Improved molecular diagnosis by the detection of exonic deletions with target gene capture and deep sequencing

Feng, Yanming; Chen, David; Wang, Guoli; Zhang, Victor Wei; Wong, Lee‐Jun C.

doi:10.1038/gim.2014.80

Cited by 67 publications

(84 citation statements)

References 30 publications

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“…A copy-number variation detection algorithm developed in house was used to evaluate the presence of large heterozygous exonic deletions. 10 HGVS guidelines were followed for mutation nomenclature (http://www.hgvs.org/mutnomen/). Nucleotide numbering uses c.1 as the A nucleotide of the ATG translation initiation codon in the reference cDNA sequence.…”

Section: Methodsmentioning

confidence: 99%

Capture-based high-coverage NGS: a powerful tool to uncover a wide spectrum of mutation types

Wang

Zhang

et al. 2016

Genetics in Medicine

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Purpose: Next-generation sequencing (NGS) has been widely applied to clinical diagnosis. Target-gene capture followed by deep sequencing provides unbiased enrichment of the target sequences, which not only accurately detects single-nucleotide variations (SNVs) and small insertion/deletions (indels) but also provides the opportunity for the identification of exonic copy-number variants (CNVs) and large genomic rearrangements. Method:Capture NGS has the ability to easily detect SNVs and small indels. However, genomic changes involving exonic deletions/ duplications and chromosomal rearrangements require more careful analysis of captured NGS data. Misaligned raw sequence reads may be more than just bad data. Some mutations that are difficult to detect are filtered by the preset analytical parameters. "Loose" filtering and alignment conditions were used for thorough analysis of the misaligned NGS reads. Additionally, using an in-house algorithm, NGS coverage depth was thoroughly analyzed to detect CNVs. Results:Using real examples, this report underscores the importance of the accessibility to raw sequence data and manual review of suspicious sequence regions to avoid false-negative results in the clinical application of NGS. Assessment of the NGS raw data generated by the use of loose filtering parameters identified several sequence aberrations, including large indels and genomic rearrangements. Furthermore, NGS coverage depth analysis identified homozygous and heterozygous deletions involving single or multiple exons. Conclusion:Our results demonstrate the power of deep NGS in the simultaneous detection of point mutations and intragenic exonic deletion in one comprehensive step.

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Section: Methodsmentioning

confidence: 99%

Capture-based high-coverage NGS: a powerful tool to uncover a wide spectrum of mutation types

Wang

Zhang

et al. 2016

Genetics in Medicine

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“…However, currently available NGS‐based detection methods are all afflicted by a high false positive rate, especially for small intragenic deletions and duplications, which increases the cost associated with orthogonal confirmation (Feng et al. 2015). …”

Section: Introductionmentioning

confidence: 99%

Next generation sequencing‐based copy number analysis reveals low prevalence of deletions and duplications in 46 genes associated with genetic cardiomyopathies

Ceyhan‐Birsoy

Pugh

Bowser

et al. 2015

Molec Gen & Gen Med

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BackgroundDiagnostic testing for genetic cardiomyopathies has undergone dramatic changes in the last decade with next generation sequencing (NGS) expanding the number of genes that can be interrogated simultaneously. Exon resolution copy number analysis is increasingly incorporated into routine diagnostic testing via cytogenomic arrays and more recently via NGS. While NGS is an attractive option for laboratories that have no access to array platforms, its higher false positive rate requires weighing the added cost incurred by orthogonal confirmation against the magnitude of the increase in diagnostic yield. Although copy number variants (CNVs) have been reported in various cardiomyopathy genes, their contribution has not been systematically studied.MethodsWe performed single exon resolution NGS‐based deletion/duplication analysis for up to 46 cardiomyopathy genes in >1400 individuals with cardiomyopathies including HCM, DCM, ARVC, RCM, and LVNC.Results and ConclusionClinically significant deletions and duplications were identified in only 9 of 1425 (0.63%) individuals. The majority of those (6/9) represented intragenic events. We conclude that the added benefit of exon level deletion/duplication analysis is low for currently known cardiomyopathy genes and may not outweigh the increased cost and complexity of incorporating it into routine diagnostic testing for these disorders.

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“…We ensured 100% coverage by fill in the low or no coverage regions such as ORF15 of RPGR gene, or regions with high GC content and/or homologous sequences, by Sanger sequencing of specifically amplified PCR products. In addition, the average coverage depth is consistently at ~1000X per base, that allows the detection of heterozygous exonic CNVs [20]. The CNV detection algorithm has been integrated into the routine analytical pipeline for clinical application that has been validated in parallel with exon targeted oligonucleotide array CGH [20].…”

Section: Discussionmentioning

confidence: 99%

“…In addition, the average coverage depth is consistently at ~1000X per base, that allows the detection of heterozygous exonic CNVs [20]. The CNV detection algorithm has been integrated into the routine analytical pipeline for clinical application that has been validated in parallel with exon targeted oligonucleotide array CGH [20]. These unique features of our panel-based NGS approach greatly improve clinical sensitivity.…”

Section: Discussionmentioning

confidence: 99%

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Retinal Diseases Caused by Mutations in Genes Not Specifically Associated with the Clinical Diagnosis

Wang

Feng

et al. 2016

PLoS ONE

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PurposeWhen seeking a confirmed molecular diagnosis in the research setting, patients with one descriptive diagnosis of retinal disease could carry pathogenic variants in genes not specifically associated with that description. However, this event has not been evaluated systematically in clinical diagnostic laboratories that validate fully all target genes to minimize false negatives/positives.MethodsWe performed targeted next-generation sequencing analysis on 207 ocular disease-related genes for 42 patients whose DNA had been tested negative for disease-specific panels of genes known to be associated with retinitis pigmentosa, Leber congenital amaurosis, or exudative vitreoretinopathy.ResultsPathogenic variants, including single nucleotide variations and copy number variations, were identified in 9 patients, including 6 with variants in syndromic retinal disease genes and 3 whose molecular diagnosis could not be distinguished easily from their submitted clinical diagnosis, accounting for 21% (9/42) of the unsolved cases.ConclusionOur study underscores the clinical and genetic heterogeneity of retinal disorders and provides valuable reference to estimate the fraction of clinical samples whose retinal disorders could be explained by genes not specifically associated with the corresponding clinical diagnosis. Our data suggest that sequencing a larger set of retinal disorder related genes can increase the molecular diagnostic yield, especially for clinically hard-to-distinguish cases.

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Improved molecular diagnosis by the detection of exonic deletions with target gene capture and deep sequencing

Cited by 67 publications

References 30 publications

Capture-based high-coverage NGS: a powerful tool to uncover a wide spectrum of mutation types

Capture-based high-coverage NGS: a powerful tool to uncover a wide spectrum of mutation types

Next generation sequencing‐based copy number analysis reveals low prevalence of deletions and duplications in 46 genes associated with genetic cardiomyopathies

Retinal Diseases Caused by Mutations in Genes Not Specifically Associated with the Clinical Diagnosis

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