Abstract
Pyrosequencing® is a widely used technology to detect gene mutations in a molecular research or diagnostics laboratory. Compared to Sanger sequencing, it is inherently more quantitative with a superior limit of detection, although it has a shorter read length and has difficulty with homopolymeric sequences.
Results of Pyrosequencing experiments are typically presented as traces with sequential peaks, called Pyrograms®. For the majority of clinical diagnostic cases, Pyrograms are straightforward to read. However, there are occasionally complex results that are uninterpretable or difficult to interpret. In this chapter, we demonstrate a computer software, named Pyromaker that has been developed to help with the analysis of Pyrograms. Pyromaker is a freely and publically available software program to assist in the recognition of patterns of mutations, interpretation of difficult or ambiguous testing results and design of an optimal strategy to detect potential mutations by generating simulated Pyrograms. In addition to help diagnostic activities, Pyromaker can also be used as a virtual and user-friendly educational tool to teach newcomers the fundamental mechanism of Pyrosequencing, and correct interpretation of actual Pyrosequencing data.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Nyren P (1987) Enzymatic method for continuous monitoring of DNA polymerase activity. Anal Biochem 167:235–238
Rothberg JM, Hinz W, Rearick TM et al (2011) An integrated semiconductor device enabling non-optical genome sequencing. Nature 475:348–352
Dufort S, Richard MJ, De Fraipont F (2009) Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues. Anal Biochem 391:166–168
Tsiatis AC, Norris-Kirby A, Rich RG et al (2010) Comparison of Sanger sequencing, Pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. J Mol Diagn 12:425–432
Chen G, Olson MT, O’neill A et al (2012) A virtual pyrogram generator to resolve complex Pyrosequencing results. J Mol Diagn 14:149–159
Olson MT, Harrington C, Beierl K et al (2014) BRAF Pyrosequencing analysis aided by a lookup table. Am J Clin Pathol 141:639–647
Harrington CT, Lin EI, Olson MT et al (2013) Fundamentals of Pyrosequencing. Arch Pathol Lab Med 137:1296–1303
Shen S, Qin D (2012) Pyrosequencing data analysis software: a useful tool for EGFR, KRAS, and BRAF mutation analysis. Diagn Pathol 7:56
Ahmadian A, Gharizadeh B, Gustafsson AC et al (2000) Single-nucleotide polymorphism analysis by Pyrosequencing. Anal Biochem 280:103–110
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Chen, G., Olson, M.T., Eshleman, J.R. (2015). Software-Based Pyrogram® Evaluation. In: Lehmann, U., Tost, J. (eds) Pyrosequencing. Methods in Molecular Biology, vol 1315. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2715-9_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2715-9_3
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2714-2
Online ISBN: 978-1-4939-2715-9
eBook Packages: Springer Protocols