.. The results of the correlation analysis suggested that family-level characterization could hide important health-state indicator taxa, and therefore a phylotype-level indicator analysis was subsequently performed on both 16S rRNA gene and cDNA data sets to identify phylotypes selleck bio indicative of specific conditions (healthy vs DSS-treated and/or wt vs STAT1?/? mice). Although 468 indicators were identified as statistically significant (P<0.05), only numerically dominant indicators, phylotypes with a mean relative abundance that was at least 0.5% greater in the condition for which they were indicators (for example, a shift from not detected to 0.5%, or of 1�C1.5%), were examined to restrict the analysis to the more abundant members of the microbiota.
In all, 61 numerically dominant indicator phylotypes were identified: 26 for healthy mice and 35 for DSS-treated mice (Figure 3). A higher number of genotype-specific indicators were identified for DSS-treated mice (63%) than for untreated mice (27%) because a relatively large number of specific indicator phylotypes were identified in DSS-treated wt mice (17/35). Roughly half of the phylotypes were indicators for both RNA and DNA template (52%), but 39% appeared as indicators only for DNA and not RNA, which is most likely attributable to deeper sequencing of 16S rRNA amplicon libraries generated from DNA than from RNA. Most indicators of healthy gut microbiota were shared between both genotypes (19/26), though genotype-specific indicators were also observed (Figure 3). Figure 3 Dominant indicator phylotypes of health state and genotype.
Dominant indicator phylotypes are presented with their relative abundance (as percent) in each sample of DNA- and RNA-based sequencing libraries. Each phylotype is annotated with the closest … All indicator phylotypes in the Ruminococcaceae and unclassified Clostridiales (that is, annotated as Clostridiales in Figure 3) were indicators for DSS treatment and all non-Bacteroidaceae Bacteroidales were indicators for untreated mice (Figure 3), which was consistent with the shifts observed at the family level (above). Most of the indicator phylotypes, however, belonged to the Lachnospiraceae and did not cluster in phylogenetic analysis according to the condition for which they were an indicator (Figure 4).
Quantitative FISH using specific probes confirmed the sequencing data for four indicator phylotypes: Akkermansia muciniphila, Mucispirillum schaedleri and two Lachnospiraceae phylotypes (Figure Cilengitide 5). A strong correlation was observed between relative abundances as measured by FISH and by sequencing libraries (R2=0.80, Supplementary Figure S8), confirming that relative shifts in abundant 16S rRNA (gene) phylotypes can be reliably monitored using the two-step PCR pyrosequencing approach (Berry et al., 2011).