A virtual pyrogram generator to resolve complex pyrosequencing results.
We report a freely available software program, Pyromaker, which generates simulated traces for pyrosequencing results based on user inputs. 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. We validated the software using the actual pyrograms for common KRAS gene mutations as well as several mutations in the BRAF, GNAS, and p53 genes. We demonstrate that all 18 possible single-base mutations within codons 12 and 13 of KRAS generate unique pyrosequencing traces and highlight the distinctions between them. We further show that all reported codon 12 and 13 complex mutations produce unique pyrograms. However, some complex mutations are indistinguishable from single-base mutations. For complicated pyrograms, Pyromaker was used in two modes, one in which hypothesis-based simulated pyrograms were pattern-matched with the actual pyrograms. In a second strategy with only the pyrogram, Pyromaker was used to identify the underlying mutation by iteratively reconstructing the mutant pyrogram. Either strategy was able to successfully identify the complex mutations, which were confirmed by cloning and sequencing. Using two examples of KRAS codon 12 mutations (specifically GGT→TTT, G12F and GGT→GAG, G12E), we report which combinations of five approaches permit unambiguous mutation identification. The most efficient approach was found to be pyrosequencing with Pyromaker.
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Guoli
G
Chen
Matthew Theodore
MT
Olson
Alan
A
O'Neill
Alexis
A
Norris
Katie
K
Beierl
Shuko
S
Harada
Marija
M
Debeljak
Keila
K
Rivera-Roman
Samantha
S
Finley
Amanda
A
Stafford
Christopher David
CD
Gocke
Ming-Tseh
MT
Lin
James Richard
JR
Eshleman
D003062:Codon / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation / Q000235:genetics*; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012984:Software; D018631:ras Proteins / Q000235:genetics*
D003062:Codon; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins