title: Improving the limit of detection for Sanger sequencing: a comparison of methodologies for KRAS variant detection.
abstract: Fluorescent dye terminator Sanger sequencing (FTSS), with detection by automated capillary electrophoresis (CE), has long been regarded as the gold standard for variant detection. However, software analysis and base-calling algorithms used to detect mutations were largely optimized for resequencing applications in which different alleles were expected as heterozygous mixtures of 50%. Increasingly, the requirements for variant detection are an analytic sensitivity for minor alleles of <20%, in particular, when assessing the mutational status of heterogeneous tumor samples. Here, we describe a simple modification to the FTSS workflow that improves the limit of detection of cell-line gDNA mixtures from 50%-20% to 5% for G>A transitions and from 50%-5% to 5% for G>C and G>T transversions. In addition, we use two different sample types to compare the limit of detection of sequence variants in codons 12 and 13 of the KRAS gene between Sanger sequencing and other methodologies including shifted termination assay (STA) detection, single-base extension (SBE), pyrosequencing (PS), high- resolution melt (HRM), and real-time PCR (qPCR).
authors.affiliation: Life Technologies, Foster City, CA, USA. colin.davidson@lifetech.com
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Davidson
authors.forename: Emily
authors.initials: E
authors.lastname: Zeringer
authors.forename: Kristen J
authors.initials: KJ
authors.lastname: Champion
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Gauthier
authors.forename: Fawn
authors.initials: F
authors.lastname: Wang
authors.forename: Jerry
authors.initials: J
authors.lastname: Boonyaratanakornkit
authors.forename: Julie R
authors.initials: JR
authors.lastname: Jones
authors.forename: Edgar
authors.initials: E
authors.lastname: Schreiber
mesh_terms: D002460:Cell Line; D004247:DNA / Q000032:analysis / Q000737:chemistry; D004252:DNA Mutational Analysis / Q000379:methods* / Q000592:standards; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes / Q000737:chemistry; D015894:Genome, Human; D060005:Genotyping Techniques / Q000379:methods / Q000592:standards; D006801:Humans; D057230:Limit of Detection; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins / Q000235:genetics*
chemical_list: D005456:Fluorescent Dyes; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins