Best practices framework for using 16S rRNA gene sequencing in poultry microbiota research

Microbiome research has shown significant potential in enhancing poultry health and productivity. Despite the increasing volume of data linking the microbiota with various host traits, challenges persist regarding the consistency and reproducibility of findings. A major underlying issue is the variability in methodologies employed across studies. As such, a need exists to establish a set of standardized guidelines that help guide experimental design, DNA extraction, sequencing, data analysis, and reporting in poultry microbiota research. Rather than advocating for a single standardized protocol, we propose a best practices framework designed to enhance methodological rigor while accommodating distinct research contexts. Such a framework emphasizes the use of appropriate positive and negative controls and improved data reporting to facilitate cross-study comparisons and reproducibility. As a companion to our previous review of the 16S rRNA gene sequencing landscape in poultry microbiota research, this manuscript represents a collaborative effort among experts from academia, industry, and government. It aims to offer a practical set of guidelines that serve as a checklist for designing, conducting, analyzing, and reporting poultry microbiota studies. These guidelines seek to improve consistency, reproducibility, and robustness of poultry microbiota research.