The commonly employed methods for sequencing DNA samples, Sanger sequencing and pyrosequencing, occasionally produce complex results that can be difficult or impossible to interpret. To improve this situation investigators at Johns Hopkins University (Baltimore, MD, USA) have developed a free software program, Pyromaker, which can more accurately evaluate such complex genetic mutations.
Pyromaker, which can be accessed online (please see Related Links below), produces simulated pyrograms based on user input including the percentage of tumor and normal cells, the wild-type sequence, the dispensation order, and any number of mutant sequences. Simulated pyrograms can aid in the analysis of complex pyrosequencing results in which various hypothesized mutations can be tested, and the resultant pyrograms can be matched with the actual pyrogram.
In a paper published in the February 10, 2012, online edition of the Journal of Molecular Diagnostics the investigators reported that they validated the software using the actual pyrograms for common KRAS gene mutations as well as several mutations in the BRAF, GNAS, and p53 genes. They demonstrated that all 18 possible single-base mutations within codons 12 and 13 of KRAS generated unique pyrosequencing traces and highlighted the distinctions between them. They further showed that all reported codon 12 and 13 complex mutations produced unique pyrograms.
Senior author Dr. James R. Eshleman, professor of pathology and oncology at Johns Hopkins University, said, “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.
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.”
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