Direct sequencing of the human microbiome readily reveals community differences

Abstract

Culture-independent studies of human microbiota by direct genomic sequencing reveal quite distinct differences among communities, indicating that improved sequencing capacity can be most wisely utilized to study more samples, rather than more sequences per sample.

Figure 1

The problem of distinguishing between sequences. (a) An investigator contemplating the problem of distinguishing between sequences from the gut of Equus asinus and the volar forearm of humans. (b) Our solution; guess the effect size based on the effect sizes reported in published studies; perform simulations based on these effect sizes as shown in Figure 2, and then acquire sufficient sequences to resolve microbial community differences of the expected magnitude. (c) When comparing the Equus asinus gut (white point) to human forearms (red and green points represent left and right arms, respectively), 100 or even 10 sequences per sample provide sufficient resolution, but one sequence per sample does not.

Figure 2

Variation in human body habitats within and between people. (a) The full dataset (approximately 1,500 sequences per sample); (b) the dataset sampled at only 10 sequences per sample, showing the same pattern; (c) the relationship between sequencing depth and the PERMANOVA component of variation. The amount of variation explained by the factors plateaus at relatively shallow sequencing depths. Note that the proportion of variation captured by differences between the samples (that is, residual variation) is still highest despite the explanatory values of the three factors examined. (d) Effect size determines the number of sequences required for sample identification. Each point in the figure represents a specific sample selected from a pair of body sites, and the number of sequences required to correctly distinguish which site the sample originated from. The point is colored according to the two body sites under consideration, the center's color represents the broad category the selected sample originated from, the border color represents the other broad category under consideration. Many body sites share the same broad category, and thus some points have the same border and center coloring. Red, external ear canal; yellow, hair; green, oral cavity; blue, gut; magenta, skin; gray, nostril. ns, not significant.

Box 1

How many sequences does it take...? .