Comparative analysis of human gut microbiota by barcoded pyrosequencing.
TL;DR: This work has employed 454-pyrosequencing of a hyper-variable region of the 16S rRNA gene in combination with sample-specific barcode sequences which enables parallel in-depth analysis of hundreds of samples with limited sample processing, and demonstrated that the method correctly describes microbial communities down to phylotypes below the genus level.
Abstract: Humans host complex microbial communities believed to contribute to health maintenance and, when in imbalance, to the development of diseases. Determining the microbial composition in patients and healthy controls may thus provide novel therapeutic targets. For this purpose, high-throughput, cost-effective methods for microbiota characterization are needed. We have employed 454-pyrosequencing of a hyper-variable region of the 16S rRNA gene in combination with sample-specific barcode sequences which enables parallel in-depth analysis of hundreds of samples with limited sample processing. In silico modeling demonstrated that the method correctly describes microbial communities down to phylotypes below the genus level. Here we applied the technique to analyze microbial communities in throat, stomach and fecal samples. Our results demonstrate the applicability of barcoded pyrosequencing as a high-throughput method for comparative microbial ecology.
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TL;DR: EdgeR as mentioned in this paper is a Bioconductor software package for examining differential expression of replicated count data, which uses an overdispersed Poisson model to account for both biological and technical variability and empirical Bayes methods are used to moderate the degree of overdispersion across transcripts, improving the reliability of inference.
Abstract: Summary: It is expected that emerging digital gene expression (DGE) technologies will overtake microarray technologies in the near future for many functional genomics applications. One of the fundamental data analysis tasks, especially for gene expression studies, involves determining whether there is evidence that counts for a transcript or exon are significantly different across experimental conditions. edgeR is a Bioconductor software package for examining differential expression of replicated count data. An overdispersed Poisson model is used to account for both biological and technical variability. Empirical Bayes methods are used to moderate the degree of overdispersion across transcripts, improving the reliability of inference. The methodology can be used even with the most minimal levels of replication, provided at least one phenotype or experimental condition is replicated. The software may have other applications beyond sequencing data, such as proteome peptide count data. Availability: The package is freely available under the LGPL licence from the Bioconductor web site (http://bioconductor.org).
29,413 citations
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...Contact: mrobinson@wehi.edu.au...
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TL;DR: The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.
Abstract: 16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of 'best available' primer pairs for Bacteria and Archaea for three amplicon size classes (100-400, 400-1000, ≥ 1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.
5,346 citations
TL;DR: The results suggest that the structure of soil bacterial communities is predictable, to some degree, across larger spatial scales, and the effect of soil pH on bacterial community composition is evident at even relatively coarse levels of taxonomic resolution.
Abstract: Soils harbor enormously diverse bacterial populations, and soil bacterial communities can vary greatly in composition across space. However, our understanding of the specific changes in soil bacterial community structure that occur across larger spatial scales is limited because most previous work has focused on either surveying a relatively small number of soils in detail or analyzing a larger number of soils with techniques that provide little detail about the phylogenetic structure of the bacterial communities. Here we used a bar-coded pyrosequencing technique to characterize bacterial communities in 88 soils from across North and South America, obtaining an average of 1,501 sequences per soil. We found that overall bacterial community composition, as measured by pairwise UniFrac distances, was significantly correlated with differences in soil pH (r = 0.79), largely driven by changes in the relative abundances of Acidobacteria, Actinobacteria, and Bacteroidetes across the range of soil pHs. In addition, soil pH explains a significant portion of the variability associated with observed changes in the phylogenetic structure within each dominant lineage. The overall phylogenetic diversity of the bacterial communities was also correlated with soil pH (R2 = 0.50), with peak diversity in soils with near-neutral pHs. Together, these results suggest that the structure of soil bacterial communities is predictable, to some degree, across larger spatial scales, and the effect of soil pH on bacterial community composition is evident at even relatively coarse levels of taxonomic resolution.
3,151 citations
TL;DR: The large-scale dynamics of the microbiome can be described by many of the tools and observations used in the study of population ecology, andiphering the metagenome and its aggregate genetic information can also be used to understand the functional properties of the microbial community.
Abstract: Interest in the role of the microbiome in human health has burgeoned over the past decade with the advent of new technologies for interrogating complex microbial communities. The large-scale dynamics of the microbiome can be described by many of the tools and observations used in the study of population ecology. Deciphering the metagenome and its aggregate genetic information can also be used to understand the functional properties of the microbial community. Both the microbiome and metagenome probably have important functions in health and disease; their exploration is a frontier in human genetics.
2,650 citations
Cites background from "Comparative analysis of human gut m..."
...pylori -negative individuals, gastric microbiota diversity is high; most of the prominent gastric phylotypes ( Streptococcus , Actinomyces, Prevotella and Gemella ) also are abundant in the oropharynx of these individual...
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TL;DR: The results of this study indicate that type 2 diabetes in humans is associated with compositional changes in intestinal microbiota and the level of glucose tolerance should be considered when linking microbiota with metabolic diseases such as obesity and developing strategies to control metabolic diseases by modifying the gut microbiota.
Abstract: Background: Recent evidence suggests that there is a link between metabolic diseases and bacterial populations in the gut The aim of this study was to assess the differences between the composition of the intestinal microbiota in humans with type 2 diabetes and non-diabetic persons as control Methods and Findings: The study included 36 male adults with a broad range of age and body-mass indices (BMIs), among which 18 subjects were diagnosed with diabetes type 2 The fecal bacterial composition was investigated by real-time quantitative PCR (qPCR) and in a subgroup of subjects (N=20) by tag-encoded amplicon pyrosequencing of the V4 region of the 16S rRNA gene The proportions of phylum Firmicutes and class Clostridia were significantly reduced in the diabetic group compared to the control group (P=003) Furthermore, the ratios of Bacteroidetes to Firmicutes as well as the ratios of Bacteroides-Prevotella group to C coccoides-E rectale group correlated positively and significantly with plasma glucose concentration (P=004) but not with BMIs Similarly, class Betaproteobacteria was highly enriched in diabetic compared to non-diabetic persons (P=002) and positively correlated with plasma glucose (P=004) Conclusions: The results of this study indicate that type 2 diabetes in humans is associated with compositional changes in intestinal microbiota The level of glucose tolerance should be considered when linking microbiota with metabolic diseases such as obesity and developing strategies to control metabolic diseases by modifying the gut microbiota
2,345 citations
References
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TL;DR: A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original.
Abstract: The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.
70,111 citations
"Comparative analysis of human gut m..." refers methods in this paper
...Each resulting unique sequence (one per group of identical sequences) was BLASTNsearched against the RDP database with default parameters....
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...Each pyrosequencing read is BLAST [24] searched against a reference database comprising .90,000 near PLoS ONE | www.plosone.org 1 July 2008 | Volume 3 | Issue 7 | e2836 full-length 16S rRNA gene sequences from the Ribosomal Database Project (RDP) [25]....
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...%) had best BLAST hits of ,90% identity to any RDP sequence (Table S2)....
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...Calculating sequence distances and grouping into phylotypes RDP sequences rendering best scoring BLAST hits to the pyrosequencing reads, as well as E. coli sequence S000380829, were downloaded in pre-aligned format from RDP and imported into ARB[33]....
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...These artificial reads were BLAST searched against the RDP database, from which the corresponding sequences first had been removed....
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TL;DR: The RDP Classifier can rapidly and accurately classify bacterial 16S rRNA sequences into the new higher-order taxonomy proposed in Bergey's Taxonomic Outline of the Prokaryotes, and the majority of the classification errors appear to be due to anomalies in the current taxonomies.
Abstract: The Ribosomal Database Project (RDP) Classifier, a naive Bayesian classifier, can rapidly and accurately classify bacterial 16S rRNA sequences into the new higher-order taxonomy proposed in Bergey's Taxonomic Outline of the Prokaryotes (2nd ed., release 5.0, Springer-Verlag, New York, NY, 2004). It provides taxonomic assignments from domain to genus, with confidence estimates for each assignment. The majority of classifications (98%) were of high estimated confidence (≥95%) and high accuracy (98%). In addition to being tested with the corpus of 5,014 type strain sequences from Bergey's outline, the RDP Classifier was tested with a corpus of 23,095 rRNA sequences as assigned by the NCBI into their alternative higher-order taxonomy. The results from leave-one-out testing on both corpora show that the overall accuracies at all levels of confidence for near-full-length and 400-base segments were 89% or above down to the genus level, and the majority of the classification errors appear to be due to anomalies in the current taxonomies. For shorter rRNA segments, such as those that might be generated by pyrosequencing, the error rate varied greatly over the length of the 16S rRNA gene, with segments around the V2 and V4 variable regions giving the lowest error rates. The RDP Classifier is suitable both for the analysis of single rRNA sequences and for the analysis of libraries of thousands of sequences. Another related tool, RDP Library Compare, was developed to facilitate microbial-community comparison based on 16S rRNA gene sequence libraries. It combines the RDP Classifier with a statistical test to flag taxa differentially represented between samples. The RDP Classifier and RDP Library Compare are available online at http://rdp.cme.msu.edu/.
16,048 citations
"Comparative analysis of human gut m..." refers methods in this paper
...phylogenetic levels, which may be more accurately done using other methods [28]....
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TL;DR: It is demonstrated through metagenomic and biochemical analyses that changes in the relative abundance of the Bacteroidetes and Firmicutes affect the metabolic potential of the mouse gut microbiota and indicates that the obese microbiome has an increased capacity to harvest energy from the diet.
Abstract: The worldwide obesity epidemic is stimulating efforts to identify host and environmental factors that affect energy balance. Comparisons of the distal gut microbiota of genetically obese mice and their lean littermates, as well as those of obese and lean human volunteers have revealed that obesity is associated with changes in the relative abundance of the two dominant bacterial divisions, the Bacteroidetes and the Firmicutes. Here we demonstrate through metagenomic and biochemical analyses that these changes affect the metabolic potential of the mouse gut microbiota. Our results indicate that the obese microbiome has an increased capacity to harvest energy from the diet. Furthermore, this trait is transmissible: colonization of germ-free mice with an 'obese microbiota' results in a significantly greater increase in total body fat than colonization with a 'lean microbiota'. These results identify the gut microbiota as an additional contributing factor to the pathophysiology of obesity.
10,126 citations
"Comparative analysis of human gut m..." refers background in this paper
...The recent demonstration that obesity is reflected in the intestinal microbial composition in both mice [30] and humans [10], and that the obesity trait is transmissible through transplantation of the microbiota [3] clearly illustrates how the microbial community can effect host physiology....
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...The gut microbiota play important roles in extracting nutrients from the diet [2,3], regulating host fat storage [4], stimulating intestinal epithelium renewal [5], and directing the maturation of the immune system [6]....
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TL;DR: A scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments with 96% coverage at 99.96% accuracy in one run of the machine is described.
Abstract: The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.
8,434 citations
"Comparative analysis of human gut m..." refers background or methods in this paper
...454-pyrosequencing has been reported to have a relatively high homopolymer insertion/deletion error rate [21] which could potentially disturb the taxonomic classifications....
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...The pooled DNA were subsequently amplified in PCR-mixture-in-oil emulsions and sequenced on different lanes of a 16-lane PicoTiterPlate on a Genome Sequencer 20 system [21] (Roche, Basel, Switzerland) at 454 Life Sciences (Branford CT) in June 2006....
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...In parallel with other groups [19,20] we have developed a method based on 454-pyrosequencing [21] for monitoring of microbial communities....
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...Thus, although insertion/deletion errors seem to occur to some extent, the application here is robust: an insertion/deletion error rate of 2% of reads [21] would affect the classification of 0....
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TL;DR: It is shown that the relative proportion of Bacteroidetes is decreased in obese people by comparison with lean people, and that this proportion increases with weight loss on two types of low-calorie diet.
Abstract: Two groups of beneficial bacteria are dominant in the human gut, the Bacteroidetes and the Firmicutes. Here we show that the relative proportion of Bacteroidetes is decreased in obese people by comparison with lean people, and that this proportion increases with weight loss on two types of low-calorie diet. Our findings indicate that obesity has a microbial component, which might have potential therapeutic implications.
7,550 citations
"Comparative analysis of human gut m..." refers background or result in this paper
...The recent demonstration that obesity is reflected in the intestinal microbial composition in both mice [30] and humans [10], and that the obesity trait is transmissible through transplantation of the microbiota [3] clearly illustrates how the microbial community can effect host physiology....
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...Interestingly, Actinobacteria were more abundant than Bacteriodetes in all six fecal samples analyzed, contrasting with prior studies....
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...[9,10,11]), whereas less has been reported from upper gastro-intestinal tract habitats (e....
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...The human lower intestine is the most densely populated microbial ecosystem known, with approximately 10(12) microorganisms/ml [1], and is considered to be dominated by the phyla Firmicutes and Bacteriodetes [9,10,11]....
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...It is possible that Bacteroidetes are under-represented in our six fecal samples, because this phylum is known to show intersubject variability [9], to vary in response to adiposity [10] and to sometimes be suppressed in inflammatory bowel disease [11]....
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