title: Diagnostic validation of a familial hypercholesterolaemia cohort provides a model for using targeted next generation DNA sequencing in the clinical setting.
abstract: Our aim was to assess the sensitivity and specificity of a next generation DNA sequencing (NGS) platform using a capture based DNA library preparation method. Data and experience gained from this diagnostic validation can be used to progress the applications of NGS in the wider molecular diagnostic setting. A technical cross-validation comparing the current molecular diagnostic gold standard methods of Sanger DNA sequencing and multiplex ligation-dependant probe amplification (MLPA) versus a customised capture based targeted re-sequencing method on a SOLiD 5500 sequencing platform was carried out using a cohort of 96 familial hypercholesterolaemia (FH) samples. We compared a total of 595 DNA variations (488 common single nucleotide polymorphisms, 73 missense mutations, 9 nonsense mutations, 3 splice site point mutations, 13 small indels, 2 multi-exonic duplications and 7 multi-exonic deletions) found previously in the 96 FH samples. DNA variation detection sensitivity and specificity were both 100% for the SOLiD 5500 NGS platform compared with Sanger sequencing and MLPA only when both LifeScope and Integrative Genomics Viewer softwares were utilised. The methods described here offer a high-quality strategy for the detection of a wide range of DNA mutations in diseases with a moderate number of well described causative genes. However, there are important issues related to the bioinformatic algorithms employed to detect small indels.
authors.affiliation: 1Department of Molecular and Clinical Genetics, Royal Prince Alfred Hospital, Camperdown 2Department of Clinical Biochemistry, Royal Prince Alfred Hospital, Camperdown 3Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Marcus
authors.initials: M
authors.lastname: Hinchcliffe
authors.forename: Huong
authors.initials: H
authors.lastname: Le
authors.forename: Anthony
authors.initials: A
authors.lastname: Fimmel
authors.forename: Laura
authors.initials: L
authors.lastname: Molloy
authors.forename: Lucinda
authors.initials: L
authors.lastname: Freeman
authors.forename: David
authors.initials: D
authors.lastname: Sullivan
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Trent
mesh_terms: D000483:Alleles; D015331:Cohort Studies; D019295:Computational Biology; D004247:DNA / Q000737:chemistry / Q000235:genetics; D005091:Exons; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000175:diagnosis* / Q000235:genetics; D025202:Molecular Diagnostic Techniques; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D022821:RNA Splice Sites; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D022821:RNA Splice Sites; D004247:DNA