The "most wanted" taxa from the human microbiome for whole genome sequencing

Abstract

The goal of the Human Microbiome Project (HMP) is to generate a comprehensive catalog of human-associated microorganisms including reference genomes representing the most common species. Toward this goal, the HMP has characterized the microbial communities at 18 body habitats in a cohort of over 200 healthy volunteers using 16S rRNA gene (16S) sequencing and has generated nearly 1,000 reference genomes from human-associated microorganisms. To determine how well current reference genome collections capture the diversity observed among the healthy microbiome and to guide isolation and future sequencing of microbiome members, we compared the HMP's 16S data sets to several reference 16S collections to create a 'most wanted' list of taxa for sequencing. Our analysis revealed that the diversity of commonly occurring taxa within the HMP cohort microbiome is relatively modest, few novel taxa are represented by these OTUs and many common taxa among HMP volunteers recur across different populations of healthy humans. Taken together, these results suggest that it should be possible to perform whole-genome sequencing on a large fraction of the human microbiome, including the 'most wanted', and that these sequences should serve to support microbiome studies across multiple cohorts. Also, in stark contrast to other taxa, the 'most wanted' organisms are poorly represented among culture collections suggesting that novel culture- and single-cell-based methods will be required to isolate these organisms for sequencing.

Figure 1. Rank Abundance curves for V1–V3 (black symbols) and V3–V5 (gray symbols) OTUs.

(A) The number of sequences in each OTU. (B) Cumulative rank abundance. For both V1–V3 and V3–V5, on the order of 10–15 OTUs captured half of all individual sequences.

Figure 2. Body habitat distribution of non-chimeric and most wanted HMP OTUs.

The distributions of 1,468 non-chimeric HMP OTUs (left panel) and 119 most wanted OTUs (right panel) are shown as phyla (outer circle) and genera (inner circle) at each of the 5 sampled body habitats. Distribution profiles were based on the habitat in which the HMP OTU was found most frequently. Bar graphs illustrate the relative proportion of HMP OTUs from each 16S variable region, shown as phyla. Color codes for all phyla and ‘most wanted’ genera with more than one representative are shown in left and right figure legends, respectively.

Figure 3. There were few novel, but many uncultured and unsequenced taxa within the HMP OTUs.

Panels A through F present results from aligning HMP OTUs to six separate 16S sequence databases, indicated. For each HMP OTU, the y-axis of each panel shows the percent identity for the best matching sequence from the queried database, as determined by the program align.seqs in Mothur . The x-axis of each panel shows the fraction of samples in which the OTU was present, at the body site of its highest prevalence. For example, a value of 0.5 means that the OTU was present in, at most, 50% of samples from a particular body site. The colors in all panels indicate assignment to priority groups for whole genome sequencing: red = highest priority, blue = medium priority, gray = low priority. Horizontal lines indicate 98% and 90% sequence identity.

Figure 4. The most prevalent HMP OTUs were also present in other human cohorts.

Databases were created from non-HMP enrolled healthy volunteers in which stool (4A), saliva (4B) and vaginal (4C) microbiomes were characterized (see Table S2). For each HMP OTU, the y-axis of each panel shows the percent identity for the best matching sequence from the queried database, as determined by the program align.seqs in Mothur . The colors in all panels indicate assignment to priority groups for whole genome sequencing: red = highest priority, blue = medium priority, gray = low priority. Horizontal lines indicate 98% and 90% sequence identity.

Figure 5. Nearly all sequenced taxa have been cultured but not all cultured taxa have been sequenced.

For each taxa, the percent identity from the best match to a human sequenced database (GOLD-Human or HMP-strains) versus the best match to a sequence database of cultured organisms (named or unnamed). The colors in all panels indicate assignment to priority groups for whole genome sequencing: red = highest priority, blue = medium priority, gray = low priority. (A) OTUs that are present in at least 20% of all samples in at least one body habitat; (B) all HMP OTUs.