Evaluation of methods for the reduction of contaminating host reads when performing shotgun metagenomic sequencing of the milk microbiome
Author
Yap, MinFeehily, 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.
Date
2020-12-10
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Yap M, Feehily C, Walsh CJ, et al. Evaluation of methods for the reduction of contaminating host reads when performing shotgun metagenomic sequencing of the milk microbiome. Scientific Reports 2020;10:21665. doi:10.1038/s41598-020-78773-6Abstract
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.Funder
Irish Dairy Levy; Science Foundation Ireland; PrecisionBiotics Ltd.Grant Number
SFI/12/RC/2273; 16/SP/3827ae974a485f413a2113503eed53cd6c53
https://doi.org/10.1038/s41598-020-78773-6
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