Choice of 16S ribosomal RNA primers affects the microbiome analysis

Authors: DARWISH, NADIA - US Department Of Agriculture (USDA); Shao, Jonathan; Schreier, Lori; Proszkowiec-Weglarz, Monika

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/26/2021
Publication Date: 6/4/2021
Citation: Darwish, N., Shao, J.Y., Schreier, L.L., Proszkowiec-Wegla, M.K. 2021. Choice of 16S ribosomal RNA primers affects the microbiome analysis. Scientific Reports. https://doi.org/10.1038/s41598-021-91387-w.
DOI: https://doi.org/10.1038/s41598-021-91387-w

Interpretive Summary: Microbiota plays important role in health, nutrition, growth and development of animal gastrointestinal tract. Microbiota can be influenced by many physiological, environmental and dietary factors. Also, the methodology used for bacterial determination can influence the results and their interpretation. Therefore, our goal was to determine the effect of different 16S rRNA primers on taxonomic composition, diversity, and predicted function of chicken cecal microbial community. Cecal digesta collected from Ross 708 birds at 1, 3 and 5 weeks of age were used for bacterial DNA isolation. Eight different primer pairs targeting different variable regions of the 16S rRNA were employed. DNA sequences were analyzed using open source platform Qiime2 and Greengenes database for alpha and beta diversity and taxonomic composition. PICRUSt was used to determine the predicted function of bacterial communities. Differences in bacterial relative abundance due to primer set were analyzed using SAS. Both alpha and beta diversities of bacterial communities were strongly influenced by primer selection. Taxonomic composition and predicted function of the microbiota were also affected by the primer set choice. Obtained results strongly suggest that the choice of 16s rRNA primers can influence microbiome analysis and data interpretation.

Technical Abstract: The aim of this study was to determine the effect of different 16S rRNA primers on taxonomic composition, diversity, and predicted function of chicken cecal microbial community. Cecal digesta collected from Ross 708 birds at 1, 3 and 5 weeks of age were used for bacterial DNA isolation. Eight different primer pairs targeting different variable regions of the 16S rRNA were employed. DNA sequences were analyzed using open source platform Qiime2 and Greengenes database for alpha and beta diversity and taxonomic composition. PICRUSt was used to determine the predicted function of bacterial communities. Differences in bacterial relative abundance due to primer set were analyzed using SAS. The average PCR amplicon size ranged from 315 bp (V3) to 769 bp (V4-V6). Alpha-diversity indexes were significantly (P<0.01) affected by the primer sets. Beta diversity analysis based on Unweighted UniFrac distance matrix showed separation of bacterial communities based on the selection of the primer set with four different clusters of bacterial communities. Taxonomic composition and predicted function of the microbiota were affected by the primer set choice. Obtained results strongly suggest that the choice of 16s rRNA primers can influence microbiome analysis and data interpretation.