Microbial DNA extraction of high-host content and low biomass samples: optimized protocol for nasopharynx metagenomic studies

Low microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization. Three depletion and three DNA extraction protocols were compared, using whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling, and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal antibiotic resistance patterns. Quality trimmed and clean sequencing reads (after removal of DNA) are provided.