Host DNA depletion methods and genome-centric metagenomics of bovine hindmilk microbiome

Bovine mastitis is a multi-etiological and complex disease, resulting in serious economic consequences for dairy farmers and industry. In recent years, the microbiological evaluation of raw milk has been investigated in-depth using next-generation sequencing approaches such as metataxonomic analysis. Despite this, host DNA is a major concern in the shotgun metagenomic sequencing of microbial communities in milk samples, and it represents a big challenge. In this study, we aimed to evaluate different methods for host DNA depletion and/or microbial DNA enrichment and assess the use of PCR-based whole genome amplification in milk samples with high somatic cell count (SCC) by using short- and long-read sequencing technologies. Our results evidenced that DNA extraction performed differently in terms of host DNA removal, impacting metagenome composition and functional profiles.. Moreover, the ratio of SCC/bacteria ultimately impacts microbial DNA yield, and samples with low SCC (SCC below 100,000 cells/mL) are the most problematic. When milk samples with high SCC (SCC above 200,000 cells/mL) underwent multiple-displacement amplification (MDA), we successfully recovered high-quality metagenome-assembled genomes (MAGs), and long-read sequencing was feasible even for samples with low DNA concentration. By associating MDA and short-read sequencing, we recovered two times more MAGs than in untreated samples, and an ongoing co-infection not reported by traditional methods was detected for mastitis pathogen. Overall, this new approach will improve the detection of mastitis-associated microorganisms and make it possible to examine host-microbiome interactions in bovine mastitis.IMPORTANCENext-generation sequencing technologies have been widely used to gain new insights into the diversity of the microbial community of milk samples and dairy products for different purposes such as microbial safety, profiling of starter cultures, and host-microbiome interactions. Milk is a complex food matrix, and additionally, the presence of host nucleic acid sequences is considered a contaminant in untargeted high-throughput sequencing studies. Therefore, genomic-centric metagenomic studies of milk samples focusing on the health-disease status in dairy cattle are still scarce, which makes it difficult to evaluate the microbial ecophysiology of bovine hindmilk. This study provides an alternative method for genome-centric metagenome studies applied to hindmilk samples with high somatic cell content, which is indispensable to examining host-microbiome interactions in bovine mastitis.