Host DNA depletion method comparison

Shotgun sequencing of microbial communities provides in-depth knowledge of the microbiome by cataloging bacterial, fungal, and viral gene content within a sample, providing an advantage over amplicon sequencing approaches that assess taxonomy but not function, and are taxonomically limited. However, mammalian DNA can dominate host-derived samples, obscuring changes in microbial populations because few DNA sequence reads are from the microbial component. We developed and optimized a novel method for enriching microbial DNA from human oral samples, and compared its efficiency and potential taxonomic bias with commercially available kits. Three commercially available host depletion kits were directly compared with size filtration and a novel method involving osmotic lysis and treatment with propidium monoazide (lyPMA) in human saliva samples. We evaluated the percentage of shotgun metagenomic sequencing reads aligning to the human genome, and taxonomic biases, compared to untreated samples. lyPMA was the most efficient method of removing host-derived sequencing reads compared to untreated sample (8.53 ± 0.10% vs 89.29 ± 0.03%). Furthermore, lyPMA treated samples exhibit the lowest taxonomic bias compared to untreated samples. Osmotic lysis followed by PMA treatment is a cost-effective, rapid and robust method for enriching microbial sequence data in shotgun metagenomics from fresh and frozen saliva samples, and may be extensible to other biomaterials.