Pyrosequencing Research Papers - Academia.edu (original) (raw)

We aimed to determine the bacterial diversity of different oral micro-niches and to assess whether saliva and plaque samples are representative of oral microbial composition. We took minute samples from each surface of the individual... more

We aimed to determine the bacterial diversity of different oral micro-niches and to assess whether saliva and plaque samples are representative of oral microbial composition. We took minute samples from each surface of the individual teeth and gingival crevices of two healthy volunteers (112 samples per donor), as well as samples from the tongue dorsum and non-stimulated and stimulated saliva. DNA was extracted from 67 selected samples of each donor, and the 16S rRNA gene was amplified by PCR and pyrosequenced to obtain, on average, over 2,700 reads per sample, which were taxonomically assigned to obtain a geographic map of bacterial diversity at each tooth and sulcus location. Analysis of the data shows considerable differences in bacterial composition between teeth at different intra-oral locations and between surfaces of the same tooth. The most pronounced differences were observed in incisors and canines, where genera like Streptococcus were found at 40% to 70% on the vestibular surfaces but were almost absent on the lingual sides. Saliva samples, especially non-stimulated saliva, were not representative of supra-and subgingival plaque in the two individuals tested. We suggest that more precise sampling is required for the proper determination of oral microbial composition and to relate that diversity to epidemiological, clinical, and etiological parameters.

The efficiency of the FT-IR technique for studying the inter- and intra biodiversity of cultivable non-Saccharomyces yeasts (NS) present indifferentmust sampleswas examined. Infirst, the capacity of the technique FT-IRto study the global... more

The efficiency of the FT-IR technique for studying the inter- and intra biodiversity of cultivable non-Saccharomyces yeasts (NS) present indifferentmust sampleswas examined. Infirst, the capacity of the technique FT-IRto study the global diversity of a given sample was compared to the pyrosequencing method, used as a reference technique. Seven different genera (Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Issatchenkia, Metschnikowia and Pichia)wereidentified by FT-IR and also by pyrosequencing. Thirty-eight other genera were identified by pyrose- quencing, but together they represented less than 6% of the average total population of 6 musts. Among the species identified, some of thempresentorganoleptic potentials inwinemaking,particularly Starmerella bacillaris (synonym Candida zemplinina). So in a second time, we evaluated the capacity of the FT-IR technique to discriminate the isolates of this species because few techniques were able to study intraspecific NS yeast biodiversity. The results obtained were validated by using a classic method as ITS sequencing. Biodiversity at strain level was high: 19 different strains were identified from 58 isolates. So, FT-IR spectroscopy seems to be an accurate and reliable method for identifying major genera present in themusts. The two biggest advantages of the FT-IR are the capacity to characterize intraspecific biodiversity of non-Saccharomyces yeasts and the possibility to discriminate a lot of strains.

One sentence summary: This study gives insight into the bacterial community composition related to iron-cycling in suboxic marine sediments based on 16S rRNA gene pyrosequencing. ABSTRACT To gain insight into the bacterial communities... more

One sentence summary: This study gives insight into the bacterial community composition related to iron-cycling in suboxic marine sediments based on 16S rRNA gene pyrosequencing. ABSTRACT To gain insight into the bacterial communities involved in iron-(Fe) cycling under marine conditions, we analysed sediments with Fe-contents (0.5–1.5 wt %) from the suboxic zone at a marine site in the Skagerrak (SK) and a brackish site in the Bothnian Bay (BB) using 16S rRNA gene pyrosequencing. Several bacterial families, including Desulfobulbaceae, Desulfuromonadaceae and Pelobacteraceae and genera, including Desulfobacter and Geobacter, known to reduce Fe were detected and showed highest abundance near the Fe(III)/Fe(II) redox boundary. Additional genera with microorganisms capable of coupling fermentation to Fe-reduction, including Clostridium and Bacillus, were observed. Also, the Fe-oxidizing families Mariprofundaceae and Gallionellaceae occurred at the SK and BB sites, respectively, supporting Fe-cycling. In contrast, the sulphate (SO 4 2−) reducing bacteria Desulfococcus and Desulfobacterium were more abundant at greater depths concurring with a decrease in Fe-reducing activity. The communities revealed by pyrosequencing, thus, match the redox stratification indicated by the geochemistry, with the known Fe-reducers coinciding with the zone of Fe-reduction. Not the intensely studied model organisms, such as Geobacter spp., but rather versatile microorganisms, including sulphate reducers and possibly unknown groups appear to be important for Fe-reduction in these marine suboxic sediments.

Olive ridley turtles, although widely distributed globally and in Indian coastal waters, have undergone declines in recent years due to anthropogenic factors, particularly fishery-related mortality. Assessment of genetic variability in... more

Olive ridley turtles, although widely distributed globally and in Indian coastal waters, have undergone declines in recent years due to anthropogenic factors, particularly fishery-related mortality. Assessment of genetic variability in existing populations is critical to the development of effective conservation strategies. Here we describe the development of six highly polymorphic microsatellite loci from a simple sequence repeat-enriched genomic DNA library of olive ridley turtle. Characterization of five of these loci using 83 individual olive ridley turtles revealed eight to 24 alleles per locus, high observed and expected heterozygosity values and broad cross-species amplifications. The sixth microsatellite was found to be monomorphic in the olive ridley samples but was polymorphic in two related marine turtle species. These microsatellites thus provide efficient genetic markers to understand the population structure, phylogeography and species relationships of olive ridley and other marine turtle species.

Objectives: Genetic variation in genes encoding for drug-metabolizing enzymes, drug targets and signaling pathways have proven to contribute significantly to differences in drug response. Pharmacogenetics is now expanding from clinical... more

Objectives: Genetic variation in genes encoding for drug-metabolizing enzymes, drug targets and signaling pathways have proven to contribute significantly to differences in drug response. Pharmacogenetics is now expanding from clinical pharmacological research to its application in clinical practice. Genotyping of patients in a routine clinical setting requires robust and reliable genotyping methods. Materials & methods: A survey of pharmacogenetic association studies for quality control samples published from 2005 to 2007 in the two most prominent pharmacogenetic journals, and development of plasmid-derived external controls. Results: Surveying journals revealed that only a minority of papers report the use of quality controls, and no standard procedures are applied. We established 12 plasmid-derived external controls and applied these in pharmacogenetic testing. Conclusion: There still is a need for quality control materials, especially for application in pharmacogenetic testing. ...

In the present study we assessed the composition of Bacteria in four biotopes, namely sediment, seawater and two sponge species (Stylissa massa and Xestospongia testudinaria) at four different reef sites in a coral reef ecosystem in west... more

In the present study we assessed the composition of Bacteria in four biotopes, namely sediment, seawater and two sponge species (Stylissa massa and Xestospongia testudinaria) at four different reef sites in a coral reef ecosystem in west Java, Indonesia. In addition to this, we used a predictive metagenomic approach to predict to what extent nitrogen metabolic pathways differ among bacterial communities in different biotopes. We observed marked differences in bacterial composition of the most abundant bacterial phyla, classes and orders among sponge species, water and sediment. Proteobacteria were by far the most abundant phylum in terms of both sequences and OTUs. Predicted counts for genes associated with the nitrogen metabolism suggest that several genes involved in the nitrogen cycle were enriched in sponge samples, including nosZ, nifD, nirK, NorB and nrfA genes Our data show that a combined barcoded pyrosequencing and predictive metagenomic- approach can provide novel insights...

We compared the gut prokaryotic communities in wild, organically-, and conventionally reared sea bream (Sparus aurata) individuals. Gut microbial communities were identified using tag pyrosequencing of the 16S rRNA genes. There were... more

We compared the gut prokaryotic communities in wild, organically-, and conventionally reared sea bream (Sparus aurata) individuals. Gut microbial communities were identified using tag pyrosequencing of the 16S rRNA genes. There were distinct prokaryotic communities in the three different fish nutritional treatments, with the bacteria dominating over the Archaea. Most of the Bacteria belonged to the Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. The number of bacterial operational taxonomic units (OTUs) was reduced from the wild to the conventionally reared fish, implying a response of the gut microorganisms to the supplied food and possibly alterations in food assimilation. The dominant bacterial OTU in all examined fish was closely related to the genus Diaphorobacter. This is the first time that a member of the β-Proteobacteria, which dominate in freshwaters, are so important in a marine fish gut. In total the majority of the few Archaea OTUs found, were related to ...

Archaea and bacteria are important drivers for nutrient transformations in soils and catalyse the production and consumption of important greenhouse gases. In this study, we investigate changes in archaeal and bacterial communities of... more

Archaea and bacteria are important drivers for nutrient transformations in soils and catalyse
the production and consumption of important greenhouse gases. In this study, we investigate
changes in archaeal and bacterial communities of four Czech grassland soils affected
by outdoor cattle husbandry. Two show short-term (3 years; STI) and long-term impact
(17 years; LTI), one is regenerating from cattle impact (REG) and a control is unaffected by
cattle (CON). Cattle manure (CMN), the source of allochthonous microbes, was collected
from the same area. We used pyrosequencing of 16S rRNA genes to assess the composition
of archaeal and bacterial communities in each soil type and CMN. Both short- and longterm
cattle impact negatively altered archaeal and bacterial diversity, leading to increase of
homogenization of microbial communities in overwintering soils over time. Moreover, strong
shifts in the prokaryotic communities were observed in response to cattle overwintering,
with the greatest impact on archaea. Oligotrophic and acidophilic microorganisms (e.g.
Thaumarchaeota, Acidobacteria, and α-Proteobacteria) dominated in CON and expressed
strong negative response to increased pH, total C and N. Whereas copiotrophic and alkalophilic
microbes (e.g. methanogenic Euryarchaeota, Firmicutes, Chloroflexi, Actinobacteria,
and Bacteroidetes) were common in LTI showing opposite trends. Crenarchaeota were
also found in LTI, though their trophic interactions remain cryptic. Firmicutes, Bacteroidetes,
Methanobacteriaceae, and Methanomicrobiaceae indicated the introduction and establishment
of faecal microbes into the impacted soils, while Chloroflexi and Methanosarcinaceae
suggested increased abundance of soil-borne microbes under altered environmental conditions.
The observed changes in prokaryotic community composition may have driven corresponding
changes in soil functioning.

The jungle crow (Corvus macrorhynchos) belongs to the order Passeriformes of bird species and is important for avian ecological and evolutionary genetics studies. However, there is limited information on the transcriptome data of this... more

The jungle crow (Corvus macrorhynchos) belongs to the order Passeriformes of bird species and is important for avian ecological and evolutionary genetics studies. However, there is limited information on the transcriptome data of this species. In the present study, we report the characterization of the lung transcriptome of the jungle crow using GS FLX Titanium XLR70. Altogether, 1 510 303 high-quality sequence reads with 581 198 230 bases was de novo assembled into 22 169 isotigs (isotig represents an individual transcript) and 784 009 singletons. Using these isotigs and 581 681 length-filtered (greater than 300 bp) singletons, 20 010 unique protein-coding genes were identified by BLASTx comparison against a nonredundant (nr) protein sequence database. Comparative analysis revealed that 46 604 (70.29%) and 51 642 (72.48%) of the assembled transcripts have significant similarity to zebra finch and chicken RefSeq proteins, respectively. As determined by GO annotation and KEGG pathway...

We present the almost complete (16,007 bp) mitochondrial genome of a Colossendeis megalonyx specimen from the Southern Ocean and discuss gene order and tRNA structure in a comparative phylogenetic context. Our data suggest a basal... more

We present the almost complete (16,007 bp) mitochondrial genome of a Colossendeis megalonyx specimen from the Southern Ocean and discuss gene order and tRNA structure in a comparative phylogenetic context. Our data suggest a basal position of the colossendeid lineage ...

Background and AimIn irritable bowel syndrome (IBS), the gut microbiota may be altered. Probiotic bacteria appear to be therapeutically effective. We characterized the mucosa-associated microbiota, and determined the clinical and... more

Background and AimIn irritable bowel syndrome (IBS), the gut microbiota may be altered. Probiotic bacteria appear to be therapeutically effective. We characterized the mucosa-associated microbiota, and determined the clinical and microbiological effects of orally administered probiotic bacteria, in patients with IBS.In irritable bowel syndrome (IBS), the gut microbiota may be altered. Probiotic bacteria appear to be therapeutically effective. We characterized the mucosa-associated microbiota, and determined the clinical and microbiological effects of orally administered probiotic bacteria, in patients with IBS.MethodsMucosal microbiota from rectal biopsies of IBS patients and controls were assessed on the V1 and V2 variable regions of the 16S ribosomal RNA gene amplified using 454 pyrosequencing. Clinical symptoms and changes in mucosal microbiota were assessed in IBS patients before and after 4 weeks of treatment with probiotic mix VSL#3.Mucosal microbiota from rectal biopsies of IBS patients and controls were assessed on the V1 and V2 variable regions of the 16S ribosomal RNA gene amplified using 454 pyrosequencing. Clinical symptoms and changes in mucosal microbiota were assessed in IBS patients before and after 4 weeks of treatment with probiotic mix VSL#3.ResultsTen IBS subjects (eight female; mean age 46 years) were included. At week 4 of probiotic therapy, six patients showed symptom improvement on global symptom assessment compared with baseline (P = 0.031). Before therapy, intestinal microbiota of IBS subjects differed significantly from that of healthy controls, with less diversity and evenness than controls (n = 9; P < 0.05), increased abundance of Bacteroidetes (P = 0.014) and Synegitestes (P = 0.017), and reduced abundance of Actinobacteria (P = 0.004). The classes Flavobacteria (P = 0.028) and Epsilonproteobacteria (P = 0.017) were less enriched in IBS. Abundance differences were largely consistent from the phylum to genus level. Probiotic treatment in IBS patients was associated with a significant reduction of the genus Bacteroides (all taxonomy levels; P < 0.05) to levels similar to that of controls.Ten IBS subjects (eight female; mean age 46 years) were included. At week 4 of probiotic therapy, six patients showed symptom improvement on global symptom assessment compared with baseline (P = 0.031). Before therapy, intestinal microbiota of IBS subjects differed significantly from that of healthy controls, with less diversity and evenness than controls (n = 9; P < 0.05), increased abundance of Bacteroidetes (P = 0.014) and Synegitestes (P = 0.017), and reduced abundance of Actinobacteria (P = 0.004). The classes Flavobacteria (P = 0.028) and Epsilonproteobacteria (P = 0.017) were less enriched in IBS. Abundance differences were largely consistent from the phylum to genus level. Probiotic treatment in IBS patients was associated with a significant reduction of the genus Bacteroides (all taxonomy levels; P < 0.05) to levels similar to that of controls.ConclusionIn this pilot study, global and deep molecular analysis demonstrates an altered mucosal microbiota composition in IBS. Probiotic leads to detectable changes in the microbiota. These effects of probiotic bacteria may contribute to their therapeutic benefit.In this pilot study, global and deep molecular analysis demonstrates an altered mucosal microbiota composition in IBS. Probiotic leads to detectable changes in the microbiota. These effects of probiotic bacteria may contribute to their therapeutic benefit.

The Western Australian rock lobster fishery has been both a highly productive and sustainable fishery. However, a recent dramatic and unexplained decline in post-larval recruitment threatens this sustainability. Our lack of knowledge of... more

The Western Australian rock lobster fishery has been both a highly productive and sustainable fishery. However, a recent dramatic and unexplained decline in post-larval recruitment threatens this sustainability. Our lack of knowledge of key processes in lobster larval ecology, such as their position in the food web, limits our ability to determine what underpins this decline. The present study uses a high-throughput amplicon sequencing approach on DNA obtained from the hepatopancreas of larvae to discover significant prey items. Two short regions of the 18S rRNA gene were amplified under the presence of lobster specific PNA to prevent lobster amplification and to improve prey amplification. In the resulting sequences either little prey was recovered, indicating that the larval gut was empty, or there was a high number of reads originating from multiple zooplankton taxa. The most abundant reads included colonial Radiolaria, Thaliacea, Actinopterygii, Hydrozoa and Sagittoidea, which supports the hypothesis that the larvae feed on multiple groups of mostly transparent gelatinous zooplankton. This hypothesis has prevailed as it has been tentatively inferred from the physiology of larvae, captive feeding trials and co-occurrence in situ. However, these prey have not been observed in the larval gut as traditional microscopic techniques cannot discern between transparent and gelatinous prey items in the gut. High-throughput amplicon sequencing of gut DNA has enabled us to classify these otherwise undetectable prey. The dominance of the colonial radiolarians among the gut contents is intriguing in that this group has been historically difficult to quantify in the water column, which may explain why they have not been connected to larval diet previously. Our results indicate that a PCR based technique is a very successful approach to identify the most abundant taxa in the natural diet of lobster larvae.

A good definition of commensal microflora and an understanding of its relation to health are essential in preventing and combating disease. We hypothesized that the species richness of human oral microflora is underestimated. Saliva and... more

A good definition of commensal microflora and an understanding of its relation to health are essential in preventing and combating disease. We hypothesized that the species richness of human oral microflora is underestimated. Saliva and supragingival plaque were sampled from 71 and 98 healthy adults, respectively. Amplicons from the V6 hypervariable region of the small-subunit ribosomal RNA gene were generated by PCR, pooled into saliva and plaque pools, and sequenced by means of the Genome Sequencer 20 system at 454 Life Sciences. Data were evaluated by taxonomic and rarefaction analyses. The 197,600 sequences generated yielded about 29,000 unique sequences, representing 22 taxonomic phyla. Grouping the sequences in operational taxonomic units (6%) yielded 3621 and 6888 species-level phylotypes in saliva and plaque, respectively. This work gives a radically new insight into the diversity of human oral microflora, which, with an estimated number of 19,000 phylotypes, is considerably...

The use of molecular markers has improved the ecological and evolutionary research in the case of clonal reproduction species, allowing the identification of boundaries among clonal modules (ramets), genetic individuals (genets), and... more

The use of molecular markers has improved the ecological and evolutionary research in the case of clonal reproduction species, allowing the identification of boundaries among clonal modules (ramets), genetic individuals (genets), and populations. Microsatellite markers were developed for Stenocereus gummosus, a columnar cactus with both sexual and clonal reproduction which is distributed in the Sonoran Desert, Mexico. 454-Pyrosequencing reads were analyzed to detect microsatellite markers. Forty primer pairs were screened to look for polymorphism. Nine loci were genotyped for two S. gummosus localities. Sampling strategy was intended to avoid collecting a genetic individual more than once, considering that clonal architecture for this cactus was previously deduced as clumped. Polymorphic loci exhibited low allele count, ranging from 2 - 7 (mean of 3.78 ± 0.62 SE); mean heterozygosity values were 0.221 and 0.234 HO and 0.408 and 0.306 HE, with FIS of 0.383 and 0.299, for peninsular and continental localities respectively. Unexpectedly, some multilocus genotypes were found repeated within locality, which were assumed as clones since data was evaluated as sufficient (clonal richness R of 0.966 and 0.897). These results were different from those previously reported: the distribution of clones might as well be intermingled, having a minimum ramet dispersion distance of 30 m. This characteristic was also consistent with the recent colonization proposed for this and other arid lands plants. A wider genetic neighborhood, due to clone dispersion might affect diversity indexes while increasing the chance of geitonogamy and mating among relatives. The markers isolation and its population characterization allowed addressing new questions about S. gummosus ecology, clonal reproduction and reproductive biology.

HIV-1 infection or systemic lupus erythematosus (SLE) disrupt B cell homeostasis, reduce memory B cells, and impair function of IgG and IgM antibodies. To determine how disturbances in B cell populations producing polyclonal antibodies... more

HIV-1 infection or systemic lupus erythematosus (SLE) disrupt B cell homeostasis, reduce memory B cells, and impair function of IgG and IgM antibodies. To determine how disturbances in B cell populations producing polyclonal antibodies relate to the IgM repertoire, the IgM transcriptome in health and disease was explored at the complementarity determining region 3 (CDRH3) sequence level. 454-deep pyrosequencing in combination with a novel analysis pipeline was applied to define populations of IGHM CDRH3 sequences based on absence or presence of somatic hypermutations (SHM) in peripheral blood B cells. HIV or SLE subjects have reduced biodiversity within their IGHM transcriptome compared to healthy subjects, mainly due to a significant decrease in the number of unique combinations of alleles, although recombination machinery was intact. While major differences between sequences without or with SHM occurred among all groups, IGHD and IGHJ allele use, CDRH3 length distribution, or gene...