This dataset contains the synthetic and empirical genomic data used to validate and demonstrate SimPhyNI (Simulated Phylogenetic Null Interactions), a framework for high-precision microbial Genome-Wide Association Studies (mGWAS) on binary traits. The data provided supports the paper’s findings regarding SimPhyNI’s ability to achieve near-zero false positive rates and high recall for both positive and negative trait associations in bacteria. The repository includes: Synthetic Validation Datasets: Binary trait matrices and corresponding phylogenetic trees used to benchmark SimPhyNI against existing methods (e.g., Pagel’s correlation, FaST-LMM, TreeWAS). These datasets include simulated "true" (both positive and negative) and neutral associations. Phage Defense System Interaction Data: A binary matrix of phage defense system presence/absence identified across a collection of Escherichia coli genomes from a prior publication. This data was used to identify synergistic and antagonistic relationships between defense mechanisms. E. coli Pangenome Association Analysis: Output files and downstream analysis of a pangenome-wide study of E. coli using a representative set of 500 genomes and over 9 million gene-gene association tests