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Updated: Jan 5, 2026
Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing
Published on: March 19, 2018
High-throughput sequencing of pooled samples to determine community-level microbiome diversity
Kathryn J Ray1, Sun Y Cotter2, Ahmed M Arzika3
1Francis I. Proctor Foundation, San Francisco; UCSF Epidemiology and Biostatistics, University of California, San Francisco.
Purpose:
Community-level interventions in cluster randomized controlled trials may alter the gut microbiome of individuals. The current method of estimating community diversities uses microbiome data obtained from multiple individual's specimens. Here we propose randomly pooling a number of microbiome samples from the same community into one sample before sequencing to estimate community-level microbiome diversity.
Methods:
We design and analyze an experiment to compare community microbiome diversity (gamma-diversity) estimates derived from 16S rRNA gene sequencing of 1) individually sequenced specimens vs. 2) pooled specimens collected from a community. Pool sizes of 10, 20, and 40 are considered. We then compare the gamma-estimates using Pearson's correlation as well as using Bland and Altman agreement analysis for three established diversity indices including richness, Simpson's and Shannon's.
Results:
The gamma-diversity estimates are highly correlated, with most being statistically significant. All correlations between all three diversity estimates are significant in the 10-pooled data. Pools comprising 40 specimens are closest to the line of agreement, but all pooled samples and individual samples fall within the 95% limits of agreement.
Conclusions:
Pooling microbiome samples before DNA amplification and metagenomics sequencing to estimate community-level diversity is a viable measure to consider in population-level association research studies.
