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51 results for “pyrosequencing”
Data from: Exploring Symbiodinium diversity and host specificity in Acropora corals from geographical extremes of Western Australia with 454 amplicon pyrosequencing
Scleractinian corals have demonstrated the ability to shuffle their endosymbiotic dinoflagellate communities (genus Symbiodinium) during periods of acute environmental stress. This has been proposed as a mechanism of acclimation, which would be increased by a diverse and flexible association with Symbiodinium. Conventional molecular techniques used to evaluate Symbiodinium diversity lack the sensitivity to capture accurate estimates of diversity and are unable to identify genetic lineages present at background levels below 10%. Next generation sequencing (NGS) offers a solution to this problem and can resolve microorganism diversity at much finer scales. Here we apply NGS to evaluate Symbiodinium diversity and host specificity in Acropora corals from contrasting regions of Western Australia. The application of 454 pyrosequencing allowed for detection of Symbiodinium operational taxonomic units (OTUs) occurring at frequencies as low as 0.001%, offering a 10 000-fold increase in sensitivity compared to traditional methods. All coral species from both regions were overwhelmingly dominated by a single clade C OTU (accounting for 98% of all recovered sequences). Only 8.5% of colonies associated with multiple clades (clades C and D, or C and G), suggesting a high level of symbiont specificity in Acropora assemblages in Western Australia. This contrasts with recent literature that have applied highly-sensitive molecular techniques and identified widespread flexibility in symbiont associations across a number of coral taxa in other geographic regions. While only 40% of the OTUs were shared between regions, the dominance of a single OTU resulted in no significant difference in Symbiodinium community structure, demonstrating that the coral-algal symbiosis can remain stable across more than 15° of latitude and a range of sea surface temperature profiles. This study validates the use of NGS platforms as tools for providing fine-scale estimates of Symbiodinium diversity and can offer critical insight into the flexibility of the coral-algal symbiosis.
Data from: Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage
Characterization and population genetic analysis of multilocus genes, such as those found in the major histocompatibility complex (MHC) is challenging in nonmodel vertebrates. The traditional method of extensive cloning and Sanger sequencing is costly and time-intensive and indirect methods of assessment often underestimate total variation. Here, we explored the suitability of 454 pyrosequencing for characterizing multilocus genes for use in population genetic studies. We compared two sample tagging protocols and two bioinformatic procedures for 454 sequencing through characterization of a 185-bp fragment of MHC DRB exon 2 in wolverines (Gulo gulo) and further compared the results with those from cloning and Sanger sequencing. We found 10 putative DRB alleles in the 88 individuals screened with between two and four alleles per individual, suggesting amplification of a duplicated DRB gene. In addition to the putative alleles, all individuals possessed an easily identifiable pseudogene. In our system, sequence variants with a frequency below 6% in an individual sample were usually artefacts. However, we found that sample preparation and data processing procedures can greatly affect variant frequencies in addition to the complexity of the multilocus system. Therefore, we recommend determining a per-amplicon-variant frequency threshold for each unique system. The extremely deep coverage obtained in our study (approximately 5000×) coupled with the semi-quantitative nature of pyrosequencing enabled us to assign all putative alleles to the two DRB loci, which is generally not possible using traditional methods. Our method of obtaining locus-specific MHC genotypes will enhance population genetic analyses and studies on disease susceptibility in nonmodel wildlife species.
Data from: Analysis of Australian fur seal diet by pyrosequencing prey DNA in faeces
DNA-based techniques have proven useful for defining trophic links in a variety of ecosystems and recently developed sequencing technologies provide new opportunities for dietary studies. We investigated the diet of Australian fur seals (Arctocephalus pusillus doriferus) by pyrosequencing prey DNA from faeces collected at three breeding colonies across the seals' range. DNA from 270 faecal samples was amplified with four polymerase chain reaction primer sets and a blocking primer was used to limit amplification of fur seal DNA. Pooled amplicons from each colony were sequenced using the Roche GS-FLX platform, generating > 20 000 sequences. Software was developed to sort and group similar sequences. A total of 54 bony fish, 4 cartilaginous fish and 4 cephalopods were identified based on the most taxonomically informative amplicons sequenced (mitochondrial 16S). The prevalence of sequences from redbait (Emmelichthys nitidus) and jack mackerel (Trachurus declivis) confirm the importance of these species in the seals' diet. A third fish species, blue mackerel (Scomber australasicus), may be a more important prey species than previously recognised. There were major differences in the proportions of prey DNA recovered in faeces from different colonies, probably reflecting differences in prey availability. Parallel hard-part analysis identified largely the same main prey species as did the DNA-based technique, but with lower species diversity and no remains from cartilaginous prey. The pyrosequencing approach presented significantly expands the capabilities of DNA-based methods of dietary analysis and is suitable for large-scale diet investigations on a broad range of animals.
Data from: Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi
Next-generation sequencing technologies have provided unprecedented insights into fungal diversity and ecology. However, intrinsic biases and insufficient quality control in next-generation methods can lead to difficult-to-detect errors in estimating fungal community richness, distributions, and composition. The aim of this study was to examine how tissue storage prior to DNA extraction, primer design, and various quality-control approaches commonly used in 454 amplicon pyrosequencing might influence ecological inferences in studies of endophytic and endolichenic fungi. We first contrast 454 data sets generated contemporaneously from subsets of the same plant and lichen tissues that were stored in CTAB buffer, dried in silica gel, or freshly frozen prior to DNA extraction. We show that storage in silica gel markedly limits the recovery of sequence data and yields a small fraction of the diversity observed by the other two methods. Using lichen mycobiont sequences as internal positive controls, we next show that despite careful filtering of raw reads and utilization of current best-practice OTU clustering methods, homopolymer errors in sequences representing rare taxa artificially increased estimates of richness ca. 15-fold in a model data set. Third, we show that inferences regarding endolichenic diversity can be improved by using a novel primer that reduces amplification of the mycobiont. Together, our results provide a rationale for selecting tissue treatment regimes prior to DNA extraction, demonstrate the efficacy of reducing mycobiont amplification in studies of the fungal microbiomes of lichen thalli, and highlight the difficulties in differentiating true information about fungal biodiversity from methodological artifacts.
Antiretroviral Resistance Detection by Ultrasensitive Pyrosequencing of the HIV-1 Genome and Virological Response to Antiretroviral Rescue Treatment
ClinicalTrials.gov study NCT01346878. IPD Sharing: Not stated. Countries: 1. Publications: 1.
Data from: Gastrointestinal microbiota of wild and inbred individuals of two house mouse subspecies assessed using high throughput parallel pyrosequencing
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Data from: Amplicon pyrosequencing reveals the soil microbial diversity associated with invasive Japanese barberry (Berberis thunbergii DC.)
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Data from: Comparative transcriptomics and gene expression in larval tiger salamander (Ambystoma tigrinum) gill and lung tissues as revealed by pyrosequencing
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Data from: Exploring Symbiodinium diversity and host specificity in Acropora corals from geographical extremes of Western Australia with 454 amplicon pyrosequencing
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Data from: Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi
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Data from: Analysis of Australian fur seal diet by pyrosequencing prey DNA in faeces
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Data from: Pyrosequencing of prey DNA in reptile faeces: analysis of earthworm consumption by slow worms
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Data from: Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage
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Data from: Diversity patterns of uncultured Haptophytes unravelled by pyrosequencing in Naples Bay
Haptophytes are a key phylum of marine protists, including ~300 described morphospecies and 80 morphogenera. We used 454 pyrosequencing on large subunit ribosomal DNA (LSU rDNA) fragments to assess the diversity from size-fractioned plankton samples collected in the Bay of Naples. One group-specific primer set targeting the LSU rDNA D1/D2 region was designed to amplify Haptophyte sequences from nucleic acid extracts (total DNA or RNA) of two size fractions (0.8–3 or 3–20 μm) and two sampling depths [subsurface, at 1 m, or deep chlorophyll maximum (DCM) at 23 m]. 454 reads were identified using a database covering the entire Haptophyta diversity currently sequenced. Our data set revealed several hundreds of Haptophyte clusters. However, most of these clusters could not be linked to taxonomically known sequences: considering OTUs97% (clusters build at a sequence identity level of 97%) on our global data set, less than 1% of the reads clustered with sequences from cultures, and less than 12% clustered with reference sequences obtained previously from cloning and Sanger sequencing of environmental samples. Thus, we highlighted a large uncharacterized environmental genetic diversity, which clearly shows that currently cultivated species poorly reflect the actual diversity present in the natural environment. Haptophyte community appeared to be significantly structured according to the depth. The highest diversity and evenness were obtained in samples from the DCM, and samples from the large size fraction (3–20 μm) taken at the DCM shared a lower proportion of common OTUs97% with the other samples. Reads from the species Chrysoculter romboideus were notably found at the DCM, while they could be detected at the subsurface. The highest proportion of totally unknown OTUs97% was collected at the DCM in the smallest size fraction (0.8–3 μm). Overall, this study emphasized several technical and theoretical barriers inherent to the exploration of the large and largely unknown diversity of unicellular eukaryotes.
Data from: De novo discovery and multiplexed amplification of microsatellite markers for black alder (Alnus glutinosa) and related species using SSR-enriched shotgun pyrosequencing.
Recent developments in sequencing technologies and bioinformatics analyses provide an unprecedented opportunity for cost and time effective high quality microsatellite marker discovery in non-model organisms for which no genomic information is available. Here, we use shotgun pyrosequencing of a microsatellite-enriched library to develop, for the first time, microsatellite markers for Alnus glutinosa, a keystone tree species of European riparian woodland communities. From a total of 17,855 short sequences, we identified 590 perfect microsatellites from which 392 had designed primers. A subset of 48 loci were tested for amplification, twelve of which were polymorphic in A. glutinosa. These twelve loci were successfully co-amplified in a single multiplex PCR experiment and validated for population genetics applications. In addition, ten and eight of these microsatellites were found to be transferable to the related A. incana and A. cordata species. The developed multiplex of 12 microsatellite markers therefore provides new opportunities for experimental evolutionary and forest genetics research in Alnus.
Data from: Analyzing reticulate relationships using CpDNA and pyrosequenced ITS1 as exemplified by Veronica subgen. Pseudolysimachium (Plantaginaceae)
Veronica subgen. Pseudolysimachium constitutes a group of about 28 species across northern Eurasia, many of them with considerable intraspecific morphological variation. This intraspecific variation may be due to large geographical distribution area, wide ecological amplitude, or widespread hybridization and polyploidization. Several recent studies using molecular data have shown that hybridization as an explanation for generating evolutionary novelties and high intraspecific variation may be more common than previously thought. Here we investigate the importance of hybridization in generating morphological variation and blurring species boundaries in V. subgen. Pseudolysimachium using analyses of cpDNA sequences from 139 individuals from 18 species and ten putative hybrids, and pyrosequenced ITS1 nrDNA sequences from 37 individuals from 16 species and four putative hybrids. In addition, we estimated ploidy levels for 42 individuals of ten species and five putative hybrids using flow cytometry. Analyses of cpDNA did not resolve phylogenetic structure (most of the species were polyphyletic). Our second approach, pyrosequencing of ITS1, generated up to nine different unique sequences per individual and phylogenetic analyses of the dataset resolved some basal nodes but, again, species were often non-monophyletic. The results are most compatible with a scenario of an East Asian origin and repeated spread across Pleistocene Eurasian steppes, known as important plant diversification center, with frequent interspecific hybridization. We compare the applicability of these molecular regions for resolving hybridizing species complexes and specifically address hypotheses of hybrid origins for several species within the subgenus. However, any population genetic, phylogeographic or other analysis of evolutionary questions in one species alone would be futile without considering introgression from related species.
Data from: Evaluation of two approaches to genotyping MHC class I in a passerine – CE-SSCP and 454 pyrosequencing
Genes of the highly dynamic major histocompatibility complex (MHC) are directly linked to individual fitness and are of high interest in evolutionary ecology and conservation genetics. Gene duplication and positive selection usually lead to high levels of polymorphism in the MHC region, making genotyping of MHC a challenging task. Here, we compare the performance of two methods for MHC class I genotyping in a passerine with highly duplicated MHC class I genes: capillary electrophoresis single strand conformation polymorphism (CE-SSCP) analysis and 454 GS FLX Titanium pyrosequencing. According to our findings, the number of MHC variants (called alleles for simplicity) detected by CE-SSCP is significantly lower than detected by 454. To resolve discrepancies between the two methods we cloned and Sanger sequenced a MHC class I amplicon for an individual with high number of alleles. We found a perfect congruence between cloning/Sanger sequencing results and 454. Thus, in case of multi-locus amplification, CE-SSCP considerably underestimates individual MHC diversity. However, numbers of alleles detected by both methods are significantly correlated, although the correlation is weak (r = 0.32). Thus, in systems with highly duplicated MHC, 454 provides more reliable information on individual diversity than CE-SSCP.
Data from: Amplicon pyrosequencing late Pleistocene permafrost: the removal of putative contaminant sequences and small-scale reproducibility
DNA sequencing of ancient permafrost samples can be used to reconstruct past plant, animal and bacterial communities. In this study, we assess the small-scale reproducibility of taxonomic composition obtained from sequencing four molecular markers (mitochondrial 12S ribosomal DNA (rDNA), prokaryote 16S rDNA, mitochondrial cox1 and chloroplast trnL intron) from two soil cores sampled 10 cm apart. In addition, sequenced control reactions were used to produce a contaminant library that was used to filter similar sequences from sample libraries. Contaminant filtering resulted in the removal of 1% of reads or 0.3% of operational taxonomic units. We found similar richness, overlap, abundance and taxonomic diversity from the 12S, 16S and trnL markers from each soil core. Jaccard dissimilarity across the two soil cores was highest for metazoan taxa detected by the 12S and cox1 markers. Taxonomic community distances were similar for each marker across the two soil cores when the chi-squared metric was used; however, the 12S and cox1 markers did not cluster well when the Goodall similarity metric was used. A comparison of plant macrofossil vs. read abundance corroborates previous work that suggests eastern Beringia was dominated by grasses and forbs during cold stages of the Pleistocene, a habitat that is restricted to isolated sites in the present-day Yukon.
Data from: Lessons learned from microsatellite development for non-model organisms using 454 pyrosequencing
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Data from: Identifying insecticide resistance genes in mosquito by combining AFLP genome scan and 454 pyrosequencing
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Allen Brain Atlas
Allen Brain Atlas is an Allen Institute collection of brain map atlases, datasets, APIs, and analysis tools covering mouse, human, and non-human primate brain resources.
Annotated Behaviour and Observability Dataset (ABODe)
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The International Brain Laboratory public data releases expose standardized mouse decision-making experiments, including Neuropixels recordings, widefield calcium imaging, behavior, and session metadata accessed through the ONE API.
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