Journal Article

Improving saliva shotgun metagenomics by chemical host DNA depletion

Zuniga, Cristal, Zengler, Karsten, Knight, Rob, Zaramela, Livia S., Marotz, Clarisse A., Sanders, Jon G.

2018

Overview

Background 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. Results 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 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. Conclusion 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 host-derived sample types.

• Publisher: BioMed Central,BioMed Central Ltd,BMC
• Subject: Azides - chemistry / Bacteria - genetics / Bioinformatics / Biomedical and Life Sciences / Biomedicine / DNA / DNA - chemistry / DNA sequencing / Fungi - genetics / Genes / Genetic research / Genomes / Genomics / High-Throughput Nucleotide Sequencing / Host depletion / Humans / Medical Microbiology / Metagenomics - methods / Methodology / Microbial Ecology / Microbial enrichment / Microbial Genetics and Genomics / Microbiology / Microbiome / Microbiota - genetics / Nucleotide sequencing / Propidium - analogs & derivatives / Propidium - chemistry / Propidium monoazide / Saliva / Saliva - microbiology / Sequence Analysis, DNA / Shotgun sequencing / Virology / Viruses - genetics