BRAF pyrosequencing analysis aided by a lookup table.
OBJECTIVE
BRAF mutations have substantial therapeutic, diagnostic, and prognostic significance, so detecting and specifying them is an important part of the workload of molecular pathology laboratories. Pyrosequencing assays are well suited for this analysis but can produce complex results. Therefore, we introduce a pyrosequencing lookup table based on Pyromaker that assists the user in generating hypotheses for solving complex pyrosequencing results.


METHODS
The lookup table contains all known mutations in the sequenced region and the positions in the dispensation sequence at which changes would occur with those mutations. We demonstrate the lookup table using a homebrew dispensation sequence for BRAF codons 596 to 605 as well as a commercially available kit-based dispensation sequence for codons 599 to 600.


RESULTS
These results demonstrate that the homebrew dispensation sequence unambiguously identifies all known BRAF mutations in this region, whereas the kit-based dispensation sequence has one unresolvable degeneracy that could be solved with the addition of two injections.


CONCLUSIONS
Using the lookup table and confirmatory virtual pyrogram, we unambiguously solved clinical pyrograms of the complex mutations V600K (c.1798_1799delGTinsAA), V600R (c.1798_1799delGTinsAG), V600D (c.1799_1800delTGinsAT), V600E (c.1799_1800delTGinsAA), and V600_K601delinsE (c.1799_1801delTGA). In addition, we used the approach to hypothesize and confirm a new mutation in human melanoma, V600_K601delinsEI (c.1799_1802delTGAAinsAAAT).
The Sol Goldman Pancreatic Cancer Research Center, Professor of Pathology and Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21231; jeshlema@jhmi.edu.
Matthew T
MT
Olson
Colleen
C
Harrington
Katie
K
Beierl
Guoli
G
Chen
Michele
M
Thiess
Alan
A
O'Neill
Janis M
JM
Taube
Martha A
MA
Zeiger
Ming-Tseh
MT
Lin
James R
JR
Eshleman
D020022:Genetic Predisposition to Disease / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D008545:Melanoma / Q000175:diagnosis / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D057089:Pathology, Molecular / Q000379:methods; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D012878:Skin Neoplasms / Q000175:diagnosis / Q000235:genetics*
D048493:Proto-Oncogene Proteins B-raf
BRAF mutations; Bioinformatics; Pyromaker; Pyrosequencing; Pyrosequencing analysis