Rare Mutation Detection: When is it not a false positive? | ESHG 2015 скачать в хорошем качестве

Rare Mutation Detection: When is it not a false positive? | ESHG 2015

Detection and quantification of mutant alleles in tumor tissue is important to cancer research. Testing for the presence of mutations in circulating free DNA (cfDNA) is one of the less invasive research methods available at this time. Digital PCR presents a research tool for mutation detection in cfDNA at a sensitivity level of 1% and below. Challenges associated with digital PCR experiments for rare allele detection include understanding the limit of detection of the assay and platform. This work compares false positive assessment strategies using the signal levels of the no-amplification cluster. Once the false positive call rate is established, this work outlines a method to determine the limit of detection of the assay and platform, at a given level of confidence. Given the number of partitions, the interrogated volume and the false call rate, the tradeoffs between sample load and sensitivity are also discussed. The mathematics outlined to calculate the theoretical limit of detection is applied on a set of assays from Thermo Fisher Scientific covering the KRAS codon mutations commonly found in tumor tissues. Experimental results showing a detection of at least 0.1% mutation rate are presented as examples. Test samples were created using both mutant plasmid and mutant genomic DNA mixed with wild-type genomic DNA at a predefined percentage.