Assessment of the human faecal microbiota: II. Reproducibility and associations of 16S rRNA pyrosequences

Abstract

Background: We conducted a pilot study of reproducibility and associations of microbial diversity and composition in faecal microbial DNA.

Methods and results: Participants (25 men and 26 women, aged 17-65 years) provided questionnaire data and multiple samples of one stool collected with two Polymedco and two Sarstedt devices preloaded with RNAlater. 16S rRNA genes in each faecal DNA aliquot were amplified, sequenced (Roche/454 Life Sciences) and assigned to taxa. Devices were compared for ease of use and reproducibility [intraclass correlation coefficient (ICC)] between duplicate aliquots on diversity and taxonomic assignment. Associations were tested by linear regression. Both collection devices were easy to use. Both alpha diversity (Shannon index) and beta diversity (UniFrac) were higher between than within duplicates (P ≤ 10(-8) ) and did not differ significantly by device (P ≥ 0·62). Reproducibility was good (ICC≥0·77) for alpha diversity and taxonomic assignment to the most abundant phyla, Firmicutes and Bacteroidetes (71·5% and 25·0% of sequences, respectively), but reproducibility was low (ICC≤0·48) for less abundant taxa. Alpha diversity was lower with nonantibiotic prescription medication (P = 0·02), with younger age (P = 0·03) and marginally with higher body mass index (P = 0·08).

Conclusions: With sampling from various parts of a stool, both devices provided good reproducibility on overall microbial diversity and classification for the major phyla, but not for minor phyla. Implementation of these methods should provide insights into how broad microbial parameters, but not necessarily rare microbes, affect risk of various conditions.

Figure 1

Reproducibility of 16S rRNA alpha diversity and relative abundance in fecal specimens obtained with two different collection devices. Shannon index of alpha diversity (A). Relative abundance of Firmicutes (B), Actinobacteria (C) and Proteobacteria (D), as aligned to the Ribosomal Data Project. The intraclass correlation coefficient (ICC) did not differ significantly between the two devices.

Figure 2

Beta diversity of 16S rRNA pyrosequence data, at the genus level, by collection device. For all pairs of samples, each cell indicates Bray-Curtis 2-way dissimilarity [minimum red, maximum black]. Red diagonal (no dissimilarity) is a sample with itself. Each participant is presented as two adjacent rows (or columns) for his or her sample pair. Several participants (blue/green rows and columns) had highly reproducible beta diversity that was distinctive from other participants' data. Lower left is a magnification of 2-way dissimilarity for selected pairs. Lower right presents relative abundance of 36 genera, with low dissimilarity in participant 59's A and B duplicates (Bray-Curtis 0.003) and high dissimilarity between A samples from participants 59 and 60 (Bray-Curtis 0.911).

Figure 3

Association of 16S rRNA alpha diversity with mutually adjusted age (left) and body mass index (right).