Evaluating the Contribution of Gut Microbiota to the Variation of Porcine Fatness with the Cecum and Fecal Samples

Next-generation sequencing technologies generate very large numbers of short reads. Even with very deep genome coverage, short read lengths cause problems in de novo assemblies. The use of paired-end libraries with a fragment size shorter than twice the read length provides an opportunity to generate much longer reads by overlapping and merging read pairs before assembling a genome. We present FLASH, a fast computational tool to extend the length of short reads by overlapping paired-end reads from fragment libraries that are sufficiently short. We tested the correctness of the tool on one million simulated read pairs, and we then applied it as a pre-processor for genome assemblies of Illumina reads from the bacterium Staphylococcus aureus and human chromosome 14. FLASH correctly extended and merged reads >99% of the time on simulated reads with an error rate of <1%. With adequately set parameters, FLASH correctly merged reads over 90% of the time even when the reads contained up to 5% errors. When FLASH was used to extend reads prior to assembly, the resulting assemblies had substantially greater N50 lengths for both contigs and scaffolds. The FLASH system is implemented in C and is freely available as open-source code at http://www.cbcb.umd.edu/software/flash. t.magoc@gmail.com.

Humans and other mammals are colonized by a vast, complex, and dynamic consortium of microorganisms. One evolutionary driving force for maintaining this metabolically active microbial society is to salvage energy from nutrients, particularly carbohydrates, that are otherwise nondigestible by the host. Much of our understanding of the molecular mechanisms by which members of the intestinal microbiota degrade complex polysaccharides comes from studies of Bacteroides thetaiotaomicron, a prominent and genetically manipulatable component of the normal human and mouse gut. Colonization of germ-free mice with B. thetaiotaomicron has shown how this anaerobe modifies many aspects of intestinal cellular differentiation/gene expression to benefit both host and microbe. These and other studies underscore the importance of understanding precisely how nutrient metabolism serves to establish and sustain symbiotic relationships between mammals and their bacterial partners.

Background & Aims While it is widely accepted that obesity is associated with low-grade systemic inflammation, the molecular origin of the inflammation remains unknown. Here, we investigated the effect of endotoxin-induced inflammation via TLR4 signaling pathway at both systemic and intestinal levels in response to a high-fat diet. Methods C57BL/6J and TLR4-deficient C57BL/10ScNJ mice were maintained on a low-fat (10 kcal % fat) diet (LFD) or a high–fat (60 kcal % fat) diet (HFD) for 8 weeks. Results HFD induced macrophage infiltration and inflammation in the adipose tissue, as well as an increase in the circulating proinflammatory cytokines. HFD increased both plasma and fecal endotoxin levels and resulted in dysregulation of the gut microbiota by increasing the Firmicutes to Bacteriodetes ratio. HFD induced the growth of Enterobecteriaceae and the production of endotoxin in vitro. Furthermore, HFD induced colonic inflammation, including the increased expression of proinflammatory cytokines, the induction of Toll-like receptor 4 (TLR4), iNOS, COX-2, and the activation of NF-κB in the colon. HFD reduced the expression of tight junction-associated proteins claudin-1 and occludin in the colon. HFD mice demonstrated higher levels of Akt and FOXO3 phosphorylation in the colon compared to the LFD mice. While the body weight of HFD-fed mice was significantly increased in both TLR4-deficient and wild type mice, the epididymal fat weight and plasma endotoxin level of HFD-fed TLR4-deficient mice were 69% and 18% of HFD-fed wild type mice, respectively. Furthermore, HFD did not increase the proinflammatory cytokine levels in TLR4-deficient mice. Conclusions HFD induces inflammation by increasing endotoxin levels in the intestinal lumen as well as in the plasma by altering the gut microbiota composition and increasing its intestinal permeability through the induction of TLR4, thereby accelerating obesity.