title: Rapid recognition of aberrant dHPLC elution profiles using the Transgenomic Navigator software.
abstract: Despite the availability of numerous technologies for detecting mutations, only a few have been formatted for automated mutation calling. Here, we evaluate the utility of the Transgenomic Navigator software to facilitate automated detection of aberrant denaturing high performance liquid chromatography (dHPLC) elution profiles. We used dHPLC to identify germline variants in MSH6, NEIL2, NEIL3, and OGG1 in 172 patients with multiple colorectal adenomas. 3,747 dHPLC profiles were analysed with the Navigator software using three levels of analysis, each differing in the degree of operator input. 43.5% (60/138) and 98.3% (59/60) of products with profiles distinct from wild type ('outliers') harboured novel variants under Level 1 and Levels 2/3 analysis conditions, respectively. We also assessed the utility of the software to rapidly detect samples carrying common polymorphisms by analysing regions of the genes that harbour polymorphisms with minor allele frequencies between 8 and 40%, therein analysing 2,784 profiles. We showed that 1573/1612 (97.6%) and 1137/1172 (97.0%) of PCR products were correctly classified as wild-type and variant, respectively (Level 3 analysis conditions). Finally, we assessed the utility of the software to detect novel variants in fragments that also harboured common polymorphisms and showed that 59/61 (96.7%) of products with profiles outlying both the wild type and polymorphism groups harboured novel variants. We conclude that the Navigator software provides an excellent tool for rapid discrimination of aberrant dHPLC elution profiles that harbour sequence variants.
authors: Institute of Medical Genetics, Cardiff University, Health Park, Cardiff, CF14 4XN, United Kingdom.
authors: James
authors: J
authors: Colley
authors: Siân
authors: S
authors: Jones
authors: Anthony R
authors: AR
authors: Dallosso
authors: Julie H
authors: JH
authors: Maynard
authors: Vikki
authors: V
authors: Humphreys
authors: Sunil
authors: S
authors: Dolwani
authors: Julian R
authors: JR
authors: Sampson
authors: Jeremy P
authors: JP
authors: Cheadle
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019295:Computational Biology / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D016678:Genome; D006801:Humans; D009691:Nucleic Acid Denaturation; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA; D012984:Software; D019076:Transgenes