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Improving the standards for gut microbiome analysis of fecal samples: insights from the field biology of Japanese macaques on Yakushima Island

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Abstract

Fecal DNA-based 16S ribosomal RNA (rRNA) gene sequencing using next-generation sequencers allows us to understand the dynamic gut microbiome adaptation of animals to their specific habitats. Conventional techniques of fecal microbiome analysis have been developed within the broad contexts defined by human biology; hence, many of these techniques are not immediately applicable to wild nonhuman primates. In order to establish a standard experimental protocol for the analysis of the gut microbiomes of wild animals, we selected the Japanese macaques (Macaca fuscata yakui) on Yakushima Island. We tested different protocols for each stage of fecal sample processing: storage, DNA extraction, and choice of the sequencing region in the bacterial 16S rRNA gene. We also analyzed the gut microbiome of captive Japanese macaques as the control. The comparison of samples obtained from identical macaques but subjected to different protocols showed that the tested storage methods (RNAlater and lysis buffer) produced effectively the same composition of bacterial operational taxonomic units (OTUs) as the standard frozen storage method, although the relative abundance of each OTU was quantitatively affected. Taxonomic assignment of the detected bacterial groups was also significantly affected by the region being sequenced, indicating that sequencing regions and the corresponding polymerase chain reaction (PCR) primer pairs for the 16S rRNA gene should be carefully selected. This study improves the current standard methods for microbiome analysis in wild nonhuman primates. Japanese macaques were shown to be a suitable model for understanding microbiome adaptation to various environments.

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Acknowledgements

We would like to thank our friends and colleagues in Yakushima. Experiment 3 of this study was performed in the Yakushima Field and Genome Science Courses of Kyoto University in spring 2014 and we thank the fellow students and lectures who joined in the courses and the administration staff for making these courses possible behind the scene. We are grateful to Dr. Hirohisa Hirai, Dr. Hiroo Imai, Dr. Nami Suzuki-Hashido, and other members of the Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University for valuable discussions. We also thank members of the Center for Human Evolution Modeling Research, Primate Research Institute, Kyoto University, particularly Mr. Takayoshi Natsume for collecting samples from captive animals. The study was financed by the Leading Graduate Program of Primatology and Wildlife Science (PWS) of Kyoto University, MEXT/JSPS KAKENHI (#12J04270 and #16K18630 to TH, #17H01911 to TH and GH, #25840170 to AS, and #25291100 to GH) and Fund for the Promotion of Joint International Research (#15KK0256 to GH).

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Authors and Affiliations

  1. Department of Wildlife Science (Nagoya Railroad Co., Ltd.), Primate Research Institute, Kyoto University, Inuyama, Aichi, 484-8506, Japan

    Takashi Hayakawa

  2. Japan Monkey Centre, Inuyama, Aichi, 484-0081, Japan

    Takashi Hayakawa

  3. Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, 606-8522, Japan

    Akiko Sawada

  4. Japan Society for the Promotion of Science, Kojimachi Business Center Building, 5-3-1, Kojimachi, Chiyoda-ku, Tokyo, 102-0083, Japan

    Akiko Sawada & Yosuke Kurihara

  5. Department of Environmental Solution Technology, Faculty of Science and Technology, Ryukoku University, Seta-Oe, Otsu, Shiga, 520-2194, Japan

    Akifumi S. Tanabe

  6. Institute for Advanced Biosciences, Keio University, 246-2 Mizukami, Kakuganji, Tsuruoka, Yamagata, 997-0052, Japan

    Shinji Fukuda

  7. Intestinal Microbiota Project, Kanagawa Institute of Industrial Science and Technology, 3-25-13 Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa, 210-0821, Japan

    Shinji Fukuda

  8. Transborder Medical Research Center, University of Tsukuba, 1-1-1, Tennodai, Tsukuba, Ibaraki, 305-8577, Japan

    Shinji Fukuda

  9. PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan

    Shinji Fukuda

  10. Wildlife Research Center, Kyoto University, 2-24 Tanaka-Sekiden-cho, Sakyo, Kyoto, 606-8203, Japan

    Takushi Kishida, Kei Matsushima, Jie Liu, Takafumi Suzumura, Hideki Sugiura & Kiyokazu Agata

  11. Ecology and Conservation Section, Primate Research Institute, Kyoto University, Inuyama, Aichi, 484-8506, Japan

    Yosuke Kurihara & Goro Hanya

  12. Research Institute in Tropical Ecology (IRET-CENAREST), Post Box 13354, Libreville, Gabon

    Etienne-Francois Akomo-Okoue

  13. Aquatic Mammals Laboratory, National Institute of Amazonian Research, Manaus, 69011-970, Brazil

    Waleska Gravena

  14. Health and Biotechnology Institute, Amazonas Federal University, Coari, 69460-000, Brazil

    Waleska Gravena

  15. Research Institute for Food and Agriculture, Ryukoku University, 1-5 Yokotani, Seta-oecho, Otsu, Shiga, 520-2194, Japan

    Makoto Kashima

  16. International Center for Island Studies Amami Station, Kagoshima University, 2-1 Naze-Yanagimachi, Amami, Kagoshima, 894-0032, Japan

    Mariko Suzuki

  17. Center for the Promotion of Interdisciplinary Education and Research, Education and Research Unit for Studies on Connectivity of Hills, Humans and Oceans (CoHHO), Kyoto University, Kitashirakawa Oiwake-cho, Sakyi-ku, Kyoto, 606-8502, Japan

    Kohmei Kadowaki

  18. Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan

    Eiji Inoue

  19. Department of Life Science, Faculty of Science, Graduate Course in Life Science, Graduate School of Science, Gakushuin University, Mejiro1-5-1, Toshima-ku, Tokyo, 171-8588, Japan

    Kiyokazu Agata

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  1. Takashi Hayakawa
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Corresponding author

Correspondence to Takashi Hayakawa.

Electronic supplementary material

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Supplementary material 1 (DOCX 168 kb)

10329_2018_671_MOESM2_ESM.csv

Supplementary material 2 Dataset S2: Operational taxonomic units (OTUs), assigned taxa, nucleotide sequences, and number of sequencing reads based on the 16S V3–V4 region. (CSV 1082 kb)

10329_2018_671_MOESM3_ESM.csv

Supplementary material 3 Dataset S1: Operational taxonomic units (OTUs), assigned taxa, nucleotide sequences, and number of sequencing reads based on the 16S V1–V2 region. (CSV 356 kb)

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Hayakawa, T., Sawada, A., Tanabe, A.S. et al. Improving the standards for gut microbiome analysis of fecal samples: insights from the field biology of Japanese macaques on Yakushima Island. Primates 59, 423–436 (2018). https://doi.org/10.1007/s10329-018-0671-x

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