Metagenomics Project

==Overview==
This paper provides an insight on how diet in infants can trickle down to immune response via the gut microbiome. The results compare breast fed infants to formula fed infants.This metagenomic study applies complex statistical methods to analyze host epithelial cell transcriptome and  gut microbiome gene expression to isolate any diffrences related to diet. They observed that variations in dietary substrate influenced gene expression of virulence genes in gut microbiota which inturn influenced host innate immune response.
==Introduction:==
Since the discovery of fast hi-thoroughput next generation sequencing techniques, a treasure trove of information has been collected over a relatively short period of time. The task that lies ahead is to combine information mining and statistical power to derive significant associations between normal and disease states. Gut microbiota has been studied by several labs. Some have published changes in bacteria colonizing the gut in infants with normal delivery as opposed to a c-section. Some have taking these studies to the next level by associating certain familes of gut microbiota to obesity, allergies, asthma, necrotizing enterocolitis and type I diabetes. Figure 2 provides a nice illustration of the good and bad bacteria in breast fed vs formula fed infants.  Review is linked here. Further, the same reivew shows how breast fed infants can be supplemented with prebiotics or probiotics to prevent diseases like obesity and colitis as seen in figure3. There is clearly a complex interplay that requires more such analysis.
==Human Gut Microbiota==
At birth, infants have a sterile gut. It is colonised by commensal bacteria during birth via the mother's birth canal. It is therefore not very suprising to find changes in the intial flora in babies with a normal delivery as opposed to a c-section. The intestines are decorated with epithelial cells that can process certain substrates and provide the first line of defence. They are constantly renewed to maintain the fitness of the gut and also to maintain the commensals. Diet clearly affects intestinal flora which in turn affects the epithelial cells. This study focuses on this aspect and presents some key observations (PMID:22546241)
==Method==
First, the group identified biomarkers in the geneome of the epithelial cells. They isolated intact intestinal epithelial cells from feces of the breast fed and formula fed infants, isolated mRNA,  identified  best gene combinations (two or three) that would highlight the differences between the two groups. They did this by linear discriminant analysis.  Table 1 shows the infants chosen for the study. Stool samples were collected and microbial DNA was isolated. In addition host epithelial cell mRNa was also isolated. The ssame study was performed in Rheus monkeys as well.
Gene expression profile of intestinal epithelial cells were analyzed. Differences in expression of 459 genes related intestinal biology, 660 genes related to immunity and defence were compared using multiple testing correlation to rule out false discoveries. A small subset of these genes were further analyzed for accuracy. This showed that genes known a-priori indeed showed expression differences in response to diet.
Shot-gun sequencing of 16S rRNA of the gut microbiome showed differences because of diet. (Figure4) This result was consistent with previous results.
Further, variation in gut metagenome by diet was analyzed by taking the sequence reads and aligning them using Rapid Annotations using Sub-systems technology (RASTv2) against the SEED database. In this database, genes are functionally annotated using 3 levels ( general to specific). A subsystem refers to functions (and hence genes) involved in a particular pathway. Diet seemed to influence virulence characteristics of these bacteria as seen in figure 5. 
Further, a canonical correlation analysis  was performed to see if diet is the underlying cause between host upregulation of intestinal biology, immunity, defence genes and gut microbial upregulation of virulence genes. This analysis was repeated three times and eleven immunity related genes with first canonical value of at least 0.85 and second canonical value of 0.5 were chosen based on significance as shown in fig6.
==Conclusion==
In this study a multivariate approach was taken to analyze two data sets simultaneously to make significant correlations on diet induced changes in host as well as commensals. They analysed each dataset seperately to reduce the number of variables first and then analyzed them both at the same time .
Metagenomic data of gut microbiome shows that there are diet influenced changes in genes involved in virulence, antibiotic resistance and secretion systems. On the host side, eleven immunity related  and mucosal defence genes showed variation. Genes that control gut motility like TACR1, bacteria mediated ROS signalling genes and epithelial homeostasis genes such as DUOX2, VAV2 and REL were upregulated in breast fed infants. Genes involved in mucosal inflammatory response KLRF1, BPIL1, ALOX5, IL1A, AOC3 were down regulated in breast fed infants. This study highlights the importance of breast feeding and the benifit of a smooth transition to establish mutualism with the established gut microbiome.