title: Analysis of the whole mitochondrial genome: translation of the Ion Torrent Personal Genome Machine system to the diagnostic bench?
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.
authors.affiliation: 1] Center for Medical Genetics, UZ Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium [2] Research Group Reproduction and Genetics, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
authors.forename: Sara
authors.initials: S
authors.lastname: Seneca
authors.affiliation: Research Group Reproduction and Genetics, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
authors.forename: Kim
authors.initials: K
authors.lastname: Vancampenhout
authors.affiliation: Department of Pediatrics, Division of Neurology and Metabolism, University Hospital Ghent, Ghent University, Ghent, Belgium.
authors.forename: Rudy
authors.initials: R
authors.lastname: Van Coster
authors.affiliation: Department of Pediatrics, Division of Neurology and Metabolism, University Hospital Ghent, Ghent University, Ghent, Belgium.
authors.forename: Joél
authors.initials: J
authors.lastname: Smet
authors.affiliation: 1] Center for Medical Genetics, UZ Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium [2] Research Group Reproduction and Genetics, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
authors.forename: Willy
authors.initials: W
authors.lastname: Lissens
authors.affiliation: Department of Pediatrics, Division of Neurology and Metabolism, University Hospital Ghent, Ghent University, Ghent, Belgium.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Vanlander
authors.affiliation: Department of Pediatrics, Division of Neurology and Metabolism, University Hospital Ghent, Ghent University, Ghent, Belgium.
authors.forename: Boel
authors.identifier: 0000000194034401
authors.initials: B
authors.lastname: De Paepe
authors.affiliation: Department of Pediatric Neurology, UZ Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
authors.forename: An
authors.initials: A
authors.lastname: Jonckheere
authors.affiliation: 1] Center for Medical Genetics, UZ Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium [2] Research Group Reproduction and Genetics, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
authors.forename: Katrien
authors.initials: K
authors.lastname: Stouffs
authors.affiliation: 1] Research Group Reproduction and Genetics, Vrije Universiteit Brussel (VUB), Brussels, Belgium [2] Department of Pediatric Neurology, UZ Brussel, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
authors.forename: Linda
authors.initials: L
authors.lastname: De Meirleir
mesh_terms: D005820:Genetic Testing / Q000379:methods / Q000592:standards; D014644:Genetic Variation; D054629:Genome, Mitochondrial; D023281:Genomics / Q000379:methods / Q000592:standards; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards; D006801:Humans; D028361:Mitochondrial Diseases / Q000175:diagnosis / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017384:Sequence Deletion