BRAF pyrosequencing analysis aided by a lookup table

Objectives: 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).