{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2024,1,23]],"date-time":"2024-01-23T18:03:38Z","timestamp":1706033018714},"reference-count":51,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2023,1,10]],"date-time":"2023-01-10T00:00:00Z","timestamp":1673308800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001656","name":"Helmholtz Young Investigator","doi-asserted-by":"publisher","award":["VH-NG-1248"]},{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["460129525"]},{"name":"Petroleum Technology Development Fund"},{"DOI":"10.13039\/501100001655","name":"German Academic Exchange Service","doi-asserted-by":"publisher","award":["91717355"]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>The recovery of metagenome-assembled genomes is biased towards the most abundant species in a given community. To improve the identification of species, even if only dominant species are recovered, we investigated the integration of flow cytometry cell sorting with bioinformatics tools to recover metagenome-assembled genomes. We used a cell culture of a wastewater microbial community as our model system. Cells were separated based on fluorescence signals via flow cytometry cell sorting into sub-communities: dominant gates, low abundant gates, and outer gates into subsets of the original community. Metagenome sequencing was performed for all groups. The unsorted community was used as control. We recovered a total of 24 metagenome-assembled genomes (MAGs) representing 11 species-level genome operational taxonomic units (gOTUs). In addition, 57 ribosomal operational taxonomic units (rOTUs) affiliated with 29 taxa at species level were reconstructed from metagenomic libraries. Our approach suggests a two-fold increase in the resolution when comparing sorted and unsorted communities. Our results also indicate that species abundance is one determinant of genome recovery from metagenomes as we can recover taxa in the sorted libraries that are not present in the unsorted community. In conclusion, a combination of cell sorting and metagenomics allows the recovery of MAGs undetected without cell sorting.<\/jats:p>","DOI":"10.3390\/microorganisms11010175","type":"journal-article","created":{"date-parts":[[2023,1,11]],"date-time":"2023-01-11T07:06:03Z","timestamp":1673420763000},"page":"175","source":"Crossref","is-referenced-by-count":5,"title":["Combining Flow Cytometry and Metagenomics Improves Recovery of Metagenome-Assembled Genomes in a Cell Culture from Activated Sludge"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"http:\/\/orcid.org\/0000-0003-1264-2003","authenticated-orcid":false,"given":"Nafi\u2019u","family":"Abdulkadir","sequence":"first","affiliation":[]},{"ORCID":"http:\/\/orcid.org\/0000-0001-8251-1960","authenticated-orcid":false,"given":"Joao Pedro","family":"Saraiva","sequence":"additional","affiliation":[]},{"given":"Florian","family":"Schattenberg","sequence":"additional","affiliation":[]},{"given":"Rodolfo Brizola","family":"Toscan","sequence":"additional","affiliation":[]},{"given":"Felipe","family":"Borim Correa","sequence":"additional","affiliation":[]},{"given":"Hauke","family":"Harms","sequence":"additional","affiliation":[]},{"ORCID":"http:\/\/orcid.org\/0000-0003-2060-6256","authenticated-orcid":false,"given":"Susann","family":"M\u00fcller","sequence":"additional","affiliation":[]},{"ORCID":"http:\/\/orcid.org\/0000-0001-6972-6692","authenticated-orcid":false,"given":"Ulisses Nunes","family":"da Rocha","sequence":"additional","affiliation":[]}],"member":"1968","published-online":{"date-parts":[[2023,1,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1144","DOI":"10.1038\/nmeth.3103","article-title":"Binning Metagenomic Contigs by Coverage and Composition","volume":"11","author":"Alneberg","year":"2014","journal-title":"Nat. Methods"},{"key":"ref_2","first-page":"D626","article-title":"TerrestrialMetagenomeDB: A Public Repository of Curated and Standardized Metadata for Terrestrial Metagenomes","volume":"48","author":"Saraiva","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_3","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":"Kasmanas","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Kallies, R., H\u00f6lzer, M., Brizola Toscan, R., Nunes da Rocha, U., Anders, J., Marz, M., and Chatzinotas, A. (2019). Evaluation of Sequencing Library Preparation Protocols for Viral Metagenomic Analysis from Pristine Aquifer Groundwaters. Viruses, 11.","DOI":"10.3390\/v11060484"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1186\/s40793-022-00449-7","article-title":"MarineMetagenomeDB: A Public Repository for Curated and Standardized Metadata for Marine Metagenomes","volume":"17","author":"Saraiva","year":"2022","journal-title":"Environ. Microbiome"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"833","DOI":"10.1038\/nbt.3935","article-title":"Shotgun Metagenomics, from Sampling to Analysis","volume":"35","author":"Quince","year":"2017","journal-title":"Nat. Biotechnol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"530","DOI":"10.1038\/s42003-021-01948-y","article-title":"High Biodiversity in a Benzene-Degrading Nitrate-Reducing Culture Is Sustained by a Few Primary Consumers","volume":"4","author":"Melkonian","year":"2021","journal-title":"Commun. Biol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1186\/s40168-022-01343-7","article-title":"Metagenomics-Resolved Genomics Provides Novel Insights into Chitin Turnover, Metabolic Specialization, and Niche Partitioning in the Octocoral Microbiome","volume":"10","author":"Kozma","year":"2022","journal-title":"Microbiome"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"533","DOI":"10.1038\/nbt.2579","article-title":"Genome Sequences of Rare, Uncultured Bacteria Obtained by Differential Coverage Binning of Multiple Metagenomes","volume":"31","author":"Albertsen","year":"2013","journal-title":"Nat. Biotechnol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1533","DOI":"10.1038\/s41564-017-0012-7","article-title":"Recovery of Nearly 8,000 Metagenome-Assembled Genomes Substantially Expands the Tree of Life","volume":"2","author":"Parks","year":"2017","journal-title":"Nat. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1186\/s13059-015-0834-7","article-title":"Metagenome-Assembled Genomes Uncover a Global Brackish Microbiome","volume":"16","author":"Hugerth","year":"2015","journal-title":"Genome Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1186\/s40168-020-0790-7","article-title":"A Pipeline for Targeted Metagenomics of Environmental Bacteria","volume":"8","author":"Grieb","year":"2020","journal-title":"Microbiome"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2009","DOI":"10.1038\/s41467-021-22203-2","article-title":"Connecting Structure to Function with the Recovery of over 1000 High-Quality Metagenome-Assembled Genomes from Activated Sludge Using Long-Read Sequencing","volume":"12","author":"Singleton","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4565","DOI":"10.1038\/s41598-019-40872-4","article-title":"Composition and Dynamics of the Activated Sludge Microbiome during Seasonal Nitrification Failure","volume":"9","author":"Johnston","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"342","DOI":"10.1128\/MMBR.00051-12","article-title":"Patterns and Processes of Microbial Community Assembly","volume":"77","author":"Nemergut","year":"2013","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1016\/j.copbio.2014.01.011","article-title":"Dynamics in the Microbial Cytome-Single Cell Analytics in Natural Systems","volume":"27","author":"Koch","year":"2014","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1038\/nprot.2012.149","article-title":"Cytometric Fingerprinting for Analyzing Microbial Intracommunity Structure Variation and Identifying Subcommunity Function","volume":"8","author":"Koch","year":"2013","journal-title":"Nat. Protoc."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1186\/s40168-020-0788-1","article-title":"Gastric Bypass Surgery in a Rat Model Alters the Community Structure and Functional Composition of the Intestinal Microbiota Independently of Weight Loss","volume":"8","author":"Haange","year":"2020","journal-title":"Microbiome"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2788","DOI":"10.1038\/s41596-020-0362-0","article-title":"Bacterial Mock Communities as Standards for Reproducible Cytometric Microbiome Analysis","volume":"15","author":"Cichocki","year":"2020","journal-title":"Nat. Protoc."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e00768-19","DOI":"10.1128\/mSystems.00768-19","article-title":"Complementary Metagenomic Approaches Improve Reconstruction of Microbial Diversity in a Forest Soil","volume":"5","author":"Alteio","year":"2020","journal-title":"mSystems"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"164","DOI":"10.1111\/1462-2920.14437","article-title":"Neutral Mechanisms and Niche Differentiation in Steady-State Insular Microbial Communities Revealed by Single Cell Analysis","volume":"21","author":"Liu","year":"2019","journal-title":"Environ. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1186\/s40168-018-0541-1","article-title":"MetaWRAP\u2014A Flexible Pipeline for Genome-Resolved Metagenomic Data Analysis","volume":"6","author":"Uritskiy","year":"2018","journal-title":"Microbiome"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"824","DOI":"10.1101\/gr.213959.116","article-title":"MetaSPAdes: A New Versatile Metagenomic Assembler","volume":"27","author":"Nurk","year":"2017","journal-title":"Genome Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e7359","DOI":"10.7717\/peerj.7359","article-title":"MetaBAT 2: An Adaptive Binning Algorithm for Robust and Efficient Genome Reconstruction from Metagenome Assemblies","volume":"7","author":"Kang","year":"2019","journal-title":"PeerJ"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"605","DOI":"10.1093\/bioinformatics\/btv638","article-title":"MaxBin 2.0: An Automated Binning Algorithm to Recover Genomes from Multiple Metagenomic Datasets","volume":"32","author":"Wu","year":"2016","journal-title":"Bioinformatics"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1043","DOI":"10.1101\/gr.186072.114","article-title":"CheckM: Assessing the Quality of Microbial Genomes Recovered from Isolates, Single Cells, and Metagenomes","volume":"25","author":"Parks","year":"2015","journal-title":"Genome Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"996","DOI":"10.1038\/nbt.4229","article-title":"A Standardized Bacterial Taxonomy Based on Genome Phylogeny Substantially Revises the Tree of Life","volume":"36","author":"Parks","year":"2018","journal-title":"Nat. Biotechnol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1925","DOI":"10.1093\/bioinformatics\/btz848","article-title":"GTDB-Tk: A Toolkit to Classify Genomes with the Genome Taxonomy Database","volume":"36","author":"Chaumeil","year":"2020","journal-title":"Bioinformatics"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Price, M.N., Dehal, P.S., and Arkin, A.P. (2010). FastTree 2\u2014Approximately Maximum-Likelihood Trees for Large Alignments. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0009490"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"W256","DOI":"10.1093\/nar\/gkz239","article-title":"Interactive Tree Of Life (ITOL) v4: Recent Updates and New Developments","volume":"47","author":"Letunic","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_31","unstructured":"da Rocha, U.N., Kasmanas, J.C., Kallies, R., Saraiva, J.P., Toscan, R.B., \u0160tefani\u010d, P., Bicalho, M.F., Correa, F.B., Ba\u015ft\u00fcrk, M.N., and Fousekis, E. (2022). MuDoGeR: Multi-Domain Genome Recovery from Metagenomes Made Easy. bioRxiv."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"5114","DOI":"10.1038\/s41467-018-07641-9","article-title":"High Throughput ANI Analysis of 90K Prokaryotic Genomes Reveals Clear Species Boundaries","volume":"9","author":"Jain","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1038\/nmeth.1923","article-title":"Fast Gapped-Read Alignment with Bowtie 2","volume":"9","author":"Langmead","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2078","DOI":"10.1093\/bioinformatics\/btp352","article-title":"1000 Genome Project Data Processing Subgroup The Sequence Alignment\/Map Format and SAMtools","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1016\/j.gde.2005.09.006","article-title":"The Microbial Pan-Genome","volume":"15","author":"Medini","year":"2005","journal-title":"Curr. Opin. Genet. Dev."},{"key":"ref_36","unstructured":"Oksanen, J., Simpson, G.L., Blanchet, F.G., Kindt, R., Legendre, P., Minchin, P.R., O\u2019Hara, R.B., Solymos, P., Stevens, M.H.H., and Szoecs, E. (2021, October 28). Eduard Szoecs and Helene Wagner (2020). Vegan: Community Ecology Package. r Package Version 2.5-7. Available online: https:\/\/github.com\/vegandevs\/vegan."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"585","DOI":"10.1093\/bioinformatics\/btx644","article-title":"MATAM: Reconstruction of Phylogenetic Marker Genes from Short Sequencing Reads in Metagenomes","volume":"34","author":"Pericard","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"D590","DOI":"10.1093\/nar\/gks1219","article-title":"The SILVA Ribosomal RNA Gene Database Project: Improved Data Processing and Web-Based Tools","volume":"41","author":"Quast","year":"2013","journal-title":"Nucleic Acids Res"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"5261","DOI":"10.1128\/AEM.00062-07","article-title":"Na\u00efve Bayesian Classifier for Rapid Assignment of RRNA Sequences into the New Bacterial Taxonomy","volume":"73","author":"Wang","year":"2007","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Shen, W., Le, S., Li, Y., and Hu, F. (2016). SeqKit: A Cross-Platform and Ultrafast Toolkit for FASTA\/Q File Manipulation. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0163962"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2194","DOI":"10.1093\/bioinformatics\/btr381","article-title":"UCHIME Improves Sensitivity and Speed of Chimera Detection","volume":"27","author":"Edgar","year":"2011","journal-title":"Bioinformatics"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"2371","DOI":"10.1093\/bioinformatics\/bty113","article-title":"Updating the 97% Identity Threshold for 16S Ribosomal RNA OTUs","volume":"34","author":"Edgar","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1613","DOI":"10.1099\/ijsem.0.001755","article-title":"Introducing EzBioCloud: A Taxonomically United Database of 16S RRNA Gene Sequences and Whole-Genome Assemblies","volume":"67","author":"Yoon","year":"2017","journal-title":"Int. J. Syst. Evol. Microbiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"766364","DOI":"10.3389\/fmicb.2021.766364","article-title":"Advances in Metagenomics and Its Application in Environmental Microorganisms","volume":"12","author":"Zhang","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1038\/s41587-020-0718-6","article-title":"A Genomic Catalog of Earth\u2019s Microbiomes","volume":"39","author":"Nayfach","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"767","DOI":"10.1038\/s41396-018-0307-6","article-title":"Quantifying the Changes in Genetic Diversity within Sequence-Discrete Bacterial Populations across a Spatial and Temporal Riverine Gradient","volume":"13","author":"Meziti","year":"2019","journal-title":"ISME J."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"e02593-20","DOI":"10.1128\/AEM.02593-20","article-title":"The Reliability of Metagenome-Assembled Genomes (MAGs) in Representing Natural Populations: Insights from Comparing MAGs against Isolate Genomes Derived from the Same Fecal Sample","volume":"87","author":"Meziti","year":"2021","journal-title":"Appl. Env. Microbiol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1101\/gr.258640.119","article-title":"Accurate and Complete Genomes from Metagenomes","volume":"30","author":"Chen","year":"2020","journal-title":"Genome Res."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1186\/s40168-016-0154-5","article-title":"Recovering Complete and Draft Population Genomes from Metagenome Datasets","volume":"4","author":"Sangwan","year":"2016","journal-title":"Microbiome"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1007\/s11390-010-9306-4","article-title":"Metagenomics: Facts and Artifacts, and Computational Challenges*","volume":"25","author":"Wooley","year":"2009","journal-title":"J. Comput. Sci. Technol."},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Mengoni, A., Bacci, G., and Fondi, M. (2021). Bacterial Pangenomics: Methods and Protocols, Methods in Molecular Biology; Springer.","DOI":"10.1007\/978-1-0716-1099-2"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/11\/1\/175\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,12,4]],"date-time":"2023-12-04T12:20:59Z","timestamp":1701692459000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/11\/1\/175"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,1,10]]},"references-count":51,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2023,1]]}},"alternative-id":["microorganisms11010175"],"URL":"http:\/\/dx.doi.org\/10.3390\/microorganisms11010175","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":["Virology","Microbiology (medical)","Microbiology"],"published":{"date-parts":[[2023,1,10]]}}}