Abstract
The microbiome is an important consideration for the conservation of endangered species. Studies provided evidence of the effect of behavior and habitat change on the microbiota of wild animals and reported various inferences. It indicates the complexity of factors influencing microbiota diversity, including incomplete sampling procedures. Data abnormality may arise due to the procedures warranting preliminary analysis, such as rarefaction, before downstream analysis. This present study demonstrated the effect of data rarefaction and aggregation on the comparison of wild rusa deer’s gut microbial diversity. Eighty-five feces samples were collected from 11 deer populations inhabiting three national parks in Java and Bali islands. Using the Illumina Nova-Seq platform, fragments of 16s rRNA gene were sequenced, and raw data of 51,389 reads corresponding to 2 domains, 22 phyla, 45 classes, 83 orders, 182 families, and 460 genera of bacteria were obtained. Data rarefaction was applied at two different library sizes (minimum and fixed) and aggregation (11 populations into 3 research sites) to investigate its effect on the microbial diversity comparison. There are significant differences in alpha diversity between populations, but not research sites, at all library sizes of rarefaction. A similar finding is also found in beta diversity. Moreover, data rarefaction and aggregation result in different values of the diversity metrics. This present study shows that statistical analysis remains a substantial concern in microbiome studies applied to conservation biology. It suggests reporting a more detailed data normalization in microbiome studies as an inherent control of suboptimal sampling, particularly when involving feces.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
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Acknowledgements
The authors are grateful to the Directorate General of Higher Education, Research, and Technology–the Ministry of Education, Culture, Research, and Technology of the Republic of Indonesia for funding this research. The authors also thank the French Ministry of Foreign Affairs and International Development (MAEDI) and the French Ministry of Higher Education and Research (MESR) for supporting this research collaboration. The authors also acknowledge the Directorate General of Nature and Ecosystem Conservation–Ministry of Environment and Forestry of the Republic of Indonesia for issuing a sample collection permit.
Funding
Authors SAS, PY, and WTA received funding from the Directorate General of Higher Education, Research, and Technology, Indonesia, and authors MdGW, AA, and JM had support from the French Ministry of Foreign Affairs and International Development and the French Ministry of Higher Education and Research.
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All authors contributed to the research conception and design. Sample preparation, data collection and analysis were performed by SS and AA. The first draft of the manuscript was written by SS and AA, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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The authors affirm that no human research participants are involved in this research, and all fecal samples were collected non-invasively. Therefore, the research ethics committee of Universitas Gadjah Mada confirmed that ethical approval is not required.
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Subrata, S.A., Yuda, P., Artama, W.T. et al. Rusa deer microbiota: the importance of preliminary data analysis for meaningful diversity comparisons. Int Microbiol (2024). https://doi.org/10.1007/s10123-024-00521-x
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DOI: https://doi.org/10.1007/s10123-024-00521-x