Host DNA Depletion Enables Successful Shotgun Metagenomics of Previously Frozen Non- cryo-Preserved Respiratory Samples

초록

INTRODUCTION: Most respiratory microbiome studies have focused on amplicon rather than metagenomics sequencing due to high host DNA content, limiting taxonomic resolution and the ability to characterize microbial community function. A few studies have focused on host DNA depletion prior to sequencing, though these studies require unfrozen samples and have evaluated single sample types. Here we evaluated the efficacy of five host DNA depletion methods on previously frozen non-cryo-preserved human bronchoalveolar lavage (BAL) fluid, nasal swabs, and sputum prior to metagenomic sequencing. METHODS: BAL, nasal swabs, and sputum samples were aliquoted and treated with 5 host depletion different methods (lyPMA, Bezonase, HostZERO, MolYsis, and QIAamp). A total of 160 untreated and treated samples as well as positive mock community and negative reagent-only controls underwent low biomass library prep (NEBNext Ultra II) and deep metagenomics sequencing on the Illumina NovaSeq 6000 platform. Raw reads underwent taxonomic and functional profiling using bioBakery3. The effect of host depletion on alpha diversity, beta diversity, and differential abundance was assessed. Possible contaminants were estimated by two methods (decontam and tinyvamp R packages). RESULTS: Median sequencing depth was 76.4 million reads per sample. Untreated nasal, sputum and BAL samples had median 94.1%, 99.2% and 99.7% host reads. Most treatment methods increased microbial reads as well as species and predicted microbial functional richness. The effect of host depletion differed by sample type. Species richness increased by 18 for BAL (MolYsis), 8 for nasal swabs (QIAamp), and 102 for sputum (HostZero). Microbial functional richness increased by 165 for BAL, 66 for nasal swabs, and 137 for sputum with the same methods. The increase in species and functional richness was due to a higher effective sequencing depth based on mediation analysis. Contamination did not contribute to the increase in species