RESULTS: Using a constraint-based regression method, the new AdvISER-PYRO-SMQ algorithm was developed in the current study and implemented into an R package. As a proof-of-concept, AdvISER-PYRO-SMQ was used to identify a set of 9 distinct point mutations affecting codon 61 of the NRAS oncogene. In parallel, a pyrosequencing assay using the Qiagen software and its AQ module was used to assess selectively the presence of a single point mutation (NRAS[Formula: see text] - Q61R-1) among the set of codon 61 mutations, and to analyze related pyrosequencing signals. AdvISER-PYRO-SMQ produced a lower limit of blank (0 %) than the AQ module of Qiagen software (5.1 %) and similar limit of detection were obtained for both software (5.6 vs 4.8 %). AdvISER-PYRO-SMQ was able to screen for the presence of 9 distinct mutations with a single pyrosequencing reaction whereas the AQ module was limited to screen a single mutation per reaction.
CONCLUSIONS: Using a constraint-based regression method enables to analyze pyrosequencing signal and to detect multiple mutations within a hotspot genomic region with an optimal compromise between sensitivity and specificity. The AdvISER-PYRO-SMQ R package provides a generic tool which can be applied on a wide range of somatic mutations. Its implementation in a Shiny web interactive application (available at https://ucl-irec-ctma.shinyapps.io/Pyrosequencing-NRAS-61/) enables its use in research or clinical routine applications.