an unknowingly affected father), 7 fetuses (43.75%) had an autosomal recessive syndrome always associated with compound heterozygosity, and 2 fetuses (12.5%) had an X-linked condition (one mother was a carrier). In addition, the highest diagnostic rate was observed in fetuses with multisystem abnormalities (38.9%, 7/18). A variant of uncertain significance was detected in 16 samples (18.8%, 16/85). CONCLUSION: Our study confirms that prenatal WES is an efficient tool for studying fetal abnormalities, although further improvements are needed to establish stronger fetal genotype-phenotype correlations.

Combined exome sequencing and deep phenotyping in highly selected fetuses with skeletal dysplasia during the first and second trimesters improves diagnostic yield.

OBJECTIVE: To investigate the genetic etiology of skeletal dysplasia in highly selected fetuses during the first and second trimesters using deep phenotyping and exome sequencing (ES). METHOD: Fetuses with short femurs were identified using the established prenatal diagnostic approach. A multidisciplinary team reviewed fetal phenotypic information (prenatal ultrasound findings, fetal postmortem, and radiographs) in a cohort of highly selected fetuses with skeletal dysplasia during the first and second trimesters. The affected families underwent multiplatform genetic tests. RESULTS: Of the 27 affected fetuses, 21 (77.8%) had pathogenic or potential pathogenic variations in the following genes: COL1A1, FGFR3, COL2A1, COL1A2, FLNB, DYNC2LI1, and TRIP11. Two fetuses had compound heterozygous mutations in DYNC2LI1 and TRIP11, respectively, and the other 19 carried de novo autosomal dominant variants. Novel variants were identified in COL1A1, COL2A1, COL1A2, DYNC2LI1, and TRIP11 in 11 fetuses. We also included the first description of the phenotype of odontochondrodysplasia in a prenatal setting. CONCLUSIONS: ES or panel sequencing offers a high diagnostic yield for fetal skeletal dysplasia during the first and second trimesters. Comprehensive and complete phenotypic information is indispensable for genetic analysis and the expansion of genotype-phenotype correlations in fetal skeletal abnormalities.

Utility of clinical exome sequencing in the evaluation of neonates with suspected genetic condition - An observational study from tertiary neonatal care unit in South India.

OBJECTIVES: To study the utility of clinical exome sequencing (CES) using next generation sequencing (NGS) in evaluating neonates with suspected genetic conditions. METHODS: This is an observational study conducted in a tertiary care neonatal unit. We included neonates with suspected genetic conditions, for whom CES were done either by direct sampling or from stored DNA. Data was collected from the Sri Ramachandra centre of excellence in perinatal health (SCOPE) case records of 2016-2019. Yield of CES, percentage of pathogenic, non-pathogenic and variant of uncertain significance (VUS) and associated disorders were studied. RESULTS: CES was done in 36 neonates. Variants were detected in 78% (28/36). However, significant variants with clinical correlation were present in 20 (56%) babies. Test was carried out from the stored sample in 10 (28%) babies. Mean turn-around time was 39 ± 7 days. Specialist was involved in 1 and treatment changes were done in 5 neonates. Five out of 8 VUS were clinically correlating. Inborn errors of metabolism were the commonest (60%). Two VUS were ascertained as likely pathogenic after parental segregation analysis. CONCLUSION: CES has a definite role in evaluation of suspected genetic conditions for diagnosis and prognostication. It also helps scientific society to build in additional evidence so that the "VUS" could be asserted as "likely pathogenic" . Our experience reiterates the importance of storing and archiving DNA of the affected child.

Dealing with uncertain results from chromosomal microarray and exome sequencing in the prenatal setting: An international cross-sectional study with healthcare professionals.

OBJECTIVES: To conduct qualitative interviews with healthcare providers working in different countries to understand their experiences of dealing with uncertain results from prenatal chromosome microarray analysis (CMA) and exome sequencing (ES). METHODS: Semi-structured interviews with 31 healthcare providers who report or return prenatal CMA and/or ES results (clinicians, genetic counsellors and clinical scientists) in six countries with differing healthcare systems; Australia (4), Denmark (5), Netherlands (6), Singapore (4), Sweden (6) and United Kingdom (6). The topic guide explored the main sources of uncertainty and their management. RESULTS: There was variation in reporting practices both between and across countries for variants of uncertain significance, however, there was broad agreement on reporting practices for incidental findings. There was also variation in who decides what results are reported (clinical scientists or clinicians). Technical limitations and lack of knowledge (to classify variants and of prenatal phenotypes) were significant challenges, as were turnaround times and lack of guidelines. CONCLUSION: Health professionals around the globe are dealing with similar sources of uncertainty, but managing them in different ways, Continued dialogue with international colleagues on ways of managing uncertain results is important to compare and contrast the benefits and limitations of the different approaches.

The diagnostic efficacy of exome data analysis using fixed neurodevelopmental gene lists: Implications for prenatal setting.

OBJECTIVE: Laboratories performing prenatal exome sequencing (ES) frequently limit analysis to predetermined gene lists. We used a diagnostic postnatal ES cohort to assess how many of the genes diagnosed are not included in a number of select fixed lists used for prenatal diagnosis. METHODS: Of 601 postnatal ES tests, pathogenic variants related to neurodevelopmental disorders were detected in 138 probands. We evaluated if causative genes were present in the following: (1) Developmental Disorders Genotype-Phenotype database list, (2) a commercial laboratory list for prenatal ES, (3) the PanelApp fetal anomalies panel, and (4) a published list used for prenatal diagnosis by ES (Prenatal Assessment of Genomes and Exomes study). RESULTS: The percentages of cases where the diagnosed gene was not included in the selected four lists were; 11.6%, 17.24%, 23.2%, and 10.9%, respectively. In 13/138 (9.4%) cases, the causative gene was not included in any of the lists; in 4/13 (∼30%) cases noninclusion was explained by a relatively recent discovery of gene-phenotype association. CONCLUSIONS: A significant number of genes related to neurocognitive phenotypes are not included in some of the lists used for prenatal ES data interpretation. These are not only genes related to recently discovered disorders, but also genes with well-established gene-phenotype.

Impact of concurrency on the performance of a whole exome sequencing pipeline.

BACKGROUND: Current high-throughput technologies-i.e. whole genome sequencing, RNA-Seq, ChIP-Seq, etc.-generate huge amounts of data and their usage gets more widespread with each passing year. Complex analysis pipelines involving several computationally-intensive steps have to be applied on an increasing number of samples. Workflow management systems allow parallelization and a more efficient usage of computational power. Nevertheless, this mostly happens by assigning the available cores to a single or few samples' pipeline at a time. We refer to this approach as naive parallel strategy (NPS). Here, we discuss an alternative approach, which we refer to as concurrent execution strategy (CES), which equally distributes the available processors across every sample's pipeline. RESULTS: Theoretically, we show that the CES results, under loose conditions, in a substantial speedup, with an ideal gain range spanning from 1 to the number of samples. Also, we observe that the CES yields even faster executions since parallelly computable tasks scale sub-linearly. Practically, we tested both strategies on a whole exome sequencing pipeline applied to three publicly available matched tumour-normal sample pairs of gastrointestinal stromal tumour. The CES achieved speedups in latency up to 2-2.4 compared to the NPS. CONCLUSIONS: Our results hint that if resources distribution is further tailored to fit specific situations, an even greater gain in performance of multiple samples pipelines execution could be achieved. For this to be feasible, a benchmarking of the tools included in the pipeline would be necessary. It is our opinion these benchmarks should be consistently performed by the tools' developers. Finally, these results suggest that concurrent strategies might also lead to energy and cost savings by making feasible the usage of low power machine clusters.

Evidence to Support the Clinical Utility of Prenatal Exome Sequencing in Evaluation of the Fetus with Congenital Anomalies: Scientific Impact Paper No. 64 [February] 2021.

Structural differences (congenital anomalies) in the makeup of the baby's heart, brain and other organs are found on antenatal ultrasound scans in up to 3% of pregnancies. These often have a genetic cause, arising because of changes in the chromosomes (which store our genetic material) or the DNA code that make up the genes. The more differences a baby has the more likely the risk of underlying genetic disease. If a structural difference is found, parents are usually offered a genetic test, which may be carried out on cells taken either from the placenta (chorionic villous sampling) or the fluid surrounding the baby (amniocentesis). At the moment, these cells are only tested for changes in the chromosomes and are only able to reveal the underlying cause in about 40% of unborn babies. Prenatal exome sequencing (ES) is a new genetic test, which, when combined with testing the DNA of both parents can find changes in the baby's genetic code. If a DNA change is found that can explain the structural changes seen on ultrasound, specific information about the underlying diagnosis can be given to the parents. Having this information can help parents make important decisions about their ongoing pregnancy, as well as help doctors to care for the mother and baby. Finding a genetic change can also help to understand how the condition has arisen and whether it might happen again in another pregnancy. It may also be possible to test for the genetic condition in future pregnancies. Although prenatal ES is an exciting new way to improve diagnosis rates for structural differences, it has some challenges. While the test is very detailed, it may not always find a genetic explanation and sometimes the results are difficult to interpret. For example, genetic changes can be found where their significance for the pregnancy is unclear. More recently, two studies have now shown that prenatal ES can find a genetic diagnosis in at least 10% of pregnancies with structural differences where standard chromosome testing has been negative. This paper reviews these studies, along with earlier evidence on ES and provides clinicians with guidance for future practice.

Efficient and flexible Integration of variant characteristics in rare variant association studies using integrated nested Laplace approximation.

Rare variants are thought to play an important role in the etiology of complex diseases and may explain a significant fraction of the missing heritability in genetic disease studies. Next-generation sequencing facilitates the association of rare variants in coding or regulatory regions with complex diseases in large cohorts at genome-wide scale. However, rare variant association studies (RVAS) still lack power when cohorts are small to medium-sized and if genetic variation explains a small fraction of phenotypic variance. Here we present a novel Bayesian rare variant Association Test using Integrated Nested Laplace Approximation (BATI). Unlike existing RVAS tests, BATI allows integration of individual or variant-specific features as covariates, while efficiently performing inference based on full model estimation. We demonstrate that BATI outperforms established RVAS methods on realistic, semi-synthetic whole-exome sequencing cohorts, especially when using meaningful biological context, such as functional annotation. We show that BATI achieves power above 70% in scenarios in which competing tests fail to identify risk genes, e.g. when risk variants in sum explain less than 0.5% of phenotypic variance. We have integrated BATI, together with five existing RVAS tests in the 'Rare Variant Genome Wide Association Study' (rvGWAS) framework for data analyzed by whole-exome or whole genome sequencing. rvGWAS supports rare variant association for genes or any other biological unit such as promoters, while allowing the analysis of essential functionalities like quality control or filtering. Applying rvGWAS to a Chronic Lymphocytic Leukemia study we identified eight candidate predisposition genes, including EHMT2 and COPS7A.

Detection of copy-number variations from NGS data using read depth information: a diagnostic performance evaluation.

The detection of copy-number variations (CNVs) from NGS data is underexploited as chip-based or targeted techniques are still commonly used. We assessed the performances of a workflow centered on CANOES, a bioinformatics tool based on read depth information. We applied our workflow to gene panel (GP) and whole-exome sequencing (WES) data, and compared CNV calls to quantitative multiplex PCR of short fluorescent fragments (QMSPF) or array comparative genomic hybridization (aCGH) results. From GP data of 3776 samples, we reached an overall positive predictive value (PPV) of 87.8%. This dataset included a complete comprehensive QMPSF comparison of four genes (60 exons) on which we obtained 100% sensitivity and specificity. From WES data, we first compared 137 samples with aCGH and filtered comparable events (exonic CNVs encompassing enough aCGH probes) and obtained an 87.25% sensitivity. The overall PPV was 86.4% following the targeted confirmation of candidate CNVs from 1056 additional WES. In addition, our CANOES-centered workflow on WES data allowed the detection of CNVs with a resolution of single exons, allowing the detection of CNVs that were missed by aCGH. Overall, switching to an NGS-only approach should be cost-effective as it allows a reduction in overall costs together with likely stable diagnostic yields. Our bioinformatics pipeline is available at: https://gitlab.bioinfo-diag.fr/nc4gpm/canoes-centered-workflow .