The importance of standardized protocol for microbiota analysis

Bacteria, the major component of chicken gastrointestinal tract (GIT) microbiota, play an important role in health, nutrition, host physiology regulation, GIT development, and growth. Recently, the microbial community profiling method based on the 16S rRNA sequencing approach has become the most popular to determine the taxonomic composition and diversity of chicken microbiota. The 16S rRNA profiling consists of many steps such as: sample collections and storage, DNA isolation, 16S primer selections, 16S rRNA PCRs, libraries preparations and indexing, sequencing, raw data analysis (pipeline or software selection), operational taxonomic unit/amplicon sequence variant picking, taxonomic database selection, diversity analysis, and statistical analysis. Currently, there is no standardized protocol for 16S determination in chicken samples. It has been shown that primer design, library preparation, DNA isolation methods, and PCR amplification artifacts can introduce unique biases that can affect community structure, richness, and microbial population analysis and lead to over- or under-representation of individual bacteria within communities. Moreover, different sequencing platforms and bioinformatics pipelines can affect the average relative abundance of microbiota and shape the taxonomic community profiles. Although the experiments in chicken microbiota studies are commonly standardized and based on identical breeds, the results are often contradictory and depend on the used animal (breed, age, sex), the experimental design (feeding and sampling), and DNA extraction and sequencing methods. Our own data indicate that the choice of 16S primers as well as taxonomic database selection has significant effects on microbiota analysis and data interpretation. Therefore, it is difficult to compare data and correlate results originating from a different protocol. The development of a standardized protocol for microbiota profiling in chickens, similar to the one used in human microbiota research, is needed to obtain comparable data sets for poultry microbiota.

Key Words: Chickens, methodology, 16S.

Presented at the 10th Symposium on Gut Health in Production of Food Animals 2022, St. Louis, USA.