Host depletion <i>in silico</i> : adaptive sampling improves metagenomic yield and characterization of the chicken gut microbiome

ABSTRACT Metagenomic sequencing is a powerful tool for microbiome research. It allows for the identification of individual taxa and their functional potential without removing the larger context of the microbial community. However, host DNA can dominate sequencing output, limiting the amount of data collected from the microbial community and, by extension, what conclusions can be confidently drawn about that community. Here, we investigate the utility of adaptive sampling to deplete host DNA in chicken cecal samples ( n = 31) during Nanopore DNA sequencing. Each sample was sequenced using two treatments: adaptive sampling depleting chicken and human DNA during sequencing (deplete) and sequencing without adaptive sampling (control). We hypothesized that the deplete treatment would reduce the proportion of host (i.e., chicken) DNA in the sequencing output and increase the output of microbial sequence data. We investigated the impact of using adaptive sampling on common downstream analyses including taxonomic identification, community composition, metagenomic assembly, and the recovery of a pathogen of interest ( Salmonella enterica serovar Infantis) in chickens under two different probiotic intervention programs. We found that adaptive sampling shortened the N50 length of chicken reads by 54% ± 5% (mean ± standard error) and increased microbial yield by 30% ± 4.4% relative to control. Assemblies generated from the deplete treatment data were significantly longer ( P &lt; 0.001) than those from the control data. Finally, using adaptive sampling resulted in greater coverage of Salmonella Infantis genomes from chickens exposed to the pathogen compared to control. IMPORTANCE Microbial infections in poultry populations represent a significant threat to food safety and security. Salmonella enterica serovar Infantis is of particular interest because of its high prevalence in broilers and its propensity for antimicrobial resistance. Metagenomic sequencing can facilitate both the identification of pathogens, like Salmonella Infantis, and their functional potential without culturing. However, in the context of food safety surveillance, the contamination of samples with DNA from the host (e.g., chicken) can limit the amount of data generated from the microbial community. This study demonstrates that adaptive sampling has great potential as a host depletion strategy in microbiome research. The successful depletion of host DNA and improvement of microbial data yield without requiring additional time and resources is particularly exciting for resource- and time-limited environments, with positive implications for pathogen surveillance and improved public health outcomes.