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133 results for “Amplicon sequencing”
Shallow shotgun sequencing of the microbiome recapitulates 16S amplicon results and provides functional insights
<p>Prevailing 16S rRNA gene-amplicon methods for characterizing the bacterial microbiome of wildlife are economical, but result in coarse taxonomic classifications, are subject to primer and 16S copy number biases, and do not allow for direct estimation of microbiome functional potential. While deep shotgun metagenomic sequencing can overcome many of these limitations, it is prohibitively expensive for large sample sets. We evaluated the ability of shallow shotgun metagenomic sequencing to characterize taxonomic and functional patterns in the fecal microbiome of a model population of feral horses (Sable Island, Canada). Since 2007, this unmanaged population has been the subject of an individual-based, long-term ecological study. Using deep shotgun metagenomic sequencing, we determined the sequencing depth required to accurately characterize the horse microbiome. In comparing conventional versus high-throughput shotgun metagenomic library preparation techniques, we validate the use of more cost-effective lab methods. Finally, we characterize similarities between 16S amplicon and shallow shotgun characterization of the microbiome and demonstrate that the latter recapitulates biological patterns first described in a published amplicon dataset. Unlike amplicon data, we further demonstrate how shallow shotgun metagenomic data provide useful insights about microbiome functional potential which support previously hypothesized diet effects in this study system.</p>
Tutorial output for Tourmaline amplicon sequence processing workflow
<p>Tutorial output for the <a href="https://github.com/aomlomics/tourmaline">Tourmaline</a> amplicon sequence processing workflow.</p> <p>Tourmaline was run on the test data provided in the directory <a href="https://github.com/aomlomics/tourmaline/tree/master/00-data">00-data</a>, which were downloaded along with the rest of the repository using this command:</p> <pre><code>git clone https://github.com/aomlomics/tourmaline</code></pre> <p>Reference data were downloaded and symlinked using these commands:</p> <pre><code>cd tourmaline/01-imported wget https://data.qiime2.org/2021.2/common/silva-138-99-seqs-515-806.qza wget https://data.qiime2.org/2021.2/common/silva-138-99-tax-515-806.qza ln -s silva-138-99-seqs-515-806.qza refseqs.qza ln -s silva-138-99-tax-515-806.qza reftax.qza</code></pre> <p>Paths in 00-data/manifest_pe.csv and 00-data/manifest_se.csv were edited to match the local paths.</p> <p>Output for all modes of the workflow were then generated in series:</p> <pre><code>conda activate qiime2-2021.2 snakemake dada2_pe_report_unfiltered snakemake dada2_pe_report_filtered snakemake dada2_se_report_unfiltered snakemake dada2_se_report_filtered snakemake deblur_se_report_unfiltered snakemake deblur_se_report_filtered</code></pre> <p> </p>
Improved library preparation protocols for amplicon sequencing-based noninvasive fetal genotyping for RHD-positive D antigen-negative alleles
<p>We aimed to simplify our fetal <i>RHD</i> genotyping protocol by changing the method to attach Illumina's sequencing adaptors to PCR products from the ligation-based method to a PCR-based method, and to improve its quantitative accuracy by introducing unique molecular indexes, which allow us to count the numbers of DNA fragments used as PCR templates and to minimize the effects of PCR and sequencing errors. Both of the newly established protocols reduced time and cost compared with our conventional protocol. Removal of PCR duplicates using UMIs reduced the frequencies of erroneously mapped sequences reads likely generated by PCR and sequencing errors. The modified protocols will help us facilitate implementing fetal <i>RHD</i> genotyping for East Asian populations into clinical practice.</p>
Population admixtures in medaka inferred by multiple arbitrary amplicon sequencing
<p>Cost-effective genotyping can be achieved by sequencing PCR amplicons. Short 3–10 base primers can arbitrarily amplify thousands of loci using only a few primers. To improve the sequencing efficiency of the multiple arbitrary amplicon sequencing (MAAS) approach, we designed new primers and examined their efficiency in sequencing and genotyping. To demonstrate the effectiveness of our method, we applied it to examine the population structure of the small freshwater fish, medaka (<em>Oryzias</em> <em>latipes</em>). We obtained 2,987 informative SNVs with no missing genotype calls for 67 individuals from 15 wild populations and three artificial strains. The estimated phylogenic and population genetic structures of the wild populations were consistent with previous studies, corroborating the accuracy of our genotyping method. We also attempted to reconstruct the genetic backgrounds of a commercial orange mutant strain, Himedaka, which has caused a genetic disturbance in wild populations. Our admixture analysis focusing on Himedaka showed that at least two wild populations had genetically contributed to the nuclear genome of this mutant strain. Our genotyping methods and results will be useful in quantitative assessments of genetic disturbance by this commercially available strain.</p>
Amplicon sequence variants (ASV) of gut pathogens in hooded cranes and domestic geese
<p>Driven by habitat loss from anthropogenic activities, wintering migratory birds forage together with poultry in paddy fields, and thus impose risks of cross transmitting pathogens. To date, there is little evidence for such risks of pathogen transmission between wild birds and poultry. Using the high-throughput sequencing, we report on detected potential pathogens of both wild hooded cranes <em>Grus monacha</em> and sympatric domestic geese <em>Anser</em> <em>anser</em> <em>domesticus</em> during the wintering period and infer the possibility of cross-species pathogen transmission. The results revealed that the number of shared amplicon sequence variants (ASVs) of potential pathogens between the gut microbiota of the two species was low during the early wintering stage (17.2%; 5 ASVs shared) but increased to 56.3% (18 ASVs shared) during the late wintering stage. That is, potential pathogens in the gut microbial communities of the two species became more similar through co-foraging in paddy fields, supporting cross-transmission of pathogens between hooded cranes and domestic geese during the wintering period. Importantly, transmission appeared to be largely from wild hooded cranes to domestic geese, although some potential pathogens may have become specialized to the domestic goose in late wintering stage. Humans are also facing the risks of contracting these potential pathogens from migratory birds through their frequent contact with domestic poultry. It is, therefore, necessary to closely monitor this pathway of pathogen transmission from wild birds to domestic animals and even to humans.</p>
Community assembly amplicon sequences, with pipeline to get asv table for "Spatial structure drives compositional convergence between nutrient environments in experimental microbial communities"
<p>Community assembly amplicon sequences, with pipeline to get asv table for "Spatial structure drives compositional convergence between nutrient environments in experimental microbial communities"</p> <p> </p> <p>compressed FASTA files for 16s amplicon sequences relating to two separate projects, "Spatial structure drives compositional convergence between nutrient environments in experimental microbial communities" and "Habitat filtering leads to phylogenetic clustering in synthetic microbial communities". DADA22 pipeline is included, which pools all samples for better accuracy. A Julia script bioinfo.jl is then used to select only the samples relevant to spatial structure project.</p> <p> </p> <p>All csv filenames are appended with "_q" indicating an increase in the stringency of quality filtering parameters (also increasing minimum hamming distance used in DADA2 algorithm to 5) to produce a taxa table with a sensible number of ASVs (given a known number of input strains) with each ASV uniquely aligning to an individual sequence from colony PCR of said input strains.</p> <p> </p>
Amplicon sequence variants by sample table from Antarctic methane seeps
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Amplicon sequence variants (ASV) of gut pathogens in hooded cranes and domestic geese
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Improved library preparation protocols for amplicon sequencing-based noninvasive fetal genotyping for RHD-positive D antigen-negative alleles
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Population admixtures in medaka inferred by multiple arbitrary amplicon sequencing
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Raw data: multispecies amplicon sequencing (Loera, Studer, and Kölliker, 2021, Molecular Ecology Resources)
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Habitats and environmental variables investigated in a diversity survey on Polynucleobacter bacteria revealed by amplicon sequencing of priB genes
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Shallow shotgun sequencing of the microbiome recapitulates 16S amplicon results and provides functional insights
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Amplicon sequencing data for genetic variants at the ASIP locus that determine grey and black fur of the brushtail possum
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A pan-cetacean MHC amplicon sequencing panel developed and evaluated in combination with genome assemblies
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Data sets for: EMA-amplicon-based sequencing of untreated and SODIS treated rainwater
<p>Data sets for the EMA-amplicon-based sequencing of untreated and treated rainwater collected from an informal settlement and rural farming community in South Africa. The data sets were used for the publication: EMA-amplicon-based sequencing informs risk assessment analysis of water treatment systems (Submitted to Science of the Total Environment).</p>
Rapid and Inexpensive Whole-Genome Sequencing of SARS-CoV2 using 1200 bp Tiled Amplicons and Oxford Nanopore Rapid Barcoding
<p>Description of 1200bp amplicon primer sets and .bed and .tsv files for SARS-CoV-2 assembly using the ARTIC bioinformatics pipeline.</p>
Coregonus spp. opsin amplicon sequence alignments
<p>Local adaptation can drive diversification of closely related species across environmental gradients and promote convergence of distantly related taxa that experience similar conditions. We examined a potential case of adaptation to novel visual environments in a species flock (Great Lakes salmonids, genus <i>Coregonus</i>) using a new amplicon genotyping protocol on the Oxford Nanopore Flongle. Five visual opsin genes were sequenced for individuals of <i>C. artedi</i>,<i> C. hoyi</i>,<i> C. kiyi</i>, and <i>C. zenithicus.</i> Comparisons revealed species-specific differences in a key spectral tuning amino acid in <i>rhodopsin</i> (Tyr261Phe substitution), suggesting local adaptation of <i>C. kiyi </i>to the blue-shifted depths of Lake Superior. Ancestral state reconstruction demonstrates that parallel evolution and "toggling" at this amino acid residue has occurred several times across the fish tree of life, resulting in identical changes to the visual systems of distantly related taxa across replicated environmental gradients. Our results suggest that ecological differences and local adaptation to distinct visual environments are strong drivers of both evolutionary parallelism and diversification.</p>
Data from: 16S rRNA amplicon sequencing for epidemiological surveys of bacteria in wildlife
The human impact on natural habitats is increasing the complexity of human-wildlife interactions and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of the large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents, including 208 Mus musculus domesticus, 189 Rattus rattus, 93 Mastomys natalensis, and 221 Mastomys erythroleucus, collected from 24 villages in Senegal. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia, Bartonella, Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia. Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species, and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of bacterial pathogens without requiring assumptions about their presence in the samples. This approach is therefore particularly suitable to continuous pathogen surveillance in the context of disease-monitoring programs.
Data from: Species tree estimation of North American chorus frogs (Hylidae: Pseudacris) with parallel tagged amplicon sequencing
The field of phylogenetics is changing rapidly with the application of high-throughput sequencing to non-model organisms. Cost-effective use of this technology for phylogenetic studies, which often include a relatively small portion of the genome but several taxa, requires strategies for genome partitioning and sequencing multiple individuals in parallel. In this study we estimated a multilocus phylogeny for the North American chorus frog genus Pseudacris using anonymous nuclear loci that were recently developed using a reduced representation library approach. We sequenced 27 nuclear loci and three mitochondrial loci for 44 individuals on 1/3 of an Illumina MiSeq run, obtaining 96.5% of the targeted amplicons at less than 20% of the cost of traditional Sanger sequencing. We found heterogeneity among gene trees, although four major clades (Trilling Frog, Fat Frog, crucifer, and West Coast) were consistently supported, and we resolved the relationships among these clades for the first time with strong support. We also found discordance between the mitochondrial and nuclear datasets that we attribute to mitochondrial introgression and a possible selective sweep. Bayesian concordance analysis in BUCKy and species tree analysis in *BEAST produced largely similar topologies, although we identify taxa that require additional investigation in order to clarify taxonomic and geographic range boundaries. Overall, we demonstrate the utility of a reduced representation library approach for marker development and parallel tagged sequencing on an Illumina MiSeq for phylogenetic studies of non-model organisms.
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OpenNeuro
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