Interpretive Summary: Weinroth Best Practices Animal Science 16S RNA profiling summary

By- Caitlin Vonderohe

The recent development of 16S rRNA gene sequencing as a method to identify microbes that live on various surfaces of the body, particularly in the gastrointestinal tract, has revolutionized the field of microbiology, and has resulted in an explosion of publications focusing on the microbiome of many species. A recent review by Weinroth et al., 2021 explored the utility of applying 16SrRNA sequencing to animal science research. 16SrRNA (16S) sequencing targets a small section of the genome and results phylogenetic classification in a relatively affordable, culture-independent manner. 

However there are important limitations to this method; 16S cannot differentiate between different species or strains, nor can it describe the metabolic potential or activity of the microbial community. Additionally, sequencing- based approaches to community characterization do not reflect the amount of bacteria present in the community, nor do they differentiate dead from living bacteria. Other considerations include study design and the complexity of microbiome analyses. While sample size estimates are becoming more feasible as additional microbiota data becomes publicly available, sample pooling should be done with care and combine samples from similar animals, particularly with regard to source, genetics and immediate environment. 

It is also important to consider where samples are collected along the gastrointestinal tract; fecal samples and swabs may not be representative of the full length of the small intestine and colon. Samples should be appropriately stored after collection and genomic DNA should be extracted from samples with uniform methods for each study. There are multiple analytical pipelines for 16S, and the choice of analytical pipeline and subsequent statistical package used to handle the mass of data should be chosen with care as they will profoundly affect the sensitivity and utility of the data collected. 

Generally, Weinroth et al. present best practices for 16SrRNA analysis that will prove highly valuable for researchers interested in microbiome research.