Abstract
Next-generation sequencing (NGS), an innovative sequencing technology that enables the successful analysis of numerous gene sequences in a massive parallel sequencing approach, has revolutionized the field of molecular biology. Although NGS was introduced in a rather recent past, the technology has already demonstrated its potential and effectiveness in many research projects, and is now on the verge of being introduced into the diagnostic setting of routine laboratories to delineate the molecular basis of genetic disease in undiagnosed patient samples. We tested a benchtop device on retrospective genomic DNA (gDNA) samples of controls and patients with a clinical suspicion of a mitochondrial DNA disorder. This Ion Torrent Personal Genome Machine platform is a high-throughput sequencer with a fast turnaround time and reasonable running costs. We challenged the chemistry and technology with the analysis and processing of a mutational spectrum composed of samples with single-nucleotide substitutions, indels (insertions and deletions) and large single or multiple deletions, occasionally in heteroplasmy. The output data were compared with previously obtained conventional dideoxy sequencing results and the mitochondrial revised Cambridge Reference Sequence (rCRS). We were able to identify the majority of all nucleotide alterations, but three false-negative results were also encountered in the data set. At the same time, the poor performance of the PGM instrument in regions associated with homopolymeric stretches generated many false-positive miscalls demanding additional manual curation of the data.
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Acknowledgements
We thank patients and physicians for their contribution to this research project. We also want to thank S Bessems, G Van Dyck, D Daneels, B Caljon and D Croes for their contribution to this work. This project was supported by grants from the Fonds voor Wetenschappelijk Onderzoek (FWO) G.0.200, the ‘Association Belge contre les Maladies Neuro-Musculaires (ABMM)’ and the Vrije Universiteit Brussel (VUB, Brussel, Belgium) with reference nos. OZR1928 and OZRMETH3.
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Seneca, S., Vancampenhout, K., Van Coster, R. et al. Analysis of the whole mitochondrial genome: translation of the Ion Torrent Personal Genome Machine system to the diagnostic bench?. Eur J Hum Genet 23, 41–48 (2015). https://doi.org/10.1038/ejhg.2014.49
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DOI: https://doi.org/10.1038/ejhg.2014.49
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