Journal
MSYSTEMS
Volume 4, Issue 1, Pages -Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/mSystems.00163-18
Keywords
ASV methods; OTU clustering; bioinformatics; microbiome
Categories
Funding
- Oregon BIRCWH (Building Interdisciplinary Research Careers in Women's Health) (U.S. National Institutes of Health [NIH]) [K12HD043488]
- National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (NIH) [1K01DK116706]
- National Eye Institute (NEI) (NIH) [R01EY029266]
- Rheumatology Research Foundation
- Spondylitis Association of America
- EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT [K12HD043488] Funding Source: NIH RePORTER
- NATIONAL EYE INSTITUTE [R01EY029266] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [K01DK116706] Funding Source: NIH RePORTER
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Microbiome community composition plays an important role in human health, and while most research to date has focused on high-microbial-biomass communities, low-biomass communities are also important. However, contamination and technical noise make determining the true community signal difficult when biomass levels are low, and the influence of varying biomass on sequence processing methods has received little attention. Here, we benchmarked six methods that infer community composition from 16S rRNA sequence reads, using samples of varying biomass. We included two operational taxonomic unit (OTU) clustering algorithms, one entropy-based method, and three more-recent amplicon sequence variant (ASV) methods. We first compared inference results from high-biomass mock communities to assess baseline performance. We then benchmarked the methods on a dilution series made from a single mock community-samples that varied only in biomass. ASVs/OTUs inferred by each method were classified as representing expected community, technical noise, or contamination. With the high-biomass data, we found that the ASV methods had good sensitivity and precision, whereas the other methods suffered in one area or in both. Inferred contamination was present only in small proportions. With the dilution series, contamination represented an increasing proportion of the data from the inferred communities, regardless of the inference method used. However, correlation between inferred contaminants and sample biomass was strongest for the ASV methods and weakest for the OTU methods. Thus, no inference method on its own can distinguish true community sequences from contaminant sequences, but ASV methods provide the most accurate characterization of community and contaminants. IMPORTANCE Microbial communities have important ramifications for human health, but determining their impact requires accurate characterization. Current technology makes microbiome sequence data more accessible than ever. However, popular software methods for analyzing these data are based on algorithms developed alongside older sequencing technology and smaller data sets and thus may not be adequate for modern, high-throughput data sets. Additionally, samples from environments where microbes are scarce present additional challenges to community characterization relative to high-biomass environments, an issue that is often ignored. We found that a new class of microbiome sequence processing tools, called amplicon sequence variant (ASV) methods, outperformed conventional methods. In samples representing low-biomass communities, where sample contamination becomes a significant confounding factor, the improved accuracy of ASV methods may allow more-robust computational identification of contaminants.
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