title: Clinical validation of targeted next-generation sequencing for inherited disorders.
abstract: BACKGROUND
  Although next-generation sequencing (NGS) can revolutionize molecular diagnostics, several hurdles remain in the implementation of this technology in clinical laboratories.
  
  
  OBJECTIVE
  To validate and implement an NGS panel for genetic diagnosis of more than 100 inherited diseases, such as neurologic conditions, congenital hearing loss and eye disorders, developmental disorders, nonmalignant diseases treated by hematopoietic cell transplantation, familial cancers, connective tissue disorders, metabolic disorders, disorders of sexual development, and cardiac disorders. The diagnostic gene panels ranged from 1 to 54 genes with most of panels containing 10 genes or fewer.
  
  
  METHODS
  We used a liquid hybridization-based, target-enrichment strategy to enrich 10 067 exons in 568 genes, followed by NGS with a HiSeq 2000 sequencing system (Illumina, San Diego, California).
  
  
  RESULTS
  We successfully sequenced 97.6% (9825 of 10 067) of the targeted exons to obtain a minimum coverage of 20× at all bases. We demonstrated 100% concordance in detecting 19 pathogenic single-nucleotide variations and 11 pathogenic insertion-deletion mutations ranging in size from 1 to 18 base pairs across 18 samples that were previously characterized by Sanger sequencing. Using 4 pairs of blinded, duplicate samples, we demonstrated a high degree of concordance (>99%) among the blinded, duplicate pairs.
  
  
  CONCLUSIONS
  We have successfully demonstrated the feasibility of using the NGS platform to multiplex genetic tests for several rare diseases and the use of cloud computing for bioinformatics analysis as a relatively low-cost solution for implementing NGS in clinical laboratories.
authors.affiliation: From the Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis (Drs Yohe, Wilson, Zhou, Spears, and Thyagarajan); the University of Minnesota Genomics Center (Mr Hauge, Mss Bunjer and Deshpande, and Dr Beckman); the Molecular Diagnostics Laboratory, Fairview-University of Minnesota Medical Center (Ms Kemmer and Messrs Bower and Schomaker); the Division of Genetics and Metabolism, University of Minnesota (Mr Bower); and the Minnesota Supercomputing Institute, University of Minnesota (Drs Onsongo and Silverstein and Mr Erdmann).
authors.forename: Sophia
authors.initials: S
authors.lastname: Yohe
authors.forename: Adam
authors.initials: A
authors.lastname: Hauge
authors.forename: Kari
authors.initials: K
authors.lastname: Bunjer
authors.forename: Teresa
authors.initials: T
authors.lastname: Kemmer
authors.forename: Matthew
authors.initials: M
authors.lastname: Bower
authors.forename: Matthew
authors.initials: M
authors.lastname: Schomaker
authors.forename: Getiria
authors.initials: G
authors.lastname: Onsongo
authors.forename: Jon
authors.initials: J
authors.lastname: Wilson
authors.forename: Jesse
authors.initials: J
authors.lastname: Erdmann
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhou
authors.forename: Archana
authors.initials: A
authors.lastname: Deshpande
authors.forename: Michael D
authors.initials: MD
authors.lastname: Spears
authors.forename: Kenneth
authors.initials: K
authors.lastname: Beckman
authors.forename: Kevin A T
authors.initials: KA
authors.lastname: Silverstein
authors.forename: Bharat
authors.initials: B
authors.lastname: Thyagarajan
mesh_terms: D019295:Computational Biology; D056915:DNA Copy Number Variations; D004252:DNA Mutational Analysis; D005091:Exons / Q000235:genetics; D005240:Feasibility Studies; D030342:Genetic Diseases, Inborn / Q000175:diagnosis* / Q000235:genetics; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000379:methods* / Q000592:standards; D014644:Genetic Variation; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D035583:Rare Diseases / Q000175:diagnosis* / Q000235:genetics; D017422:Sequence Analysis, DNA