Assessment of Microbial Richness in Pelagic Sediment of Andaman Sea by Bacterial Tag Encoded FLX Titanium Amplicon Pyrosequencing (bTEFAP).
Ontology highlight
ABSTRACT:
SUBMITTER: Sundarakrishnan B
PROVIDER: S-EPMC3516664 | biostudies-literature | 2012 Dec
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Bacterial diversity and archaeal diversity in metagenome of the Lonar soda lake sediment were assessed by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). Metagenome comprised 5093 sequences with 2,531,282 bp and 53 ± 2% G + C content. Metagenome sequence data are available at NCBI under the Bioproject database with accession no. PRJNA218849. Metagenome sequence represented the presence of 83.1% bacterial and 10.5% archaeal origin. A total of 14 different bacteria demonstrating 57 species were recorded with dominating species like Coxiella burnetii (17%), Fibrobacter intestinalis (12%) and Candidatus Cloacamonas acidaminovorans (11%). Occurrence of two archaeal phyla representing 24 species, among them Methanosaeta harundinacea (35%), Methanoculleus chikugoensis (12%) and Methanolinea tarda (11%) were dominating species. Significant presence of 11% sequences as an unclassified indicated the possibilities for unknown novel prokaryotes from the metagenome.
| S-EPMC4535755 | biostudies-literature
Project description:BACKGROUND: Approximately 1 out of every 100 individuals has some form of venous insufficiency, which can lead to chronic venous disease and Venous Leg Ulcer (VLU). There are known underlying pathologies which contribute to the chronic nature of VLU including biofilm phenotype infections. RESULTS: Using pyrosequencing based approaches we evaluated VLU to characterize their microbial ecology. Results show that VLU infections are polymicrobial with no single bacterium colonizing the wounds. The most ubiquitous and predominant organisms include a previously uncharacterized bacteroidales, various anaerobes, Staphylococcus, Corynebacterium, and Serratia. Topological analysis of VLU show some notable differences in bacterial populations across the surface of the wounds highlighting the importance of sampling techniques during diagnostics. Metagenomics provide a preliminary indication that there may be protozoa, fungi and possibly an undescribed virus associated with these wounds. CONCLUSION: The polymicrobial nature of VLU and previous research on diabetic foot ulcers and surgical site infections suggest that the future of therapy for such wounds lies in the core of the logical and proven multiple concurrent strategy approach, which has been termed "biofilm-based wound care" and the use of individualized therapeutics rather than in a single treatment modality.
| S-EPMC2773781 | biostudies-literature
Project description:BackgroundThe microbiota of an animal's intestinal tract plays important roles in the animal's overall health, productivity and well-being. There is still a scarcity of information on the microbial diversity in the gut of livestock species such as cattle. The primary reason for this lack of data relates to the expense of methods needed to generate such data. Here we have utilized a bacterial tag-encoded FLX 16s rDNA amplicon pyrosequencing (bTEFAP) approach that is able to perform diversity analyses of gastrointestinal populations. bTEFAP is relatively inexpensive in terms of both time and labor due to the implementation of a novel tag priming method and an efficient bioinformatics pipeline. We have evaluated the microbiome from the feces of 20 commercial, lactating dairy cows.ResultsUbiquitous bacteria detected from the cattle feces included Clostridium, Bacteroides, Porpyhyromonas, Ruminococcus, Alistipes, Lachnospiraceae, Prevotella, Lachnospira, Enterococcus, Oscillospira, Cytophage, Anaerotruncus, and Acidaminococcus spp. Foodborne pathogenic bacteria were detected in several of the cattle, a total of 4 cows were found to be positive for Salmonella spp (tentative enterica) and 6 cows were positive for Campylobacter spp. (tentative lanienae).ConclusionUsing bTEFAP we have examined the microbiota in the feces of cattle. As these methods continue to mature we will better understand the ecology of the major populations of bacteria the lower intestinal tract. This in turn will allow for a better understanding of ways in which the intestinal microbiome contributes to animal health, productivity and wellbeing.
| S-EPMC2515157 | biostudies-literature
Project description:BackgroundThe rapid evolution of 454 GS-FLX sequencing technology has not been accompanied by a reassessment of the quality and accuracy of the sequences obtained. Current strategies for decision-making and error-correction are based on an initial analysis by Huse et al. in 2007, for the older GS20 system based on experimental sequences. We analyze here the quality of 454 sequencing data and identify factors playing a role in sequencing error, through the use of an extensive dataset for Roche control DNA fragments.ResultsWe obtained a mean error rate for 454 sequences of 1.07%. More importantly, the error rate is not randomly distributed; it occasionally rose to more than 50% in certain positions, and its distribution was linked to several experimental variables. The main factors related to error are the presence of homopolymers, position in the sequence, size of the sequence and spatial localization in PT plates for insertion and deletion errors. These factors can be described by considering seven variables. No single variable can account for the error rate distribution, but most of the variation is explained by the combination of all seven variables.ConclusionsThe pattern identified here calls for the use of internal controls and error-correcting base callers, to correct for errors, when available (e.g. when sequencing amplicons). For shotgun libraries, the use of both sequencing primers and deep coverage, combined with the use of random sequencing primer sites should partly compensate for even high error rates, although it may prove more difficult than previous thought to distinguish between low-frequency alleles and errors.
| S-EPMC3116506 | biostudies-literature
Project description:Understanding microbial populations in hospital environments is crucial for improving human health. Hospital-acquired infections are an increasing problem in intensive care units (ICU). In this work we present an exploration of bacterial diversity at inanimate surfaces of the ICU wards of the University Hospital A Coruña (Spain), as an example of confined hospital environment subjected to selective pressure, taking the entrance hall of the hospital, an open and crowded environment, as reference. Surface swab samples were collected from both locations and recovered DNA used as template to amplify a hypervariable region of the bacterial 16S rRNA gene. Sequencing of the amplicons was performed at the Roche 454 Sequencing Center using GS-FLX Titanium procedures. Reads were pre-processed and clustered into OTUs (operational taxonomic units), which were further classified. A total of 16 canonical bacterial phyla were detected in both locations. Members of the phyla Firmicutes (mainly Staphylococcus and Streptococcus) and Actinobacteria (mainly Micrococcaceae, Corynebacteriaceae and Brevibacteriaceae) were over-represented in the ICU with respect to the Hall. The phyllum Proteobacteria was also well represented in the ICU, mainly by members of the families Enterobacteriaceae, Methylobacteriaceae and Sphingomonadaceae. In the Hall sample, the phyla Proteobacteria, Bacteroidetes, Deinococcus-Thermus and Cyanobacteria were over-represented with respect to the ICU. Over-representation of Proteobacteria was mainly due to the high abundance of Enterobacteriaceae members. The presented results demonstrate that bacterial diversity differs at the ICU and entrance hall locations. Reduced diversity detected at ICU, relative to the entrance hall, can be explained by its confined character and by the existence of antimicrobial selective pressure. This is the first study using deep sequencing techniques made in hospital wards showing substantial hospital microbial diversity.
| S-EPMC3430676 | biostudies-literature
Project description:Contaminated aquifer (Dusseldorf-Flinger, Germany) templates extracted from 5 sediment depths ranging between 6.4 and 8.4 m below ground and over 3 years of sampling were amplified for amplicon pyrosequencing using the primers Ba27f (5’-aga gtt tga tcm tgg ctc ag-3’) and Ba519r (5’- tat tac cgc ggc kgc tg-3’), extended as amplicon fusion primers with respective primer A or B adapters, key sequence and multiplex identifiers (MID) as recommended by 454/Roche. Amplicons were purified and pooled as specified by the manufacturer. Emulsion PCR (emPCR), purification of DNA-enriched beads and sequencing run were performed following protocols and using a 2nd generation pyrosequencer (454 GS FLX Titanium, Roche) as recommended by the developer. Quality filtering of the pyrosequencing reads was performed using the automatic amplicon pipeline of the GS Run Processor (Roche), with a slight modification concerning the valley filter (vfScanAllFlows false instead of TiOnly) to extract the sequences. Demultiplexed raw reads were furhter trimmed for quality and lenght (>250 bp).
2012-11-11 | GSE25345 | GEO
Project description:BackgroundMicrosatellites are markers of choice in population genetics and genomics, as they provide useful insight into patterns and processes as diverse as genome evolutionary dynamics and demographic processes. The acquisition of microsatellites through multiplex-enriched libraries and 454 GS-FLX Titanium pyrosequencing is a promising new tool for the isolation of new markers in unknown genomes. This approach can also be used to evaluate the extent to which microsatellite-enriched libraries are representative of the genome from which they were isolated. In this study, we deciphered potential discrepancies in microsatellite content recovery for two reference genomes (Apis mellifera and Danio rerio), selected on the basis of their extreme heterogeneity in terms of the proportions and distributions of microsatellites on chromosomes.ResultsThe A. mellifera genome, in particular, was found to be highly heterogeneous, due to extremely high rates of recombination, with hotspots, but the only bias consistently introduced into pyrosequenced multiplex-enriched libraries concerned sequence length, with the overrepresentation of sequences 160 to 320 bp in length. Other deviations from expected proportions or distributions of motifs on chromosomes were observed, but the significance and intensity of these deviations was mostly limited. Furthermore, no consistent adverse competition between multiplexed probes was observed during the motif enrichment phase.ConclusionsThis approach therefore appears to be a promising strategy for improving the development of microsatellites, as it introduces no major bias in terms of the proportions and distribution of microsatellites.
| S-EPMC3091709 | biostudies-literature
Project description:Contaminated aquifer (Dusseldorf-Flinger, Germany) templates extracted from 5 sediment depths ranging between 6.4 and 8.4 m below ground and over 3 years of sampling were amplified for amplicon pyrosequencing using the primers Ba27f (5’-aga gtt tga tcm tgg ctc ag-3’) and Ba519r (5’- tat tac cgc ggc kgc tg-3’), extended as amplicon fusion primers with respective primer A or B adapters, key sequence and multiplex identifiers (MID) as recommended by 454/Roche. Amplicons were purified and pooled as specified by the manufacturer. Emulsion PCR (emPCR), purification of DNA-enriched beads and sequencing run were performed following protocols and using a 2nd generation pyrosequencer (454 GS FLX Titanium, Roche) as recommended by the developer. Quality filtering of the pyrosequencing reads was performed using the automatic amplicon pipeline of the GS Run Processor (Roche), with a slight modification concerning the valley filter (vfScanAllFlows false instead of TiOnly) to extract the sequences. Demultiplexed raw reads were furhter trimmed for quality and lenght (>250 bp). 15 samples examined in total from important plume zones of the aquifer sampled in Feb. 2006, Sep. 2008 and Jun. 2009 (5 every year of sampling).
2012-11-11 | E-GEOD-25345 | biostudies-arrayexpress
Project description:BackgroundLycoris aurea, also called Golden Magic Lily, is an ornamentally and medicinally important species of the Amaryllidaceae family. To date, the sequencing of its whole genome is unavailable as a non-model organism. Transcriptomic information is also scarce for this species. In this study, we performed de novo transcriptome sequencing to produce the first comprehensive expressed sequence tag (EST) dataset for L. aurea using high-throughput sequencing technology.Methodology and principal findingsTotal RNA was isolated from leaves with sodium nitroprusside (SNP), salicylic acid (SA), or methyl jasmonate (MeJA) treatment, stems, and flowers at the bud, blooming, and wilting stages. Equal quantities of RNA from each tissue and stage were pooled to construct a cDNA library. Using 454 pyrosequencing technology, a total of 937,990 high quality reads (308.63 Mb) with an average read length of 329 bp were generated. Clustering and assembly of these reads produced a non-redundant set of 141,111 unique sequences, comprising 24,604 contigs and 116,507 singletons. All of the unique sequences were involved in the biological process, cellular component and molecular function categories by GO analysis. Potential genes and their functions were predicted by KEGG pathway mapping and COG analysis. Based on our sequence analysis and published literatures, many putative genes involved in Amaryllidaceae alkaloids synthesis, including PAL, TYDC OMT, NMT, P450, and other potentially important candidate genes, were identified for the first time in this Lycoris. Furthermore, 6,386 SSRs and 18,107 high-confidence SNPs were identified in this EST dataset.ConclusionsThe transcriptome provides an invaluable new data for a functional genomics resource and future biological research in L. aurea. The molecular markers identified in this study will provide a material basis for future genetic linkage and quantitative trait loci analyses, and will provide useful information for functional genomic research in future.
| S-EPMC3621892 | biostudies-literature
Project description:BACKGROUND: Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. RESULTS: Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus. CONCLUSIONS: This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts are expected as our understanding of its microbiota expands. Increased awareness of the role R. microplus can play in the transmission of pathogenic bacteria will enhance our ability to mitigate its economic impact on animal agriculture globally. This recognition should be included as part of analyses to assess the risk for re-invasion of areas like the United States of America where R. microplus was eradicated.
| S-EPMC3025832 | biostudies-other