Res Sq. 2024 Jan 23:rs.3.rs-3638876. doi: 10.21203/rs.3.rs-3638876/v1. Preprint.
Background Most respiratory microbiome studies have focused on amplicon rather than metagenomics sequencing due to high host DNA content. We evaluated efficacy of five host DNA depletion methods on previously frozen human bronchoalveolar lavage (BAL), nasal swabs, and sputum prior to metagenomic sequencing. Results Median sequencing depth was 76.4 million reads per sample. Untreated nasal, sputum and BAL samples had 94.1%, 99.2%, and 99.7% host-reads. The effect of host depletion differed by sample type. Most treatment methods increased microbial reads, species richness and predicted functional richness; the increase in species and predicted functional richness was mediated by higher effective sequencing depth. For BAL and nasal samples, most methods did not change Morisita-Horn dissimilarity suggesting limited bias introduced by host depletion. Conclusions Metagenomics sequencing without host depletion will underestimate microbial diversity of most respiratory samples due to shallow effective sequencing depth and is not recommended. Optimal host depletion methods vary by sample type.