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Translating sanger-based routine DNA diagnostics into generic massive parallel ion semiconductor sequencing.
- Source :
-
Clinical chemistry [Clin Chem] 2015 Jan; Vol. 61 (1), pp. 154-62. Date of Electronic Publication: 2014 Oct 01. - Publication Year :
- 2015
-
Abstract
- Background: 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.<br />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.<br />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.<br />Conclusions: Ion semiconductor sequencing can be a first choice mutation scanning technique, independent of the genes analyzed.<br /> (© 2014 American Association for Clinical Chemistry.)
- Subjects :
- DNA genetics
DNA Mutational Analysis
High-Throughput Nucleotide Sequencing instrumentation
Humans
Molecular Diagnostic Techniques instrumentation
Polymerase Chain Reaction
Reproducibility of Results
Robotics
Semiconductors
Sensitivity and Specificity
Sequence Analysis, DNA instrumentation
DNA analysis
High-Throughput Nucleotide Sequencing methods
Molecular Diagnostic Techniques methods
Sequence Analysis, DNA methods
Subjects
Details
- Language :
- English
- ISSN :
- 1530-8561
- Volume :
- 61
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Clinical chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 25274553
- Full Text :
- https://doi.org/10.1373/clinchem.2014.225250