• Evaluation of methods for the reduction of contaminating host reads when performing shotgun metagenomic sequencing of the milk microbiome

      Yap, Min; Feehily, Conor; Walsh, Calum J.; Fenelon, Mark; Murphy, Eileen F.; McAuliffe, Fionnuala M.; van Sinderen, Douwe; O’Toole, Paul W.; O’Sullivan, Orla; Cotter, Paul D.; et al. (Springer, 2020-12-10)
      Shotgun metagenomic sequencing is a valuable tool for the taxonomic and functional profiling of microbial communities. However, this approach is challenging in samples, such as milk, where a low microbial abundance, combined with high levels of host DNA, result in inefficient and uneconomical sequencing. Here we evaluate approaches to deplete host DNA or enrich microbial DNA prior to sequencing using three commercially available kits. We compared the percentage of microbial reads obtained from each kit after shotgun metagenomic sequencing. Using bovine and human milk samples, we determined that host depletion with the MolYsis complete5 kit significantly improved microbial sequencing depth compared to other approaches tested. Importantly, no biases were introduced. Additionally, the increased microbial sequencing depth allowed for further characterization of the microbiome through the generation of metagenome-assembled genomes (MAGs). Furthermore, with the use of a mock community, we compared three common classifiers and determined that Kraken2 was the optimal classifier for these samples. This evaluation shows that microbiome analysis can be performed on both bovine and human milk samples at a much greater resolution without the need for more expensive deep-sequencing approaches.
    • Seasonality and Geography Have a Greater Influence than the Use of Chlorine-Based Cleaning Agents on the Microbiota of Bulk Tank Raw Milk

      Yap, Min; Gleeson, David; O’Toole, Paul W.; O'Sullivan, Orla; Cotter, Paul D.; Irish Dairy Levy (American Society for Microbiology, 2021-10-28)
      Cleaning of the production environment is vital to ensure the safety and quality of dairy products. Although cleaning with chlorine-based agents is widely adopted, it has been associated with detrimental effects on milk quality and safety, which has garnered increasing interest in chlorine-free cleaning. However, the influence of these methods on the milk microbiota is not well documented. This study investigated the factors that influence the raw milk microbiota, with a focus on the differences when chlorine-based and chlorine-free cleaning of milking equipment are used. Bulk tank raw milk was sampled during three sampling months (April, August, and November), from farms across Ireland selected to capture the use of different cleaning methods, i.e., exclusively chlorine-based (n = 51) and chlorine-free cleaning (n = 92) and farms that used chlorine-free agents for the bulk tank and chlorine-based cleaning agents for the rest of the equipment (n = 28). Shotgun metagenomic analysis revealed the significant influence of seasonal and geographic factors on the bulk tank milk microbiota, indicated by differences in diversity, taxonomic composition, and functional characteristics. Taxonomic and functional profiles of samples collected in November clustered separately from those of samples collected in other months. In contrast, cleaning methods only accounted for 1% of the variation in the bulk tank milk bacterial community, and samples collected from farms using chlorine-based versus chlorine-free cleaning did not differ significantly, suggesting that the chlorine-free approaches used did not negatively impact microbiological quality. This study shows the value of shotgun metagenomics in advancing our knowledge of the raw milk microbiota.