Cohort-HMM marker recruitment with per-OG orthology QC for phylogenomic supermatrices

OrthoFinder’s all-vs-all DIAMOND step systematically misses single-copy orthogroups (SC OGs) at deep taxonomic divergence: a marker recovered cleanly within a tightly defined cohort is dropped when the same marker is searched against phylum-broad metagenome-assembled genome (MAG) sets, because pairwise sequence similarity falls below DIAMOND’s detection threshold even when the underlying ortholog is present. The result is biased dropout — supermatrices that retain genomes near the cohort but lose genomes from the deeper, more diverged corners of the same phylum. We describe a two-stage cohort-HMM recruitment pipeline (per-OG profile HMMs built from cohort alignments, then hmmsearch against the broader proteome set) followed by an independent per-OG gene-tree QC step that classifies each recruited hit relative to the cohort’s most recent common ancestor (MRCA) descendant set, with a per-MAG paralog-rate filter applied before supermatrix concatenation. We characterize the pipeline across three taxonomic ranks. At phylum scale ( Omnitrophota , 97 cohort OGs, 714 NCBI MAGs), the recruitment recovers MAGs that the OrthoFinder-only supermatrix would otherwise drop, and the QC identifies 2 deep-peripheral MAGs — divergent genomes whose per-OG tips repeatedly place outside the cohort MRCA descendant set despite being orthologs — that the per-MAG filter removes. At family scale ( Pelagibacteraceae , 146 cohort OGs, 366 NCBI MAGs) and at genus scale ( Actinomarina , 289 cohort OGs, 23 NCBI MAGs), the per-tip paralog-candidate rate drops to 0.0 %. The pipeline addresses two independent failure modes. Cohort paralog density breaks strict-SC OG discovery at the cohort step (the family-rank case, where every candidate marker has at least one cohort species carrying multiple copies; the relaxed cohort criterion supplies the marker set and HMM recruitment disambiguates which copy each NCBI MAG contributes). DIAMOND-reach attrition breaks OG assignment for the most divergent NCBI MAGs (the phylum-rank case, where pairwise similarities fall below DIAMOND’s detection threshold; HMM recruitment recovers the dropouts and the per-OG QC step filters residual paralog candidates). At genus rank both modes are inactive and OrthoFinder suffices directly; HMM recruitment runs but finds no new orthologs. Code and per-case data products are released as a community resource at Zenodo (DOI 10.5281/zenodo.20422348).