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Sample Collection, DNA Extraction, and Library Construction Protocols of the Human Microbiome Studies in the International Human Phenome Project

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Abstract

The human microbiome plays a crucial role in human health. In the past decade, advances in high-throughput sequencing technologies and analytical software have significantly improved our knowledge of the human microbiome. However, most studies concerning the human microbiome did not provide repeatable protocols to guide the sample collection, handling, and processing procedures, which impedes obtaining valid and timely microbial taxonomic and functional results. This protocol provides detailed operation methods of human microbial sample collection, DNA extraction, and library construction for both the amplicon sequencing-based measurements of the microbial samples from the human nasal cavity, oral cavity, and skin, as well as the shotgun metagenomic sequencing-based measurements of the human stool samples among adult participants. This study intends to develop practical procedure standards to improve the reproducibility of microbiota profiling of human samples.

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Data Availability Statements

Raw sequence data are available in the National Omics Data Encyclopedia under the project ID OEP003565 (https://www.biosino.org/node/project/detail/OEP003565), with sample IDs OES23343-OES23362 (for the skin samples in Fig. 3) and OES23395-OES23397 (for the pure strain sample in Fig. 4).

Abbreviations

SCFAs:

Short-chain fatty acids

PCR:

Polymerase chain reaction

SCF-1:

Specimen collection fluid-1

rRNA:

Ribosomal RNA

ITS:

Internal transcribed spacer

QIIME2:

Quantitative insights into microbial ecology 2

DADA2:

Divisive Amplicon Denoising Algorithm 2

ASVs:

Amplicon sequence variants

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Acknowledgements

We thank all the participants enrolled for their generous supports. We thank Ms. Yimeng Wu from the College of Foreign Languages and Literature at Fudan University for the language editing and proofreading of this article.

Funding

This study was supported by the National Key Research and Development Program of China (2021YFA1301000) and the Shanghai Municipal Science and Technology Major Project (2017SHZDZX01).

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Authors and Affiliations

  1. State Key Laboratory of Genetic Engineering, Human Phenome Institute, and School of Life Sciences, Fudan University, 2005 Songhu Road, Shanghai, 200433, China

    Yetong Wang, Yanni Pu, Xuemei Wu, Yujie Pan, Chen Luo, Guoping Zhao & Yan Zheng

  2. Department of Cardiology, Shanghai Institute of Cardiovascular Disease, Zhongshan Hospital, Fudan University, 1609 Xietu Road, Shanghai, 200032, China

    Yan Zheng

  3. Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, School of Life Sciences, Fudan University, 2005 Songhu Road, Shanghai, 200433, China

    Ruyi Zhang, Danqi Wang, Yanren Wang & Zhexue Quan

  4. Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China

    Guoping Zhao

Authors
  1. Yetong Wang
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  2. Ruyi Zhang
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  3. Yanni Pu
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  4. Danqi Wang
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  10. Zhexue Quan
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  11. Yan Zheng
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Contributions

All authors contributed to the study conception and design. YTW, RYZ, YNP, YJP, CL, YRW, XMW and DQW completed the whole procedures of sample collection, DNA extraction, library construction and pilot studies of the protocol. Data collection and analysis were performed by DQW and YRW. The first draft of the manuscript was written by YTW and all authors commented on previous versions of the manuscript. YZ, ZXQ and GPZ provided insightful suggestions to improve the framework and enrich the content of the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Zhexue Quan or Yan Zheng.

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Ethics approval

This study was approved by the Ethics Committees of the School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, China, and carried out following the principles of the Declaration of Helsinki.

Consent to participate

Written informed consent was obtained from each participant before enrollment.

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Not applicable.

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Supplementary file1 (DOCX 3612 KB)

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Wang, Y., Zhang, R., Pu, Y. et al. Sample Collection, DNA Extraction, and Library Construction Protocols of the Human Microbiome Studies in the International Human Phenome Project. Phenomics 3, 300–308 (2023). https://doi.org/10.1007/s43657-023-00097-y

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