New Virtual Tool May Provide More Accurate Diagnosis of Genetic Mutations

New technology described in The Journal of Molecular Diagnostics

Philadelphia, PA, February 6, 2012 – DNA sequencing to detect genetic mutations can aid in the diagnosis and selection of treatment for cancer. Current methods of testing DNA samples, Sanger sequencing and pyrosequencing, occasionally produce complex results that can be difficult or impossible to interpret. Scientists at the Johns Hopkins University School of Medicine have developed a free software program, Pyromaker, that can more accurately identify such complex genetic mutations.

Pyromaker is a web-based application that 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. Pyromaker calculates the relative mutant and wild-type allele percentages and then uses these to generate the expected signal at each point in the dispensation sequence. The final result is a virtual trace of the expected pyrogram.

The researchers validated Pyromaker against actual pyrograms containing common mutations in the KRAS gene, which plays an important role in the pathogenesis of a variety of tumors. The actual pyrograms and virtual pyrograms were quantitatively identical for all mutations tested.

They then demonstrated that all codon 12 and 13 single and complex mutations generate unique pyrograms. However, some complex mutations were indistinguishable from single base mutations, indicating that complex mutations may be underreported. Working with two complex pyrograms that were difficult to interpret initially, the researchers identified five approaches to resolve them: Sanger sequencing alone, hypothesis testing with Pyromaker, Pyromaker iterative mutation re-creation, melting curve analysis, and TA cloning with Sanger sequencing.

Senior author James R. Eshleman, MD, PhD, Professor of Pathology and Oncology, Associate Director, Molecular Diagnostics Laboratory, Johns Hopkins University School of Medicine, 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 strategies were able to successfully identify the complex mutations.

TA cloning and sequencing also provided unequivocal interpretation, but this method is labor intensive, risks plasmid contamination of the laboratory, may delay reporting, and is not routinely used in most clinical diagnostic laboratories.

“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,” notes Dr. Eshleman. “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 at

The article is “A Virtual Pyrogram Generator to Resolve Complex Pyrosequencing Results,” by G. Chen, M.T. Olson, A. O’Neill, A. Norris-Kirby, K. Beierl, S. Harada, M. Debeljak, K. Rivera-Roman, S. Finley, A. Stafford, C.D. Gocke, M-T. Lin, and J. R. Eshleman (doi: 10.1016/j.jmoldx.2011.12.001). The Journal of Molecular Diagnostics, Volume 14, Issue 2 (March 2012) published by Elsevier.

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Notes for editors
Full text of the article is available to credentialed journalists upon request; contact David Sampson at + 1 215 239 3171 or Journalists wishing to interview the authors may contact James R. Eshleman, MD, PhD, at + 1 410 955 3511 or

About The Journal of Molecular Diagnostics
The Journal of Molecular Diagnostics, the official publication of the Association for Molecular Pathology, co-published by the American Society for Investigative Pathology, seeks to publish high quality original papers on scientific advances in the translation and validation of molecular discoveries in medicine into the clinical diagnostic setting, and the description and application of technological advances in the field of molecular diagnostic medicine. The editors welcome for review articles that contain: novel discoveries or clinicopathologic correlations including studies in oncology, infectious diseases, inherited diseases, predisposition to disease, or the description of polymorphisms linked to disease states or normal variations; the application of diagnostic methodologies in clinical trials; or the development of new or improved molecular methods for diagnosis or monitoring of disease or disease predisposition.

The Journal of Molecular Diagnostics, with an Impact Factor of 4.219, ranks among the Top 10 journals in Pathology, according to Thomson Reuters Journal Citation Reports® 2010.

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