biostudies-literatureQu EBNational Science Foundation Graduate Research Fellowship ProgramNational Institutes of Health Office of the DirectorNational Institutes of HealthNIGMS NIH HHSDSMNational Science Foundation116134https://www.ebi.ac.uk/biostudies/studies/S-EPMC12452263biostudies-literatureUnknown44(8)Genetically distinct strains of a species can vary widely in phenotype, reducing the utility of species-resolved microbiome measurements for detecting associations with health or disease. While metagenomics theoretically provides information on all strains in a sample, current strain-resolved analysis methods face a tradeoff: de novo genotyping approaches can detect novel strains but struggle when applied to strain-rich or low-coverage samples, while reference database methods work robustly across sample types but are insensitive to novel diversity. We present PHLAME, a method that bridges this divide by combining the advantages of reference database approaches with novelty awareness. PHLAME explicitly defines clades at multiple phylogenetic levels and introduces a probabilistic, mutation-Cell reportsIntraspecies associations from strain-rich metagenome samples.PMC12452263R35 GM1562821DP2GM140922R35GM156282DP2 GM140922Mancuso CLieberman TDQu EBBaker JSKhadka VTripp ADMarkey LfalseIntraspecies associations from strain-rich metagenome samples.Genetically distinct strains of a species can vary widely in phenotype, reducing the utility of species-resolved microbiome measurements for detecting associations with health or disease. While metagenomics theoretically provides information on all strains in a sample, current strain-resolved analysis methods face a tradeoff: de novo genotyping approaches can detect novel strains but struggle when applied to strain-rich or low-coverage samples, while reference database methods work robustly across sample types but are insensitive to novel diversity. We present PHLAME, a method that bridges this divide by combining the advantages of reference database approaches with novelty awareness. PHLAME explicitly defines clades at multiple phylogenetic levels and introduces a probabilistic, mutation-2025-01-01T00:00:00Z2025 Aug2026-06-03T19:31:47.182Z2026-04-30T03:12:08.97ZS-EPMC124522634081106310.1016/j.celrep.2025.116134