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Gene-centric metagenomics analysis of feline intestinal microbiome using 454 junior pyrosequencing.

ABSTRACT: The feline gastrointestinal microbiota have direct influence on feline health and also human health as a reservoir for potential zoonotic pathogens and antibiotic resistant bacterial strains. In order to describe the feline gastrointestinal microbial diversity, fecal samples from cats have been characterized using both culture-dependent and culture-independent methods. However, data correlating total microbial composition and their functions are lacking. Present descriptive study evaluated both phylogenetic and metabolic diversity of the feline intestinal microbiota using GS Junior titanium shotgun pyrosequencing. A total of 152,494 pyrosequencing reads (5405 assembled contigs) were generated and classified into both phylogenetic and metabolic profiles of the feline intestinal microbiota. The Bacteroides/Chlorobi group was the most predominant bacterial phylum comprising ~68% of total classified diversity, followed by Firmicutes (~13%) and Proteobacteria (~6%) respectively. Archaea, fungi and viruses made up the minor communities in the overall microbial diversity. Interestingly, this study also identified a range of potential enteric zoonotic pathogens (0.02-1.25%) and genes involved in antimicrobial resistance (0.02-0.7%) in feline fecal materials. Based on clustering among nine gastrointestinal metagenomes from five different monogastric hosts (dog, human, mice, cat and chicken), the cat metagenome clustered closely together with chicken in both phylogenetic and metabolic level (>80%). Future studies are required to provide deeper understandings on both intrinsic and extrinsic effects such as impact of age, genetics and dietary interventions on the composition of the feline gastrointestinal microbiome.

SUBMITTER: Tun HM

PROVIDER: S-EPMC7114314 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Gene-centric metagenomics analysis of feline intestinal microbiome using 454 junior pyrosequencing.

Tun Hein Min HM Brar Manreetpal Singh MS Khin Nina N Jun Li L Hui Raymond Kin-Hi RK Dowd Scot E SE Leung Frederick Chi-Ching FC

Journal of microbiological methods 20120112 3

The feline gastrointestinal microbiota have direct influence on feline health and also human health as a reservoir for potential zoonotic pathogens and antibiotic resistant bacterial strains. In order to describe the feline gastrointestinal microbial diversity, fecal samples from cats have been characterized using both culture-dependent and culture-independent methods. However, data correlating total microbial composition and their functions are lacking. Present descriptive study evaluated both ...[more]

PMID: 22265636

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Project description:AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome. METHODS:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral, esophageal, and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species. Candidate primers evaluated were from the European rRNA database. To assess the effect of sequence length on accuracy of classification, 16S rRNA genes of various lengths were created by trimming the full length sequences. Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs. The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP). The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes, represented by 36 phyla. RESULTS:Truncation to 100 nucleotides (nt) downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87 (39.7%) of the 219 sequences, compared with misclassification of only 29 (13.2%) sequences with truncation to 350 nt. Among 350-nt sequence reads within various regions of the 16S rRNA gene, the reverse read of an amplicon generated using the 343F/798R primers had the least (8.2%) effect on classification. In comparison, truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0% of the 219 sequences. The 343F/798R amplicon accurately assigned 91.8% of the 219 sequences at the species level. Weighted by abundance of the species in the esophageal dataset, the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage (92%). Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species. Assuming that a typical polymerase chain reaction can tolerate 2 mismatches between a primer and a template, the modified 347F and 803R primers should be able to anneal 98% and 99.6% of all 16S rRNA genes in the RDP database. CONCLUSION:347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.

Project description:BackgroundLeymus chinensis (Trin.) Tzvel. is a high saline-alkaline tolerant forage grass genus of the tribe Gramineae family, which also plays an important role in protection of natural environment. To date, little is known about the saline-alkaline tolerance of L. chinensis on the molecular level. To better understand the molecular mechanism of saline-alkaline tolerance in L. chinensis, 454 pyrosequencing was used for the transcriptome study.ResultsWe used Roche-454 massive parallel pyrosequencing technology to sequence two different cDNA libraries that were built from the two samples of control and under saline-alkaline treatment (optimal stress concentration-Hoagland solution with 100 mM NaCl and 200 mM NaHCO(3)). A total of 363,734 reads in control group and 526,267 reads in treatment group with an average length of 489 bp and 493 bp were obtained, respectively. The reads were assembled into 104,105 unigenes with MIRA sequence assemable software, among which, 73,665 unigenes were in control group, 88,016 unigenes in treatment group and 57,576 unigenes in both groups. According to the comparative expression analysis between the two groups with the threshold of "log2 Ratio ?1", there were 36,497 up-regulated unegenes and 18,218 down-regulated unigenes predicted to be the differentially expressed genes. After gene annotation and pathway enrichment analysis, most of them were involved in stress and tolerant function, signal transduction, energy production and conversion, and inorganic ion transport. Furthermore, 16 of these differentially expressed genes were selected for real-time PCR validation, and they were successfully confirmed with the results of 454 pyrosequencing.ConclusionsThis work is the first time to study the transcriptome of L. chinensis under saline-alkaline treatment based on the 454-FLX massively parallel DNA sequencing platform. It also deepened studies on molecular mechanisms of saline-alkaline in L. chinensis, and constituted a database for future studies.

Project description:BackgroundThe Black Sea is the largest brackish water body in the world, although it is connected to the Mediterranean Sea and presents an upper water layer similar to some regions of the former, albeit with lower salinity and temperature. Despite its well-known hydrology and physicochemical features, this enormous water mass remains poorly studied at the microbial genomics level.ResultsWe have sampled its different water masses and analyzed the microbiome by shotgun and genome-resolved metagenomics, generating a large number of metagenome-assembled genomes (MAGs) from them. We found various similarities with previously described Black Sea metagenomic datasets, that show remarkable stability in its microbiome. Our datasets are also comparable to other marine anoxic water columns like the Cariaco Basin. The oxic zone resembles to standard marine (e.g. Mediterranean) photic zones, with Cyanobacteria (Synechococcus but a conspicuously absent Prochlorococcus), and photoheterotrophs domination (largely again with marine relatives). The chemocline presents very different characteristics from the oxic surface with many examples of chemolithotrophic metabolism (Thioglobus) and facultatively anaerobic microbes. The euxinic anaerobic zone presents, as expected, features in common with the bottom of meromictic lakes with a massive dominance of sulfate reduction as energy-generating metabolism, a few (but detectable) methanogenesis marker genes, and a large number of "dark matter" streamlined genomes of largely unpredictable ecology.ConclusionsThe Black Sea oxic zone presents many similarities to the global ocean while the redoxcline and euxinic water masses have similarities to other similar aquatic environments of marine (Cariaco Basin or other Black Sea regions) or freshwater (meromictic monimolimnion strata) origin. The MAG collection represents very well the different types of metabolisms expected in this kind of environment. We are adding critical information about this unique and important ecosystem and its microbiome.

Dataset Information

Gene-centric metagenomics analysis of feline intestinal microbiome using 454 junior pyrosequencing.

Publications

Gene-centric metagenomics analysis of feline intestinal microbiome using 454 junior pyrosequencing.

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