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Updated: May 1, 2026

Pyrosequencing: A Simple Method for Accurate Genotyping
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BRAF pyrosequencing analysis aided by a lookup table.

Matthew T Olson1, Colleen Harrington, Katie Beierl

  • 1The Sol Goldman Pancreatic Cancer Research Center, Professor of Pathology and Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21231; jeshlema@jhmi.edu.

American Journal of Clinical Pathology
|April 10, 2014
PubMed
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A new pyrosequencing lookup table aids molecular pathology labs in identifying BRAF mutations. This tool helps solve complex pyrosequencing results, improving diagnostic accuracy for BRAF mutations.

Area of Science:

  • Molecular Pathology
  • Genetics
  • Biotechnology

Background:

  • BRAF mutations are crucial for cancer diagnosis, prognosis, and therapy.
  • Pyrosequencing is a valuable method for detecting these mutations but can yield complex results.
  • Accurate identification of BRAF mutations is essential for patient management.

Purpose of the Study:

  • To introduce a pyrosequencing lookup table to aid in interpreting complex BRAF mutation results.
  • To provide a tool for molecular pathology laboratories to improve the accuracy of BRAF mutation detection.
  • To facilitate hypothesis generation for solving challenging pyrosequencing data.

Main Methods:

  • Development of a pyrosequencing lookup table using Pyromaker software.
  • Inclusion of all known mutations and their corresponding dispensation sequence changes.
Keywords:
BRAF mutationsBioinformaticsPyromakerPyrosequencingPyrosequencing analysis

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  • Demonstration using both homebrew and commercial kit-based BRAF codon sequencing (596-605 and 599-600).

    • Main Results:

    • The developed lookup table successfully identified all known BRAF mutations in the tested region using a homebrew assay.
    • A commercial kit-based assay showed an unresolvable degeneracy, which could be overcome by adding two injections.
    • The lookup table and virtual pyrograms unambiguously solved complex BRAF mutations, including V600K, V600R, V600D, V600E, and V600_K601delinsE.

    Conclusions:

    • The pyrosequencing lookup table effectively aids in solving complex BRAF mutation pyrograms.
    • This approach facilitated the identification and confirmation of a novel BRAF mutation (V600_K601delinsEI) in human melanoma.
    • The tool enhances the diagnostic capabilities of molecular pathology labs for BRAF mutation analysis.