IsoPairFinder: A tool for biochemical pathway discovery using stable isotope tracing metabolomics

Summary: The functional annotation of microbial genes lags far behind genome sequencing, leaving critical gaps in our knowledge of metabolic pathways. While integrating genetic manipulation with stable isotope tracing (SIT) metabolomics holds promise for pathway discovery, existing tools lack specialized capabilities for gene perturbation experiments. To address this need, we developed IsoPairFinder, a computational tool that identifies pathway intermediates by analyzing paired unlabeled (12C) and isotope-labeled (13C) metabolomics data from gene-edited microbes. By prioritizing substrate-specific feature pairs, IsoPairFinder efficiently prioritizes biologically relevant intermediates. Implemented as an open-source R package and integrated into the GNPS2 ecosystem, IsoPairFinder provides an accessible platform for the research community to accelerate novel pathway discovery and validation. Availability and implementation: IsoPairFinder is freely available as an open-source R package (https://github.com/DoddLab/IsoPairFinder) and as a web-based workflow in the GNPS2 ecosystem (https://gnps2.org/workflowinput?workflowname=isopairfinder_nextflow_workflow). Comprehensive tutorials are provided at https://doddlab.github.io/IsoPairFinder_Tutorials. These implementations support both command-line and graphical analysis workflows.