Choice of microbiota database affects data analysis and interpretation in chicken cecal microbiota

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

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/13/2022
Publication Date: 5/21/2022
Citation: Campos, P.M., Darwish, N., Shao, J.Y., Proszkowiec-Wegla, M.K. 2022. Choice of microbiota database affects data analysis and interpretation in chicken cecal microbiota. Poultry Science. https://doi.org/10.1016/j.psj.2022.101971.
DOI: https://doi.org/10.1016/j.psj.2022.101971

Interpretive Summary: The gastrointestinal microbiota plays an important role in poultry health and production. Recently, the effects of diet and disease on the microbiota are researched to improve poultry health and production efficiency. Microbiota are characterized using DNA sequencing and analyzed using open-source platforms, which incorporate public databases such as Greengenes, the ribosomal database project (RDP), and SILVA to identify groups of bacteria (assign taxonomies) based on DNA sequences. Many chicken microbiota studies continue to rely on the Greengenes database, which has not been updated since 2013. To determine whether a choice of database could affect results, this study compared the cecal microbiota results obtained on the QIIME 2 platform using the Greengenes, RDP, and SILVA databases. The results of microbiota analysis were compared between the three databases to show whether database choice would affect which groups of bacteria were identified to be in greater relative abundance in groups of chickens. Some notable differences between databases were observed in results, in particular the ability of SILVA database to classify members of the bacterial family Lachnospiraceae into separate genera, while these members remained in one group of unclassified Lachnospiraceae through Greengenes and RDP. Additionally, the relative abundance of unclassified Lachnospiraceae in SILVA results was significantly lower than in RDP results. Our results show the choice of taxonomic database can influence the results of a microbiota study at the genus level, potentially affecting the interpretation of the results. With the growing interest in probiotics to limit the effects of poultry diseases, the use of the SILVA database is recommended over Greengenes, as more specific classifications at the genus level may provide more accurate interpretations of changes in the microbiota.

Technical Abstract: The chicken microbiota is often analyzed to address questions about the effects of diet or disease on poultry health. To analyze the microbiota, bioinformatic platforms such as QIIME 2 and mothur are used, which incorporate public taxonomic databases such as Greengenes, the ribosomal database project (RDP), and SILVA to assign taxonomies to bacterial sequences. Many chicken microbiota studies continue to incorporate the Greengenes database, which has not been updated since 2013. To determine whether a choice of database could affect results, this study compared the results of bioinformatic analyses obtained using the Greengenes, RDP, and SILVA databases on a cecal luminal microbiome dataset. The QIIME 2 platform was used to process 16S bacterial sequences and assign taxonomies with Greengenes, RDP, and SILVA. Linear Discriminant Analysis Effect Size (LEfSe) was performed, allowing for the comparison of taxonomies considered significantly differentially abundant between the three databases. Some notable differences between databases were observed in results, in particular the ability of SILVA database to classify members of the family Lachnospiraceae into separate genera, while these members remained in one group of unclassified Lachnospiraceae through Greengenes and RDP. LEfSe analyses showed that the SILVA database produced more differentially abundant genera, in large part due to the classification of these separate Lachnospiraceae genera. Additionally, the relative abundance of unclassified Lachnospiraceae in SILVA results was significantly lower than in RDP results. Our results show the choice of taxonomic database can influence the results of a microbiota study at the genus level, potentially affecting the interpretation of the results. The use of the SILVA database is recommended over Greengenes in chicken microbiota studies, as more specific classifications at the genus level may provide more accurate interpretations of changes in the microbiota.