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  • Oxford University Press (OUP)  (3)
  • Nelen, Marcel R  (3)
  • 1
    Online Resource
    Online Resource
    Is the $1000 Genome as Near as We Think? A Cost Analysis of Next-Generation Sequencing
    Oxford University Press (OUP) ; 2016
    In:  Clinical Chemistry Vol. 62, No. 11 ( 2016-11-01), p. 1458-1464
    Details
    In: Clinical Chemistry, Oxford University Press (OUP), Vol. 62, No. 11 ( 2016-11-01), p. 1458-1464
    Abstract: The substantial technological advancements in next-generation sequencing (NGS), combined with dropping costs, have allowed for a swift diffusion of NGS applications in clinical settings. Although several commercial parties report to have broken the $1000 barrier for sequencing an entire human genome, a valid cost overview for NGS is currently lacking. This study provides a complete, transparent and up-to-date overview of the total costs of different NGS applications. METHODS Cost calculations for targeted gene panels (TGP), whole exome sequencing (WES) and whole genome sequencing (WGS) were based on the Illumina NextSeq500, HiSeq4000, and HiSeqX5 platforms, respectively. To anticipate future developments, sensitivity analyses are performed. RESULTS Per-sample costs were €1669 for WGS, € 792 for WES and €333 for TGP. To reach the coveted $1000 genome, not only is the long-term and efficient use of the sequencing equipment needed, but also large reductions in capital costs and especially consumable costs are also required. CONCLUSIONS WES and TGP are considerably lower-cost alternatives to WGS. However, this does not imply that these NGS approaches should be preferred in clinical practice, since this should be based on the tradeoff between costs and the expected clinical utility of the approach chosen. The results of the present study contribute to the evaluation of such tradeoffs.
    Type of Medium: Online Resource
    ISSN: 0009-9147 , 1530-8561
    URL: Article
    DOI: 10.1373/clinchem.2016.258632
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2016
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  • 2
    Online Resource
    Online Resource
    Translating Sanger-Based Routine DNA Diagnostics into Generic Massive Parallel Ion Semiconductor Sequencing
    Oxford University Press (OUP) ; 2015
    In:  Clinical Chemistry Vol. 61, No. 1 ( 2015-01-01), p. 154-162
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    In: Clinical Chemistry, Oxford University Press (OUP), Vol. 61, No. 1 ( 2015-01-01), p. 154-162
    Abstract: Dideoxy-based chain termination sequencing developed by Sanger is the gold standard sequencing approach and allows clinical diagnostics of disorders with relatively low genetic heterogeneity. Recently, new next generation sequencing (NGS) technologies have found their way into diagnostic laboratories, enabling the sequencing of large targeted gene panels or exomes. The development of benchtop NGS instruments now allows the analysis of single genes or small gene panels, making these platforms increasingly competitive with Sanger sequencing. METHODS We developed a generic automated ion semiconductor sequencing work flow that can be used in a clinical setting and can serve as a substitute for Sanger sequencing. Standard amplicon-based enrichment remained identical to PCR for Sanger sequencing. A novel postenrichment pooling strategy was developed, limiting the number of library preparations and reducing sequencing costs up to 70% compared to Sanger sequencing. RESULTS A total of 1224 known pathogenic variants were analyzed, yielding an analytical sensitivity of 99.92% and specificity of 99.99%. In a second experiment, a total of 100 patient-derived DNA samples were analyzed using a blind analysis. The results showed an analytical sensitivity of 99.60% and specificity of 99.98%, comparable to Sanger sequencing. CONCLUSIONS Ion semiconductor sequencing can be a first choice mutation scanning technique, independent of the genes analyzed.
    Type of Medium: Online Resource
    ISSN: 0009-9147 , 1530-8561
    URL: Article
    DOI: 10.1373/clinchem.2014.225250
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2015
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  • 3
    Online Resource
    Online Resource
    BRCA Testing by Single-Molecule Molecular Inversion Probes
    Oxford University Press (OUP) ; 2017
    In:  Clinical Chemistry Vol. 63, No. 2 ( 2017-02-01), p. 503-512
    Details
    In: Clinical Chemistry, Oxford University Press (OUP), Vol. 63, No. 2 ( 2017-02-01), p. 503-512
    Abstract: Despite advances in next generation DNA sequencing (NGS), NGS-based single gene tests for diagnostic purposes require improvements in terms of completeness, quality, speed, and cost. Single-molecule molecular inversion probes (smMIPs) are a technology with unrealized potential in the area of clinical genetic testing. In this proof-of-concept study, we selected 2 frequently requested gene tests, those for the breast cancer genes BRCA1 and BRCA2, and developed an automated work flow based on smMIPs. METHODS The BRCA1 and BRCA2 smMIPs were validated using 166 human genomic DNA samples with known variant status. A generic automated work flow was built to perform smMIP-based enrichment and sequencing for BRCA1, BRCA2, and the checkpoint kinase 2 (CHEK2) c.1100del variant. RESULTS Pathogenic and benign variants were analyzed in a subset of 152 previously BRCA-genotyped samples, yielding an analytical sensitivity and specificity of 100%. Following automation, blind analysis of 65 in-house samples and 267 Norwegian samples correctly identified all true-positive variants ( & gt;3000), with no false positives. Consequent to process optimization, turnaround times were reduced by 60% to currently 10–15 days. Copy number variants were detected with an analytical sensitivity of 100% and an analytical specificity of 88%. CONCLUSIONS smMIP-based genetic testing enables automated and reliable analysis of the coding sequences of BRCA1 and BRCA2. The use of single-molecule tags, double-tiled targeted enrichment, and capturing and sequencing in duplo, in combination with automated library preparation and data analysis, results in a robust process and reduces routine turnaround times. Furthermore, smMIP-based copy number variation analysis could make independent copy number variation tools like multiplex ligation-dependent probes amplification dispensable.
    Type of Medium: Online Resource
    ISSN: 0009-9147 , 1530-8561
    URL: Article
    DOI: 10.1373/clinchem.2016.263897
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2017
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