title: Fungal ITS1 Deep-Sequencing Strategies to Reconstruct the Composition of a 26-Species Community and Evaluation of the Gut Mycobiota of Healthy Japanese Individuals.
abstract: The study of mycobiota remains relatively unexplored due to the lack of sufficient available reference strains and databases compared to those of bacterial microbiome studies. Deep sequencing of Internal Transcribed Spacer (ITS) regions is thede factostandard for fungal diversity analysis. However, results are often biased because of the wide variety of sequence lengths in the ITS regions and the complexity of high-throughput sequencing (HTS) technologies. In this study, a curated ITS database, ntF-ITS1, was constructed. This database can be utilized for the taxonomic assignment of fungal community members. We evaluated the efficacy of strategies for mycobiome analysis by using this database and characterizing a mock fungal community consisting of 26 species representing 15 genera using ITS1 sequencing with three HTS platforms: Illumina MiSeq (MiSeq), Ion Torrent Personal Genome Machine (IonPGM), and Pacific Biosciences (PacBio). Our evaluation demonstrated that PacBio's circular consensus sequencing with greater than 8 full-passes most accurately reconstructed the composition of the mock community. Using this strategy for deep-sequencing analysis of the gut mycobiota in healthy Japanese individuals revealed two major mycobiota types: a single-species type composed ofCandida albicansorSaccharomyces cerevisiaeand a multi-species type. In this study, we proposed the best possible processing strategies for the three sequencing platforms, of which, the PacBio platform allowed for the most accurate estimation of the fungal community. The database and methodology described here provide critical tools for the emerging field of mycobiome studies.
authors.affiliation: Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University Suita, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Motooka
authors.affiliation: Laboratory of Immune Regulation, Department of Microbiology and Immunology, Graduate School of Medicine, WPI Immunology Frontier Research Center, Osaka UniversitySuita, Japan; Department of Respiratory Medicine, Allergy and Rheumatic Diseases, Graduate School of Medicine, Osaka UniversitySuita, Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Fujimoto
authors.affiliation: Division of Bio-resources, Medical Mycology Research Center, Chiba University Chiba, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Tanaka
authors.affiliation: Division of Bio-resources, Medical Mycology Research Center, Chiba University Chiba, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yaguchi
authors.affiliation: Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka UniversitySuita, Japan; Department of Bacteriology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama UniversityOkayama, Japan.
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Gotoh
authors.affiliation: Laboratory of Immune Regulation, Department of Microbiology and Immunology, Graduate School of Medicine, WPI Immunology Frontier Research Center, Osaka UniversitySuita, Japan; Department of Respiratory Medicine, Allergy and Rheumatic Diseases, Graduate School of Medicine, Osaka UniversitySuita, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Maeda
authors.affiliation: Laboratory of Immune Regulation, Department of Microbiology and Immunology, Graduate School of Medicine, WPI Immunology Frontier Research Center, Osaka University Suita, Japan.
authors.forename: Yoki
authors.initials: Y
authors.lastname: Furuta
authors.affiliation: Laboratory of Immune Regulation, Department of Microbiology and Immunology, Graduate School of Medicine, WPI Immunology Frontier Research Center, Osaka University Suita, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kurakawa
authors.affiliation: Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University Suita, Japan.
authors.forename: Naohisa
authors.initials: N
authors.lastname: Goto
authors.affiliation: Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University Suita, Japan.
authors.forename: Teruo
authors.initials: T
authors.lastname: Yasunaga
authors.affiliation: Department of Respiratory Medicine, Allergy and Rheumatic Diseases, Graduate School of Medicine, Osaka University Suita, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Narazaki
authors.affiliation: Department of Respiratory Medicine, Allergy and Rheumatic Diseases, Graduate School of Medicine, Osaka University Suita, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kumanogoh
authors.affiliation: Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University Suita, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Horii
authors.affiliation: Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University Suita, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Iida
authors.affiliation: Laboratory of Immune Regulation, Department of Microbiology and Immunology, Graduate School of Medicine, WPI Immunology Frontier Research Center, Osaka University Suita, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Takeda
authors.affiliation: Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University Suita, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Nakamura
keywords: fungi; high-throughtput sequencing (HTS); internal transcribed spacer (ITS); mycobiota; the gut mycobiota in Japanese