Yeast metagenomics: analytical challenges in the analysis of the eukaryotic microbiome

Abstract

Even if their impact is often underestimated, yeasts and yeast-like fungi represent the most prevalent eukaryotic members of microbial communities on Earth. They play numerous roles in natural ecosystems and in association with their hosts. They are involved in the food industry and pharmaceutical production, but they can also cause diseases in other organisms, making the understanding of their biology mandatory. The ongoing loss of biodiversity due to overexploitation of environmental resources is a growing concern in many countries. Therefore, it becomes crucial to understand the ecology and evolutionary history of these organisms to systematically classify them. To achieve this, it is essential that our knowledge of the mycobiota reaches a level similar to that of the bacterial communities. To overcome the existing challenges in the study of fungal communities, the first step should be the establishment of standardized techniques for the correct identification of species, even from complex matrices, both in wet lab practices and in bioinformatic tools.

Keywords

Yeasts / fungi / microbiome / microbial eukaryotes / eukaryome / ngs / metagenomics / taxonomy

References

[1] Bik HM,Creer S,Knight R.Sequencing our way towards understanding global eukaryotic biodiversity.Trends Ecol Evol2012;27:233-43 PMCID:PMC3311718

[2] Rodriguez RJ,Arnold AE.Fungal endophytes: diversity and functional roles.New Phytol2009;182:314-30

[3] Akin DE.Role of rumen fungi in fiber degradation.J Dairy Sci1990;73:3023-32

[4] Kamoun S,Jones JD.The Top 10 oomycete pathogens in molecular plant pathology.Mol Plant Pathol2015;16:413-34 PMCID:PMC6638381

[5] Haque R.Human intestinal parasites.J Health Popul Nutr2007;25:387-91 PMCID:PMC2754014

[6] Laforest-Lapointe I.Microbial eukaryotes: a missing link in gut microbiome studies.mSystems2018;3:e00201-17 PMCID:PMC5850078

[7] Parfrey LW,Lauber CL.Communities of microbial eukaryotes in the mammalian gut within the context of environmental eukaryotic diversity.Front Microbiol2014;5:298 PMCID:PMC4063188

[8] Sonnenburg ED,Tikhonov M,Wingreen NS.Diet-induced extinctions in the gut microbiota compound over generations.Nature2016;529:212-5 PMCID:PMC4850918

[9] Caron DA,Allen AE.Probing the evolution, ecology and physiology of marine protists using transcriptomics.Nat Rev Microbiol2017;15:6-20

[10] Brussaard L,Brown GG.Soil biodiversity for agricultural sustainability.Agr Ecosyst Environ2007;121:233-44

[11] James TY,Hittinger CT.Toward a fully resolved fungal tree of life.Annu Rev Microbiol2020;74:291-313

[12] Shen XX,Kominek J.Tempo and mode of genome evolution in the budding yeast subphylum.Cell2018;175:1533-45.e20 PMCID:PMC6291210

[13] Hernández-Santos N.Through the scope darkly: the gut mycobiome comes into focus.Cell Host Microbe2017;22:728-9 PMCID:PMC5964996

[14] Alou M, Naud S, Khelaifia S, Bonnet M, Lagier JC, Raoult D. State of the art in the culture of the human microbiota: new interests and strategies.Clin Microbiol Rev2020;34:e00129-19 PMCID:PMC7605308

[15] Vu D,Szöke S.DNA barcoding analysis of more than 9000 yeast isolates contributes to quantitative thresholds for yeast species and genera delimitation.Stud Mycol2016;85:91-105 PMCID:PMC5192050

[16] Makimura K.Species identification system for dermatophytes based on the DNA sequences of nuclear ribosomal internal transcribed spacer 1.Nihon Ishinkin Gakkai Zasshi2001;42:61-7

[17] Leaw SN,Sun HF,Bouchara JP.Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions.J Clin Microbiol2006;44:693-9 PMCID:PMC1393093

[18] Del Campo J,Herranz M.Validation of a universal set of primers to study animal-associated microeukaryotic communities.Environ Microbiol2019;21:3855-61

[19] del Campo J, Bass D, Keeling PJ, Bennett A. The eukaryome: diversity and role of microeukaryotic organisms associated with animal hosts.Functional Ecology2020;34:2045-54

[20] Parfrey LW,Knight R.Microbial eukaryotes in the human microbiome: ecology, evolution, and future directions.Front Microbiol2011;2:153 PMCID:PMC3135866

[21] Andersen LO,Stensvold CR.Waiting for the human intestinal Eukaryotome.ISME J2013;7:1253-5 PMCID:PMC3695289

[22] Franco-Duarte R,Gomes AC,de Sousa B.Genotyping of Saccharomyces cerevisiae strains by interdelta sequence typing using automated microfluidics.Electrophoresis2011;32:1447-55

[23] Lücking R,Robbertse B.Unambiguous identification of fungi: where do we stand and how accurate and precise is fungal DNA barcoding?.IMA Fungus2020;11:14 PMCID:PMC7353689

[24] Schoch CL,Huhndorf S.Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi.Proc Natl Acad Sci U S A2012;109:6241-6 PMCID:PMC3341068

[25] Knight R,Taylor BC.Best practices for analysing microbiomes.Nat Rev Microbiol2018;16:410-22

[26] Gillevet PM,Torzilli AP.Analyzing salt-marsh fungal diversity: comparing ARISA fingerprinting with clone sequencing and pyrosequencing.Fungal Ecology2009;2:160-7

[27] Ghannoum MA,Mukherjee PK.Characterization of the oral fungal microbiome (mycobiome) in healthy individuals.PLoS Pathog2010;6:e1000713 PMCID:PMC2795202

[28] Kurtzman CP.1 Saccharomycotina and taphrinomycotina: the yeasts and yeastlike fungi of the ascomycota. In: Mclaughlin DJ, Spatafora JW, editors. Systematics and Evolution. Berlin: Springer Berlin Heidelberg; 2015. p. 3-33.

[29] Kurtzman CP,Boekhout T.Chapter 1 - Definition, classification and nomenclature of the yeasts. In: The Yeasts. Elsevier; 2011. p. 3-5.

[30] Li Y,Chang Y.A genome-scale phylogeny of the kingdom fungi.Curr Biol2021;31:1653-65.e5 PMCID:PMC8347878

[31] Żymańczyk-duda E,Klimek-ochab M,Zerka A.Yeast as a versatile tool in biotechnology. In: Morata A, Loira I, editors. Yeast - Industrial Applications. InTech; 2017.

[32] Boekhout T,Begerow D.The evolving species concepts used for yeasts: from phenotypes and genomes to speciation networks.Fungal Divers2021;109:27-55 PMCID:PMC8550739

[33] Hawksworth DL.The magnitude of fungal diversity: the 1.5 million species estimate revisited.Mycol Res2001;105:1422-32

[34] Blackwell M.The fungi: 1, 2, 3 ... 5.1 million species?.Am J Bot2011;98:426-38

[35] Hawksworth DL.Fungal diversity revisited: 2.2 to 3.8 million species.Microbiol Spectr2017;5:

[36] Cheek M,Kirk P.New scientific discoveries: plants and fungi.Plants People Planet2020;2:371-88

[37] Lücking R,Robbertse B.Fungal taxonomy and sequence-based nomenclature.Nat Microbiol2021;6:540-8 PMCID:PMC10116568

[38] Huseyin CE,Cotter PD.Forgotten fungi-the gut mycobiome in human health and disease.FEMS Microbiol Rev2017;41:479-511

[39] Naranjo-Ortiz MA.Fungal evolution: diversity, taxonomy and phylogeny of the Fungi.Biol Rev Camb Philos Soc2019;94:2101-37 PMCID:PMC6899921

[40] Suhr MJ.The human gut mycobiome: pitfalls and potentials - a mycologist’s perspective.Mycologia2015;107:1057-73

[41] Hinsu A,Joshi A.To culture or not to culture: a snapshot of culture-dependent and culture-independent bacterial diversity from peanut rhizosphere.PeerJ2021;9:e12035 PMCID:PMC8418214

[42] Strati F,Stefanini I.Age and gender affect the composition of fungal population of the human gastrointestinal tract.Front Microbiol2016;7:1227 PMCID:PMC4971113

[43] Browne HP,Anonye BO.Culturing of 'unculturable' human microbiota reveals novel taxa and extensive sporulation.Nature2016;533:543-6 PMCID:PMC4890681

[44] Gutleben J,van Elsas JD,Overmann J.The multi-omics promise in context: from sequence to microbial isolate.Crit Rev Microbiol2018;44:212-29

[45] Borges FM,Sarmiento MRA.Fungal diversity of human gut microbiota among eutrophic, overweight, and obese individuals based on aerobic culture-dependent approach.Curr Microbiol2018;75:726-35

[46] Hamad I,Azhar EI.Culturomics and amplicon-based metagenomic approaches for the study of fungal population in human gut microbiota.Sci Rep2017;7:16788 PMCID:PMC5711903

[47] Huseyin CE,O’Sullivan O,Scanlan PD.The fungal frontier: a comparative analysis of methods used in the study of the human gut mycobiome.Front Microbiol2017;8:1432 PMCID:PMC5534473

[48] Aimanianda V,Simenel C,Delepierre M.Cell wall beta-(1,6)-glucan of Saccharomyces cerevisiae: structural characterization and in situ synthesis.J Biol Chem2009;284:13401-12 PMCID:PMC2679440

[49] Valiante V,Föge M.The Aspergillus fumigatus cell wall integrity signaling pathway: drug target, compensatory pathways, and virulence.Front Microbiol2015;6:325 PMCID:PMC4399325

[50] Gow NAR,Munro CA.The fungal cell wall: structure, biosynthesis, and function.Microbiol Spectr2017;5:

[51] Machová E,Kogan G.Effect of ultrasonic treatment on the molecular weight of carboxymethylated chitin-glucan complex from Aspergillus niger.Ultrason Sonochem1999;5:169-72

[52] Mendonça A,Franco-Duarte R.Correction to: optimization of a quantitative PCR methodology for detection of Aspergillus spp. and Rhizopus arrhizus.Mol Diagn Ther2022;26:527 PMCID:PMC9629307

[53] Turnbaugh PJ,Hamady M,Knight R.The human microbiome project.Nature2007;449:804-10

[54] Stefanini I,Legras JL.Role of social wasps in Saccharomyces cerevisiae ecology and evolution.Proc Natl Acad Sci U S A2012;109:13398-403 PMCID:PMC3421210

[55] Abdelrhman KF,Mancusi C,Serena F.A first insight into the gut microbiota of the sea turtle caretta caretta.Front Microbiol2016;7:1060 PMCID:PMC4935691

[56] Abdelrhman KF,Marras B.Exploring the bacterial gut microbiota of supralittoral talitrid amphipods.Res Microbiol2017;168:74-84

[57] Ramazzotti M.Chapter 5 - 16S rRNA-based taxonomy profiling in the metagenomics era. In: Nagarajan M, editor. Metagenomics. Academic Press; 2018. p. 103-19.

[58] Arranz V,Aguirre JD.MARES, a replicable pipeline and curated reference database for marine eukaryote metabarcoding.Sci Data2020;7:209 PMCID:PMC7334202

[59] Frøslev TG,Bruun HH.Algorithm for post-clustering curation of DNA amplicon data yields reliable biodiversity estimates.Nat Commun2017;8:1188 PMCID:PMC5662604

[60] Callahan BJ,Rosen MJ,Johnson AJ.DADA2: high-resolution sample inference from Illumina amplicon data.Nat Methods2016;13:581-3 PMCID:PMC4927377

[61] Nilsson RH,Bahram M,Baldrian P.Mycobiome diversity: high-throughput sequencing and identification of fungi.Nat Rev Microbiol2019;17:95-109

[62] Nilsson RH,Ryberg M,Larsson KH.Intraspecific ITS variability in the kingdom fungi as expressed in the international sequence databases and its implications for molecular species identification.Evol Bioinform Online2008;4:193-201 PMCID:PMC2614188

[63] Ali NABM,Morales RF,Chotirmall SH.Optimisation and benchmarking of targeted amplicon sequencing for mycobiome analysis of respiratory specimens.Int J Mol Sci2019;20:4991 PMCID:PMC6829331

[64] Bokulich NA.Improved selection of internal transcribed spacer-specific primers enables quantitative, ultra-high-throughput profiling of fungal communities.Appl Environ Microbiol2013;79:2519-26 PMCID:PMC3623200

[65] Tedersoo L.Fungal identification biases in microbiome projects.Environ Microbiol Rep2016;8:774-9

[66] Franco-Duarte R,Gulis V,Pascoal C.ITS rDNA barcodes clarify molecular diversity of aquatic hyphomycetes.Microorganisms2022;10:1569 PMCID:PMC9415940

[67] Bradshaw MJ,Rokas A.Extensive intragenomic variation in the internal transcribed spacer region of fungi.iScience2023;26:107317 PMCID:PMC10387565

[68] Bellemain E,Brochmann C,Taberlet P.ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases.BMC Microbiol2010;10:189 PMCID:PMC2909996

[69] Mbareche H,Bilodeau G.Comparison of the performance of ITS1 and ITS2 as barcodes in amplicon-based sequencing of bioaerosols.PeerJ2020;8:e8523 PMCID:PMC7032056

[70] Hoggard M,Wong G.Characterizing the human mycobiota: a comparison of small subunit rRNA, ITS1, ITS2, and large subunit rRNA genomic targets.Front Microbiol2018;9:2208 PMCID:PMC6157398

[71] Peterson SW.Ribosomal RNA sequence divergence among sibling species of yeasts.Syst Appl Microbiol1991;14:124-9

[72] Kurtzman CP.Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences.Antonie Van Leeuwenhoek1998;73:331-71

[73] Tang J,Brown J,Funari VA.Mycobiome: approaches to analysis of intestinal fungi.J Immunol Methods2015;421:112-21 PMCID:PMC4451377

[74] Filippis F, Laiola M, Blaiotta G, Ercolini D. Different amplicon targets for sequencing-based studies of fungal diversity.Appl Environ Microbiol2017;83:e00905-17 PMCID:PMC5561290

[75] Kiss L.Limits of nuclear ribosomal DNA internal transcribed spacer (ITS) sequences as species barcodes for Fungi.Proc Natl Acad Sci U S A2012;109:E1811; author reply E1812 PMCID:PMC3390822

[76] Stielow JB,Seifert KA.One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes.Persoonia2015;35:242-63

[77] James TY,Schoch CL.Reconstructing the early evolution of fungi using a six-gene phylogeny.Nature2006;443:818-22

[78] Matheny PB,Ammirati JF.Using RPB1 sequences to improve phylogenetic inference among mushrooms (Inocybe, Agaricales).Am J Bot2002;89:688-98

[79] Meyer W,Hoang MTV.Database establishment for the secondary fungal DNA barcode translational elongation factor 1α (TEF1α)¹.Genome2019;62:160-9

[80] Větrovský T,Žifčáková L,Baldrian P.The rpb2 gene represents a viable alternative molecular marker for the analysis of environmental fungal communities.Mol Ecol Resour2016;16:388-401

[81] Morrison GA,Lee GC.Nanopore sequencing of the fungal intergenic spacer sequence as a potential rapid diagnostic assay.J Clin Microbiol2020;58:e01972-20 PMCID:PMC7685901

[82] Geiser DM,Taylor JW.Evolutionary relationships in Aspergillus section Fumigati inferred from partial β-tubulin and hydrophobin DNA sequences.Mycologia1998;90:831-45

[83] Hu T,Monos D.Next-generation sequencing technologies: an overview.Hum Immunol2021;82:801-11

[84] Quince C,Simpson JT,Segata N.Shotgun metagenomics, from sampling to analysis.Nat Biotechnol2017;35:833-44

[85] Morgan XC.Meta’omic analytic techniques for studying the intestinal microbiome.Gastroenterology2014;146:1437-48.e1

[86] Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome.Nature2012;486:207-14 PMCID:PMC3564958

[87] Nash AK,Wong MC.The gut mycobiome of the Human Microbiome Project healthy cohort.Microbiome2017;5:153 PMCID:PMC5702186

[88] Hoang MTV,Hu Y,Meyer W.Long-reads-based metagenomics in clinical diagnosis with a special focus on fungal infections.Front Microbiol2021;12:708550 PMCID:PMC8770865

[89] Pollard MO,Mentzer AJ,Sandhu MS.Long reads: their purpose and place.Hum Mol Genet2018;27:R234-41 PMCID:PMC6061690

[90] Mantere T,Hoischen A.Long-read sequencing emerging in medical genetics.Front Genet2019;10:426 PMCID:PMC6514244

[91] Sui Y,Droby S,Wu X.Genome sequence, assembly, and characterization of the antagonistic yeast candida oleophila used as a biocontrol agent against post-harvest diseases.Front Microbiol2020;11:295 PMCID:PMC7052047

[92] Cuomo CA,Yang B,Forche A.Whole genome sequence of the heterozygous clinical isolate candida krusei 81-B-5.G32017;7:2883-9 PMCID:PMC5592916

[93] Luo R,Workman R.First draft genome sequence of the pathogenic fungus Lomentospora prolificans (Formerly Scedosporium prolificans).G32017;7:3831-6 PMCID:PMC5677167

[94] Vale-Silva L,Tran VDT.Comparative genomics of two sequential candida glabrata clinical isolates.G32017;7:2413-26 PMCID:PMC5555451

[95] Panthee S,Ishijima SA,Sekimizu K.Utilization of hybrid assembly approach to determine the genome of an opportunistic pathogenic fungus, candida albicans TIMM 1768.Genome Biol Evol2018;10:2017-22 PMCID:PMC6097704

[96] Rhodes J,Farrer RA.Genomic epidemiology of the UK outbreak of the emerging human fungal pathogen Candida auris.Emerg Microbes Infect2018;7:43 PMCID:PMC5874254

[97] Morand SC,Iltis A.Complete genome sequence of Malassezia restricta CBS 7877, an opportunist pathogen involved in dandruff and seborrheic dermatitis.Microbiol Resour Announc2019;8:e01543-18 PMCID:PMC6368656

[98] Schultzhaus Z,Wang Z.Genome sequence of the black yeast exophiala lecanii-corni.Microbiol Resour Announc2019;8:e01709-18 PMCID:PMC6406120

[99] Pchelin IM,Churina MA.Whole genome sequence of first Candida auris strain, isolated in Russia.Med Mycol2020;58:414-6

[100] Arnaud MB,Costanzo MC.The aspergillus genome database, a curated comparative genomics resource for gene, protein and sequence information for the Aspergillus research community.Nucleic Acids Res2010;38:D420-7 PMCID:PMC2808984

[101] Ratnasingham S.bold: The barcode of life data system (http://www.barcodinglife.org).Mol Ecol Notes2007;7:355-64 PMCID:PMC1890991

[102] Inglis DO,Binkley J.The Candida genome database incorporates multiple Candida species: multispecies search and analysis tools with curated gene and protein information for Candida albicans and Candida glabrata.Nucleic Acids Res2012;40:D667-74 PMCID:PMC3245171

[103] Güldener U,Kastenmüller G.CYGD: the comprehensive yeast genome database.Nucleic Acids Res2005;33:D364-8 PMCID:PMC540007

[104] Stajich JE,Brunk BP.FungiDB: an integrated functional genomics database for fungi.Nucleic Acids Res2012;40:D675-81 PMCID:PMC3245123

[105] Grossetête S,Lespinet O.FUNGIpath: a tool to assess fungal metabolic pathways predicted by orthology.BMC Genomics2010;11:81 PMCID:PMC2829015

[106] Geiser DM,Kang S.FUSARIUM-ID v. 1.0: a DNA sequence database for identifying fusarium.Eur J Plant Pathol2004;110:473-9

[107] O’donnell K,Rinaldi MG.Internet-accessible DNA sequence database for identifying fusaria from human and animal infections.J Clin Microbiol2010;48:3708-18 PMCID:PMC2953079

[108] Irinyi L,Garcia-Hermoso D.International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database - the quality controlled standard tool for routine identification of human and animal pathogenic fungi.Med Mycol2015;53:313-37

[109] Ahrendt SR,Haridas S.MycoCosm, the JGI’s fungal genome portal for comparative genomic and multiomics data analyses. In: Martin F, Uroz S, editors. Microbial Environmental Genomics (MEG). New York: Springer US; 2023. p. 271-91.

[110] Wood V,McDowall MD.PomBase: a comprehensive online resource for fission yeast.Nucleic Acids Res2012;40:D695-9 PMCID:PMC3245111

[111] Cherry JM,Amundsen C.Saccharomyces genome database: the genomics resource of budding yeast.Nucleic Acids Res2012;40:D700-5 PMCID:PMC3245034

[112] Abarenkov K,Larsson KH.The UNITE database for molecular identification of fungi - recent updates and future perspectives.New Phytol2010;186:281-5

[113] Prakash PY,Halliday C,Robert V.Online databases for taxonomy and identification of pathogenic fungi and proposal for a cloud-based dynamic data network platform.J Clin Microbiol2017;55:1011-24 PMCID:PMC5377827

[114] Kuczynski J,Walters WA.Experimental and analytical tools for studying the human microbiome.Nat Rev Genet2011;13:47-58 PMCID:PMC5119550

[115] Schloss PD,Ryabin T.Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities.Appl Environ Microbiol2009;75:7537-41 PMCID:PMC2786419

[116] Gweon HS,Taylor J.PIPITS: an automated pipeline for analyses of fungal internal transcribed spacer sequences from the Illumina sequencing platform.Methods Ecol Evol2015;6:973-80 PMCID:PMC4981123

[117] Rognes T,Nichols B,Mahé F.VSEARCH: a versatile open source tool for metagenomics.PeerJ2016;4:e2584 PMCID:PMC5075697

[118] Mysara M,Leys N,Monsieurs P.From reads to operational taxonomic units: an ensemble processing pipeline for MiSeq amplicon sequencing data.Gigascience2017;6:1-10 PMCID:PMC5466709

[119] Bolyen E,Dillon MR.Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2.Nat Biotechnol2019;37:852-7 PMCID:PMC7015180

[120] He Y,Jiang XT.Stability of operational taxonomic units: an important but neglected property for analyzing microbial diversity.Microbiome2015;3:20 PMCID:PMC4438525

[121] Callahan BJ,Holmes SP.Exact sequence variants should replace operational taxonomic units in marker-gene data analysis.ISME J2017;11:2639-43 PMCID:PMC5702726

[122] Chiarello M,Villéger S.Ranking the biases: the choice of OTUs vs. ASVs in 16S rRNA amplicon data analysis has stronger effects on diversity measures than rarefaction and OTU identity threshold.PLoS One2022;17:e0264443 PMCID:PMC8870492

[123] Wood DE.Kraken: ultrafast metagenomic sequence classification using exact alignments.Genome Biol2014;15:R46 PMCID:PMC4053813

[124] Caporaso JG,Stombaugh J.QIIME allows analysis of high-throughput community sequencing data.Nat Methods2010;7:335-6 PMCID:PMC3156573

[125] Abarenkov K,Nilsson RH.PlutoF - a web based workbench for ecological and taxonomic research, with an online implementation for fungal ITS sequences.Evol Bioinform Online2010;6:EBO.S6271 PMCID:PMC3023303

[126] Kumar S,Mevik BH.CLOTU: an online pipeline for processing and clustering of 454 amplicon reads into OTUs followed by taxonomic annotation.BMC Bioinform2011;12:182 PMCID:PMC3120705

[127] White JR,White O,Fricke WF.CloVR-ITS: automated internal transcribed spacer amplicon sequence analysis pipeline for the characterization of fungal microbiota.Microbiome2013;1:6 PMCID:PMC3869194

[128] Albanese D,De Filippo C,Donati C.MICCA: a complete and accurate software for taxonomic profiling of metagenomic data.Sci Rep2015;5:9743 PMCID:PMC4649890

[129] Fosso B,Marzano M.BioMaS: a modular pipeline for Bioinformatic analysis of Metagenomic AmpliconS.BMC Bioinform2015;16:203 PMCID:PMC4486701

[130] Odom AR,Castro-Nallar E,Johnson WE.Metagenomic profiling pipelines improve taxonomic classification for 16S amplicon sequencing data.Sci Rep2023;13:13957 PMCID:PMC10460424

[131] Edgar RC.UPARSE: highly accurate OTU sequences from microbial amplicon reads.Nat Methods2013;10:996-8

[132] Edgar RC.Error filtering, pair assembly and error correction for next-generation sequencing reads.Bioinformatics2015;31:3476-82

[133] Truong DT,Tickle TL.MetaPhlAn2 for enhanced metagenomic taxonomic profiling.Nat Methods2015;12:902-3

[134] Wood DE,Langmead B.Improved metagenomic analysis with Kraken 2.Genome Biol2019;20:257 PMCID:PMC6883579

[135] Buchfink B,Huson DH.Fast and sensitive protein alignment using DIAMOND.Nat Methods2015;12:59-60

[136] Olson ND,Hill CM.Metagenomic assembly through the lens of validation: recent advances in assessing and improving the quality of genomes assembled from metagenomes.Brief Bioinform2019;20:1140-50 PMCID:PMC6781575

[137] Jünemann S,Jaenicke S.Bioinformatics for NGS-based metagenomics and the application to biogas research.J Biotechnol2017;261:10-23

[138] Nurk S,Korobeynikov A.metaSPAdes: a new versatile metagenomic assembler.Genome Res2017;27:824-34 PMCID:PMC5411777

[139] Li D,Luo R,Lam TW.MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph.Bioinformatics2015;31:1674-6

[140] Peng Y,Yiu SM.IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth.Bioinformatics2012;28:1420-8

[141] Kang DD,Kirton E.MetaBAT 2: an adaptive binning algorithm for robust and efficient genome reconstruction from metagenome assemblies.PeerJ2019;7:e7359 PMCID:PMC6662567

[142] Alneberg J,de Bruijn I.Binning metagenomic contigs by coverage and composition.Nat Methods2014;11:1144-6

[143] Wu YW,Singer SW.MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets.Bioinformatics2016;32:605-7

[144] Uritskiy GV,Taylor J.MetaWRAP-a flexible pipeline for genome-resolved metagenomic data analysis.Microbiome2018;6:158 PMCID:PMC6138922

[145] Kanehisa M.KEGG: kyoto encyclopedia of genes and genomes.Nucleic Acids Res2000;28:27-30 PMCID:PMC102409