Cancer diagnosis and selecting the most appropriate treatment can be made easier by identifying genetic mutations through DNA sequencing. Present test methods for DNA samples, Sanger sequencing and pyrosequencing can sometimes produce complex results that can be difficult or impossible to interpret, however, scientists at the Johns Hopkins University School of Medicine have developed a free software program named Pyromaker, which can assist in accurately identifying such complex genetic mutations.
Pyromaker, a web-based application produces simulated pyrograms based on user input. The pyrograms display various functions, including the percentage of tumor and normal cells, the wild-type sequence, the dispensation order, and any number of mutant sequences. The program calculates the relative mutant and wild-type allele percentages from which it generates the expected signal at each point in the dispensation sequence. The result is a virtual trace of the expected pyrogram.
Pyromaker was validated against actual pyrograms containing common mutations in the KRAS gene that serves an important function in the pathogenesis of several different tumors. In comparison to the actual programs, the virtual pyrograms were quantitatively identical for all mutations tested.
The researchers then demonstrated that all codon 12 and 13 single and complex mutations produce unique programs. Some complex mutations however were difficult to distinguish from single base mutations, which suggests that complex mutations may be underreported. The researchers worked with two complex pyrograms that were difficult to interpret at first. In order to resolve this, they came up with five strategies consisting of Pyromaker iterative mutation re-creation, Sanger sequencing alone, hypothesis testing with Pyromaker, melting curve analysis, and TA cloning with Sanger sequencing.
James R. Eshleman, MD, PhD, Professor of Pathology and Oncology, Associate Director, Molecular Diagnostics Laboratory, Johns Hopkins University School of Medicine, who is the senior author of the study, explains, “User-directed hypothesis testing allows for generating virtual traces that can be compared to the actual data to clarify ambiguous results from pyrosequencing and the Sanger method. Alternatively, Pyromaker can quickly and efficiently test the possibilities that can explain a complicated polysequencing result.” Both methods successfully identified the complex mutations.
Although TA cloning and sequencing also offer an unequivocal interpretation, the approach is has various drawbacks as it is labor intensive and may delay reporting, there is a risk of plasmid contamination of the laboratory, and is also not used frequently in most clinical diagnostic laboratories.
Dr. Eshleman concludes:
“Although pyrosequencing and Sanger sequencing are both powerful tools to resolve most mutations, for certain complex cases, neither of them alone is enough to provide a definitive interpretation. Additional methods, such as Pyromaker analysis or TA cloning and sequencing, allow one to definitively diagnose the variant allele. Pyromaker is available free online and can be accessed from any computer with internet access. Iterative Pyromaker analysis is the least expensive and fastest method to resolve these cases.”
Pyromaker has been made freely available here.
Written by Petra Rattue