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

Abstract

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.

Crosstalk between Gut and Brain in Alzheimer’s Disease: The Role of Gut Microbiota Modulation Strategies

“…Thus, recent studies have been shifting towards the use of high-throughput data techniques for obtaining quantitative and qualitative information of metabolites, DNA, mRNA transcripts, and proteins of the microbial groups in the microbiome [ 152 ]. Shotgun metagenomic sequencing helps analyze the whole genomic DNA of bacteria to understand and identify the functional potential of microbial genes [ 155 ]. Additionally, meta-omic approaches can be helpful in providing a comprehensive functional view of microorganisms and their role within the microbiome [ 156 ].…”

A review of the resistome within the digestive tract of livestock

“…Trimmomatic [ 45 ] and Cutadapt [ 46 ] are frequently used tools for adapter trimming and read filtering. Non-microbial host DNA can be a major concern for sequencing-based microbiome analysis, especially for samples that are low in microbial biomass [ 47 , 48 ]. It has been shown that samples with high amounts of host DNA coupled with reduced sequencing depths lower the sensitivity of microbiome profiling within metagenomic datasets [ 49 ].…”

“…Host DNA can be depleted either experimentally or bioinformatically. For example, the use of MolYsis complete5 kit was shown to effectively remove host DNA in bovine milk samples [ 48 ]. In addition to experimental methods, several bioinformatic tools/resources such as MG-RAST [ 50 ] and TopHat2 [ 51 ] can be further used to filter bovine DNA and RNA sequences from metagenomic or metatranscriptomic datasets [ 52 , 53 ].…”

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“…Interestingly, the detection of host nucleic acids via mNGS may be useful, as samples with lower host nucleic acid content have been shown to be associated with higher rates of false-negative COVID-19 RT-PCR tests, presumably due to sample quality (42). Regardless, methods to enrich for pathogen sequences or deplete host DNA to increase sensitivity have been examined (43)(44)(45), and may prove useful for future clinical metagenomics studies.…”