title: Translating sanger-based routine DNA diagnostics into generic massive parallel ion semiconductor sequencing.
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.
  
  
  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.
authors.affiliation: Department of Human Genetics and.
authors.forename: Adinda
authors.initials: A
authors.lastname: Diekstra
authors.affiliation: Department of Human Genetics and.
authors.forename: Ermanno
authors.initials: E
authors.lastname: Bosgoed
authors.affiliation: Department of Human Genetics and.
authors.forename: Alwin
authors.initials: A
authors.lastname: Rikken
authors.affiliation: Department of Human Genetics and.
authors.forename: Bart
authors.initials: B
authors.lastname: van Lier
authors.affiliation: Department of Human Genetics and.
authors.forename: Erik-Jan
authors.initials: EJ
authors.lastname: Kamsteeg
authors.affiliation: Department of Human Genetics and.
authors.forename: Marloes
authors.initials: M
authors.lastname: Tychon
authors.affiliation: Department of Human Genetics and.
authors.forename: Ronny C
authors.initials: RC
authors.lastname: Derks
authors.affiliation: Department of Human Genetics and.
authors.forename: Ronald A
authors.initials: RA
authors.lastname: van Soest
authors.affiliation: Department of Human Genetics and.
authors.forename: Arjen R
authors.initials: AR
authors.lastname: Mensenkamp
authors.affiliation: Department of Human Genetics and Donders Center for Neurosciences, Radboud university medical center, Nijmegen, the Netherlands.
authors.forename: Hans
authors.initials: H
authors.lastname: Scheffer
authors.affiliation: Department of Human Genetics and.
authors.forename: Kornelia
authors.initials: K
authors.lastname: Neveling
authors.affiliation: Department of Human Genetics and marcel.nelen@radboudumc.nl.
authors.forename: Marcel R
authors.initials: MR
authors.lastname: Nelen
mesh_terms: D004247:DNA / Q000032:analysis* / Q000235:genetics; D004252:DNA Mutational Analysis; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012371:Robotics; D012666:Semiconductors; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*
chemical_list: D004247:DNA