Michael A. Mahowald, Federico E. Rey, Henning Seedorf, Peter J. Turnbaugh, Robert S. Fulton, Aye Wollam, Neha Shah, Chunyan Wang, Vincent Magrini, Richard K. Wilson, Brandi L. Cantarel, Pedro M. Coutinho, Bernard Henrissat, Lara W. Crock, Alison Russell, Nathan C. Verberkmoes, Robert L. Hettich, Jeffrey I. Gordon.
PNAS, Early edition | doi:10.1073/pnas.0901529106 | PMID:19321416
The adult human distal gut microbial community is typically dominated by 2 bacterial phyla (divisions), the Firmicutes and the Bacteroidetes. Little is known about the factors that govern the interactions between their members. Here, we examine the niches of representatives of both phyla in vivo. Finished genome sequences were generated from Eubacterium rectale and E. eligens, which belong to Clostridium Cluster XIVa, one of the most common gut Firmicute clades. Comparison of these and 25 other gut Firmicutes and Bacteroidetes indicated that the Firmicutes possess smaller genomes and a disproportionately smaller number of glycan-degrading enzymes. Germ-free mice were then colonized with E. rectale and/or a prominent human gut Bacteroidetes, Bacteroides thetaiotaomicron, followed by whole-genome transcriptional profiling, high-resolution proteomic analysis, and biochemical assays of microbial–microbial and microbial–host interactions. B. thetaiotaomicron adapts to E. rectale by up-regulating expression of a variety of polysaccharide utilization loci encoding numerous glycoside hydrolases, and by signaling the host to produce mucosal glycans that it, but not E. rectale, can access. E. rectale adapts to B. thetaiotaomicron by decreasing production of its glycan-degrading enzymes, increasing expression of selected amino acid and sugar transporters, and facilitating glycolysis by reducing levels of NADH, in part via generation of butyrate from acetate, which in turn is used by the gut epithelium. This simplified model of the human gut microbiota illustrates niche specialization and functional redundancy within members of its major bacterial phyla, and the importance of host glycans as a nutrient foundation that ensures ecosystem stability.
Xiaoming Liu, Taylor J. Maxwell, Eric Boerwinkle, Yun-Xin Fu.
Molecular Biology and Evolution, MBE Advance Access | doi:10.1093/molbev/msp059 | PMID:19318520
One challenge of analyzing samples of DNA sequences is to account for the non-negligible polymorphisms produced by error when the sequencing error rate is high or the sample size is large. Specifically, those artificial sequence variations will bias the observed SNP frequency spectrum, which in turn may further bias the estimators of the population mutation rate Θ = 4Nµ for diploids. In this paper, we propose a new approach based on the generalized least squares (GLS) method to estimate Θ, given a SNP frequency spectrum in a random sample of DNA sequences from a population. With this approach, error rate e can be either known or unknown. In the latter case ε can be estimated given an estimation of Θ. Using coalescent simulation, we compared our estimators with other estimators of Θ. The results showed the GLS estimators are more efficient than other Θ estimators with error, and the estimation of ε is usable in practice when the Θ per bp is small. We demonstrate the application of the estimators with 10kb noncoding region sequence sampled from a human population and provide suggestions for choosing Θ estimators with error
BRUCE E. DEAGLE, ROGER KIRKWOOD, SIMON N. JARMAN.
Molecular Ecology 18, 2022-2038 (2009) | doi:10.1111/j.1365-294X.2009.04158.x | PMID:19317847
DNA-based techniques have proven useful for defining trophic links in a variety of ecosystems and recently developed sequencing technologies provide new opportunities for dietary studies. We investigated the diet of Australian fur seals (Arctocephalus pusillus doriferus) by pyrosequencing prey DNA from faeces collected at three breeding colonies across the seals' range. DNA from 270 faecal samples was amplified with four polymerase chain reaction primer sets and a blocking primer was used to limit amplification of fur seal DNA. Pooled amplicons from each colony were sequenced using the Roche GS-FLX platform, generating > 20,000 sequences. Software was developed to sort and group similar sequences. A total of 54 bony fish, 4 cartilaginous fish and 4 cephalopods were identified based on the most taxonomically informative amplicons sequenced (mitochondrial 16S). The prevalence of sequences from redbait (Emmelichthys nitidus) and jack mackerel (Trachurus declivis) confirm the importance of these species in the seals' diet. A third fish species, blue mackerel (Scomber australasicus), may be a more important prey species than previously recognised. There were major differences in the proportions of prey DNA recovered in faeces from different colonies, probably reflecting differences in prey availability. Parallel hard-part analysis identified largely the same main prey species as did the DNA-based technique, but with lower species diversity and no remains from cartilaginous prey. The pyrosequencing approach presented significantly expands the capabilities of DNA-based methods of dietary analysis and is suitable for large-scale diet investigations on a broad range of animals.
Olivier Harismendy, Kelly A. Frazer.
BioTechniques 46, 229–231 (2009) | doi 10.2144/000113082 | PMID:19317667
One approach for high-throughput population-based sequencing of targeted intervals in the human genome is to amplify the regions using long-range PCR (LR-PCR) followed by sequencing with next-generation sequencing (NGS) technologies. Utilizing this method, we have observed that the 50 bp located at the amplicon ends account for more than 50% of the sequenced bases and that the sequence coverage depth of base pairs within an amplicon is highly variable. Here we propose an explanation for the overrepresentation of the amplicon ends and show that the use of 5′-blocked primers for the LR-PCR reaction reduces their overrepresentation. Furthermore, we demonstrate that using a 600-bp library insert size rather than the standard 200-bp insert size results in more uniform sequence coverage depth. The capability to increase sequence coverage uniformity greatly improves the effective throughput of NGS platforms.
Quentin C. Santana, Martin P. A. Coetzee, Emma T. Steenkamp, Osmond X. Mlonyeni, Gifty N. A. Hammond, Michael J. Wingfield, Brenda D. Wingfield.
BioTechniques 46, 217–223 (2009) | doi 10.2144/000113085 | PMID:19317665
Robust molecular markers such as microsatellites are important tools used to understand the dynamics of natural populations, but their identification and development are typically time consuming and labor intensive. The recent emergence of so-called next-generation sequencing raised the question as to whether this new technology might be applied to microsatellite development. Following this view, we considered whether deep sequencing using the 454 Life Sciences/Roche GS-FLX genome sequencing system could lead to a rapid protocol to develop microsatellite primers as markers for genetic studies. For this purpose, genomic DNA was sourced from three unrelated organisms: a fungus (the pine pathogen Fusarium circinatum), an insect (the pine-damaging wasp Sirex noctilio), and the wasp's associated nematode parasite (Deladenus siricidicola). Two methods, FIASCO (fast isolation by AFLP of sequences containing repeats) and ISSR-PCR (inter-simple sequence repeat PCR), were used to generate microsatellite-enriched DNA for the 454 libraries. From the resulting 1.2–1.7 megabases of DNA sequence data, we were able to identify 873 microsatellites that have sufficient flanking sequence available for primer design and potential amplification. This approach to microsatellite discovery was substantially more rapid, effective, and economical than other methods, and this study has shown that pyrosequencing provides an outstanding new technology that can be applied to this purpose.
