title: Illumina sequencing of 15 deafness genes using fragmented amplicons.
abstract: BACKGROUND
  Resequencing of deafness related genes using GS FLX massive parallel sequencing of PCR amplicons spanning selected genes has previously been reported as a successful strategy to discover causal variants. The amplicon lengths were designed to be smaller than the sequencing read length of GS FLX technology, but are longer than Illumina sequencing technology read lengths. Fragmentation is thus required to sequence these amplicons using high throughput Illumina technology.
  
  
  METHODS
  We performed Illumina sequencing in 4 patients on 563 multiplexed amplicons covering the exons of 15 genes involved in the hearing process. After exploring several fragmentation strategies, the amplicons were fragmented using Covaris sonication prior to library preparation. CLC genomic workbench was used to analyze the data.
  
  
  RESULTS
  We achieve an excellent coverage with more than 99% of the amplicons bases covered. All variants that were previously validated using Sanger sequencing, were also called in this study. Variant calling revealed less false positive and false negative results compared to the previous study. For each patient, several variants were found that are reported by ClinVar as possible hearing loss variants.
  
  
  CONCLUSIONS
  Migration from GS FLX amplicon sequencing to Illumina amplicon sequencing is straightforward and leads to more accurate results.
authors.affiliation: Laboratory for Pharmaceutical Biotechnology, Ghent University, Ottergemsesteenweg 460, Ghent 9000, Belgium. Filip.VanNieuwerburgh@UGent.be.
authors.forename: Filip
authors.initials: F
authors.lastname: Van Nieuwerburgh
authors.forename: Sarah
authors.initials: S
authors.lastname: De Keulenaer
authors.forename: Joachim
authors.initials: J
authors.lastname: De Schrijver
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
authors.forename: Wim
authors.initials: W
authors.lastname: Van Criekinge
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Coucke
authors.forename: Dieter
authors.initials: D
authors.lastname: Deforce
mesh_terms: D003638:Deafness / Q000235:genetics*; D005784:Gene Amplification; D020022:Genetic Predisposition to Disease; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods*
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