Abstract
BackgroundShotgun 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.ResultsThree 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 of those not aligning, compared to untreated samples. lyPMA was the most efficient method of removing host-derived sequencing reads compared to untreated sample (8.53 ± 0.10% versus 89.29 ± 0.03%). Furthermore, lyPMA-treated samples exhibit the lowest taxonomic bias compared to untreated samples.ConclusionOsmotic 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 host-derived sample types.
Highlights
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
Because buccal epithelial cells are on average 50 μm wide, whereas a typical bacterial coccus is ~ 1 μm, we passed saliva samples across a 5-μm filter and analyzed the filtrate and residue compared to the raw sample
We designed a Quantitative polymerase chain reaction (qPCR) assay to evaluate the percentage of host DNA relative to untreated sample using a human-specific primer against the PTGER2 gene [19]
Summary
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. In the past decade, sequencing costs have plummeted, and 16S rRNA gene amplicon sequencing has become a nearly ubiquitous tool used to characterize bacterial populations from a wide range of environments and host systems [1, 2]. This technique has revealed that bacteria inhabit a far greater range of human body sites than previously believed, including many long presumed to be sterile (e.g., urine [3], breast milk [4], blood [5], and atherosclerotic plaque [6]).
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