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To test this hypothesis, we assessed MAG recovery in three in silico microbial communities composed of 42 species with the same richness but different sample species abundance, sequencing depth, and taxonomic distribution profiles using three different pipelines for MAG recovery. The pipeline developed by Parks and colleagues (8K) generated the highest number of MAGs and the lowest number of true positives per community profile. The pipeline by Karst and colleagues (DT) showed the most accurate results (~ 92%), outperforming the 8K and Multi-Metagenome pipeline (MM) developed by Albertsen and collaborators. Sequencing depth influenced the accurate recovery of genomes when using the 8K and MM, even with contrasting patterns: the MM pipeline recovered more MAGs found in the original communities when employing sequencing depths up to 60 million reads, while the 8K recovered more true positives in communities sequenced above 60 million reads. DT showed the best species recovery from the same genus, even though close-related species have a low recovery rate in all pipelines. Our results highlight that more bins do not translate to the actual community composition and that sequencing depth plays a role in MAG recovery and increased community resolution. Even low MAG recovery error rates can significantly impact biological inferences. Our data indicates that the scientific community should curate their findings from MAG recovery, especially when asserting novel species or metabolic traits.<\/jats:p>","DOI":"10.1371\/journal.pcbi.1012530","type":"journal-article","created":{"date-parts":[[2024,10,22]],"date-time":"2024-10-22T17:39:04Z","timestamp":1729618744000},"page":"e1012530","update-policy":"https:\/\/doi.org\/10.1371\/journal.pcbi.corrections_policy","source":"Crossref","is-referenced-by-count":9,"title":["Simulation of 69 microbial communities indicates sequencing depth and false positives are major drivers of bias in prokaryotic metagenome-assembled genome recovery"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6972-6692","authenticated-orcid":true,"given":"Ulisses","family":"Rocha","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jonas Coelho","family":"Kasmanas","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rodolfo","family":"Toscan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Danilo S.","family":"Sanches","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stefania","family":"Magnusdottir","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8251-1960","authenticated-orcid":true,"given":"Joao Pedro","family":"Saraiva","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2024,10,22]]},"reference":[{"issue":"1","key":"pcbi.1012530.ref001","doi-asserted-by":"crossref","first-page":"e01078","DOI":"10.1128\/mSystems.01078-20","article-title":"Complementary Roles of Wood-Inhabiting Fungi and Bacteria Facilitate Deadwood Decomposition.","volume":"6","author":"V Tl\u00c1skal","year":"2021","journal-title":"mSystems."},{"key":"pcbi.1012530.ref002","article-title":"Paraquat Degradation From Contaminated Environments: Current Achievements and Perspectives.","author":"Y Huang","year":"2019","journal-title":"Front Microbiol [Internet]."},{"issue":"7","key":"pcbi.1012530.ref003","doi-asserted-by":"crossref","first-page":"1768","DOI":"10.1038\/s41396-018-0084-2","article-title":"Decomposer food web in a deciduous forest shows high share of generalist microorganisms and importance of microbial biomass recycling.","volume":"12","author":"R L\u00f3pez-Mond\u00e9jar","year":"2018","journal-title":"ISME JJul"},{"issue":"4","key":"pcbi.1012530.ref004","doi-asserted-by":"crossref","first-page":"1953","DOI":"10.1111\/gcb.14962","article-title":"Microbial carbon limitation: The need for integrating microorganisms into our understanding of ecosystem carbon cycling.","volume":"26","author":"JL Soong","year":"2020","journal-title":"Global Change Biology"},{"issue":"D1","key":"pcbi.1012530.ref005","first-page":"D626","article-title":"TerrestrialMetagenomeDB: a public repository of curated and standardized metadata for terrestrial metagenomes","volume":"48","author":"FB Corr\u00eaa","year":"2020","journal-title":"Nucleic Acids Research"},{"issue":"D1","key":"pcbi.1012530.ref006","doi-asserted-by":"crossref","first-page":"D743","DOI":"10.1093\/nar\/gkaa1031","article-title":"HumanMetagenomeDB: a public repository of curated and standardized metadata for human metagenomes","volume":"49","author":"JC Kasmanas","year":"2021","journal-title":"Nucleic Acids Research"},{"issue":"1","key":"pcbi.1012530.ref007","first-page":"1","article-title":"MarineMetagenomeDB: a public repository for curated and standardized metadata for marine metagenomes.","volume":"17","author":"MK Nata\u2019ala","year":"2022","journal-title":"Environmental Microbiomes"},{"key":"pcbi.1012530.ref008","author":"NCBI","year":"2023","journal-title":"Home\u2014Assembly\u2014NCBI [Internet]"},{"key":"pcbi.1012530.ref009","author":"JGI GOLD","year":"2023","journal-title":"JGI GOLD | Statistics [Internet]"},{"issue":"3","key":"pcbi.1012530.ref010","first-page":"e02918","article-title":"Genome-centric analyses of 165 metagenomes show that mobile genetic elements are crucial for the transmission of antimicrobial resistance genes to pathogens in activated sludge and wastewater. 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