title: Next generation massively parallel sequencing of targeted exomes to identify genetic mutations in primary ciliary dyskinesia: implications for application to clinical testing.
abstract: OBJECTIVE
  Advances in genetic sequencing technology have the potential to enhance testing for genes associated with genetically heterogeneous clinical syndromes, such as primary ciliary dyskinesia. The objective of this study was to investigate the performance characteristics of exon-capture technology coupled with massively parallel sequencing for clinical diagnostic evaluation.
  
  
  METHODS
  We performed a pilot study of four individuals with a variety of previously identified primary ciliary dyskinesia mutations. We designed a custom array (NimbleGen) to capture 2089 exons from 79 genes associated with primary ciliary dyskinesia or ciliary function and sequenced the enriched material using the GS FLX Titanium (Roche 454) platform. Bioinformatics analysis was performed in a blinded fashion in an attempt to detect the previously identified mutations and validate the process.
  
  
  RESULTS
  Three of three substitution mutations and one of three small insertion/deletion mutations were readily identified using this methodology. One small insertion mutation was clearly observed after adjusting the bioinformatics handling of previously described SNPs. This process failed to detect two known mutations: one single-nucleotide insertion and a whole-exon deletion. Additional retrospective bioinformatics analysis revealed strong sequence-based evidence for the insertion but failed to detect the whole-exon deletion. Numerous other variants were also detected, which may represent potential genetic modifiers of the primary ciliary dyskinesia phenotype.
  
  
  CONCLUSIONS
  We conclude that massively parallel sequencing has considerable potential for both research and clinical diagnostics, but further development is required before widespread adoption in a clinical setting.
authors.affiliation: Departments of Genetics, the University of North Carolina Chapel Hill, North Carolina 27599-7264, USA. jsberg@med.unc.edu
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Berg
authors.forename: James P
authors.initials: JP
authors.lastname: Evans
authors.forename: Margaret W
authors.initials: MW
authors.lastname: Leigh
authors.forename: Heymut
authors.initials: H
authors.lastname: Omran
authors.forename: Chris
authors.initials: C
authors.lastname: Bizon
authors.forename: Ketan
authors.initials: K
authors.lastname: Mane
authors.forename: Michael R
authors.initials: MR
authors.lastname: Knowles
authors.forename: Karen E
authors.initials: KE
authors.lastname: Weck
authors.forename: Maimoona A
authors.initials: MA
authors.lastname: Zariwala
mesh_terms: D000293:Adolescent; D000328:Adult; D001483:Base Sequence; D002648:Child; D005091:Exons; D005260:Female; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007619:Kartagener Syndrome / Q000175:diagnosis* / Q000235:genetics*; D008297:Male; D009154:Mutation; D010375:Pedigree; D010865:Pilot Projects; D012680:Sensitivity and Specificity; D016415:Sequence Alignment