Fungal ITS1 deep-sequencing strategies to reconstruct the composition of a 26-species community and evaluation of the gut mycobiota of healthy Japanese individuals

Daisuke Motooka, Kosuke Fujimoto, Reiko Tanaka, Takashi Yaguchi, Kazuyoshi Gotoh, Yuichi Maeda, Yoki Furuta, Takashi Kurakawa, Naohisa Goto, Teruo Yasunaga, Masashi Narazaki, Atsushi Kumanogoh, Toshihiro Horii, Tetsuya Iida, Kiyoshi Takeda, Shota Nakamura

Research output: Contribution to journalArticle

28 Citations (Scopus)

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 the de facto standard 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 of Candida albicans or Saccharomyces cerevisiae and 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.

Original languageEnglish
Article number238
JournalFrontiers in Microbiology
Volume8
Issue numberFEB
DOIs
Publication statusPublished - Feb 15 2017

Keywords

  • Fungi
  • High-throughtput sequencing (HTS)
  • Internal transcribed spacer (ITS)
  • Mycobiota
  • The gut mycobiota in Japanese

ASJC Scopus subject areas

  • Microbiology
  • Microbiology (medical)

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    Motooka, D., Fujimoto, K., Tanaka, R., Yaguchi, T., Gotoh, K., Maeda, Y., Furuta, Y., Kurakawa, T., Goto, N., Yasunaga, T., Narazaki, M., Kumanogoh, A., Horii, T., Iida, T., Takeda, K., & Nakamura, S. (2017). Fungal ITS1 deep-sequencing strategies to reconstruct the composition of a 26-species community and evaluation of the gut mycobiota of healthy Japanese individuals. Frontiers in Microbiology, 8(FEB), [238]. https://doi.org/10.3389/fmicb.2017.00238