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Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing.

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This study presents a standardized protocol for 16S rRNA amplicon sequencing to analyze the human gut microbiome. It addresses batch effects and provides a robust method for microbial composition analysis.

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Area of Science:

  • Microbiology
  • Genomics
  • Bioinformatics

Background:

  • The human intestinal microbiome is crucial for health, influencing immunity and metabolism.
  • Dysbiosis, an imbalance in gut microbiota, is linked to various diseases.
  • High-throughput sequencing, including 16S rRNA amplicon sequencing, enables gut microbial composition analysis.

Purpose of the Study:

  • To present a standardized, robust, and reproducible protocol for 16S rRNA amplicon sequencing of the human gut microbiome.
  • To address and minimize batch effects common in microbiome studies.
  • To provide a comprehensive guide from sample collection to data analysis.

Main Methods:

  • A column-free, direct-PCR approach for simultaneous DNA extraction and amplification of the V4 region of the 16S rRNA gene.
  • Integration of widely adopted protocols for fecal sample collection, processing, and sequencing.
  • Utilized QIIME 2 (version 2017.7.0) and DADA2 for data analysis, including a provided script.

Main Results:

  • The protocol enables efficient handling and DNA extraction from large numbers of fecal samples.
  • It facilitates the identification and quantification of different microbial taxa using 16S rRNA gene sequence variations.
  • The proposed method aims to minimize batch effects, enhancing data reproducibility.

Conclusions:

  • This step-by-step protocol offers a user-friendly, detailed, and reproducible method for 16S rRNA amplicon sequencing.
  • It empowers researchers to initiate robust gut microbiome analysis.
  • Standardization of this protocol can improve the reliability and comparability of microbiome research findings.