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To date, meta-omic approaches use high-throughput sequencing technologies, which produce a huge amount of data, thus challenging modern computers. Here we present MetaTrans, an efficient open-source pipeline to analyze the structure and functions of active microbial communities using the power of multi-threading computers. The pipeline is designed to perform two types of RNA-Seq analyses: taxonomic and gene expression. It performs quality-control assessment, rRNA removal, maps reads againstdoi:10.1038/srep26447 pmid:27211518 pmcid:PMC4876386 fatcat:bdqoscfvgjatdharwhwshe7idq
more »... tional databases and also handles differential gene expression analysis. Its efficacy was validated by analyzing data from synthetic mock communities, data from a previous study and data generated from twelve human fecal samples. Compared to an existing web application server, MetaTrans shows more efficiency in terms of runtime (around 2 hours per million of transcripts) and presents adapted tools to compare gene expression levels. It has been tested with a human gut microbiome database but also proposes an option to use a general database in order to analyze other ecosystems. For the installation and use of the pipeline, we provide a detailed guide at the following website (www.metatrans.org). In the last decade, next-generation sequencing technologies have allowed sequencing at a very low-cost and have thus boosted the use of meta-omic approaches to study microbial communities. To date, the main challenge is to develop, create, and optimize reliable tools that take advantage of current multi-threading computers to analyze the huge amount of data generated by high-throughput sequencing technologies. Over the last decade, the human microbiome has been the focus of important international consortia such as the Human Microbiome Project, a NIH initiative, and MetaHIT, a European consortium. These consortia have deposited catalogues of microbial genes in an unprecedented amount 1,2 . Metagenomics aims at cataloging the genes present in a sample, while the study of RNAs, called metatranscriptomics, provides an opportunity to gain insights into the functionality of microbial communities. By assessing the genes expressed by the microbial community, metatranscriptomics gives a mechanistic understanding of inter-community relationships and the crosstalk between a microbial community and its host 3,4 . Previous transcriptomic 5 and metatranscriptomic 6-8 studies developed various approaches to analyze RNA-Seq experiments; however, the particularity of distinct experimental methods hinders the development of a generic pipeline that covers all possible scenarios. It is important that such tools be not only flexible and adaptable but also efficient, both in terms of runtime and memory footprint. Here we present a downloadable, open-source, effective and efficient metatranscriptomic pipeline developed for a paired-end RNA-Seq analysis and easily adaptable to other high-throughput experiments. Given the rapid emergence of research into metatranscriptomics, additional bioinformatics tools are likely to be developed for specific tasks in the near future and will probably serve to improve our pipeline. Thus, we designed the pipeline in order to facilitate the inclusion of such third-party tools in each of its stages. Our pipeline was designed to perform two types of RNA-Seq analyses, namely those addressing 16S rRNA taxonomy and gene expression. To test the present metatranscriptomic pipeline, we analyzed synthetic mock communities, twelve fecal samples collected from eight individuals obtained from a previous study 9 and from an unpublished one. For four individuals, stool samples were collected and intestinal volume of gas was measured before and after a flatulogenic diet challenge of three days. In the present study, we believe that combining 16S rDNA, 16S rRNA and mRNA data can provide a new perspective of the factors involved in the origin of flatulence. Using these samples, we extracted total RNA, performed an rRNA removal step in a set of four samples
Objective A decade of microbiome studies has linked IBD to an alteration in the gut microbial community of genetically predisposed subjects. However, existing profiles of gut microbiome dysbiosis in adult IBD patients are inconsistent among published studies, and did not allow the identification of microbial signatures for CD and UC. Here, we aimed to compare the faecal microbiome of CD with patients having UC and with non-IBD subjects in a longitudinal study. Design We analysed a cohort ofdoi:10.1136/gutjnl-2016-313235 pmid:28179361 pmcid:PMC5531220 fatcat:mmpbxeomunebpiqlp4zm5bj3dq
more »... non-IBD and IBD faecal samples from four countries (Spain, Belgium, the UK and Germany), applied a 16S rRNA sequencing approach and analysed a total dataset of 115 million sequences. Results In the Spanish cohort, dysbiosis was found significantly greater in patients with CD than with UC, as shown by a more reduced diversity, a less stable microbial community and eight microbial groups were proposed as a specific microbial signature for CD. Tested against the whole cohort, the signature achieved an overall sensitivity of 80% and a specificity of 94%, 94%, 89% and 91% for the detection of CD versus healthy controls, patients with anorexia, IBS and UC, respectively. Conclusions Although UC and CD share many epidemiologic, immunologic, therapeutic and clinical features, our results showed that they are two distinct subtypes of IBD at the microbiome level. For the first time, we are proposing microbiomarkers to discriminate between CD and non-CD independently of geographical regions. To cite: Pascal V, Pozuelo M, Borruel N, et al. Gut 2017;66:813-822. ► Additional material is published online only. To view please visit the journal online (http://dx.
Diet is recognised as the main driver of changes in gut microbiota. However, linking habitual dietary intake to microbiome composition and activity remains a challenge, leaving most microbiome studies with little or no dietary information. To fill this knowledge gap, we conducted two consecutive studies (n = 84: a first pilot study (n = 40) to build a web-based, semi-quantitative simplified FFQ (sFFQ) based on three 24-h dietary recalls (24HRs); a second study (n = 44) served to validate thedoi:10.3390/nu13092978 pmid:34578856 fatcat:lx673n33lvbhdkje2m7f76hntm
more »... ly developed sFFQ using three 24HRs as reference method and to relate gut microbiome profiling (16S rRNA gene) with the extracted dietary and lifestyle data. Relative validation analysis provided acceptable classification and agreement for 13 out of 24 (54%) food groups and 20 out of 29 nutrients (69%) based on intraclass correlation coefficient, cross-classification, Spearman's correlation, Wilcoxon test, and Bland–Altman. Microbiome analysis showed that higher diversity was positively associated with age, vaginal birth, and intake of fruit. In contrast, microbial diversity was negatively associated with BMI, processed meats, ready-to-eat meals, sodium, and saturated fat. Our analysis also revealed a correlation between food groups or nutrients and microbial composition. Overall, we provide the first dietary assessment tool to be validated and correlated with microbiome data for population studies.
The structure and function of human gut microbiota is currently inferred from metagenomic and metatranscriptomic analyses. Recovery of intact DNA and RNA is therefore a critical step in these studies. Here, we evaluated how different storage conditions of fecal samples affect the quality of extracted nucleic acids and the stability of their microbial communities. Results: We assessed the quality of genomic DNA and total RNA by microcapillary electrophoresis and analyzed the bacterial communitydoi:10.1186/1471-2180-12-158 pmid:22846661 pmcid:PMC3489833 fatcat:i6br3op4svc3vjs6wlak5nz2yu
more »... tructure by pyrosequencing the 16S rRNA gene. DNA and RNA started to fragment when samples were kept at room temperature for more than 24 h. The use of RNAse inhibitors diminished RNA degradation but this protection was not consistent among individuals. DNA and RNA degradation also occurred when frozen samples were defrosted for a short period (1 h) before nucleic acid extraction. The same conditions that affected DNA and RNA integrity also altered the relative abundance of most taxa in the bacterial community analysis. In this case, intra-individual variability of microbial diversity was larger than inter-individual one. Conclusions: Though this preliminary work explored a very limited number of parameters, the results suggest that storage conditions of fecal samples affect the integrity of DNA and RNA and the composition of their microbial community. For optimal preservation, stool samples should be kept at room temperature and brought at the laboratory within 24 h after collection or be stored immediately at −20°C in a home freezer and transported afterwards in a freezer pack to ensure that they do not defrost at any time. Mixing the samples with RNAse inhibitors outside the laboratory is not recommended since proper homogenization of the stool is difficult to monitor.
Eukaryotic topoisomerase II (topo II) is the essential decatenase of newly replicated chromosomes and the main relaxase of nucleosomal DNA. Apart from these general tasks, topo II participates in more specialized functions. In mammals, topo IIa interacts with specific RNA polymerases and chromatin-remodeling complexes, whereas topo IIb regulates developmental genes in conjunction with chromatin remodeling and heterochromatin transitions. Here we show that in budding yeast, topo II regulates thedoi:10.1093/nar/gkt707 pmid:23935120 pmcid:PMC3814376 fatcat:tlfr6td6ebbt7bpqxyuuvbcufe
more »... expression of specific gene subsets. To uncover this, we carried out a genomic transcription run-on shortly after the thermal inactivation of topo II. We identified a modest number of genes not involved in the general stress response but strictly dependent on topo II. These genes present distinctive functional and structural traits in comparison with the genome average. Yeast topo II is a positive regulator of genes with well-defined promoter architecture that associates to chromatin remodeling complexes; it is a negative regulator of genes extremely hypo-acetylated with complex promoters and undefined nucleosome positioning, many of which are involved in polyamine transport. These findings indicate that yeast topo II operates on singular chromatin architectures to activate or repress DNA transcription and that this activity produces functional responses to ensure chromatin stability.
et al.. Processing faecal samples: a step forward for standards in microbial community analysis. BMC Microbiology, Abstract Background: The microbial community analysis of stools requires optimised and standardised protocols for their collection, homogenisation, microbial disruption and nucleic acid extraction. Here we examined whether different layers of the stool are equally representative of the microbiome. We also studied the effect of stool water content, which typically increases indoi:10.1186/1471-2180-14-112 pmid:24884524 pmcid:PMC4021188 fatcat:k3sdt6p57zgydg3a44ikhe5h2y
more »... oeic samples, and of a microbial disruption method on DNA integrity and, therefore, on providing an unbiased microbial composition analysis. Results: We collected faecal samples from healthy subjects and performed microbial composition analysis by pyrosequencing the V4 region of the 16S rRNA gene. To examine the effect of stool structure, we compared the inner and outer layers of the samples (N = 8). Both layers presented minor differences in microbial composition and abundance at the species level. These differences did not significantly bias the microbial community specific to an individual. To evaluate the effect of stool water content and bead-beating, we used various volumes of a water-based salt solution and beads of distinct weights before nucleic acid extraction (N = 4). The different proportions of water did not affect the UniFrac-based clustering of samples from the same subject However, the use or omission of a bead-beating step produced different proportions of Gram-positive and Gram-negative bacteria and significant changes in the UniFrac-based clustering of the samples. Conclusion: The degree of hydration and homogenisation of faecal samples do not significantly alter their microbial community composition. However, the use of bead-beating is critical for the proper detection of Gram-positive bacteria such as Blautia and Bifidobacterium.
From birth onwards, the human gut microbiota rapidly increases in diversity and reaches an adult-like stage at three years of age. After this age, the composition may fluctuate in response to external factors such as antibiotics. Previous studies have shown that resilience is not complete months after cessation of the antibiotic intake. However, little is known about the short-term effects of antibiotic intake on the gut microbial community. Here we examined the load and composition of thedoi:10.1371/journal.pone.0095476 pmid:24748167 pmcid:PMC3991704 fatcat:6j2rmjclbvdhdenqu5guf4d6ui
more »... microbiota immediately after treatment in 21 patients, who received broad-spectrum antibiotics such as fluoroquinolones and b-lactams. A fecal sample was collected from all participants before treatment and one week after for microbial load and community composition analyses by quantitative PCR and pyrosequencing of the 16S rRNA gene, respectively. Fluoroquinolones and b-lactams significantly decreased microbial diversity by 25% and reduced the core phylogenetic microbiota from 29 to 12 taxa. However, at the phylum level, these antibiotics increased the Bacteroidetes/ Firmicutes ratio (p = 0.0007, FDR = 0.002). At the species level, our findings unexpectedly revealed that both antibiotic types increased the proportion of several unknown taxa belonging to the Bacteroides genus, a Gram-negative group of bacteria (p = 0.0003, FDR,0.016). Furthermore, the average microbial load was affected by the treatment. Indeed, the b-lactams increased it significantly by two-fold (p = 0.04). The maintenance of or possible increase detected in microbial load and the selection of Gram-negative over Gram-positive bacteria breaks the idea generally held about the effect of broad-spectrum antibiotics on gut microbiota.
The gut microbiome of animals is emerging as an important factor influencing ecological and evolutionary processes. A major bottleneck in obtaining microbiome data from large numbers of samples is the time-consuming laboratory procedures required, specifically the isolation of DNA and generation of amplicon libraries. Recently, direct PCR kits have been developed that circumvent conventional DNA extraction steps, thereby streamlining the laboratory process by reducing preparation time anddoi:10.1128/msystems.00132-17 pmid:29181448 pmcid:PMC5698494 fatcat:gilr4vjzefab7j7b2t3d2y45ku
more »... However, the reliability and efficacy of direct PCR for measuring host microbiomes have not yet been investigated other than in humans with 454 sequencing. Here, we conduct a comprehensive evaluation of the microbial communities obtained with direct PCR and the widely used Mo Bio PowerSoil DNA extraction kit in five distinct gut sample types (ileum, cecum, colon, feces, and cloaca) from 20 juvenile ostriches, using 16S rRNA Illumina MiSeq sequencing. We found that direct PCR was highly comparable over a range of measures to the DNA extraction method in cecal, colon, and fecal samples. However, the two methods significantly differed in samples with comparably low bacterial biomass: cloacal and especially ileal samples. We also sequenced 100 replicate sample pairs to evaluate repeatability during both extraction and PCR stages and found that both methods were highly consistent for cecal, colon, and fecal samples ( r s > 0.7) but had low repeatability for cloacal ( r s = 0.39) and ileal ( r s = −0.24) samples. This study indicates that direct PCR provides a fast, cheap, and reliable alternative to conventional DNA extraction methods for retrieving 16S rRNA data, which can aid future gut microbiome studies. IMPORTANCE The microbial communities of animals can have large impacts on their hosts, and the number of studies using high-throughput sequencing to measure gut microbiomes is rapidly increasing. However, the library preparation procedure in microbiome research is both costly and time-consuming, especially for large numbers of samples. We investigated a cheaper and faster direct PCR method designed to bypass the DNA isolation steps during 16S rRNA library preparation and compared it with a standard DNA extraction method. We used both techniques on five different gut sample types collected from 20 juvenile ostriches and sequenced samples with Illumina MiSeq. The methods were highly comparable and highly repeatable in three sample types with high microbial biomass (cecum, colon, and feces), but larger differences and low repeatability were found in the microbiomes obtained from the ileum and cloaca. These results will help microbiome researchers assess library preparation procedures and plan their studies accordingly.
Faecal microbiota transplantation (FMT) is a novel potential therapy for inflammatory bowel diseases, but it is poorly characterised. We evaluated the performance of the mouse and rat as a pre-clinical model for human microbiota engraftment. We then characterised the effect of a single human stool transfer (HST) on a humanised model of DSS-induced colitis. Colonic and faecal microbial communities were analysed using the 16S rRNA approach and clinical manifestations were assessed in adoi:10.1016/j.ebiom.2019.10.002 pmid:31628021 pmcid:PMC6838378 fatcat:kkzp4kx3kvfo3dywsg65p3desq
more »... l setting. The microbial community of rats showed greater similarity to that of humans, while the microbiome of mice showed less similarity to that of humans. Moreover, rats captured more human microbial species than mice after a single HST. Using the rat model, we showed that HST compensated faecal dysbiosis by restoring alpha-diversity and by increasing the relative abundance of health-related microbial genera. To some extent, HST also modulated the microbial composition of colonic tissue. These faecal and colonic microbial communities alterations led to a relative restoration of colon length, and a significant decrease in both epithelium damage and disease severity. Remarkably, stopping inflammation by removing DSS before HST caused a faster and greater recovery of both microbiome and clinical manifestation features. Our results indicate that the rat outperforms the mouse as a model for human microbiota engraftment and show that the efficacy of HST can be enhanced when inflammation stimulation is withdrawn. Finally, our findings support a new therapeutic strategy based on the use FMT combined with anti-inflammatory drugs.
By transporting one DNA double helix (T-segment) through a double-strand break in another (G-segment), topoisomerase II reduces fractions of DNA catenanes, knots and supercoils to below equilibrium values. How DNA segments are selected to simplify the equilibrium DNA topology is enigmatic, and the biological relevance of this activity is unclear. Here we examined the transit of the T-segment across the three gates of topoisomerase II (entry N-gate, DNA-gate and exit C-gate). Our experimentaldoi:10.1093/nar/gkt1037 pmid:24185700 pmcid:PMC3919613 fatcat:s2dknp6qp5dnjbfvad6ianemim
more »... ults uncovered that DNA transport probability is determined not only during the capture of a T-segment at the N-gate. When a captured T-segment has crossed the DNA-gate, it can backtrack to the N-gate instead of exiting by the C-gate. When such backtracking is precluded by locking the N-gate or by removing the C-gate, topoisomerase II no longer simplifies equilibrium DNA topology. Therefore, we conclude that the C-gate enables a post-DNA passage proofreading mechanism, which challenges the release of passed T-segments to either complete or cancel DNA transport. This proofreading activity not only clarifies how type-IIA topoisomerases simplify the equilibrium topology of DNA in free solution, but it may explain also why these enzymes are able to solve the topological constraints of intracellular DNA without randomly entangling adjacent chromosomal regions.
The pathophysiology of irritable bowel syndrome (IBS) remains unclear. Here we investigated the microbiome of a large cohort of patients to identify specific signatures for IBS subtypes. We examined the microbiome of 113 patients with IBS and 66 healthy controls. A subset of these participants provided two samples one month apart. We analyzed a total of 273 fecal samples, generating more than 20 million 16S rRNA sequences. In patients with IBS, a significantly lower microbial diversity wasdoi:10.1038/srep12693 pmid:26239401 pmcid:PMC4523847 fatcat:rbqqgiednve6lcrfndq2ougcbe
more »... iated with a lower relative abundance of butyrate-producing bacteria (P = 0.002; q < 0.06), in particular in patients with IBS-D and IBS-M. IBS patients who did not receive any treatment harboured a lower abundance of Methanobacteria compared to healthy controls (P = 0.005; q = 0.05). Furthermore, significant correlations were observed between several bacterial taxa and sensation of flatulence and abdominal pain (P < 0.05). Altogether, our findings showed that IBS-M and IBS-D patients are characterized by a reduction of butyrate producing bacteria, known to improve intestinal barrier function, and a reduction of methane producing microorganisms a major mechanism of hydrogen disposal in the human colon, which could explain excess of abdominal gas in IBS. increase epithelial permeability, activate nociceptive sensory pathways, and dysregulate the enteric nervous system 5 . Supplementary Table S1 reports the methods and main results gathered from 24 studies on IBS and microbiome using culture-independent techniques. Over the past ten years, most of these studies have used 16S rRNA gene (16S) surveys through quantitative specific polymerase chain reaction (qPCR), denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), fluorescent in situ hybridization (FISH) or cloning, and Sanger sequencing to characterize the microbiome of patients with IBS. Only since 2011 have a few studies used high-throughput techniques, such as 16S phylogenetic microarray, 16S and shotgun pyrosequencing, and metatranscriptomics. Results from those studies showed several common trends, as well as inconsistencies in the microbial signatures of patients with IBS or subtypes of this condition. Among the trends, patients show dysbiotic microbiota, which can be characterized at various phylogenetic levels. At the phylum level, a higher proportion of Proteobacteria 6,7 has been reported in patients compared to healthy controls. At the genus level, a higher count or proportion of Veillonella 8-10 , Lactobacillus 9-12 and Ruminococcus 7,8,12,13 , has been associated with IBS. In contrast, a lower count of Bifidobacterium 10,13-15 , Faecalibacterium 13,16 , and methanogens 13,17 was encountered in patients with this condition. Among the inconsistencies, three studies reported a higher ratio of Firmicutes/Bacteroidetes in patients 6,13,18 , while the opposite was found in one study 19 . Lower 20 and higher 21 counts of Eubacterium rectal were found in two studies. Also, it has been proposed that IBS involves a higher count of Clostridium coccoides 8,20 , while other authors reported a lower count of this species. Despite the replacement of culture methods by more powerful molecular techniques, these studies described small sample sizes, ranging from 2 to 62 IBS patients and did not consider confounding factors such as medications for IBS symptoms. Using a high number of healthy controls (n = 66) and IBS patients (n = 113), two-time points for 94 participants, and a deep sequencing coverage of the 16S rRNA gene, we sought to determine: (a) whether dysbiosis occurs in patients with IBS and, if so, the phylogenetic level that defines it; (b) whether the three IBS subtypes can be distinguished by microbial community clustering; (c) whether microbial diversity is lower in patients compared to healthy controls and, if so, which bacteria are absent; (d) the temporal stability of the microbial community in IBS patients; (e) the effect of medication on the microbiota of IBS patients and (f) the correlation between microbiota and the patients' symptoms.
A major function of the gut microbiota is to provide colonization resistance, wherein pathogens are inhibited or suppressed below infectious levels. However, the fraction of gut microbiota required for colonization resistance remains unclear. We used culturomics to isolate a gut microbiota culture collection comprising 1,590 isolates belonging to 102 species. This culture collection represents 34.57% of the taxonomic diversity and 70% functional capacity, as estimated by metagenomic sequencingdoi:10.1128/msystems.00620-19 pmid:32019832 pmcid:PMC7002114 fatcat:hvdd7h76hvfdxkdfdfm7vtki5a
more »... f the fecal samples used for culture. Using whole-genome sequencing, we characterized species representatives from this collection and predicted their phenotypic traits, further characterizing isolates by defining nutrient utilization profiles and short-chain fatty acid production. When screened with a coculture assay, 66 species in our culture collection inhibited Clostridioides difficile. Several phenotypes, particularly, growth rate, production of SCFAs, and the utilization of mannitol, sorbitol, or succinate, correlated with C. difficile inhibition. We used a combinatorial community assembly approach to formulate defined bacterial mixes inhibitory to C. difficile. We tested 256 combinations and found that both species composition and blend size were important in inhibition. Our results show that the interaction of bacteria with one another in a mix and with other members of gut commensals must be investigated to design defined bacterial mixes for inhibiting C. difficile in vivo. IMPORTANCE Antibiotic treatment causes instability of gut microbiota and the loss of colonization resistance, thus allowing pathogens such as Clostridioides difficile to colonize and causing recurrent infection and mortality. Although fecal microbiome transplantation has been shown to be an effective treatment for C. difficile infection (CDI), a more desirable approach would be the use of a defined mix of inhibitory gut bacteria. The C. difficile-inhibiting species and bacterial combinations identified herein improve the understanding of the ecological interactions controlling colonization resistance against C. difficile and could aid in the design of defined bacteriotherapy as a nonantibiotic alternative against CDI.
Hyperlipidemia has been intensively focused on by researchers around the world owing to its major contribution to cardiovascular diseases. Various evidence reveals that women are more susceptible than male counterparts to dyslipidemia, making sex-dependent therapeutic strategies and drugs urgently needed. In the present work, we demonstrate that DNJ, the main active component of mulberry leaves, exerts an obvious female-preferential antihyperlipidemic effect through specifically enrichingdoi:10.1128/msystems.00313-20 pmid:33024047 fatcat:nuyncui5nzcnzc6wsasfmyosqu
more »... ansia and Clostridium XIVa and elevating an active microbial metabolite, indole-3-propionic acid (IPA), in female mice. Moreover, we have corroborated the potent lipid-lowering efficacy of IPA both in vitro and in vivo. These findings not only indicate a potential mechanism by which gut microbes and their metabolites confer the beneficial role of DNJ in ameliorating hyperlipidemia but also provide an in-depth theoretical basis for therapeutic exploitation of DNJ as a female-specific intervention against hyperlipidemia.
In Manichanh et al. ... In Manichanh et al. (18) , taxonomic analysis was based on a multiple alignment (ClustalW). ...doi:10.1093/nar/gkn496 pmid:18682527 pmcid:PMC2532719 fatcat:esx6s5vqqfdddeepqjlff2g3hq
Goal: To determine the effect of a prebiotic chicory-derived inulintype fructan on the tolerance of intestinal gas.doi:10.1097/mcg.0000000000000723 pmid:27680592 pmcid:PMC5499961 fatcat:wdjanayu4fg43hajadvmtwuixa
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