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).
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Davidson CJ,Zeringer E,Champion KJ,Gauthier MP,Wang F,Boonyaratanakornkit J,Jones JR,Schreiber Edoi
10.2144/000113913subject
Has Abstractpub_date
2012-09-01 00:00:00pages
182-8issue
3eissn
0736-6205issn
1940-9818pii
000113913journal_volume
53pub_type
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