Richness estimates for each body site.

<p>Estimated number of species for each body site using both the V1–V3 and the V3–V5 tags computed with CatchAll. Numbers in parentheses are upper and lower confidence limits. The stool samples have the highest estimate of total richness, followed by the oral sites, particularly the plaque and tonsils. The skin and the vaginal sites have the lowest estimated richness.</p

Core OTUs present in at least seven of the nine oral sites.

<p>While each 16S amplification (V1–V3 and V3–V5) has 6 core OTUs, there are differences between the amplifications, with Pasteurellaceae and Lactobacillales identified with the V3–V5 tags but not the V1–V3 tags, and <i>Leptotrichia</i> and <i>Granulicatella</i> (a member of the Lactobacillales order) identified with the V1–V3 tags but not the V3–V5 tags. <i>Fucobacterium</i>, <i>Gemella</i>, <i>Streptococcus</i>, and <i>Veillonella</i> were identified in both sets of OTUs.</p

Comparison of OTU abundance and prevalence across all samples.

<p>The OTU rank is defined by the total number of sequences across all samples, with the most abundant ranked first (on the left). In panel A, OTU prevalence, the fraction of samples containing that OTU, is compared to OTU rank abundance. The most abundant OTUs appeared in a higher percentage of samples than the less abundant OTUs. In panel B, the cumulative abundance of OTUs as a function of OTU rank abundance showing that the 100 most abundant OTUs accounted for almost all of the sequence reads in both the V1–V3 and the V3–V5 amplifications. Panels C and D (V1–V3 and V3–V5 respectively) show the OTU prevalence rank against the OTU rank, with the most abundant OTUs tending also to be the most prevalent OTUs.</p

Body site preference of distinct OTUs from individual taxa.

<p>The use of genus or family names alone can imply a broad colonization of one organism across body sites. Using 3% OTUs, we can discern a high degree of site specialization of distinct organisms within these taxonomic groups, especially within the oral cavity. Not all OTUs assigned to these taxa were graphed, and only OTUs with greater than 100 tags were included. The area under each curve sums to 100% of the occurrence of that OTU across the body sites. Body site labels are: saliva (sv), supragingival plaque (supp), subgingival plaque (subp), keratinized gingiva (kg), tongue dorsum (td), hard palate (hp), buccal mucosa (bm), palatine tonsils (pt), throat (th), anterior nares (an), stool (st), left and right antecubital fossae (laf, raf), left and right retroauricular creases (lrc, rrc), mid-vagina (mv), posterior fornix (pf), and vaginal introitus (vi).</p

Size of the 95% core microbiome by body site.

<p>The oral cavity sites show the greatest number of core OTUs with both the V1–V3 and the V3–V5 tags, followed by the stool, the anterior nares, then the skin and the vaginal sites. Core OTUs are defined as those OTUs appearing in at least 95% of all samples for a given body site. Body site labels in order are: saliva (SV), supragingival plaque (SUPP), hard palate (HP), palatine tonsils (PT), tongue dorsum (TD), throat (TH), buccal mucosa (BM), subgingival plaque (SUBP), keratinized gingiva (KG), anterior nares (AN), stool (ST), right (RAF) and left (LAF) antecubital fossae, left (LRC) and right (RRC) retroauricular creases, posterior fornix (PF), mid-vagina (MV), and vaginal introitus (VI).</p

PCoA of Biome Types in stool and vaginal midpoint samples.

<p>Panels A and B are principle coordinates analyses of stool samples based on the RDP taxonomy and using Morisita-Horn distance. With the V3–V5 data, the <i>Bacteroides</i>-dominated subjects are segregated from both the <i>Ruminococcus</i>-dominated samples and the <i>Prevotella</i>-dominated samples. The <i>Alistipes</i> and <i>Oscillibacter</i> samples overlap with the other biome types. The <i>Bacteroides</i> and Clostridiales show greater overlap with the V1–V3 taxonomy (Panel B), while <i>Prevotella</i> is still segregated but not well separated. With both V3–V5 and V1–V3 data, the intra-biome type distances are as great as inter-biome type distances. At the OTU-level (Panels C and D) the <i>Bacteroides</i> and Clostridiales biome types have much greater overlap. The Prevotellaceae biome type has complete overlap with the other two biome types in the V3–V5 OTU data (Panel C) but mild segregation with V1–V3. At the OTU-level, the intra-biome type distances are greater than the inter-biome type distances. Panels E and F are the PCoA results for the mid-vagina samples, V3–V5 and V1–V3 respectively. The V3–V5 OTUs did not differentiate the <i>Lactobacillus</i> species, but show that while most subjects fall under the <i>Lactobacillus</i>-dominated type, there are also types dominated by either Bifidobacteriaceae or other taxa. The V1–V3 OTUs separated the subjects by <i>Lactobacillus</i> sub-types. Not all of the subjects classified as Bifidobacteriaceae with V3–V5 had corresponding samples large enough (>1000 tags) to be included in the V1–V3 plot and vice-versa.</p

OTU richness estimates for each body site.

<p>Estimated richness calculated using CatchAll <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034242#pone.0034242-Bunge1" target="_blank">[16]</a> with both the V1–V3 and the V3–V5 tag data for each body site. Bars represent the upper and lower confidence bounds provided by Catchall. Both sets of rRNA tags provided similar estimates. The stool samples showed the most richness with the oral samples having the next greatest richness, followed by the skin and vaginal samples.</p

Relative abundance of OTUs.

<p>For each OTU, the relative abundance is plotted for each sample in which the OTU is present. The OTUs with the highest number of total sequences are ranked first and plotted at the leftmost side, and the OTUs with the lowest total number of sequences are ranked last and plotted toward the right. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034242#pone-0034242-g003" target="_blank">Figure 3A</a> shows the V1–V3 OTUs and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034242#pone-0034242-g003" target="_blank">Figure 3B</a> the V3–V5 OTUs. Even OTUs that are among the top 10 most abundant span at least 3 orders of magnitude of relative abundances from less than 0.01% to more than 10%.</p

NMDS of early and late onset NEC and controls at the genus level.

<p>The difference between NEC and controls is displayed for early onset NEC (Fig. 2A) and late onset NEC (Fig. 2B) with their controls by NMDS plot. Each dot represents one sample. Green dots represent the controls and red dots represent the NEC samples. Early NEC subjects and control subjects have a clear separation at second week of the life. The distinction between late onset NEC and controls is less obvious except at the third week of life.</p

Microbiota progression before early onset NEC and late onset NEC at class and genus level.

<p>The relative abundances of four most dominant classes (Fig. 3A-early onset at class level, 3B-late onset at class level) and the genera (Fig. 3C-early onset at genus level, 3D-late onset at genus level) that are significantly different between NEC and controls are plotted at 7–9 days, 4–6 days and 1–3 days prior to NEC onset. In early onset NEC category, 3,6, 8 NEC samples were included at 7–9 days, 4–6 days and 1–3 days; 8, 5, 6 control samples were included in the above time points. In late onset NEC category, 7, 7,10 NEC samples and 6, 8, 4 control samples were included in the above time points. Red and green of the boxplots indicate NEC and control samples, respectively. The asterisks indicate the significant difference between NEC and control.</p