Metagenomic Characterization of Intestinal Regions in Pigs With Contrasting Feed Efficiency

Measures of RNA abundance are important for many areas of biology and often obtained from high-throughput RNA sequencing methods such as Illumina sequence data. These measures need to be normalized to remove technical biases inherent in the sequencing approach, most notably the length of the RNA species and the sequencing depth of a sample. These biases are corrected in the widely used reads per kilobase per million reads (RPKM) measure. Here, we argue that the intended meaning of RPKM is a measure of relative molar RNA concentration (rmc) and show that for each set of transcripts the average rmc is a constant, namely the inverse of the number of transcripts mapped. Further, we show that RPKM does not respect this invariance property and thus cannot be an accurate measure of rmc. We propose a slight modification of RPKM that eliminates this inconsistency and call it TPM for transcripts per million. TPM respects the average invariance and eliminates statistical biases inherent in the RPKM measure.

Background In a series of studies of the gut microbiome, “enterotypes” have been used to classify gut microbiome samples that cluster together in ordination analyses. Initially, three distinct enterotypes were described, although later studies reduced this to two clusters, one dominated by Bacteroides or Clostridiales species found more commonly in Western (American and Western European) subjects and the other dominated by Prevotella more often associated with non-Western subjects. The two taxa, Bacteroides and Prevotella, have been presumed to represent consistent underlying microbial communities, but no one has demonstrated the presence of additional microbial taxa across studies that can define these communities. Results We analyzed the combined microbiome data from five previous studies with samples across five continents. We clearly demonstrate that there are no consistent bacterial taxa associated with either Bacteroides- or Prevotella-dominated communities across the studies. By increasing the number and diversity of samples, we found gradients of both Bacteroides and Prevotella and a lack of the distinct clusters in the principal coordinate plots originally proposed in the “enterotypes” hypothesis. The apparent segregation of the samples seen in many ordination plots is due to the differences in the samples’ Prevotella and Bacteroides abundances and does not represent consistent microbial communities within the “enterotypes” and is not associated with other taxa across studies. The projections we see are consistent with a continuum of values created from a simple mixture of Bacteroides and Prevotella; these two biomarkers are significantly correlated to the projection axes. We suggest that previous findings citing Bacteroides- and Prevotella-dominated clusters are the result of an artifact caused by the greater relative abundance of these two taxa over other taxa in the human gut and the sparsity of Prevotella abundant samples. Conclusions We believe that the term “enterotypes” is misleading because it implies both an underlying consistency of community taxa and a clear separation of sets of human gut samples, neither of which is supported by the broader data. We propose the use of “biomarker” as a more accurate description of these and other taxa that correlate with diet, lifestyle, and disease state. Electronic supplementary material The online version of this article (doi:10.1186/s40168-016-0160-7) contains supplementary material, which is available to authorized users.

Disturbance of the beneficial gut microbial community is a potential collateral effect of antibiotics, which have many uses in animal agriculture (disease treatment or prevention and feed efficiency improvement). Understanding antibiotic effects on bacterial communities at different intestinal locations is essential to realize the full benefits and consequences of in-feed antibiotics. In this study, we defined the lumenal and mucosal bacterial communities from the small intestine (ileum) and large intestine (cecum and colon) plus feces, and characterized the effects of in-feed antibiotics (chlortetracycline, sulfamethazine and penicillin (ASP250)) on these communities. 16S rRNA gene sequence and metagenomic analyses of bacterial membership and functions revealed dramatic differences between small and large intestinal locations, including enrichment of Firmicutes and phage-encoding genes in the ileum. The large intestinal microbiota encoded numerous genes to degrade plant cell wall components, and these genes were lacking in the ileum. The mucosa-associated ileal microbiota harbored greater bacterial diversity than the lumen but similar membership to the mucosa of the large intestine, suggesting that most gut microbes can associate with the mucosa and might serve as an inoculum for the lumen. The collateral effects on the microbiota of antibiotic-fed animals caused divergence from that of control animals, with notable changes being increases in Escherichia coli populations in the ileum, Lachnobacterium spp. in all gut locations, and resistance genes to antibiotics not administered. Characterizing the differential metabolic capacities and response to perturbation at distinct intestinal locations will inform strategies to improve gut health and food safety.