Benchmarking Microbiome Methods for Improved Metagenomic Sequencing of Lower Respiratory Tract Samples

Bronchoalveolar lavage is a common diagnostic strategy used to sample immune cells and microbiota of the lower respiratory tract. Metagenomic sequencing of raw bronchoalveolar lavage fluid reveals a low biomass microbial community often obscured by abundant host DNA. Here, we set out to determine an optimal strategy to robustly purify microbial DNA in excess of human DNA using a combinatorial lysis, host-depletion, DNA extraction approach. We identify strategies that improve metagenomic sequencing success that are commensurate with purification challenges that reduce signal from respiratory microbiota. Finally, we present a general framework to further improve microbial sequencing of lower respiratory tract samples in a robust and reproducible manner.