High-throughput Sequencing of Pooled Samples to Determine Community-Level Microbiome Diversity

Abstract Background 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 versus 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 & 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 3 diversity estimates are significant in the 10-pooled data. Pools comprised of 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 prior to DNA amplification and metagenomics sequencing to estimate community-level diversity is a viable measure to consider in population-level association research studies.