Increasing ecological inference from high throughput sequencing of fungi in the environment through a tagging approach

Treesearch

D. Lee Taylor; Michael G. Booth; Jack W. McFarland; Ian C. Herriott; Niall J. Lennon; Chad Nusbaum; Thomas G. Marr

2008-01-01

High throughput sequencing methods are widely used in analyses of microbial diversity but are generally applied to small numbers of samples, which precludes charaterization of patterns of microbial diversity across space and time. We have designed a primer-tagging approach that allows pooling and subsequent sorting of numerous samples, which is directed to...

Massively parallel rRNA gene sequencing exacerbates the potential for biased community diversity comparisons due to variable library sizes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gihring, Thomas; Green, Stefan; Schadt, Christopher Warren

2011-01-01

Technologies for massively parallel sequencing are revolutionizing microbial ecology and are vastly increasing the scale of ribosomal RNA (rRNA) gene studies. Although pyrosequencing has increased the breadth and depth of possible rRNA gene sampling, one drawback is that the number of reads obtained per sample is difficult to control. Pyrosequencing libraries typically vary widely in the number of sequences per sample, even within individual studies, and there is a need to revisit the behaviour of richness estimators and diversity indices with variable gene sequence library sizes. Multiple reports and review papers have demonstrated the bias in non-parametric richness estimators (e.g.more » Chao1 and ACE) and diversity indices when using clone libraries. However, we found that biased community comparisons are accumulating in the literature. Here we demonstrate the effects of sample size on Chao1, ACE, CatchAll, Shannon, Chao-Shen and Simpson's estimations specifically using pyrosequencing libraries. The need to equalize the number of reads being compared across libraries is reiterated, and investigators are directed towards available tools for making unbiased diversity comparisons.« less

Insights into Deep-Sea Sediment Fungal Communities from the East Indian Ocean Using Targeted Environmental Sequencing Combined with Traditional Cultivation

PubMed Central

Zhang, Xiao-yong; Tang, Gui-ling; Xu, Xin-ya; Nong, Xu-hua; Qi, Shu-Hua

2014-01-01

The fungal diversity in deep-sea environments has recently gained an increasing amount attention. Our knowledge and understanding of the true fungal diversity and the role it plays in deep-sea environments, however, is still limited. We investigated the fungal community structure in five sediments from a depth of ∼4000 m in the East India Ocean using a combination of targeted environmental sequencing and traditional cultivation. This approach resulted in the recovery of a total of 45 fungal operational taxonomic units (OTUs) and 20 culturable fungal phylotypes. This finding indicates that there is a great amount of fungal diversity in the deep-sea sediments collected in the East Indian Ocean. Three fungal OTUs and one culturable phylotype demonstrated high divergence (89%–97%) from the existing sequences in the GenBank. Moreover, 44.4% fungal OTUs and 30% culturable fungal phylotypes are new reports for deep-sea sediments. These results suggest that the deep-sea sediments from the East India Ocean can serve as habitats for new fungal communities compared with other deep-sea environments. In addition, different fungal community could be detected when using targeted environmental sequencing compared with traditional cultivation in this study, which suggests that a combination of targeted environmental sequencing and traditional cultivation will generate a more diverse fungal community in deep-sea environments than using either targeted environmental sequencing or traditional cultivation alone. This study is the first to report new insights into the fungal communities in deep-sea sediments from the East Indian Ocean, which increases our knowledge and understanding of the fungal diversity in deep-sea environments. PMID:25272044

Characterization of an endogenous retrovirus class in elephants and their relatives

PubMed Central

Greenwood, Alex D; Englbrecht, Claudia C; MacPhee, Ross DE

2004-01-01

Background Endogenous retrovirus-like elements (ERV-Ls, primed with tRNA leucine) are a diverse group of reiterated sequences related to foamy viruses and widely distributed among mammals. As shown in previous investigations, in many primates and rodents this class of elements has remained transpositionally active, as reflected by increased copy number and high sequence diversity within and among taxa. Results Here we examine whether proviral-like sequences may be suitable molecular probes for investigating the phylogeny of groups known to have high element diversity. As a test we characterized ERV-Ls occurring in a sample of extant members of superorder Uranotheria (Asian and African elephants, manatees, and hyraxes). The ERV-L complement in this group is even more diverse than previously suspected, and there is sequence evidence for active expansion, particularly in elephantids. Many of the elements characterized have protein coding potential suggestive of activity. Conclusions In general, the evidence supports the hypothesis that the complement had a single origin within basal Uranotheria. PMID:15476555

Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons

PubMed Central

Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.; Lamson, Jacob S.; He, Jennifer; Hoover, Cindi A.; Blow, Matthew J.; Bristow, James; Butland, Gareth

2015-01-01

ABSTRACT Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative d-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. PMID:25968644

Broad Surveys of DNA Viral Diversity Obtained through Viral Metagenomics of Mosquitoes

PubMed Central

Ng, Terry Fei Fan; Willner, Dana L.; Lim, Yan Wei; Schmieder, Robert; Chau, Betty; Nilsson, Christina; Anthony, Simon; Ruan, Yijun; Rohwer, Forest; Breitbart, Mya

2011-01-01

Viruses are the most abundant and diverse genetic entities on Earth; however, broad surveys of viral diversity are hindered by the lack of a universal assay for viruses and the inability to sample a sufficient number of individual hosts. This study utilized vector-enabled metagenomics (VEM) to provide a snapshot of the diversity of DNA viruses present in three mosquito samples from San Diego, California. The majority of the sequences were novel, suggesting that the viral community in mosquitoes, as well as the animal and plant hosts they feed on, is highly diverse and largely uncharacterized. Each mosquito sample contained a distinct viral community. The mosquito viromes contained sequences related to a broad range of animal, plant, insect and bacterial viruses. Animal viruses identified included anelloviruses, circoviruses, herpesviruses, poxviruses, and papillomaviruses, which mosquitoes may have obtained from vertebrate hosts during blood feeding. Notably, sequences related to human papillomaviruses were identified in one of the mosquito samples. Sequences similar to plant viruses were identified in all mosquito viromes, which were potentially acquired through feeding on plant nectar. Numerous bacteriophages and insect viruses were also detected, including a novel densovirus likely infecting Culex erythrothorax. Through sampling insect vectors, VEM enables broad survey of viral diversity and has significantly increased our knowledge of the DNA viruses present in mosquitoes. PMID:21674005

Influence of Seasonality on the Genetic Diversity of Vibrio parahaemolyticus in New Hampshire Shellfish Waters as Determined by Multilocus Sequence Analysis

PubMed Central

Ellis, Crystal N.; Schuster, Brian M.; Striplin, Megan J.; Jones, Stephen H.; Whistler, Cheryl A.

2012-01-01

Risk of gastric infection with Vibrio parahaemolyticus increases with favorable environmental conditions and population shifts that increase prevalence of infective strains. Genetic analysis of New Hampshire strains revealed a unique population with some isolates similar to outbreak-causing strains and high-level diversity that increased as waters warmed. PMID:22407686

Influence of seasonality on the genetic diversity of Vibrio parahaemolyticus in New Hampshire shellfish waters as determined by multilocus sequence analysis.

PubMed

Ellis, Crystal N; Schuster, Brian M; Striplin, Megan J; Jones, Stephen H; Whistler, Cheryl A; Cooper, Vaughn S

2012-05-01

Risk of gastric infection with Vibrio parahaemolyticus increases with favorable environmental conditions and population shifts that increase prevalence of infective strains. Genetic analysis of New Hampshire strains revealed a unique population with some isolates similar to outbreak-causing strains and high-level diversity that increased as waters warmed.

Assessing Diversity of DNA Structure-Related Sequence Features in Prokaryotic Genomes

PubMed Central

Huang, Yongjie; Mrázek, Jan

2014-01-01

Prokaryotic genomes are diverse in terms of their nucleotide and oligonucleotide composition as well as presence of various sequence features that can affect physical properties of the DNA molecule. We present a survey of local sequence patterns which have a potential to promote non-canonical DNA conformations (i.e. different from standard B-DNA double helix) and interpret the results in terms of relationships with organisms' habitats, phylogenetic classifications, and other characteristics. Our present work differs from earlier similar surveys not only by investigating a wider range of sequence patterns in a large number of genomes but also by using a more realistic null model to assess significant deviations. Our results show that simple sequence repeats and Z-DNA-promoting patterns are generally suppressed in prokaryotic genomes, whereas palindromes and inverted repeats are over-represented. Representation of patterns that promote Z-DNA and intrinsic DNA curvature increases with increasing optimal growth temperature (OGT), and decreases with increasing oxygen requirement. Additionally, representations of close direct repeats, palindromes and inverted repeats exhibit clear negative trends with increasing OGT. The observed relationships with environmental characteristics, particularly OGT, suggest possible evolutionary scenarios of structural adaptation of DNA to particular environmental niches. PMID:24408877

Strain-Level Diversity of Secondary Metabolism in Streptomyces albus

PubMed Central

Seipke, Ryan F.

2015-01-01

Streptomyces spp. are robust producers of medicinally-, industrially- and agriculturally-important small molecules. Increased resistance to antibacterial agents and the lack of new antibiotics in the pipeline have led to a renaissance in natural product discovery. This endeavor has benefited from inexpensive high quality DNA sequencing technology, which has generated more than 140 genome sequences for taxonomic type strains and environmental Streptomyces spp. isolates. Many of the sequenced streptomycetes belong to the same species. For instance, Streptomyces albus has been isolated from diverse environmental niches and seven strains have been sequenced, consequently this species has been sequenced more than any other streptomycete, allowing valuable analyses of strain-level diversity in secondary metabolism. Bioinformatics analyses identified a total of 48 unique biosynthetic gene clusters harboured by Streptomyces albus strains. Eighteen of these gene clusters specify the core secondary metabolome of the species. Fourteen of the gene clusters are contained by one or more strain and are considered auxiliary, while 16 of the gene clusters encode the production of putative strain-specific secondary metabolites. Analysis of Streptomyces albus strains suggests that each strain of a Streptomyces species likely harbours at least one strain-specific biosynthetic gene cluster. Importantly, this implies that deep sequencing of a species will not exhaust gene cluster diversity and will continue to yield novelty. PMID:25635820

spads 1.0: a toolbox to perform spatial analyses on DNA sequence data sets.

PubMed

Dellicour, Simon; Mardulyn, Patrick

2014-05-01

SPADS 1.0 (for 'Spatial and Population Analysis of DNA Sequences') is a population genetic toolbox for characterizing genetic variability within and among populations from DNA sequences. In view of the drastic increase in genetic information available through sequencing methods, spads was specifically designed to deal with multilocus data sets of DNA sequences. It computes several summary statistics from populations or groups of populations, performs input file conversions for other population genetic programs and implements locus-by-locus and multilocus versions of two clustering algorithms to study the genetic structure of populations. The toolbox also includes two MATLAB and r functions, GDISPAL and GDIVPAL, to display differentiation and diversity patterns across landscapes. These functions aim to generate interpolating surfaces based on multilocus distance and diversity indices. In the case of multiple loci, such surfaces can represent a useful alternative to multiple pie charts maps traditionally used in phylogeography to represent the spatial distribution of genetic diversity. These coloured surfaces can also be used to compare different data sets or different diversity and/or distance measures estimated on the same data set. © 2013 John Wiley & Sons Ltd.

Genomic Diversity and Evolution of the Lyssaviruses

PubMed Central

Delmas, Olivier; Holmes, Edward C.; Talbi, Chiraz; Larrous, Florence; Dacheux, Laurent; Bouchier, Christiane; Bourhy, Hervé

2008-01-01

Lyssaviruses are RNA viruses with single-strand, negative-sense genomes responsible for rabies-like diseases in mammals. To date, genomic and evolutionary studies have most often utilized partial genome sequences, particularly of the nucleoprotein and glycoprotein genes, with little consideration of genome-scale evolution. Herein, we report the first genomic and evolutionary analysis using complete genome sequences of all recognised lyssavirus genotypes, including 14 new complete genomes of field isolates from 6 genotypes and one genotype that is completely sequenced for the first time. In doing so we significantly increase the extent of genome sequence data available for these important viruses. Our analysis of these genome sequence data reveals that all lyssaviruses have the same genomic organization. A phylogenetic analysis reveals strong geographical structuring, with the greatest genetic diversity in Africa, and an independent origin for the two known genotypes that infect European bats. We also suggest that multiple genotypes may exist within the diversity of viruses currently classified as ‘Lagos Bat’. In sum, we show that rigorous phylogenetic techniques based on full length genome sequence provide the best discriminatory power for genotype classification within the lyssaviruses. PMID:18446239

Microbial dynamics in upflow anaerobic sludge blanket (UASB) bioreactor granules in response to short-term changes in substrate feed

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kovacik, William P.; Scholten, Johannes C.; Culley, David E.

2010-08-01

The complexity and diversity of the microbial communities in biogranules from an upflow anaerobic sludge blanket (UASB) bioreactor were determined in response to short-term changes in substrate feeds. The reactor was fed simulated brewery wastewater (SBWW) (70% ethanol, 15% acetate, 15% propionate) for 1.5 months (phase 1), acetate / sulfate for 2 months (phase 2), acetate-alone for 3 months (phase 3), and then a return to SBWW for 2 months (phase 4). Performance of the reactor remained relatively stable throughout the experiment as shown by COD removal and gas production. 16S rDNA, methanogen-associated mcrA and sulfate reducer-associated dsrAB genes weremore » PCR amplified, then cloned and sequenced. Sequence analysis of 16S clone libraries showed a relatively simple community composed mainly of the methanogenic Archaea (Methanobacterium and Methanosaeta), members of the Green Non-Sulfur (Chloroflexi) group of Bacteria, followed by fewer numbers of Syntrophobacter, Spirochaeta, Acidobacteria and Cytophaga-related Bacterial sequences. Methanogen-related mcrA clone libraries were dominated throughout by Methanobacter and Methanospirillum related sequences. Although not numerous enough to be detected in our 16S rDNA libraries, sulfate reducers were detected in dsrAB clone libraries, with sequences related to Desulfovibrio and Desulfomonile. Community diversity levels (Shannon-Weiner index) generally decreased for all libraries in response to a change from SBWW to acetate-alone feed. But there was a large transitory increase noted in 16S diversity at the two-month sampling on acetate-alone, entirely related to an increase in Bacterial diversity. Upon return to SBWW conditions in phase 4, all diversity measures returned to near phase 1 levels.« less

Genetic diversity and demographic instability in Riftia pachyptila tubeworms from eastern Pacific hydrothermal vents

PubMed Central

2011-01-01

Background Deep-sea hydrothermal vent animals occupy patchy and ephemeral habitats supported by chemosynthetic primary production. Volcanic and tectonic activities controlling the turnover of these habitats contribute to demographic instability that erodes genetic variation within and among colonies of these animals. We examined DNA sequences from one mitochondrial and three nuclear gene loci to assess genetic diversity in the siboglinid tubeworm, Riftia pachyptila, a widely distributed constituent of vents along the East Pacific Rise and Galápagos Rift. Results Genetic differentiation (FST) among populations increased with geographical distances, as expected under a linear stepping-stone model of dispersal. Low levels of DNA sequence diversity occurred at all four loci, allowing us to exclude the hypothesis that an idiosyncratic selective sweep eliminated mitochondrial diversity alone. Total gene diversity declined with tectonic spreading rates. The southernmost populations, which are subjected to superfast spreading rates and high probabilities of extinction, are relatively homogenous genetically. Conclusions Compared to other vent species, DNA sequence diversity is extremely low in R. pachyptila. Though its dispersal abilities appear to be effective, the low diversity, particularly in southern hemisphere populations, is consistent with frequent local extinction and (re)colonization events. PMID:21489281

Genetic diversity and demographic instability in Riftia pachyptila tubeworms from eastern Pacific hydrothermal vents

USGS Publications Warehouse

Coykendall, D.K.; Johnson, S.B.; Karl, S.A.; Lutz, R.A.; Vrijenhoek, R.C.

2011-01-01

Background: Deep-sea hydrothermal vent animals occupy patchy and ephemeral habitats supported by chemosynthetic primary production. Volcanic and tectonic activities controlling the turnover of these habitats contribute to demographic instability that erodes genetic variation within and among colonies of these animals. We examined DNA sequences from one mitochondrial and three nuclear gene loci to assess genetic diversity in the siboglinid tubeworm, Riftia pachyptila, a widely distributed constituent of vents along the East Pacific Rise and Galpagos Rift. Results: Genetic differentiation (FST) among populations increased with geographical distances, as expected under a linear stepping-stone model of dispersal. Low levels of DNA sequence diversity occurred at all four loci, allowing us to exclude the hypothesis that an idiosyncratic selective sweep eliminated mitochondrial diversity alone. Total gene diversity declined with tectonic spreading rates. The southernmost populations, which are subjected to superfast spreading rates and high probabilities of extinction, are relatively homogenous genetically. Conclusions: Compared to other vent species, DNA sequence diversity is extremely low in R. pachyptila. Though its dispersal abilities appear to be effective, the low diversity, particularly in southern hemisphere populations, is consistent with frequent local extinction and (re)colonization events. ?? 2011 Coykendall et al; licensee BioMed Central Ltd.

Molecular analysis of meso- and thermophilic microbiota associated with anaerobic biowaste degradation

PubMed Central

2012-01-01

Background Microbial anaerobic digestion (AD) is used as a waste treatment process to degrade complex organic compounds into methane. The archaeal and bacterial taxa involved in AD are well known, whereas composition of the fungal community in the process has been less studied. The present study aimed to reveal the composition of archaeal, bacterial and fungal communities in response to increasing organic loading in mesophilic and thermophilic AD processes by applying 454 amplicon sequencing technology. Furthermore, a DNA microarray method was evaluated in order to develop a tool for monitoring the microbiological status of AD. Results The 454 sequencing showed that the diversity and number of bacterial taxa decreased with increasing organic load, while archaeal i.e. methanogenic taxa remained more constant. The number and diversity of fungal taxa increased during the process and varied less in composition with process temperature than bacterial and archaeal taxa, even though the fungal diversity increased with temperature as well. Evaluation of the microarray using AD sample DNA showed correlation of signal intensities with sequence read numbers of corresponding target groups. The sensitivity of the test was found to be about 1%. Conclusions The fungal community survives in anoxic conditions and grows with increasing organic loading, suggesting that Fungi may contribute to the digestion by metabolising organic nutrients for bacterial and methanogenic groups. The microarray proof of principle tests suggest that the method has the potential for semiquantitative detection of target microbial groups given that comprehensive sequence data is available for probe design. PMID:22727142

Pros and Cons of Ion-Torrent Next Generation Sequencing versus Terminal Restriction Fragment Length Polymorphism T-RFLP for Studying the Rumen Bacterial Community

PubMed Central

de la Fuente, Gabriel; Belanche, Alejandro; Girwood, Susan E.; Pinloche, Eric; Wilkinson, Toby; Newbold, C. Jamie

2014-01-01

The development of next generation sequencing has challenged the use of other molecular fingerprinting methods used to study microbial diversity. We analysed the bacterial diversity in the rumen of defaunated sheep following the introduction of different protozoal populations, using both next generation sequencing (NGS: Ion Torrent PGM) and terminal restriction fragment length polymorphism (T-RFLP). Although absolute number differed, there was a high correlation between NGS and T-RFLP in terms of richness and diversity with R values of 0.836 and 0.781 for richness and Shannon-Wiener index, respectively. Dendrograms for both datasets were also highly correlated (Mantel test = 0.742). Eighteen OTUs and ten genera were significantly impacted by the addition of rumen protozoa, with an increase in the relative abundance of Prevotella, Bacteroides and Ruminobacter, related to an increase in free ammonia levels in the rumen. Our findings suggest that classic fingerprinting methods are still valuable tools to study microbial diversity and structure in complex environments but that NGS techniques now provide cost effect alternatives that provide a far greater level of information on the individual members of the microbial population. PMID:25051490

Palynological composition of a Lower Cretaceous South American tropical sequence: climatic implications and diversity comparisons with other latitudes.

PubMed

Mejia-Velasquez, Paula J; Dilcher, David L; Jaramillo, Carlos A; Fortini, Lucas B; Manchester, Steven R

2012-11-01

Reconstruction of floristic patterns during the early diversification of angiosperms is impeded by the scarce fossil record, especially in tropical latitudes. Here we collected quantitative palynological data from a stratigraphic sequence in tropical South America to provide floristic and climatic insights into such tropical environments during the Early Cretaceous. We reconstructed the floristic composition of an Aptian-Albian tropical sequence from central Colombia using quantitative palynology (rarefied species richness and abundance) and used it to infer its predominant climatic conditions. Additionally, we compared our results with available quantitative data from three other sequences encompassing 70 floristic assemblages to determine latitudinal diversity patterns. Abundance of humidity indicators was higher than that of aridity indicators (61% vs. 10%). Additionally, we found an angiosperm latitudinal diversity gradient (LDG) for the Aptian, but not for the Albian, and an inverted LDG of the overall diversity for the Albian. Angiosperm species turnover during the Albian, however, was higher in humid tropics. There were humid climates in northwestern South America during the Aptian-Albian interval contrary to the widespread aridity expected for the tropical belt. The Albian inverted overall LDG is produced by a faster increase in per-sample angiosperm and pteridophyte diversity in temperate latitudes. However, humid tropical sequences had higher rates of floristic turnover suggesting a higher degree of morphological variation than in temperate regions.

Palynological composition of a Lower Cretaceous South American tropical sequence: Climatic implications and diversity comparisons with other latitudes.

USGS Publications Warehouse

Mejia-Velasquez, Paula J.; Dilcher, David L.; Jaramillo, Carlos A.; Fortini, Lucas B.; Manchester, Steven R.

2012-01-01

Premise of the study: Reconstruction of floristic patterns during the early diversification of angiosperms is impeded by the scarce fossil record, especially in tropical latitudes. Here we collected quantitative palynological data from a stratigraphic sequence in tropical South America to provide floristic and climatic insights into such tropical environments during the Early Cretaceous. Methods: We reconstructed the floristic composition of an Aptian-Albian tropical sequence from central Colombia using quantitative palynology (rarefied species richness and abundance) and used it to infer its predominant climatic conditions. Additionally, we compared our results with available quantitative data from three other sequences encompassing 70 floristic assemblages to determine latitudinal diversity patterns. Key results: Abundance of humidity indicators was higher than that of aridity indicators (61% vs. 10%). Additionally, we found an angiosperm latitudinal diversity gradient (LDG) for the Aptian, but not for the Albian, and an inverted LDG of the overall diversity for the Albian. Angiosperm species turnover during the Albian, however, was higher in humid tropics. Conclusions: There were humid climates in northwestern South America during the Aptian-Albian interval contrary to the widespread aridity expected for the tropical belt. The Albian inverted overall LDG is produced by a faster increase in per-sample angiosperm and pteridophyte diversity in temperate latitudes. However, humid tropical sequences had higher rates of floristic turnover suggesting a higher degree of morphological variation than in temperate regions.

The Effects of Spatial Diversity and Imperfect Channel Estimation on Wideband MC-DS-CDMA and MC-CDMA

DTIC Science & Technology

2009-10-01

In our previous work, we compared the theoretical bit error rates of multi-carrier direct sequence code division multiple access (MC- DS - CDMA ) and...consider only those cases where MC- CDMA has higher frequency diversity than MC- DS - CDMA . Since increases in diversity yield diminishing gains, we conclude

Recombination enhances HIV-1 envelope diversity by facilitating the survival of latent genomic fragments in the plasma virus population

DOE Office of Scientific and Technical Information (OSTI.GOV)

Immonen, Taina T.; Conway, Jessica M.; Romero-Severson, Ethan O.

HIV-1 is subject to immune pressure exerted by the host, giving variants that escape the immune response an advantage. Virus released from activated latent cells competes against variants that have continually evolved and adapted to host immune pressure. Nevertheless, there is increasing evidence that virus displaying a signal of latency survives in patient plasma despite having reduced fitness due to long-term immune memory. We investigated the survival of virus with latent envelope genomic fragments by simulating within-host HIV-1 sequence evolution and the cycling of viral lineages in and out of the latent reservoir. Our model incorporates a detailed mutation processmore » including nucleotide substitution, recombination, latent reservoir dynamics, diversifying selection pressure driven by the immune response, and purifying selection pressure asserted by deleterious mutations. We evaluated the ability of our model to capture sequence evolution in vivo by comparing our simulated sequences to HIV-1 envelope sequence data from 16 HIV-infected untreated patients. Empirical sequence divergence and diversity measures were qualitatively and quantitatively similar to those of our simulated HIV-1 populations, suggesting that our model invokes realistic trends of HIV-1 genetic evolution. Moreover, reconstructed phylogenies of simulated and patient HIV-1 populations showed similar topological structures. Our simulation results suggest that recombination is a key mechanism facilitating the persistence of virus with latent envelope genomic fragments in the productively infected cell population. Recombination increased the survival probability of latent virus forms approximately 13-fold. Prevalence of virus with latent fragments in productively infected cells was observed in only 2% of simulations when we ignored recombination, while the proportion increased to 27% of simulations when we allowed recombination. We also found that the selection pressures exerted by different fitness landscapes influenced the shape of phylogenies, diversity trends, and survival of virus with latent genomic fragments. Furthermore, our model predicts that the persistence of latent genomic fragments from multiple different ancestral origins increases sequence diversity in plasma for reasonable fitness landscapes.« less

Recombination enhances HIV-1 envelope diversity by facilitating the survival of latent genomic fragments in the plasma virus population

DOE PAGES

Immonen, Taina T.; Conway, Jessica M.; Romero-Severson, Ethan O.; ...

2015-12-22

HIV-1 is subject to immune pressure exerted by the host, giving variants that escape the immune response an advantage. Virus released from activated latent cells competes against variants that have continually evolved and adapted to host immune pressure. Nevertheless, there is increasing evidence that virus displaying a signal of latency survives in patient plasma despite having reduced fitness due to long-term immune memory. We investigated the survival of virus with latent envelope genomic fragments by simulating within-host HIV-1 sequence evolution and the cycling of viral lineages in and out of the latent reservoir. Our model incorporates a detailed mutation processmore » including nucleotide substitution, recombination, latent reservoir dynamics, diversifying selection pressure driven by the immune response, and purifying selection pressure asserted by deleterious mutations. We evaluated the ability of our model to capture sequence evolution in vivo by comparing our simulated sequences to HIV-1 envelope sequence data from 16 HIV-infected untreated patients. Empirical sequence divergence and diversity measures were qualitatively and quantitatively similar to those of our simulated HIV-1 populations, suggesting that our model invokes realistic trends of HIV-1 genetic evolution. Moreover, reconstructed phylogenies of simulated and patient HIV-1 populations showed similar topological structures. Our simulation results suggest that recombination is a key mechanism facilitating the persistence of virus with latent envelope genomic fragments in the productively infected cell population. Recombination increased the survival probability of latent virus forms approximately 13-fold. Prevalence of virus with latent fragments in productively infected cells was observed in only 2% of simulations when we ignored recombination, while the proportion increased to 27% of simulations when we allowed recombination. We also found that the selection pressures exerted by different fitness landscapes influenced the shape of phylogenies, diversity trends, and survival of virus with latent genomic fragments. Furthermore, our model predicts that the persistence of latent genomic fragments from multiple different ancestral origins increases sequence diversity in plasma for reasonable fitness landscapes.« less

Clonal evolution in breast cancer revealed by single nucleus genome sequencing.

PubMed

Wang, Yong; Waters, Jill; Leung, Marco L; Unruh, Anna; Roh, Whijae; Shi, Xiuqing; Chen, Ken; Scheet, Paul; Vattathil, Selina; Liang, Han; Multani, Asha; Zhang, Hong; Zhao, Rui; Michor, Franziska; Meric-Bernstam, Funda; Navin, Nicholas E

2014-08-14

Sequencing studies of breast tumour cohorts have identified many prevalent mutations, but provide limited insight into the genomic diversity within tumours. Here we developed a whole-genome and exome single cell sequencing approach called nuc-seq that uses G2/M nuclei to achieve 91% mean coverage breadth. We applied this method to sequence single normal and tumour nuclei from an oestrogen-receptor-positive (ER(+)) breast cancer and a triple-negative ductal carcinoma. In parallel, we performed single nuclei copy number profiling. Our data show that aneuploid rearrangements occurred early in tumour evolution and remained highly stable as the tumour masses clonally expanded. In contrast, point mutations evolved gradually, generating extensive clonal diversity. Using targeted single-molecule sequencing, many of the diverse mutations were shown to occur at low frequencies (<10%) in the tumour mass. Using mathematical modelling we found that the triple-negative tumour cells had an increased mutation rate (13.3×), whereas the ER(+) tumour cells did not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer.

De novo assembly of mitochondrial genomes provides insights into genetic diversity and molecular evolution in wild boars and domestic pigs.

PubMed

Ni, Pan; Bhuiyan, Ali Akbar; Chen, Jian-Hai; Li, Jingjin; Zhang, Cheng; Zhao, Shuhong; Du, Xiaoyong; Li, Hua; Yu, Hui; Liu, Xiangdong; Li, Kui

2018-06-01

Up to date, the scarcity of publicly available complete mitochondrial sequences for European wild pigs hampers deeper understanding about the genetic changes following domestication. Here, we have assembled 26 de novo mtDNA sequences of European wild boars from next generation sequencing (NGS) data and downloaded 174 complete mtDNA sequences to assess the genetic relationship, nucleotide diversity, and selection. The Bayesian consensus tree reveals the clear divergence between the European and Asian clade and a very small portion (10 out of 200 samples) of maternal introgression. The overall nucleotides diversities of the mtDNA sequences have been reduced following domestication. Interestingly, the selection efficiencies in both European and Asian domestic pigs are reduced, probably caused by changes in both selection constraints and maternal population size following domestication. This study suggests that de novo assembled mitogenomes can be a great boon to uncover the genetic turnover following domestication. Further investigation is warranted to include more samples from the ever-increasing amounts of NGS data to help us to better understand the process of domestication.

Systematization of the protein sequence diversity in enzymes related to secondary metabolic pathways in plants, in the context of big data biology inspired by the KNApSAcK motorcycle database.

PubMed

Ikeda, Shun; Abe, Takashi; Nakamura, Yukiko; Kibinge, Nelson; Hirai Morita, Aki; Nakatani, Atsushi; Ono, Naoaki; Ikemura, Toshimichi; Nakamura, Kensuke; Altaf-Ul-Amin, Md; Kanaya, Shigehiko

2013-05-01

Biology is increasingly becoming a data-intensive science with the recent progress of the omics fields, e.g. genomics, transcriptomics, proteomics and metabolomics. The species-metabolite relationship database, KNApSAcK Core, has been widely utilized and cited in metabolomics research, and chronological analysis of that research work has helped to reveal recent trends in metabolomics research. To meet the needs of these trends, the KNApSAcK database has been extended by incorporating a secondary metabolic pathway database called Motorcycle DB. We examined the enzyme sequence diversity related to secondary metabolism by means of batch-learning self-organizing maps (BL-SOMs). Initially, we constructed a map by using a big data matrix consisting of the frequencies of all possible dipeptides in the protein sequence segments of plants and bacteria. The enzyme sequence diversity of the secondary metabolic pathways was examined by identifying clusters of segments associated with certain enzyme groups in the resulting map. The extent of diversity of 15 secondary metabolic enzyme groups is discussed. Data-intensive approaches such as BL-SOM applied to big data matrices are needed for systematizing protein sequences. Handling big data has become an inevitable part of biology.

Cyanobacterial Diversity in Microbial Mats from the Hypersaline Lagoon System of Araruama, Brazil: An In-depth Polyphasic Study.

PubMed

Ramos, Vitor M C; Castelo-Branco, Raquel; Leão, Pedro N; Martins, Joana; Carvalhal-Gomes, Sinda; Sobrinho da Silva, Frederico; Mendonça Filho, João G; Vasconcelos, Vitor M

2017-01-01

Microbial mats are complex, micro-scale ecosystems that can be found in a wide range of environments. In the top layer of photosynthetic mats from hypersaline environments, a large diversity of cyanobacteria typically predominates. With the aim of strengthening the knowledge on the cyanobacterial diversity present in the coastal lagoon system of Araruama (state of Rio de Janeiro, Brazil), we have characterized three mat samples by means of a polyphasic approach. We have used morphological and molecular data obtained by culture-dependent and -independent methods. Moreover, we have compared different classification methodologies and discussed the outcomes, challenges, and pitfalls of these methods. Overall, we show that Araruama's lagoons harbor a high cyanobacterial diversity. Thirty-six unique morphospecies could be differentiated, which increases by more than 15% the number of morphospecies and genera already reported for the entire Araruama system. Morphology-based data were compared with the 16S rRNA gene phylogeny derived from isolate sequences and environmental sequences obtained by PCR-DGGE and pyrosequencing. Most of the 48 phylotypes could be associated with the observed morphospecies at the order level. More than one third of the sequences demonstrated to be closely affiliated (best BLAST hit results of ≥99%) with cyanobacteria from ecologically similar habitats. Some sequences had no close relatives in the public databases, including one from an isolate, being placed as "loner" sequences within different orders. This hints at hidden cyanobacterial diversity in the mats of the Araruama system, while reinforcing the relevance of using complementary approaches to study cyanobacterial diversity.

Cyanobacterial Diversity in Microbial Mats from the Hypersaline Lagoon System of Araruama, Brazil: An In-depth Polyphasic Study

PubMed Central

Ramos, Vitor M. C.; Castelo-Branco, Raquel; Leão, Pedro N.; Martins, Joana; Carvalhal-Gomes, Sinda; Sobrinho da Silva, Frederico; Mendonça Filho, João G.; Vasconcelos, Vitor M.

2017-01-01

Microbial mats are complex, micro-scale ecosystems that can be found in a wide range of environments. In the top layer of photosynthetic mats from hypersaline environments, a large diversity of cyanobacteria typically predominates. With the aim of strengthening the knowledge on the cyanobacterial diversity present in the coastal lagoon system of Araruama (state of Rio de Janeiro, Brazil), we have characterized three mat samples by means of a polyphasic approach. We have used morphological and molecular data obtained by culture-dependent and -independent methods. Moreover, we have compared different classification methodologies and discussed the outcomes, challenges, and pitfalls of these methods. Overall, we show that Araruama's lagoons harbor a high cyanobacterial diversity. Thirty-six unique morphospecies could be differentiated, which increases by more than 15% the number of morphospecies and genera already reported for the entire Araruama system. Morphology-based data were compared with the 16S rRNA gene phylogeny derived from isolate sequences and environmental sequences obtained by PCR-DGGE and pyrosequencing. Most of the 48 phylotypes could be associated with the observed morphospecies at the order level. More than one third of the sequences demonstrated to be closely affiliated (best BLAST hit results of ≥99%) with cyanobacteria from ecologically similar habitats. Some sequences had no close relatives in the public databases, including one from an isolate, being placed as “loner” sequences within different orders. This hints at hidden cyanobacterial diversity in the mats of the Araruama system, while reinforcing the relevance of using complementary approaches to study cyanobacterial diversity. PMID:28713360

A comprehensive insight into bacterial virulence in drinking water using 454 pyrosequencing and Illumina high-throughput sequencing.

PubMed

Huang, Kailong; Zhang, Xu-Xiang; Shi, Peng; Wu, Bing; Ren, Hongqiang

2014-11-01

In order to comprehensively investigate bacterial virulence in drinking water, 454 pyrosequencing and Illumina high-throughput sequencing were used to detect potential pathogenic bacteria and virulence factors (VFs) in a full-scale drinking water treatment and distribution system. 16S rRNA gene pyrosequencing revealed high bacterial diversity in the drinking water (441-586 operational taxonomic units). Bacterial diversity decreased after chlorine disinfection, but increased after pipeline distribution. α-Proteobacteria was the most dominant taxonomic class. Alignment against the established pathogen database showed that several types of putative pathogens were present in the drinking water and Pseudomonas aeruginosa had the highest abundance (over 11‰ of total sequencing reads). Many pathogens disappeared after chlorine disinfection, but P. aeruginosa and Leptospira interrogans were still detected in the tap water. High-throughput sequencing revealed prevalence of various pathogenicity islands and virulence proteins in the drinking water, and translocases, transposons, Clp proteases and flagellar motor switch proteins were the predominant VFs. Both diversity and abundance of the detectable VFs increased after the chlorination, and decreased after the pipeline distribution. This study indicates that joint use of 454 pyrosequencing and Illumina sequencing can comprehensively characterize environmental pathogenesis, and several types of putative pathogens and various VFs are prevalent in drinking water. Copyright © 2014 Elsevier Inc. All rights reserved.

Bacterial diversity in a glacier foreland of the high Arctic.

PubMed

Schütte, Ursel M E; Abdo, Zaid; Foster, James; Ravel, Jacques; Bunge, John; Solheim, Bjørn; Forney, Larry J

2010-03-01

Over the past 100 years, Arctic temperatures have increased at almost twice the global average rate. One consequence is the acceleration of glacier retreat, exposing new habitats that are colonized by microorganisms whose diversity and function are unknown. Here, we characterized bacterial diversity along two approximately parallel chronosequences in an Arctic glacier forefield that span six time points following glacier retreat. We assessed changes in phylotype richness, evenness and turnover rate through the analysis of 16S rRNA gene sequences recovered from 52 samples taken from surface layers along the chronosequences. An average of 4500 sequences was obtained from each sample by 454 pyrosequencing. Using parametric methods, it was estimated that bacterial phylotype richness was high, and that it increased significantly from an average of 4000 (at a threshold of 97% sequence similarity) at locations exposed for 5 years to an average of 7050 phylotypes per 0.5 g of soil at sites that had been exposed for 150 years. Phylotype evenness also increased over time, with an evenness of 0.74 for 150 years since glacier retreat reflecting large proportions of rare phylotypes. The bacterial species turnover rate was especially high between sites exposed for 5 and 19 years. The level of bacterial diversity present in this High Arctic glacier foreland was comparable with that found in temperate and tropical soils, raising the question whether global patterns of bacterial species diversity parallel that of plants and animals, which have been found to form a latitudinal gradient and be lower in polar regions compared with the tropics.

Diversity and Activity of Alternative Nitrogenases in Sequenced Genomes and Coastal Environments

PubMed Central

McRose, Darcy L.; Zhang, Xinning; Kraepiel, Anne M. L.; Morel, François M. M.

2017-01-01

The nitrogenase enzyme, which catalyzes the reduction of N2 gas to NH4+, occurs as three separate isozyme that use Mo, Fe-only, or V. The majority of global nitrogen fixation is attributed to the more efficient ‘canonical’ Mo-nitrogenase, whereas Fe-only and V-(‘alternative’) nitrogenases are often considered ‘backup’ enzymes, used when Mo is limiting. Yet, the environmental distribution and diversity of alternative nitrogenases remains largely unknown. We searched for alternative nitrogenase genes in sequenced genomes and used PacBio sequencing to explore the diversity of canonical (nifD) and alternative (anfD and vnfD) nitrogenase amplicons in two coastal environments: the Florida Everglades and Sippewissett Marsh (MA). Genome-based searches identified an additional 25 species and 10 genera not previously known to encode alternative nitrogenases. Alternative nitrogenase amplicons were found in both Sippewissett Marsh and the Florida Everglades and their activity was further confirmed using newly developed isotopic techniques. Conserved amino acid sequences corresponding to cofactor ligands were also analyzed in anfD and vnfD amplicons, offering insight into environmental variants of these motifs. This study increases the number of available anfD and vnfD sequences ∼20-fold and allows for the first comparisons of environmental Mo-, Fe-only, and V-nitrogenase diversity. Our results suggest that alternative nitrogenases are maintained across a range of organisms and environments and that they can make important contributions to nitrogenase diversity and nitrogen fixation. PMID:28293220

Diversity and Activity of Alternative Nitrogenases in Sequenced Genomes and Coastal Environments.

PubMed

McRose, Darcy L; Zhang, Xinning; Kraepiel, Anne M L; Morel, François M M

2017-01-01

The nitrogenase enzyme, which catalyzes the reduction of N 2 gas to NH 4 + , occurs as three separate isozyme that use Mo, Fe-only, or V. The majority of global nitrogen fixation is attributed to the more efficient 'canonical' Mo-nitrogenase, whereas Fe-only and V-('alternative') nitrogenases are often considered 'backup' enzymes, used when Mo is limiting. Yet, the environmental distribution and diversity of alternative nitrogenases remains largely unknown. We searched for alternative nitrogenase genes in sequenced genomes and used PacBio sequencing to explore the diversity of canonical ( nifD ) and alternative ( anfD and vnfD ) nitrogenase amplicons in two coastal environments: the Florida Everglades and Sippewissett Marsh (MA). Genome-based searches identified an additional 25 species and 10 genera not previously known to encode alternative nitrogenases. Alternative nitrogenase amplicons were found in both Sippewissett Marsh and the Florida Everglades and their activity was further confirmed using newly developed isotopic techniques. Conserved amino acid sequences corresponding to cofactor ligands were also analyzed in anfD and vnfD amplicons, offering insight into environmental variants of these motifs. This study increases the number of available anfD and vnfD sequences ∼20-fold and allows for the first comparisons of environmental Mo-, Fe-only, and V-nitrogenase diversity. Our results suggest that alternative nitrogenases are maintained across a range of organisms and environments and that they can make important contributions to nitrogenase diversity and nitrogen fixation.

Impact of sequencing depth on the characterization of the microbiome and resistome.

PubMed

Zaheer, Rahat; Noyes, Noelle; Ortega Polo, Rodrigo; Cook, Shaun R; Marinier, Eric; Van Domselaar, Gary; Belk, Keith E; Morley, Paul S; McAllister, Tim A

2018-04-12

Developments in high-throughput next generation sequencing (NGS) technology have rapidly advanced the understanding of overall microbial ecology as well as occurrence and diversity of specific genes within diverse environments. In the present study, we compared the ability of varying sequencing depths to generate meaningful information about the taxonomic structure and prevalence of antimicrobial resistance genes (ARGs) in the bovine fecal microbial community. Metagenomic sequencing was conducted on eight composite fecal samples originating from four beef cattle feedlots. Metagenomic DNA was sequenced to various depths, D1, D0.5 and D0.25, with average sample read counts of 117, 59 and 26 million, respectively. A comparative analysis of the relative abundance of reads aligning to different phyla and antimicrobial classes indicated that the relative proportions of read assignments remained fairly constant regardless of depth. However, the number of reads being assigned to ARGs as well as to microbial taxa increased significantly with increasing depth. We found a depth of D0.5 was suitable to describe the microbiome and resistome of cattle fecal samples. This study helps define a balance between cost and required sequencing depth to acquire meaningful results.

Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

DOE PAGES

Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.; ...

2015-05-12

Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with anymore » transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.« less

Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.

Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with anymore » transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.« less

High‑throughput sequencing analyses of oral microbial diversity in healthy people and patients with dental caries and periodontal disease.

PubMed

Chen, Tingtao; Shi, Yan; Wang, Xiaolei; Wang, Xin; Meng, Fanjing; Yang, Shaoguo; Yang, Jian; Xin, Hongbo

2017-07-01

Recurrence of oral diseases caused by antibiotics has brought about an urgent requirement to explore the oral microbial diversity in the human oral cavity. In the present study, the high‑throughput sequencing method was adopted to compare the microbial diversity of healthy people and oral patients and sequence analysis was performed by UPARSE software package. The Venn results indicated that a mean of 315 operational taxonomic units (OTUs) was obtained, and 73, 64, 53, 19 and 18 common OTUs belonging to Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria and Fusobacteria, respectively, were identified in healthy people. Moreover, the reduction of Firmicutes and the increase of Proteobacteria in the children group, and the increase of Firmicutes and the reduction of Proteobacteria in the youth and adult groups, indicated that the age bracket and oral disease had largely influenced the tooth development and microbial development in the oral cavity. In addition, the traditional 'pathogenic bacteria' of Firmicutes, Proteobacteria and Bacteroidetes (accounted for >95% of the total sequencing number in each group) indicated that the 'harmful' bacteria may exert beneficial effects on oral health. Therefore, the data will provide certain clues for curing some oral diseases by the strategy of adjusting the disturbed microbial compositions in oral disease to healthy level.

Distribution and Diversity of Pathogenic Leptospira Species in Peri-domestic Surface Waters from South Central Chile.

PubMed

Mason, Meghan R; Encina, Carolina; Sreevatsan, Srinand; Muñoz-Zanzi, Claudia

2016-08-01

Leptospirosis is a neglected zoonosis affecting animals and humans caused by infection with Leptospira. The bacteria can survive outside of hosts for long periods of time in soil and water. While identification of Leptospira species from human cases and animal reservoirs are increasingly reported, little is known about the diversity of pathogenic Leptospira species in the environment and how surveillance of the environment might be used for monitoring and controlling disease. Water samples (n = 104) were collected from the peri-domestic environment of 422 households from farms, rural villages, and urban slums participating in a broader study on the eco-epidemiology of leptospirosis in the Los Rios Region, Chile, between October 2010 and April 2012. The secY region of samples, previously detected as pathogenic Leptospira by PCR, was amplified and sequenced. Sequences were aligned using ClustalW in MEGA, and a minimum spanning tree was created in PHYLOViZ using the goeBURST algorithm to assess sequence similarity. Sequences from four clinical isolates, 17 rodents, and 20 reference strains were also included in the analysis. Overall, water samples contained L. interrogans, L. kirschneri, and L. weilii, with descending frequency. All species were found in each community type. The distribution of the species differed by the season in which the water samples were obtained. There was no evidence that community-level prevalence of Leptospira in dogs, rodents, or livestock influenced pathogen diversity in the water samples. This study reports the presence of pathogenic Leptospira in the peri-domestic environment of households in three community types and the differences in Leptospira diversity at the community level. Systematic environmental surveillance of Leptospira can be used for detecting changes in pathogen diversity and to identify and monitor contaminated areas where an increased risk of human infection exists.

Structural diversity of domain superfamilies in the CATH database.

PubMed

Reeves, Gabrielle A; Dallman, Timothy J; Redfern, Oliver C; Akpor, Adrian; Orengo, Christine A

2006-07-14

The CATH database of domain structures has been used to explore the structural variation of homologous domains in 294 well populated domain structure superfamilies, each containing at least three sequence diverse relatives. Our analyses confirm some previously detected trends relating sequence divergence to structural variation but for a much larger dataset and in some superfamilies the new data reveal exceptional structural variation. Use of a new algorithm (2DSEC) to analyse variability in secondary structure compositions across a superfamily sheds new light on how structures evolve. 2DSEC detects inserted secondary structures that embellish the core of conserved secondary structures found throughout the superfamily. Analysis showed that for 56% of highly populated superfamilies (>9 sequence diverse relatives), there are twofold or more increases in the numbers of secondary structures in some relatives. In some families fivefold increases occur, sometimes modifying the fold of the domain. Manual inspection of secondary structure insertions or embellishments in 48 particularly variable superfamilies revealed that although these insertions were usually discontiguous in the sequence they were often co-located in 3D resulting in a larger structural motif that often modified the geometry of the active site or the surface conformation promoting diverse domain partnerships and protein interactions. These observations, supported by automatic analysis of all well populated CATH families, suggest that accretion of small secondary structure insertions may provide a simple mechanism for evolving new functions in diverse relatives. Some layered domain architectures (e.g. mainly-beta and alpha-beta sandwiches) that recur highly in the genomes more frequently exploit these types of embellishments to modify function. In these architectures, aggregation occurs most often at the edges, top or bottom of the beta-sheets. Information on structural variability across domain superfamilies has been made available through the CATH Dictionary of Homologous Structures (DHS).

PuLSE: Quality control and quantification of peptide sequences explored by phage display libraries.

PubMed

Shave, Steven; Mann, Stefan; Koszela, Joanna; Kerr, Alastair; Auer, Manfred

2018-01-01

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use.

Microbial eukaryote diversity in the marine oxygen minimum zone off northern Chile.

PubMed

Parris, Darren J; Ganesh, Sangita; Edgcomb, Virginia P; DeLong, Edward F; Stewart, Frank J

2014-01-01

Molecular surveys are revealing diverse eukaryotic assemblages in oxygen-limited ocean waters. These communities may play pivotal ecological roles through autotrophy, feeding, and a wide range of symbiotic associations with prokaryotes. We used 18S rRNA gene sequencing to provide the first snapshot of pelagic microeukaryotic community structure in two cellular size fractions (0.2-1.6 μm, >1.6 μm) from seven depths through the anoxic oxygen minimum zone (OMZ) off northern Chile. Sequencing of >154,000 amplicons revealed contrasting patterns of phylogenetic diversity across size fractions and depths. Protist and total eukaryote diversity in the >1.6 μm fraction peaked at the chlorophyll maximum in the upper photic zone before declining by ~50% in the OMZ. In contrast, diversity in the 0.2-1.6 μm fraction, though also elevated in the upper photic zone, increased four-fold from the lower oxycline to a maximum at the anoxic OMZ core. Dinoflagellates of the Dinophyceae and endosymbiotic Syndiniales clades dominated the protist assemblage at all depths (~40-70% of sequences). Other protist groups varied with depth, with the anoxic zone community of the larger size fraction enriched in euglenozoan flagellates and acantharean radiolarians (up to 18 and 40% of all sequences, respectively). The OMZ 0.2-1.6 μm fraction was dominated (11-99%) by Syndiniales, which exhibited depth-specific variation in composition and total richness despite uniform oxygen conditions. Metazoan sequences, though confined primarily to the 1.6 μm fraction above the OMZ, were also detected within the anoxic zone where groups such as copepods increased in abundance relative to the oxycline and upper OMZ. These data, compared to those from other low-oxygen sites, reveal variation in OMZ microeukaryote composition, helping to identify clades with potential adaptations to oxygen-depletion.

Microbial eukaryote diversity in the marine oxygen minimum zone off northern Chile

PubMed Central

Parris, Darren J.; Ganesh, Sangita; Edgcomb, Virginia P.; DeLong, Edward F.; Stewart, Frank J.

2014-01-01

Molecular surveys are revealing diverse eukaryotic assemblages in oxygen-limited ocean waters. These communities may play pivotal ecological roles through autotrophy, feeding, and a wide range of symbiotic associations with prokaryotes. We used 18S rRNA gene sequencing to provide the first snapshot of pelagic microeukaryotic community structure in two cellular size fractions (0.2–1.6 μm, >1.6 μm) from seven depths through the anoxic oxygen minimum zone (OMZ) off northern Chile. Sequencing of >154,000 amplicons revealed contrasting patterns of phylogenetic diversity across size fractions and depths. Protist and total eukaryote diversity in the >1.6 μm fraction peaked at the chlorophyll maximum in the upper photic zone before declining by ~50% in the OMZ. In contrast, diversity in the 0.2–1.6 μm fraction, though also elevated in the upper photic zone, increased four-fold from the lower oxycline to a maximum at the anoxic OMZ core. Dinoflagellates of the Dinophyceae and endosymbiotic Syndiniales clades dominated the protist assemblage at all depths (~40–70% of sequences). Other protist groups varied with depth, with the anoxic zone community of the larger size fraction enriched in euglenozoan flagellates and acantharean radiolarians (up to 18 and 40% of all sequences, respectively). The OMZ 0.2–1.6 μm fraction was dominated (11–99%) by Syndiniales, which exhibited depth-specific variation in composition and total richness despite uniform oxygen conditions. Metazoan sequences, though confined primarily to the 1.6 μm fraction above the OMZ, were also detected within the anoxic zone where groups such as copepods increased in abundance relative to the oxycline and upper OMZ. These data, compared to those from other low-oxygen sites, reveal variation in OMZ microeukaryote composition, helping to identify clades with potential adaptations to oxygen-depletion. PMID:25389417

Single sea urchin phagocytes express messages of a single sequence from the diverse Sp185/333 gene family in response to bacterial challenge.

PubMed

Majeske, Audrey J; Oren, Matan; Sacchi, Sandro; Smith, L Courtney

2014-12-01

Immune systems in animals rely on fast and efficient responses to a wide variety of pathogens. The Sp185/333 gene family in the purple sea urchin, Strongylocentrotus purpuratus, consists of an estimated 50 (±10) members per genome that share a basic gene structure but show high sequence diversity, primarily due to the mosaic appearance of short blocks of sequence called elements. The genes show significantly elevated expression in three subpopulations of phagocytes responding to marine bacteria. The encoded Sp185/333 proteins are highly diverse and have central effector functions in the immune system. In this study we report the Sp185/333 gene expression in single sea urchin phagocytes. Sea urchins challenged with heat-killed marine bacteria resulted in a typical increase in coelomocyte concentration within 24 h, which included an increased proportion of phagocytes expressing Sp185/333 proteins. Phagocyte fractions enriched from coelomocytes were used in limiting dilutions to obtain samples of single cells that were evaluated for Sp185/333 gene expression by nested RT-PCR. Amplicon sequences showed identical or nearly identical Sp185/333 amplicon sequences in single phagocytes with matches to six known Sp185/333 element patterns, including both common and rare element patterns. This suggested that single phagocytes show restricted expression from the Sp185/333 gene family and infers a diverse, flexible, and efficient response to pathogens. This type of expression pattern from a family of immune response genes in single cells has not been identified previously in other invertebrates. Copyright © 2014 by The American Association of Immunologists, Inc.

Evolutionary trajectory of Pack-MULEs is determined by their epigenetic status

USDA-ARS?s Scientific Manuscript database

Acquisition and rearrangement of host genes by transposable elements is one mechanism to increase gene diversity. The rice genome is replete in such sequences and while ~3,000 Pack- Mutator-like transposable elements containing gene sequences (Pack-MULEs) have been identified, their function remains...

Trophic and Non-Trophic Interactions in a Biodiversity Experiment Assessed by Next-Generation Sequencing

PubMed Central

Tiede, Julia; Wemheuer, Bernd; Traugott, Michael; Daniel, Rolf; Tscharntke, Teja; Ebeling, Anne; Scherber, Christoph

2016-01-01

Plant diversity affects species richness and abundance of taxa at higher trophic levels. However, plant diversity effects on omnivores (feeding on multiple trophic levels) and their trophic and non-trophic interactions are not yet studied because appropriate methods were lacking. A promising approach is the DNA-based analysis of gut contents using next generation sequencing (NGS) technologies. Here, we integrate NGS-based analysis into the framework of a biodiversity experiment where plant taxonomic and functional diversity were manipulated to directly assess environmental interactions involving the omnivorous ground beetle Pterostichus melanarius. Beetle regurgitates were used for NGS-based analysis with universal 18S rDNA primers for eukaryotes. We detected a wide range of taxa with the NGS approach in regurgitates, including organisms representing trophic, phoretic, parasitic, and neutral interactions with P. melanarius. Our findings suggest that the frequency of (i) trophic interactions increased with plant diversity and vegetation cover; (ii) intraguild predation increased with vegetation cover, and (iii) neutral interactions with organisms such as fungi and protists increased with vegetation cover. Experimentally manipulated plant diversity likely affects multitrophic interactions involving omnivorous consumers. Our study therefore shows that trophic and non-trophic interactions can be assessed via NGS to address fundamental questions in biodiversity research. PMID:26859146

Genome-wide diversity and association mapping for capsaicinoids and fruit weight in Capsicum annuum L

USDA-ARS?s Scientific Manuscript database

Accumulated capsaicinoid content and increased fruit size are traits resulting from Capsicum annuum domestication. In this study, we used a diverse collection of domesticated and wild C. annuum to generate 66,960 SNPs using genotyping by sequencing. Principal component analysis and identity by state...

Genomic distribution and estimation of nucleotide diversity in natural populations: perspectives from the collared flycatcher (Ficedula albicollis) genome.

PubMed

Dutoit, Ludovic; Burri, Reto; Nater, Alexander; Mugal, Carina F; Ellegren, Hans

2017-07-01

Properly estimating genetic diversity in populations of nonmodel species requires a basic understanding of how diversity is distributed across the genome and among individuals. To this end, we analysed whole-genome resequencing data from 20 collared flycatchers (genome size ≈1.1 Gb; 10.13 million single nucleotide polymorphisms detected). Genomewide nucleotide diversity was almost identical among individuals (mean = 0.00394, range = 0.00384-0.00401), but diversity levels varied extensively across the genome (95% confidence interval for 200-kb windows = 0.0013-0.0053). Diversity was related to selective constraint such that in comparison with intergenic DNA, diversity at fourfold degenerate sites was reduced to 85%, 3' UTRs to 82%, 5' UTRs to 70% and nondegenerate sites to 12%. There was a strong positive correlation between diversity and chromosome size, probably driven by a higher density of targets for selection on smaller chromosomes increasing the diversity-reducing effect of linked selection. Simulations exploring the ability of sequence data from a small number of genetic markers to capture the observed diversity clearly demonstrated that diversity estimation from finite sampling of such data is bound to be associated with large confidence intervals. Nevertheless, we show that precision in diversity estimation in large outbred population benefits from increasing the number of loci rather than the number of individuals. Simulations mimicking RAD sequencing showed that this approach gives accurate estimates of genomewide diversity. Based on the patterns of observed diversity and the performed simulations, we provide broad recommendations for how genetic diversity should be estimated in natural populations. © 2016 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

Rhizobial characterization in revegetated areas after bauxite mining.

PubMed

Borges, Wardsson Lustrino; Prin, Yves; Ducousso, Marc; Le Roux, Christine; de Faria, Sergio Miana

2016-01-01

Little is known regarding how the increased diversity of nitrogen-fixing bacteria contributes to the productivity and diversity of plants in complex communities. However, some authors have shown that the presence of a diverse group of nodulating bacteria is required for different plant species to coexist. A better understanding of the plant symbiotic organism diversity role in natural ecosystems can be extremely useful to define recovery strategies of environments that were degraded by human activities. This study used ARDRA, BOX-PCR fingerprinting and sequencing of the 16S rDNA gene to assess the diversity of root nodule nitrogen-fixing bacteria in former bauxite mining areas that were replanted in 1981, 1985, 1993, 1998, 2004 and 2006 and in a native forest. Among the 12 isolates for which the 16S rDNA gene was partially sequenced, eight, three and one isolate(s) presented similarity with sequences of the genera Bradyrhizobium, Rhizobium and Mesorhizobium, respectively. The richness, Shannon and evenness indices were the highest in the area that was replanted the earliest (1981) and the lowest in the area that was replanted most recently (2006). Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Viral quasispecies inference from 454 pyrosequencing

PubMed Central

2013-01-01

Background Many potentially life-threatening infectious viruses are highly mutable in nature. Characterizing the fittest variants within a quasispecies from infected patients is expected to allow unprecedented opportunities to investigate the relationship between quasispecies diversity and disease epidemiology. The advent of next-generation sequencing technologies has allowed the study of virus diversity with high-throughput sequencing, although these methods come with higher rates of errors which can artificially increase diversity. Results Here we introduce a novel computational approach that incorporates base quality scores from next-generation sequencers for reconstructing viral genome sequences that simultaneously infers the number of variants within a quasispecies that are present. Comparisons on simulated and clinical data on dengue virus suggest that the novel approach provides a more accurate inference of the underlying number of variants within the quasispecies, which is vital for clinical efforts in mapping the within-host viral diversity. Sequence alignments generated by our approach are also found to exhibit lower rates of error. Conclusions The ability to infer the viral quasispecies colony that is present within a human host provides the potential for a more accurate classification of the viral phenotype. Understanding the genomics of viruses will be relevant not just to studying how to control or even eradicate these viral infectious diseases, but also in learning about the innate protection in the human host against the viruses. PMID:24308284

Effort versus reward: preparing samples for fungal community characterization in high-throughput sequencing surveys of soils

USDA-ARS?s Scientific Manuscript database

Next generation fungal amplicon sequencing is being used with increasing frequency to study fungal diversity in various ecosystems; however, the influence of sample preparation on the characterization of fungal community is poorly understood. We investigated the effects of four procedural modificati...

DISSECTING THE QUASAR MAIN SEQUENCE: INSIGHT FROM HOST GALAXY PROPERTIES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Jiayi; Shen, Yue

2015-05-01

The diverse properties of broad-line quasars appear to follow a well-defined main sequence along which the optical Fe ii strength increases. It has been suggested that this sequence is mainly driven by the Eddington ratio (L/L{sub Edd}) of the black hole (BH) accretion. Shen and Ho demonstrated with quasar clustering analysis that the average BH mass decreases with increasing Fe ii strength when quasar luminosity is fixed, consistent with this suggestion. Here we perform an independent test by measuring the stellar velocity dispersion σ{sub *} (hence, the BH mass via the M–σ{sub *} relation) from decomposed host spectra in low-redshiftmore » Sloan Digital Sky Survey quasars. We found that at fixed quasar luminosity, σ{sub *} systematically decreases with increasing Fe ii strength, confirming that the Eddington ratio increases with Fe ii strength. We also found that at fixed luminosity and Fe ii strength, there is little dependence of σ{sub *} on the broad Hβ FWHM. These new results reinforce the framework that the Eddington ratio and orientation govern most of the diversity seen in broad-line quasar properties.« less

Genome-wide-analyses of Listeria monocytogenes from food-processing plants reveal clonal diversity and date the emergence of persisting sequence types.

PubMed

Knudsen, Gitte M; Nielsen, Jesper Boye; Marvig, Rasmus L; Ng, Yin; Worning, Peder; Westh, Henrik; Gram, Lone

2017-08-01

Whole genome sequencing is increasing used in epidemiology, e.g. for tracing outbreaks of food-borne diseases. This requires in-depth understanding of pathogen emergence, persistence and genomic diversity along the food production chain including in food processing plants. We sequenced the genomes of 80 isolates of Listeria monocytogenes sampled from Danish food processing plants over a time-period of 20 years, and analysed the sequences together with 10 public available reference genomes to advance our understanding of interplant and intraplant genomic diversity of L. monocytogenes. Except for three persisting sequence types (ST) based on Multi Locus Sequence Typing being ST7, ST8 and ST121, long-term persistence of clonal groups was limited, and new clones were introduced continuously, potentially from raw materials. No particular gene could be linked to the persistence phenotype. Using time-based phylogenetic analyses of the persistent STs, we estimate the L. monocytogenes evolutionary rate to be 0.18-0.35 single nucleotide polymorphisms/year, suggesting that the persistent STs emerged approximately 100 years ago, which correlates with the onset of industrialization and globalization of the food market. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Alterations of microbiota in urine from women with interstitial cystitis

PubMed Central

2012-01-01

Background Interstitial Cystitis (IC) is a chronic inflammatory condition of the bladder with unknown etiology. The aim of this study was to characterize the microbial community present in the urine from IC female patients by 454 high throughput sequencing of the 16S variable regions V1V2 and V6. The taxonomical composition, richness and diversity of the IC microbiota were determined and compared to the microbial profile of asymptomatic healthy female (HF) urine. Results The composition and distribution of bacterial sequences differed between the urine microbiota of IC patients and HFs. Reduced sequence richness and diversity were found in IC patient urine, and a significant difference in the community structure of IC urine in relation to HF urine was observed. More than 90% of the IC sequence reads were identified as belonging to the bacterial genus Lactobacillus, a marked increase compared to 60% in HF urine. Conclusion The 16S rDNA sequence data demonstrates a shift in the composition of the bacterial community in IC urine. The reduced microbial diversity and richness is accompanied by a higher abundance of the bacterial genus Lactobacillus, compared to HF urine. This study demonstrates that high throughput sequencing analysis of urine microbiota in IC patients is a powerful tool towards a better understanding of this enigmatic disease. PMID:22974186

Patterns of microbial diversity along a salinity gradient in the Guerrero Negro solar saltern, Baja CA Sur, Mexico

PubMed Central

Dillon, Jesse G.; Carlin, Mark; Gutierrez, Abraham; Nguyen, Vivian; McLain, Nathan

2013-01-01

The goal of this study was to use environmental sequencing of 16S rRNA and bop genes to compare the diversity of planktonic bacteria and archaea across ponds with increasing salinity in the Exportadora de Sal (ESSA) evaporative saltern in Guerrero Negro, Baja CA S., Mexico. We hypothesized that diverse communities of heterotrophic bacteria and archaea would be found in the ESSA ponds, but that bacterial diversity would decrease relative to archaea at the highest salinities. Archaeal 16S rRNA diversity was higher in Ponds 11 and 12 (370 and 380 g l−1 total salts, respectively) compared to Pond 9 (180 g l−1 total salts). Both Pond 11 and 12 communities had high representation (47 and 45% of clones, respectively) by Haloquadratum walsbyi-like (99% similarity) lineages. The archaeal community in Pond 9 was dominated (79%) by a single uncultured phylotype with 99% similarity to sequences recovered from the Sfax saltern in Tunisia. This pattern was mirrored in bop gene diversity with greater numbers of highly supported phylotypes including many Haloquadratum-like sequences from the two highest salinity ponds. In Pond 9, most bop sequences, were not closely related to sequences in databases. Bacterial 16S rRNA diversity was higher than archaeal in both Pond 9 and Pond 12 samples, but not Pond 11, where a non-Salinibacter lineage within the Bacteroidetes >98% similar to environmental clones recovered from Lake Tuz in Turkey and a saltern in Chula Vista, CA was most abundant (69% of community). This OTU was also the most abundant in Pond 12, but only represented 14% of clones in the more diverse pond. The most abundant OTU in Pond 9 (33% of community) was 99% similar to an uncultured gammaproteobacterial clone from the Salton Sea. Results suggest that the communities of saltern bacteria and archaea vary even in ponds with similar salinity and further investigation into the ecology of diverse, uncultured halophile communities is warranted. PMID:24391633

SNPGenie: estimating evolutionary parameters to detect natural selection using pooled next-generation sequencing data.

PubMed

Nelson, Chase W; Moncla, Louise H; Hughes, Austin L

2015-11-15

New applications of next-generation sequencing technologies use pools of DNA from multiple individuals to estimate population genetic parameters. However, no publicly available tools exist to analyse single-nucleotide polymorphism (SNP) calling results directly for evolutionary parameters important in detecting natural selection, including nucleotide diversity and gene diversity. We have developed SNPGenie to fill this gap. The user submits a FASTA reference sequence(s), a Gene Transfer Format (.GTF) file with CDS information and a SNP report(s) in an increasing selection of formats. The program estimates nucleotide diversity, distance from the reference and gene diversity. Sites are flagged for multiple overlapping reading frames, and are categorized by polymorphism type: nonsynonymous, synonymous, or ambiguous. The results allow single nucleotide, single codon, sliding window, whole gene and whole genome/population analyses that aid in the detection of positive and purifying natural selection in the source population. SNPGenie version 1.2 is a Perl program with no additional dependencies. It is free, open-source, and available for download at https://github.com/hugheslab/snpgenie. nelsoncw@email.sc.edu or austin@biol.sc.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The others: our biased perspective of eukaryotic genomes

PubMed Central

del Campo, Javier; Sieracki, Michael E.; Molestina, Robert; Keeling, Patrick; Massana, Ramon; Ruiz-Trillo, Iñaki

2015-01-01

Understanding the origin and evolution of the eukaryotic cell and the full diversity of eukaryotes is relevant to many biological disciplines. However, our current understanding of eukaryotic genomes is extremely biased, leading to a skewed view of eukaryotic biology. We argue that a phylogeny-driven initiative to cover the full eukaryotic diversity is needed to overcome this bias. We encourage the community: (i) to sequence a representative of the neglected groups available at public culture collections, (ii) to increase our culturing efforts, and (iii) to embrace single cell genomics to access organisms refractory to propagation in culture. We hope that the community will welcome this proposal, explore the approaches suggested, and join efforts to sequence the full diversity of eukaryotes. PMID:24726347

Toward an Understanding of Changes in Diversity Associated with Fecal Microbiome Transplantation Based on 16S rRNA Gene Deep Sequencing

PubMed Central

Shahinas, Dea; Silverman, Michael; Sittler, Taylor; Chiu, Charles; Kim, Peter; Allen-Vercoe, Emma; Weese, Scott; Wong, Andrew; Low, Donald E.; Pillai, Dylan R.

2012-01-01

ABSTRACT Fecal microbiome transplantation by low-volume enema is an effective, safe, and inexpensive alternative to antibiotic therapy for patients with chronic relapsing Clostridium difficile infection (CDI). We explored the microbial diversity of pre- and posttransplant stool specimens from CDI patients (n = 6) using deep sequencing of the 16S rRNA gene. While interindividual variability in microbiota change occurs with fecal transplantation and vancomycin exposure, in this pilot study we note that clinical cure of CDI is associated with an increase in diversity and richness. Genus- and species-level analysis may reveal a cocktail of microorganisms or products thereof that will ultimately be used as a probiotic to treat CDI. PMID:23093385

Antibiotics reduce genetic diversity of core species in the honeybee gut microbiome.

PubMed

Raymann, Kasie; Bobay, Louis-Marie; Moran, Nancy A

2018-04-01

The gut microbiome plays a key role in animal health, and perturbing it can have detrimental effects. One major source of perturbation to microbiomes, in humans and human-associated animals, is exposure to antibiotics. Most studies of how antibiotics affect the microbiome have used amplicon sequencing of highly conserved 16S rRNA sequences, as in a recent study showing that antibiotic treatment severely alters the species-level composition of the honeybee gut microbiome. But because the standard 16S rRNA-based methods cannot resolve closely related strains, strain-level changes could not be evaluated. To address this gap, we used amplicon sequencing of protein-coding genes to assess effects of antibiotics on fine-scale genetic diversity of the honeybee gut microbiota. We followed the population dynamics of alleles within two dominant core species of the bee gut community, Gilliamella apicola and Snodgrassella alvi, following antibiotic perturbation. Whereas we observed a large reduction in genetic diversity in G. apicola, S. alvi diversity was mostly unaffected. The reduction in G. apicola diversity accompanied an increase in the frequency of several alleles, suggesting resistance to antibiotic treatment. We find that antibiotic perturbation can cause major shifts in diversity and that the extent of these shifts can vary substantially across species. Thus, antibiotics impact not only species composition, but also allelic diversity within species, potentially affecting hosts if variants with particular functions are reduced or eliminated. Overall, we show that amplicon sequencing of protein-coding genes, without clustering into operational taxonomic units, provides an accurate picture of the fine-scale dynamics of microbial communities over time. © 2017 John Wiley & Sons Ltd.

Genome sequence analysis of five Canadian isolates of strawberry mottle virus reveals extensive intra-species diversity and a longer RNA2 with increased coding capacity compared to a previously characterized European isolate.

PubMed

Bhagwat, Basdeo; Dickison, Virginia; Ding, Xinlun; Walker, Melanie; Bernardy, Michael; Bouthillier, Michel; Creelman, Alexa; DeYoung, Robyn; Li, Yinzi; Nie, Xianzhou; Wang, Aiming; Xiang, Yu; Sanfaçon, Hélène

2016-06-01

In this study, we report the genome sequence of five isolates of strawberry mottle virus (family Secoviridae, order Picornavirales) from strawberry field samples with decline symptoms collected in Eastern Canada. The Canadian isolates differed from the previously characterized European isolate 1134 in that they had a longer RNA2, resulting in a 239-amino-acid extension of the C-terminal region of the polyprotein. Sequence analysis suggests that reassortment and recombination occurred among the isolates. Phylogenetic analysis revealed that the Canadian isolates are diverse, grouping in two separate branches along with isolates from Europe and the Americas.

Responses of the soil fungal communities to the co-invasion of two invasive species with different cover classes.

PubMed

Wang, C; Zhou, J; Liu, J; Jiang, K; Xiao, H; Du, D

2018-01-01

Soil fungal communities play an important role in the successful invasion of non-native species. It is common for two or more invasive plant species to co-occur in invaded ecosystems. This study aimed to determine the effects of co-invasion of two invasive species (Erigeron annuus and Solidago canadensis) with different cover classes on soil fungal communities using high-throughput sequencing. Invasion of E. annuus and/or S. canadensis had positive effects on the sequence number, operational taxonomic unit (OTU) richness, Shannon diversity, abundance-based cover estimator (ACE index) and Chao1 index of soil fungal communities, but negative effects on the Simpson index. Thus, invasion of E. annuus and/or S. canadensis could increase diversity and richness of soil fungal communities but decrease dominance of some members of these communities, in part to facilitate plant further invasion, because high soil microbial diversity could increase soil functions and plant nutrient acquisition. Some soil fungal species grow well, whereas others tend to extinction after non-native plant invasion with increasing invasion degree and presumably time. The sequence number, OTU richness, Shannon diversity, ACE index and Chao1 index of soil fungal communities were higher under co-invasion of E. annuus and S. canadensis than under independent invasion of either individual species. The co-invasion of the two invasive species had a positive synergistic effect on diversity and abundance of soil fungal communities, partly to build a soil microenvironment to enhance competitiveness of the invaders. The changed diversity and community under co-invasion could modify resource availability and niche differentiation within the soil fungal communities, mediated by differences in leaf litter quality and quantity, which can support different fungal/microbial species in the soil. © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.

Association of high-risk sexual behaviour with diversity of the vaginal microbiota and abundance of Lactobacillus

PubMed Central

Wessels, Jocelyn M.; Lajoie, Julie; Vitali, Danielle; Omollo, Kenneth; Kimani, Joshua; Oyugi, Julius; Cheruiyot, Juliana; Kimani, Makubo; Mungai, John N.; Akolo, Maureen; Stearns, Jennifer C.; Surette, Michael G.; Fowke, Keith R.

2017-01-01

Objective To compare the vaginal microbiota of women engaged in high-risk sexual behaviour (sex work) with women who are not engaged in high-risk sexual behaviour. Diverse vaginal microbiota, low in Lactobacillus species, like those in bacterial vaginosis (BV), are associated with increased prevalence of sexually transmitted infections (STIs) and human immunodeficiency virus (HIV) acquisition. Although high-risk sexual behaviour increases risk for STIs, the vaginal microbiota of sex workers is understudied. Methods A retrospective cross-sectional study was conducted comparing vaginal microbiota of women who are not engaged in sex work (non-sex worker controls, NSW, N = 19) and women engaged in sex work (female sex workers, FSW, N = 48), using Illumina sequencing (16S rRNA, V3 region). Results Bacterial richness and diversity were significantly less in controls, than FSW. Controls were more likely to have Lactobacillus as the most abundant genus (58% vs. 17%; P = 0.002) and composition of their vaginal microbiota differed from FSW (PERMANOVA, P = 0.001). Six microbiota clusters were detected, including a high diversity cluster with three sub-clusters, and 55% of women with low Nugent Scores fell within this cluster. High diversity was observed by 16S sequencing in FSW, regardless of Nugent Scores, suggesting that Nugent Score may not be capable of capturing the diversity present in the FSW vaginal microbiota. Conclusions High-risk sexual behaviour is associated with diversity of the vaginal microbiota and lack of Lactobacillus. These factors could contribute to increased risk of STIs and HIV in women engaged in high-risk sexual behaviour. PMID:29095928

Association of high-risk sexual behaviour with diversity of the vaginal microbiota and abundance of Lactobacillus.

PubMed

Wessels, Jocelyn M; Lajoie, Julie; Vitali, Danielle; Omollo, Kenneth; Kimani, Joshua; Oyugi, Julius; Cheruiyot, Juliana; Kimani, Makubo; Mungai, John N; Akolo, Maureen; Stearns, Jennifer C; Surette, Michael G; Fowke, Keith R; Kaushic, Charu

2017-01-01

To compare the vaginal microbiota of women engaged in high-risk sexual behaviour (sex work) with women who are not engaged in high-risk sexual behaviour. Diverse vaginal microbiota, low in Lactobacillus species, like those in bacterial vaginosis (BV), are associated with increased prevalence of sexually transmitted infections (STIs) and human immunodeficiency virus (HIV) acquisition. Although high-risk sexual behaviour increases risk for STIs, the vaginal microbiota of sex workers is understudied. A retrospective cross-sectional study was conducted comparing vaginal microbiota of women who are not engaged in sex work (non-sex worker controls, NSW, N = 19) and women engaged in sex work (female sex workers, FSW, N = 48), using Illumina sequencing (16S rRNA, V3 region). Bacterial richness and diversity were significantly less in controls, than FSW. Controls were more likely to have Lactobacillus as the most abundant genus (58% vs. 17%; P = 0.002) and composition of their vaginal microbiota differed from FSW (PERMANOVA, P = 0.001). Six microbiota clusters were detected, including a high diversity cluster with three sub-clusters, and 55% of women with low Nugent Scores fell within this cluster. High diversity was observed by 16S sequencing in FSW, regardless of Nugent Scores, suggesting that Nugent Score may not be capable of capturing the diversity present in the FSW vaginal microbiota. High-risk sexual behaviour is associated with diversity of the vaginal microbiota and lack of Lactobacillus. These factors could contribute to increased risk of STIs and HIV in women engaged in high-risk sexual behaviour.

Diversity of Basidiomycetes in Michigan Agricultural Soils▿

PubMed Central

Lynch, Michael D. J.; Thorn, R. Greg

2006-01-01

We analyzed the communities of soil basidiomycetes in agroecosystems that differ in tillage history at the Kellogg Biological Station Long-Term Ecological Research site near Battle Creek, Michigan. The approach combined soil DNA extraction through a bead-beating method modified to increase recovery of fungal DNA, PCR amplification with basidiomycete-specific primers, cloning and restriction fragment length polymorphism screening of mixed PCR products, and sequencing of unique clones. Much greater diversity was detected than was anticipated in this habitat on the basis of culture-based methods or surveys of fruiting bodies. With “species” defined as organisms yielding PCR products with ≥99% identity in the 5′ 650 bases of the nuclear large-subunit ribosomal DNA, 241 “species” were detected among 409 unique basidiomycete sequences recovered. Almost all major clades of basidiomycetes from basidiomycetous yeasts and other heterobasidiomycetes through polypores and euagarics (gilled mushrooms and relatives) were represented, with a majority from the latter clade. Only 24 of 241 “species” had 99% or greater sequence similarity to named reference sequences in GenBank, and several clades with multiple “species” could not be identified at the genus level by phylogenetic comparisons with named sequences. The total estimated “species” richness for this 11.2-ha site was 367 “species” of basidiomycetes. Since >99% of the study area has not been sampled, the accuracy of our diversity estimate is uncertain. Replication in time and space is required to detect additional diversity and the underlying community structure. PMID:16950900

Archaeal and bacterial diversity in two hot springs from geothermal regions in Bulgaria as demostrated by 16S rRNA and GH-57 genes.

PubMed

Stefanova, Katerina; Tomova, Iva; Tomova, Anna; Radchenkova, Nadja; Atanassov, Ivan; Kambourova, Margarita

2015-12-01

Archaeal and bacterial diversity in two Bulgarian hot springs, geographically separated with different tectonic origin and different temperature of water was investigated exploring two genes, 16S rRNA and GH-57. Archaeal diversity was significantly higher in the hotter spring Levunovo (LV) (82°C); on the contrary, bacterial diversity was higher in the spring Vetren Dol (VD) (68°C). The analyzed clones from LV library were referred to twenty eight different sequence types belonging to five archaeal groups from Crenarchaeota and Euryarchaeota. A domination of two groups was observed, Candidate Thaumarchaeota and Methanosarcinales. The majority of the clones from VD were referred to HWCG (Hot Water Crenarchaeotic Group). The formation of a group of thermophiles in the order Methanosarcinales was suggested. Phylogenetic analysis revealed high numbers of novel sequences, more than one third of archaeal and half of the bacterial phylotypes displayed similarity lower than 97% with known ones. The retrieved GH-57 gene sequences showed a complex phylogenic distribution. The main part of the retrieved homologous GH-57 sequences affiliated with bacterial phyla Bacteroidetes, Deltaproteobacteria, Candidate Saccharibacteria and affiliation of almost half of the analyzed sequences is not fully resolved. GH-57 gene analysis allows an increased resolution of the biodiversity assessment and in depth analysis of specific taxonomic groups. [Int Microbiol 18(4):217-223 (2015)]. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

High bacterial diversity of biological soil crusts in water tracks over permafrost in the high arctic polar desert.

PubMed

Steven, Blaire; Lionard, Marie; Kuske, Cheryl R; Vincent, Warwick F

2013-01-01

In this study we report the bacterial diversity of biological soil crusts (biocrusts) inhabiting polar desert soils at the northern land limit of the Arctic polar region (83° 05 N). Employing pyrosequencing of bacterial 16S rRNA genes this study demonstrated that these biocrusts harbor diverse bacterial communities, often as diverse as temperate latitude communities. The effect of wetting pulses on the composition of communities was also determined by collecting samples from soils outside and inside of permafrost water tracks, hill slope flow paths that drain permafrost-affected soils. The intermittent flow regime in the water tracks was correlated with altered relative abundance of phylum level taxonomic bins in the bacterial communities, but the alterations varied between individual sampling sites. Bacteria related to the Cyanobacteria and Acidobacteria demonstrated shifts in relative abundance based on their location either inside or outside of the water tracks. Among cyanobacterial sequences, the proportion of sequences belonging to the family Oscillatoriales consistently increased in relative abundance in the samples from inside the water tracks compared to those outside. Acidobacteria showed responses to wetting pulses in the water tracks, increasing in abundance at one site and decreasing at the other two sites. Subdivision 4 acidobacterial sequences tended to follow the trends in the total Acidobacteria relative abundance, suggesting these organisms were largely responsible for the changes observed in the Acidobacteria. Taken together, these data suggest that the bacterial communities of these high latitude polar biocrusts are diverse but do not show a consensus response to intermittent flow in water tracks over high Arctic permafrost.

Endophyte Microbiome Diversity in Micropropagated Atriplex canescens and Atriplex torreyi var griffithsii

PubMed Central

Lucero, Mary E.; Unc, Adrian; Cooke, Peter; Dowd, Scot; Sun, Shulei

2011-01-01

Microbial diversity associated with micropropagated Atriplex species was assessed using microscopy, isolate culturing, and sequencing. Light, electron, and confocal microscopy revealed microbial cells in aseptically regenerated leaves and roots. Clone libraries and tag-encoded FLX amplicon pyrosequencing (TEFAP) analysis amplified sequences from callus homologous to diverse fungal and bacterial taxa. Culturing isolated some seed borne endophyte taxa which could be readily propagated apart from the host. Microbial cells were observed within biofilm-like residues associated with plant cell surfaces and intercellular spaces. Various universal primers amplified both plant and microbial sequences, with different primers revealing different patterns of fungal diversity. Bacterial and fungal TEFAP followed by alignment with sequences from curated databases revealed 7 bacterial and 17 ascomycete taxa in A. canescens, and 5 bacterial taxa in A. torreyi. Additional diversity was observed among isolates and clone libraries. Micropropagated Atriplex retains a complex, intimately associated microbiome which includes diverse strains well poised to interact in manners that influence host physiology. Microbiome analysis was facilitated by high throughput sequencing methods, but primer biases continue to limit recovery of diverse sequences from even moderately complex communities. PMID:21437280

A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules

PubMed Central

Popova, Blagovesta; Schubert, Steffen; Bulla, Ingo; Buchwald, Daniela; Kramer, Wilfried

2015-01-01

A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis. PMID:26355961

A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules.

PubMed

Popova, Blagovesta; Schubert, Steffen; Bulla, Ingo; Buchwald, Daniela; Kramer, Wilfried

2015-01-01

A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis.

Unique transposon landscapes are pervasive across Drosophila melanogaster genomes

PubMed Central

Rahman, Reazur; Chirn, Gung-wei; Kanodia, Abhay; Sytnikova, Yuliya A.; Brembs, Björn; Bergman, Casey M.; Lau, Nelson C.

2015-01-01

To understand how transposon landscapes (TLs) vary across animal genomes, we describe a new method called the Transposon Insertion and Depletion AnaLyzer (TIDAL) and a database of >300 TLs in Drosophila melanogaster (TIDAL-Fly). Our analysis reveals pervasive TL diversity across cell lines and fly strains, even for identically named sub-strains from different laboratories such as the ISO1 strain used for the reference genome sequence. On average, >500 novel insertions exist in every lab strain, inbred strains of the Drosophila Genetic Reference Panel (DGRP), and fly isolates in the Drosophila Genome Nexus (DGN). A minority (<25%) of transposon families comprise the majority (>70%) of TL diversity across fly strains. A sharp contrast between insertion and depletion patterns indicates that many transposons are unique to the ISO1 reference genome sequence. Although TL diversity from fly strains reaches asymptotic limits with increasing sequencing depth, rampant TL diversity causes unsaturated detection of TLs in pools of flies. Finally, we show novel transposon insertions negatively correlate with Piwi-interacting RNA (piRNA) levels for most transposon families, except for the highly-abundant roo retrotransposon. Our study provides a useful resource for Drosophila geneticists to understand how transposons create extensive genomic diversity in fly cell lines and strains. PMID:26578579

Molecular sequence typing reveals genotypic diversity among Escherichia coli isolates recovered from a cantaloupe packinghouse in Northwestern Mexico

USDA-ARS?s Scientific Manuscript database

The increase in the consumption of fresh produce in the United States has correlated with a rise in the number of reported foodborne illnesses. To identify potential risk factors associated with post-harvest practices, the present study employed multilocus sequence typing (MLST) for the genotypic c...

Distribution and Diversity of Pathogenic Leptospira Species in Peri-domestic Surface Waters from South Central Chile

PubMed Central

Mason, Meghan R.; Encina, Carolina; Sreevatsan, Srinand; Muñoz-Zanzi, Claudia

2016-01-01

Background Leptospirosis is a neglected zoonosis affecting animals and humans caused by infection with Leptospira. The bacteria can survive outside of hosts for long periods of time in soil and water. While identification of Leptospira species from human cases and animal reservoirs are increasingly reported, little is known about the diversity of pathogenic Leptospira species in the environment and how surveillance of the environment might be used for monitoring and controlling disease. Methods and Findings Water samples (n = 104) were collected from the peri-domestic environment of 422 households from farms, rural villages, and urban slums participating in a broader study on the eco-epidemiology of leptospirosis in the Los Rios Region, Chile, between October 2010 and April 2012. The secY region of samples, previously detected as pathogenic Leptospira by PCR, was amplified and sequenced. Sequences were aligned using ClustalW in MEGA, and a minimum spanning tree was created in PHYLOViZ using the goeBURST algorithm to assess sequence similarity. Sequences from four clinical isolates, 17 rodents, and 20 reference strains were also included in the analysis. Overall, water samples contained L. interrogans, L. kirschneri, and L. weilii, with descending frequency. All species were found in each community type. The distribution of the species differed by the season in which the water samples were obtained. There was no evidence that community-level prevalence of Leptospira in dogs, rodents, or livestock influenced pathogen diversity in the water samples. Conclusions This study reports the presence of pathogenic Leptospira in the peri-domestic environment of households in three community types and the differences in Leptospira diversity at the community level. Systematic environmental surveillance of Leptospira can be used for detecting changes in pathogen diversity and to identify and monitor contaminated areas where an increased risk of human infection exists. PMID:27529550

Resource availability controls fungal diversity across a plant diversity gradient

USGS Publications Warehouse

Waldrop, M.P.; Zak, D.R.; Blackwood, C.B.; Curtis, C.D.; Tilman, D.

2006-01-01

Despite decades of research, the ecological determinants of microbial diversity remain poorly understood. Here, we test two alternative hypotheses concerning the factors regulating fungal diversity in soil. The first states that higher levels of plant detritus production increase the supply of limiting resources (i.e. organic substrates) thereby increasing fungal diversity. Alternatively, greater plant diversity increases the range of organic substrates entering soil, thereby increasing the number of niches to be filled by a greater array of heterotrophic fungi. These two hypotheses were simultaneously examined in experimental plant communities consisting of one to 16 species that have been maintained for a decade. We used ribosomal intergenic spacer analysis (RISA), in combination with cloning and sequencing, to quantify fungal community composition and diversity within the experimental plant communities. We used soil microbial biomass as a temporally integrated measure of resource supply. Plant diversity was unrelated to fungal diversity, but fungal diversity was a unimodal function of resource supply. Canonical correspondence analysis (CCA) indicated that plant diversity showed a relationship to fungal community composition, although the occurrence of RISA bands and operational taxonomic units (OTUs) did not differ among the treatments. The relationship between fungal diversity and resource availability parallels similar relationships reported for grasslands, tropical forests, coral reefs, and other biotic communities, strongly suggesting that the same underlying mechanisms determine the diversity of organisms at multiple scales. ?? 2006 Blackwell Publishing Ltd/CNRS.

First insight into dead wood protistan diversity: a molecular sampling of bright-spored Myxomycetes (Amoebozoa, slime-moulds) in decaying beech logs.

PubMed

Clissmann, Fionn; Fiore-Donno, Anna Maria; Hoppe, Björn; Krüger, Dirk; Kahl, Tiemo; Unterseher, Martin; Schnittler, Martin

2015-06-01

Decaying wood hosts a large diversity of seldom investigated protists. Environmental sequencing offers novel insights into communities, but has rarely been applied to saproxylic protists. We investigated the diversity of bright-spored wood-inhabiting Myxomycetes by environmental sequencing. Myxomycetes have a complex life cycle culminating in the formation of mainly macroscopic fruiting bodies, highly variable in shape and colour that are often found on decaying logs. Our hypothesis was that diversity of bright-spored Myxomycetes would increase with decay. DNA was extracted from wood chips collected from 17 beech logs of varying decay stages from the Hainich-Dün region in Central Germany. We obtained 260 partial small subunit ribosomal RNA gene sequences of bright-spored Myxomycetes that were assembled into 29 OTUs, of which 65% were less than 98% similar to those in the existing database. The OTU richness revealed by molecular analysis surpassed that of a parallel inventory of fruiting bodies. We tested several environmental variables and identified pH, rather than decay stage, as the main structuring factor of myxomycete distribution. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

How many novel eukaryotic 'kingdoms'? Pitfalls and limitations of environmental DNA surveys

PubMed Central

Berney, Cédric; Fahrni, José; Pawlowski, Jan

2004-01-01

Background Over the past few years, the use of molecular techniques to detect cultivation-independent, eukaryotic diversity has proven to be a powerful approach. Based on small-subunit ribosomal RNA (SSU rRNA) gene analyses, these studies have revealed the existence of an unexpected variety of new phylotypes. Some of them represent novel diversity in known eukaryotic groups, mainly stramenopiles and alveolates. Others do not seem to be related to any molecularly described lineage, and have been proposed to represent novel eukaryotic kingdoms. In order to review the evolutionary importance of this novel high-level eukaryotic diversity critically, and to test the potential technical and analytical pitfalls and limitations of eukaryotic environmental DNA surveys (EES), we analysed 484 environmental SSU rRNA gene sequences, including 81 new sequences from sediments of the small river, the Seymaz (Geneva, Switzerland). Results Based on a detailed screening of an exhaustive alignment of eukaryotic SSU rRNA gene sequences and the phylogenetic re-analysis of previously published environmental sequences using Bayesian methods, our results suggest that the number of novel higher-level taxa revealed by previously published EES was overestimated. Three main sources of errors are responsible for this situation: (1) the presence of undetected chimeric sequences; (2) the misplacement of several fast-evolving sequences; and (3) the incomplete sampling of described, but yet unsequenced eukaryotes. Additionally, EES give a biased view of the diversity present in a given biotope because of the difficult amplification of SSU rRNA genes in some taxonomic groups. Conclusions Environmental DNA surveys undoubtedly contribute to reveal many novel eukaryotic lineages, but there is no clear evidence for a spectacular increase of the diversity at the kingdom level. After re-analysis of previously published data, we found only five candidate lineages of possible novel high-level eukaryotic taxa, two of which comprise several phylotypes that were found independently in different studies. To ascertain their taxonomic status, however, the organisms themselves have now to be identified. PMID:15176975

Sensitive Next-Generation Sequencing Method Reveals Deep Genetic Diversity of HIV-1 in the Democratic Republic of the Congo.

PubMed

Rodgers, Mary A; Wilkinson, Eduan; Vallari, Ana; McArthur, Carole; Sthreshley, Larry; Brennan, Catherine A; Cloherty, Gavin; de Oliveira, Tulio

2017-03-15

As the epidemiological epicenter of the human immunodeficiency virus (HIV) pandemic, the Democratic Republic of the Congo (DRC) is a reservoir of circulating HIV strains exhibiting high levels of diversity and recombination. In this study, we characterized HIV specimens collected in two rural areas of the DRC between 2001 and 2003 to identify rare strains of HIV. The env gp41 region was sequenced and characterized for 172 HIV-positive specimens. The env sequences were predominantly subtype A (43.02%), but 7 other subtypes (33.14%), 20 circulating recombinant forms (CRFs; 11.63%), and 20 unclassified (11.63%) sequences were also found. Of the rare and unclassified subtypes, 18 specimens were selected for next-generation sequencing (NGS) by a modified HIV-switching mechanism at the 5' end of the RNA template (SMART) method to obtain full-genome sequences. NGS produced 14 new complete genomes, which included pure subtype C ( n = 2), D ( n = 1), F1 ( n = 1), H ( n = 3), and J ( n = 1) genomes. The two subtype C genomes and one of the subtype H genomes branched basal to their respective subtype branches but had no evidence of recombination. The remaining 6 genomes were complex recombinants of 2 or more subtypes, including subtypes A1, F, G, H, J, and K and unclassified fragments, including one subtype CRF25 isolate, which branched basal to all CRF25 references. Notably, all recombinant subtype H fragments branched basal to the H clade. Spatial-geographical analysis indicated that the diverse sequences identified here did not expand globally. The full-genome and subgenomic sequences identified in our study population significantly increase the documented diversity of the strains involved in the continually evolving HIV-1 pandemic. IMPORTANCE Very little is known about the ancestral HIV-1 strains that founded the global pandemic, and very few complete genome sequences are available from patients in the Congo Basin, where HIV-1 expanded early in the global pandemic. By sequencing a subgenomic fragment of the HIV-1 envelope from study participants in the DRC, we identified rare variants for complete genome sequencing. The basal branching of some of the complete genome sequences that we recovered suggests that these strains are more closely related to ancestral HIV-1 strains than to previously reported strains and is evidence that the local diversification of HIV in the DRC continues to outpace the diversity of global strains decades after the emergence of the pandemic. Copyright © 2017 Rodgers et al.

Mapping membrane activity in undiscovered peptide sequence space using machine learning

PubMed Central

Fulan, Benjamin M.; Wong, Gerard C. L.

2016-01-01

There are some ∼1,100 known antimicrobial peptides (AMPs), which permeabilize microbial membranes but have diverse sequences. Here, we develop a support vector machine (SVM)-based classifier to investigate ⍺-helical AMPs and the interrelated nature of their functional commonality and sequence homology. SVM is used to search the undiscovered peptide sequence space and identify Pareto-optimal candidates that simultaneously maximize the distance σ from the SVM hyperplane (thus maximize its “antimicrobialness”) and its ⍺-helicity, but minimize mutational distance to known AMPs. By calibrating SVM machine learning results with killing assays and small-angle X-ray scattering (SAXS), we find that the SVM metric σ correlates not with a peptide’s minimum inhibitory concentration (MIC), but rather its ability to generate negative Gaussian membrane curvature. This surprising result provides a topological basis for membrane activity common to AMPs. Moreover, we highlight an important distinction between the maximal recognizability of a sequence to a trained AMP classifier (its ability to generate membrane curvature) and its maximal antimicrobial efficacy. As mutational distances are increased from known AMPs, we find AMP-like sequences that are increasingly difficult for nature to discover via simple mutation. Using the sequence map as a discovery tool, we find a unexpectedly diverse taxonomy of sequences that are just as membrane-active as known AMPs, but with a broad range of primary functions distinct from AMP functions, including endogenous neuropeptides, viral fusion proteins, topogenic peptides, and amyloids. The SVM classifier is useful as a general detector of membrane activity in peptide sequences. PMID:27849600

Association of coral algal symbionts with a diverse viral community responsive to heat shock.

PubMed

Brüwer, Jan D; Agrawal, Shobhit; Liew, Yi Jin; Aranda, Manuel; Voolstra, Christian R

2017-08-17

Stony corals provide the structural foundation of coral reef ecosystems and are termed holobionts given they engage in symbioses, in particular with photosynthetic dinoflagellates of the genus Symbiodinium. Besides Symbiodinium, corals also engage with bacteria affecting metabolism, immunity, and resilience of the coral holobiont, but the role of associated viruses is largely unknown. In this regard, the increase of studies using RNA sequencing (RNA-Seq) to assess gene expression provides an opportunity to elucidate viral signatures encompassed within the data via careful delineation of sequence reads and their source of origin. Here, we re-analyzed an RNA-Seq dataset from a cultured coral symbiont (Symbiodinium microadriaticum, Clade A1) across four experimental treatments (control, cold shock, heat shock, dark shock) to characterize associated viral diversity, abundance, and gene expression. Our approach comprised the filtering and removal of host sequence reads, subsequent phylogenetic assignment of sequence reads of putative viral origin, and the assembly and analysis of differentially expressed viral genes. About 15.46% (123 million) of all sequence reads were non-host-related, of which <1% could be classified as archaea, bacteria, or virus. Of these, 18.78% were annotated as virus and comprised a diverse community consistent across experimental treatments. Further, non-host related sequence reads assembled into 56,064 contigs, including 4856 contigs of putative viral origin that featured 43 differentially expressed genes during heat shock. The differentially expressed genes included viral kinases, ubiquitin, and ankyrin repeat proteins (amongst others), which are suggested to help the virus proliferate and inhibit the algal host's antiviral response. Our results suggest that a diverse viral community is associated with coral algal endosymbionts of the genus Symbiodinium, which prompts further research on their ecological role in coral health and resilience.

Analysis of ATP6 sequence diversity in the Triticum-Aegilops group of species reveals the crucial role of rearrangement in mitochondrial genome evolution

USDA-ARS?s Scientific Manuscript database

Mutation and chromosomal rearrangements are the two main forces of increasing genetic diversity for natural selection to act upon, and ultimately drive the evolutionary process. Although genome evolution is a function of both forces, simultaneously, the ratio of each can be varied among different ge...

Rhizosphere bacteriome of the medicinal plant Sapindus saponaria L. revealed by pyrosequencing.

PubMed

Garcia, A; Polonio, J C; Polli, A D; Santos, C M; Rhoden, S A; Quecine, M C; Azevedo, J L; Pamphile, J A

2016-11-03

Sapindus saponaria L. of Sapindaceae family is popularly known as soldier soap and is found in Central and South America. A study of such medicinal plants might reveal a more complex diversity of microorganisms as compared to non-medicinal plants, considering their metabolic potential and the chemical communication between their natural microbiota. Rhizosphere is a highly diverse microbial habitat with respect to both the diversity of species and the size of the community. Rhizosphere bacteriome associated with medicinal plant S. saponaria is still poorly known. The objective of this study was to assess the rhizosphere microbiome of the medicinal plant S. saponaria using pyrosequencing, a culture-independent approach that is increasingly being used to estimate the number of bacterial species present in different environments. In their rhizosphere microbiome, 26 phyla were identified from 5089 sequences of 16S rRNA gene, with a predominance of Actinobacteria (33.54%), Acidobacteria (22.62%), and Proteobacteria (24.72%). The rarefaction curve showed a linear increase, with 2660 operational taxonomic units at 3% distance sequence dissimilarity, indicating that the rhizosphere microbiome associated with S. saponaria was highly diverse with groups of bacteria important for soil management, which could be further exploited for agricultural and biotechnological purposes.

Design of Protein Multi-specificity Using an Independent Sequence Search Reduces the Barrier to Low Energy Sequences

PubMed Central

Sevy, Alexander M.; Jacobs, Tim M.; Crowe, James E.; Meiler, Jens

2015-01-01

Computational protein design has found great success in engineering proteins for thermodynamic stability, binding specificity, or enzymatic activity in a ‘single state’ design (SSD) paradigm. Multi-specificity design (MSD), on the other hand, involves considering the stability of multiple protein states simultaneously. We have developed a novel MSD algorithm, which we refer to as REstrained CONvergence in multi-specificity design (RECON). The algorithm allows each state to adopt its own sequence throughout the design process rather than enforcing a single sequence on all states. Convergence to a single sequence is encouraged through an incrementally increasing convergence restraint for corresponding positions. Compared to MSD algorithms that enforce (constrain) an identical sequence on all states the energy landscape is simplified, which accelerates the search drastically. As a result, RECON can readily be used in simulations with a flexible protein backbone. We have benchmarked RECON on two design tasks. First, we designed antibodies derived from a common germline gene against their diverse targets to assess recovery of the germline, polyspecific sequence. Second, we design “promiscuous”, polyspecific proteins against all binding partners and measure recovery of the native sequence. We show that RECON is able to efficiently recover native-like, biologically relevant sequences in this diverse set of protein complexes. PMID:26147100

Analysis of bacterial and archaeal diversity in coastal microbial mats using massive parallel 16S rRNA gene tag sequencing.

PubMed

Bolhuis, Henk; Stal, Lucas J

2011-11-01

Coastal microbial mats are small-scale and largely closed ecosystems in which a plethora of different functional groups of microorganisms are responsible for the biogeochemical cycling of the elements. Coastal microbial mats play an important role in coastal protection and morphodynamics through stabilization of the sediments and by initiating the development of salt-marshes. Little is known about the bacterial and especially archaeal diversity and how it contributes to the ecological functioning of coastal microbial mats. Here, we analyzed three different types of coastal microbial mats that are located along a tidal gradient and can be characterized as marine (ST2), brackish (ST3) and freshwater (ST3) systems. The mats were sampled during three different seasons and subjected to massive parallel tag sequencing of the V6 region of the 16S rRNA genes of Bacteria and Archaea. Sequence analysis revealed that the mats are among the most diverse marine ecosystems studied so far and consist of several novel taxonomic levels ranging from classes to species. The diversity between the different mat types was far more pronounced than the changes between the different seasons at one location. The archaeal community for these mats have not been studied before and revealed a strong reaction on a short period of draught during summer resulting in a massive increase in halobacterial sequences, whereas the bacterial community was barely affected. We concluded that the community composition and the microbial diversity were intrinsic of the mat type and depend on the location along the tidal gradient indicating a relation with salinity.

Genetic diversity of Grapevine virus A in Washington and California vineyards.

PubMed

Alabi, Olufemi J; Al Rwahnih, Maher; Mekuria, Tefera A; Naidu, Rayapati A

2014-05-01

Grapevine virus A (GVA; genus Vitivirus, family Betaflexiviridae) has been implicated with the Kober stem grooving disorder of the rugose wood disease complex. In this study, 26 isolates of GVA recovered from wine grape (Vitis vinifera) cultivars from California and Washington were analyzed for their genetic diversity. An analysis of a portion of the RNA-dependent RNA polymerase (RdRp) and complete coat protein (CP) sequences revealed intra- and inter-isolate sequence diversity. Our results indicated that both RdRp and CP are under strong negative selection based on the normalized values for the ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site. A global phylogenetic analysis of CP sequences revealed segregation of virus isolates into four major clades with no geographic clustering. In contrast, the RdRp-based phylogenetic tree indicated segregation of GVA isolates from California and Washington into six clades, independent of geographic origin or cultivar. Phylogenetic network coupled with recombination analyses showed putative recombination events in both RdRp and CP sequence data sets, with more of these events located in the CP sequence. The preponderance of divergent variants of GVA co-replicating within individual grapevines could increase viral genotypic complexity with implications for phylogenetic analysis and evolutionary history of the virus. The knowledge of genetic diversity of GVA generated in this study will provide a foundation for elucidating the epidemiological characteristics of virus populations at different scales and implementing appropriate management strategies for minimizing the spread of genetic variants of the virus by vectors and via planting materials supplied to nurseries and grape growers.

High Bacterial Diversity of Biological Soil Crusts in Water Tracks over Permafrost in the High Arctic Polar Desert

DOE PAGES

Steven, Blaire; Lionard, Marie; Kuske, Cheryl R.; ...

2013-08-13

In this paper we report the bacterial diversity of biological soil crusts (biocrusts) inhabiting polar desert soils at the northern land limit of the Arctic polar region (83° 05 N). Employing pyrosequencing of bacterial 16S rRNA genes this study demonstrated that these biocrusts harbor diverse bacterial communities, often as diverse as temperate latitude communities. The effect of wetting pulses on the composition of communities was also determined by collecting samples from soils outside and inside of permafrost water tracks, hill slope flow paths that drain permafrost-affected soils. The intermittent flow regime in the water tracks was correlated with altered relativemore » abundance of phylum level taxonomic bins in the bacterial communities, but the alterations varied between individual sampling sites. Bacteria related to the Cyanobacteria and Acidobacteria demonstrated shifts in relative abundance based on their location either inside or outside of the water tracks. Among cyanobacterial sequences, the proportion of sequences belonging to the family Oscillatoriales consistently increased in relative abundance in the samples from inside the water tracks compared to those outside. Acidobacteria showed responses to wetting pulses in the water tracks, increasing in abundance at one site and decreasing at the other two sites. Subdivision 4 acidobacterial sequences tended to follow the trends in the total Acidobacteria relative abundance, suggesting these organisms were largely responsible for the changes observed in the Acidobacteria. Finally, taken together, these data suggest that the bacterial communities of these high latitude polar biocrusts are diverse but do not show a consensus response to intermittent flow in water tracks over high Arctic permafrost.« less

High Bacterial Diversity of Biological Soil Crusts in Water Tracks over Permafrost in the High Arctic Polar Desert

PubMed Central

Steven, Blaire; Lionard, Marie; Kuske, Cheryl R.; Vincent, Warwick F.

2013-01-01

In this study we report the bacterial diversity of biological soil crusts (biocrusts) inhabiting polar desert soils at the northern land limit of the Arctic polar region (83° 05 N). Employing pyrosequencing of bacterial 16S rRNA genes this study demonstrated that these biocrusts harbor diverse bacterial communities, often as diverse as temperate latitude communities. The effect of wetting pulses on the composition of communities was also determined by collecting samples from soils outside and inside of permafrost water tracks, hill slope flow paths that drain permafrost-affected soils. The intermittent flow regime in the water tracks was correlated with altered relative abundance of phylum level taxonomic bins in the bacterial communities, but the alterations varied between individual sampling sites. Bacteria related to the Cyanobacteria and Acidobacteria demonstrated shifts in relative abundance based on their location either inside or outside of the water tracks. Among cyanobacterial sequences, the proportion of sequences belonging to the family Oscillatoriales consistently increased in relative abundance in the samples from inside the water tracks compared to those outside. Acidobacteria showed responses to wetting pulses in the water tracks, increasing in abundance at one site and decreasing at the other two sites. Subdivision 4 acidobacterial sequences tended to follow the trends in the total Acidobacteria relative abundance, suggesting these organisms were largely responsible for the changes observed in the Acidobacteria. Taken together, these data suggest that the bacterial communities of these high latitude polar biocrusts are diverse but do not show a consensus response to intermittent flow in water tracks over high Arctic permafrost. PMID:23967218

High-Throughput Sequencing: A Roadmap Toward Community Ecology

PubMed Central

Poisot, Timothée; Péquin, Bérangère; Gravel, Dominique

2013-01-01

High-throughput sequencing is becoming increasingly important in microbial ecology, yet it is surprisingly under-used to generate or test biogeographic hypotheses. In this contribution, we highlight how adding these methods to the ecologist toolbox will allow the detection of new patterns, and will help our understanding of the structure and dynamics of diversity. Starting with a review of ecological questions that can be addressed, we move on to the technical and analytical issues that will benefit from an increased collaboration between different disciplines. PMID:23610649

Genome-wide diversity and differentiation in New World populations of the human malaria parasite Plasmodium vivax

PubMed Central

de Oliveira, Thais C.; Rodrigues, Priscila T.; Menezes, Maria José; Gonçalves-Lopes, Raquel M.; Bastos, Melissa S.; Lima, Nathália F.; Barbosa, Susana; Gerber, Alexandra L.; Loss de Morais, Guilherme; Berná, Luisa; Phelan, Jody; Robello, Carlos; de Vasconcelos, Ana Tereza R.

2017-01-01

Background The Americas were the last continent colonized by humans carrying malaria parasites. Plasmodium falciparum from the New World shows very little genetic diversity and greater linkage disequilibrium, compared with its African counterparts, and is clearly subdivided into local, highly divergent populations. However, limited available data have revealed extensive genetic diversity in American populations of another major human malaria parasite, P. vivax. Methods We used an improved sample preparation strategy and next-generation sequencing to characterize 9 high-quality P. vivax genome sequences from northwestern Brazil. These new data were compared with publicly available sequences from recently sampled clinical P. vivax isolates from Brazil (BRA, total n = 11 sequences), Peru (PER, n = 23), Colombia (COL, n = 31), and Mexico (MEX, n = 19). Principal findings/Conclusions We found that New World populations of P. vivax are as diverse (nucleotide diversity π between 5.2 × 10−4 and 6.2 × 10−4) as P. vivax populations from Southeast Asia, where malaria transmission is substantially more intense. They display several non-synonymous nucleotide substitutions (some of them previously undescribed) in genes known or suspected to be involved in antimalarial drug resistance, such as dhfr, dhps, mdr1, mrp1, and mrp-2, but not in the chloroquine resistance transporter ortholog (crt-o) gene. Moreover, P. vivax in the Americas is much less geographically substructured than local P. falciparum populations, with relatively little between-population genome-wide differentiation (pairwise FST values ranging between 0.025 and 0.092). Finally, P. vivax populations show a rapid decline in linkage disequilibrium with increasing distance between pairs of polymorphic sites, consistent with very frequent outcrossing. We hypothesize that the high diversity of present-day P. vivax lineages in the Americas originated from successive migratory waves and subsequent admixture between parasite lineages from geographically diverse sites. Further genome-wide analyses are required to test the demographic scenario suggested by our data. PMID:28759591

Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing.

PubMed

Miyazaki, Nobuo; Kiyose, Norihiko; Akazawa, Yoko; Takashima, Mizuki; Hagihara, Yosihisa; Inoue, Naokazu; Matsuda, Tomonari; Ogawa, Ryu; Inoue, Seiya; Ito, Yuji

2015-09-01

The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Mining on scorpion venom biodiversity.

PubMed

Rodríguez de la Vega, Ricardo C; Schwartz, Elisabeth F; Possani, Lourival D

2010-12-15

Scorpion venoms are complex mixtures of dozens or even hundreds of distinct proteins, many of which are inter-genome active elements. Fifty years after the first scorpion toxin sequences were determined, chromatography-assisted purification followed by automated protein sequencing or gene cloning, on a case-by-case basis, accumulated nearly 250 amino acid sequences of scorpion venom components. A vast majority of the available sequences correspond to proteins adopting a common three-dimensional fold, whose ion channel modulating functions have been firmly established or could be confidently inferred. However, the actual molecular diversity contained in scorpion venoms -as revealed by bioassay-driven purification, some unexpected activities of "canonical" neurotoxins and even serendipitous discoveries- is much larger than those "canonical" toxin types. In the last few years mining into the molecular diversity contained in scorpion has been assisted by high-throughput Mass Spectrometry techniques and large-scale DNA sequencing, collectively accounting for the more than twofold increase in the number of known sequences of scorpion venom components (now reaching 500 unique sequences). This review, from a comparative perspective, deals with recent data obtained by proteomic and transcriptomic studies on scorpion venoms and venom glands. Altogether, these studies reveal a large contribution of non canonical venom components, which would account for more than half of the total protein diversity of any scorpion venom. On top of aiding at the better understanding of scorpion venom biology, whether in the context of venom function or within the venom gland itself, these "novel" venom components certainly are an interesting source of bioactive proteins, whose characterization is worth pursuing. Copyright © 2009 Elsevier Ltd. All rights reserved.

Soil bacterial diversity patterns and drivers along an elevational gradient on Shennongjia Mountain, China

PubMed Central

Zhang, Yuguang; Cong, Jing; Lu, Hui; Li, Guangliang; Xue, Yadong; Deng, Ye; Li, Hui; Zhou, Jizhong; Li, Diqiang

2015-01-01

Understanding biological diversity elevational pattern and the driver factors are indispensable to develop the ecological theories. Elevational gradient may minimize the impact of environmental factors and is the ideal places to study soil microbial elevational patterns. In this study, we selected four typical vegetation types from 1000 to 2800 m above the sea level on the northern slope of Shennongjia Mountain in central China, and analysed the soil bacterial community composition, elevational patterns and the relationship between soil bacterial diversity and environmental factors by using the 16S rRNA Illumina sequencing and multivariate statistical analysis. The results revealed that the dominant bacterial phyla were Acidobacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Verrucomicrobia, which accounted for over 75% of the bacterial sequences obtained from tested samples, and the soil bacterial operational taxonomic unit (OTU) richness was a significant monotonous decreasing (P < 0.01) trend with the elevational increasing. The similarity of soil bacterial population composition decreased significantly (P < 0.01) with elevational distance increased as measured by the Jaccard and Bray–Curtis index. Canonical correspondence analysis and Mantel test analysis indicated that plant diversity and soil pH were significantly correlated (P < 0.01) with the soil bacterial community. Therefore, the soil bacterial diversity on Shennongjia Mountain had a significant and different elevational pattern, and plant diversity and soil pH may be the key factors in shaping the soil bacterial spatial pattern. PMID:26032124

Necessary Sequencing Depth and Clustering Method to Obtain Relatively Stable Diversity Patterns in Studying Fish Gut Microbiota.

PubMed

Xiao, Fanshu; Yu, Yuhe; Li, Jinjin; Juneau, Philippe; Yan, Qingyun

2018-05-25

The 16S rRNA gene is one of the most commonly used molecular markers for estimating bacterial diversity during the past decades. However, there is no consistency about the sequencing depth (from thousand to millions of sequences per sample), and the clustering methods used to generate OTUs may also be different among studies. These inconsistent premises make effective comparisons among studies difficult or unreliable. This study aims to examine the necessary sequencing depth and clustering method that would be needed to ensure a stable diversity patterns for studying fish gut microbiota. A total number of 42 samples dataset of Siniperca chuatsi (carnivorous fish) gut microbiota were used to test how the sequencing depth and clustering may affect the alpha and beta diversity patterns of fish intestinal microbiota. Interestingly, we found that the sequencing depth (resampling 1000-11,000 per sample) and the clustering methods (UPARSE and UCLUST) did not bias the estimates of the diversity patterns during the fish development from larva to adult. Although we should acknowledge that a suitable sequencing depth may differ case by case, our finding indicates that a shallow sequencing such as 1000 sequences per sample may be also enough to reflect the general diversity patterns of fish gut microbiota. However, we have shown in the present study that strict pre-processing of the original sequences is required to ensure reliable results. This study provides evidences to help making a strong scientific choice of the sequencing depth and clustering method for future studies on fish gut microbiota patterns, but at the same time reducing as much as possible the costs related to the analysis.

Impact of treated wastewater for irrigation on soil microbial communities.

PubMed

Ibekwe, A M; Gonzalez-Rubio, A; Suarez, D L

2018-05-01

The use of treated wastewater (TWW) for irrigation has been suggested as an alternative to use of fresh water because of the increasing scarcity of fresh water in arid and semiarid regions of the world. However, significant barriers exist to widespread adoption due to some potential contaminants that may have adverse effects on soil quality and or public health. In this study, we investigated the abundance and diversity of bacterial communities and the presence of potential pathogenic bacterial sequences in TWW in comparison to synthetic fresh water (SFW) using pyrosequencing. The results were analyzed using UniFrac coupled with principal coordinate analysis (PCoA) to compare diversity and abundance of different bacterial groups in TWW irrigated soils to soils treated with SFW. Shannon diversity index values (H') suggest that microbial diversity was not significantly different (P<0.086) between soils with TWW and SFW. Pyrosequencing detected sequences of 17 bacterial phyla with Proteobacteria (32.1%) followed by Firmicutes (26.5%) and Actinobacteria (14.3%). Most of the sequences associated with nitrifying bacteria, nitrogen-fixing bacteria, carbon degraders, denitrifying bacteria, potential pathogens, and fecal indicator bacteria were more abundant in TWW than in SFW. Therefore, TWW effluent may contain bacterial that may be very active in many soil functions as well as some potential pathogens. Published by Elsevier B.V.

'Candidatus Phytoplasma phoenicium' associated with almond witches'-broom disease: from draft genome to genetic diversity among strain populations.

PubMed

Quaglino, Fabio; Kube, Michael; Jawhari, Maan; Abou-Jawdah, Yusuf; Siewert, Christin; Choueiri, Elia; Sobh, Hana; Casati, Paola; Tedeschi, Rosemarie; Lova, Marina Molino; Alma, Alberto; Bianco, Piero Attilio

2015-07-30

Almond witches'-broom (AlmWB), a devastating disease of almond, peach and nectarine in Lebanon, is associated with 'Candidatus Phytoplasma phoenicium'. In the present study, we generated a draft genome sequence of 'Ca. P. phoenicium' strain SA213, representative of phytoplasma strain populations from different host plants, and determined the genetic diversity among phytoplasma strain populations by phylogenetic analyses of 16S rRNA, groEL, tufB and inmp gene sequences. Sequence-based typing and phylogenetic analysis of the gene inmp, coding an integral membrane protein, distinguished AlmWB-associated phytoplasma strains originating from diverse host plants, whereas their 16S rRNA, tufB and groEL genes shared 100 % sequence identity. Moreover, dN/dS analysis indicated positive selection acting on inmp gene. Additionally, the analysis of 'Ca. P. phoenicium' draft genome revealed the presence of integral membrane proteins and effector-like proteins and potential candidates for interaction with hosts. One of the integral membrane proteins was predicted as BI-1, an inhibitor of apoptosis-promoting Bax factor. Bioinformatics analyses revealed the presence of putative BI-1 in draft and complete genomes of other 'Ca. Phytoplasma' species. The genetic diversity within 'Ca. P. phoenicium' strain populations in Lebanon suggested that AlmWB disease could be associated with phytoplasma strains derived from the adaptation of an original strain to diverse hosts. Moreover, the identification of a putative inhibitor of apoptosis-promoting Bax factor (BI-1) in 'Ca. P. phoenicium' draft genome and within genomes of other 'Ca. Phytoplasma' species suggested its potential role as a phytoplasma fitness-increasing factor by modification of the host-defense response.

Dynamic Evolution of Pathogenicity Revealed by Sequencing and Comparative Genomics of 19 Pseudomonas syringae Isolates

PubMed Central

Romanchuk, Artur; Chang, Jeff H.; Mukhtar, M. Shahid; Cherkis, Karen; Roach, Jeff; Grant, Sarah R.; Jones, Corbin D.; Dangl, Jeffery L.

2011-01-01

Closely related pathogens may differ dramatically in host range, but the molecular, genetic, and evolutionary basis for these differences remains unclear. In many Gram- negative bacteria, including the phytopathogen Pseudomonas syringae, type III effectors (TTEs) are essential for pathogenicity, instrumental in structuring host range, and exhibit wide diversity between strains. To capture the dynamic nature of virulence gene repertoires across P. syringae, we screened 11 diverse strains for novel TTE families and coupled this nearly saturating screen with the sequencing and assembly of 14 phylogenetically diverse isolates from a broad collection of diseased host plants. TTE repertoires vary dramatically in size and content across all P. syringae clades; surprisingly few TTEs are conserved and present in all strains. Those that are likely provide basal requirements for pathogenicity. We demonstrate that functional divergence within one conserved locus, hopM1, leads to dramatic differences in pathogenicity, and we demonstrate that phylogenetics-informed mutagenesis can be used to identify functionally critical residues of TTEs. The dynamism of the TTE repertoire is mirrored by diversity in pathways affecting the synthesis of secreted phytotoxins, highlighting the likely role of both types of virulence factors in determination of host range. We used these 14 draft genome sequences, plus five additional genome sequences previously reported, to identify the core genome for P. syringae and we compared this core to that of two closely related non-pathogenic pseudomonad species. These data revealed the recent acquisition of a 1 Mb megaplasmid by a sub-clade of cucumber pathogens. This megaplasmid encodes a type IV secretion system and a diverse set of unknown proteins, which dramatically increases both the genomic content of these strains and the pan-genome of the species. PMID:21799664

Dynamics of soil diazotrophic community structure, diversity, and functioning during the cropping period of cotton (Gossypium hirsutum).

PubMed

Rai, Sandhya; Singh, Dileep Kumar; Annapurna, Kannepalli

2015-01-01

The soil sampled at different growth stages along the cropping period of cotton were analyzed using various molecular tools: restriction fragment length polymorphism (RFLP), terminal restriction length polymorphism (T-RFLP), and cloning-sequencing. The cluster analysis of the diazotrophic community structure of early sampled soil (0, 15, and 30 days) was found to be more closely related to each other than the later sampled one. Phylogenetic and diversity analysis of sequences obtained from the first (0 Day; C0) and last soil sample (180 day; C180) confirmed the data. The phylogenetic analysis revealed that C0 was having more unique sequences than C180 (presence of γ-Proteobacteria exclusively in C0). A relatively higher richness of diazotrophic community sequences was observed in C0 (S(ACE) : 30.76; S(Chao1) : 20.94) than C180 (S(ACE) : 18.00; S(Chao1) : 18.00) while the evenness component of Shannon diversity index increased from C0 (0.97) to C180 (1.15). The impact of routine agricultural activities was more evident based on diazotrophic activity (measured by acetylene reduction assay) than its structure and diversity. The nitrogenase activity of C0 (1264.85 ± 35.7 ηmol of ethylene production g(-1) dry soil h(-1) ) was statistically higher when compared to all other values (p < 0.05). There was no correlation found between diazotrophic community structure/diversity and N2 fixation rates. Thus, considerable functional redundancy of nifH was concluded to be existing at the experimental site. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microbial community analysis of the hypersaline water of the Dead Sea using high-throughput amplicon sequencing.

PubMed

Jacob, Jacob H; Hussein, Emad I; Shakhatreh, Muhamad Ali K; Cornelison, Christopher T

2017-10-01

Amplicon sequencing using next-generation technology (bTEFAP ® ) has been utilized in describing the diversity of Dead Sea microbiota. The investigated area is a well-known salt lake in the western part of Jordan found in the lowest geographical location in the world (more than 420 m below sea level) and characterized by extreme salinity (approximately, 34%) in addition to other extreme conditions (low pH, unique ionic composition different from sea water). DNA was extracted from Dead Sea water. A total of 314,310 small subunit RNA (SSU rRNA) sequences were parsed, and 288,452 sequences were then clustered. For alpha diversity analysis, sample was rarefied to 3,000 sequences. The Shannon-Wiener index curve plot reached a plateau at approximately 3,000 sequences indicating that sequencing depth was sufficient to capture the full scope of microbial diversity. Archaea was found to be dominating the sequences (52%), whereas Bacteria constitute 45% of the sequences. Altogether, prokaryotic sequences (which constitute 97% of all sequences) were found to predominate. The findings expand on previous studies by using high-throughput amplicon sequencing to describe the microbial community in an environment which in recent years has been shown to hide some interesting diversity. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

PCR Primers for Metazoan Nuclear 18S and 28S Ribosomal DNA Sequences

PubMed Central

Machida, Ryuji J.; Knowlton, Nancy

2012-01-01

Background Metagenetic analyses, which amplify and sequence target marker DNA regions from environmental samples, are increasingly employed to assess the biodiversity of communities of small organisms. Using this approach, our understanding of microbial diversity has expanded greatly. In contrast, only a few studies using this approach to characterize metazoan diversity have been reported, despite the fact that many metazoan species are small and difficult to identify or are undescribed. One of the reasons for this discrepancy is the availability of universal primers for the target taxa. In microbial studies, analysis of the 16S ribosomal DNA is standard. In contrast, the best gene for metazoan metagenetics is less clear. In the present study, we have designed primers that amplify the nuclear 18S and 28S ribosomal DNA sequences of most metazoan species with the goal of providing effective approaches for metagenetic analyses of metazoan diversity in environmental samples, with a particular emphasis on marine biodiversity. Methodology/Principal Findings Conserved regions suitable for designing PCR primers were identified using 14,503 and 1,072 metazoan sequences of the nuclear 18S and 28S rDNA regions, respectively. The sequence similarity of both these newly designed and the previously reported primers to the target regions of these primers were compared for each phylum to determine the expected amplification efficacy. The nucleotide diversity of the flanking regions of the primers was also estimated for genera or higher taxonomic groups of 11 phyla to determine the variable regions within the genes. Conclusions/Significance The identified nuclear ribosomal DNA primers (five primer pairs for 18S and eleven for 28S) and the results of the nucleotide diversity analyses provide options for primer combinations for metazoan metagenetic analyses. Additionally, advantages and disadvantages of not only the 18S and 28S ribosomal DNA, but also other marker regions as targets for metazoan metagenetic analyses, are discussed. PMID:23049971

Low diversity in the mitogenome of sperm whales revealed by next-generation sequencing

Treesearch

Alana Alexander; Debbie Steel; Beth Slikas; Kendra Hoekzema; Colm Carraher; Matthew Parks; Richard Cronn; C. Scott Baker

2012-01-01

Large population sizes and global distributions generally associate with high mitochondrial DNA control region (CR) diversity. The sperm whale (Physeter macrocephalus) is an exception, showing low CR diversity relative to other cetaceans; however, diversity levels throughout the remainder of the sperm whale mitogenome are unknown. We sequenced 20...

Comparison of the Diversity of Basidiomycetes from Dead Wood of the Manchurian fir (Abies holophylla) as Evaluated by Fruiting Body Collection, Mycelial Isolation, and 454 Sequencing.

PubMed

Jang, Yeongseon; Jang, Seokyoon; Min, Mihee; Hong, Joo-Hyun; Lee, Hanbyul; Lee, Hwanhwi; Lim, Young Woon; Kim, Jae-Jin

2015-10-01

In this study, three different methods (fruiting body collection, mycelial isolation, and 454 sequencing) were implemented to determine the diversity of wood-inhabiting basidiomycetes from dead Manchurian fir (Abies holophylla). The three methods recovered similar species richness (26 species from fruiting bodies, 32 species from mycelia, and 32 species from 454 sequencing), but Fisher's alpha, Shannon-Wiener, Simpson's diversity indices of fungal communities indicated fruiting body collection and mycelial isolation displayed higher diversity compared with 454 sequencing. In total, 75 wood-inhabiting basidiomycetes were detected. The most frequently observed species were Heterobasidion orientale (fruiting body collection), Bjerkandera adusta (mycelial isolation), and Trichaptum fusco-violaceum (454 sequencing). Only two species, Hymenochaete yasudae and Hypochnicium karstenii, were detected by all three methods. This result indicated that Manchurian fir harbors a diverse basidiomycetous fungal community and for complete estimation of fungal diversity, multiple methods should be used. Further studies are required to understand their ecology in the context of forest ecosystems.

[Detection and diversity analysis of rumen methanogens in the co-cultures with anaerobic fungi].

PubMed

Cheng, Yan-fen; Mao, Sheng-yong; Pei, Cai-xia; Liu, Jian-xin; Zhu, Wei-yun

2006-12-01

Rumen methanogen diversity in the co-cultures with anaerobic fungi from goat rumen was analyzed. Mix-cultures of anaerobic fungi and methanogens were obtained from goat rumen using anaerobic fungal medium and the addition of penicillin and streptomycin and then subcultured 62 times by transferring cultures every 3 - 4d. Total DNA from the original rumen fluid and subcultured fungal cultures was used for PCR/DGGE and RFLP analysis. 16S rDNA of clones corresponding to representative OTUs were sequenced. Results showed that the diversity index (Shannon index) of the methanogens generated from DGGE profiles reduced from 1.32 to 0.99 from rumen fluid to fungal culture after 45 subculturing, with the lowest similarity of DGGE profiles at 34.7%. The Shannon index increased from 0.99 to 1.15 from the fungal culture after 45 subculturing to that after 62 subculturing, with the lowest similarity at 89.2% . A total of 5 OTUs were obtained from 69. clones using RFLP analysis and six clones representing the 5 OTUs respectively were sequenced. Of the 5 OTUs, three had their cloned 16S rDNA sequences most closely related to uncultured archaeal symbiont PA202 with the same similarity of 95 %, but had not closely related to any identified culturable methanogen. The rest two OTUs had their cloned 16S rDNA sequences sharing the same closest relative, uncultured rumen methanogen 956, with the same similarity of 97% .Their 16S rDNA sequences of these two OTUs also showed 97% similar to the closest identified culturable methanogen Methanobrevibacter sp. NT7. In conclusion, diverse yet unidentified rumen methanogen species exist in the co-cultures with anaerobic fungi isolated from the goat rumen.

Functional evolution and structural conservation in chimeric cytochromes p450: calibrating a structure-guided approach.

PubMed

Otey, Christopher R; Silberg, Jonathan J; Voigt, Christopher A; Endelman, Jeffrey B; Bandara, Geethani; Arnold, Frances H

2004-03-01

Recombination generates chimeric proteins whose ability to fold depends on minimizing structural perturbations that result when portions of the sequence are inherited from different parents. These chimeric sequences can display functional properties characteristic of the parents or acquire entirely new functions. Seventeen chimeras were generated from two CYP102 members of the functionally diverse cytochrome p450 family. Chimeras predicted to have limited structural disruption, as defined by the SCHEMA algorithm, displayed CO binding spectra characteristic of folded p450s. Even this small population exhibited significant functional diversity: chimeras displayed altered substrate specificities, a wide range in thermostabilities, up to a 40-fold increase in peroxidase activity, and ability to hydroxylate a substrate toward which neither parent heme domain shows detectable activity. These results suggest that SCHEMA-guided recombination can be used to generate diverse p450s for exploring function evolution within the p450 structural framework.

Exploring fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing

NASA Astrophysics Data System (ADS)

Zhang, Xiao-Yong; Wang, Guang-Hua; Xu, Xin-Ya; Nong, Xu-Hua; Wang, Jie; Amin, Muhammad; Qi, Shu-Hua

2016-10-01

The present study investigated the fungal diversity in four different deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS1). A total of 40,297 fungal ITS1 sequences clustered into 420 operational taxonomic units (OTUs) with 97% sequence similarity and 170 taxa were recovered from these sediments. Most ITS1 sequences (78%) belonged to the phylum Ascomycota, followed by Basidiomycota (17.3%), Zygomycota (1.5%) and Chytridiomycota (0.8%), and a small proportion (2.4%) belonged to unassigned fungal phyla. Compared with previous studies on fungal diversity of sediments from deep-sea environments by culture-dependent approach and clone library analysis, the present result suggested that Illumina sequencing had been dramatically accelerating the discovery of fungal community of deep-sea sediments. Furthermore, our results revealed that Sordariomycetes was the most diverse and abundant fungal class in this study, challenging the traditional view that the diversity of Sordariomycetes phylotypes was low in the deep-sea environments. In addition, more than 12 taxa accounted for 21.5% sequences were found to be rarely reported as deep-sea fungi, suggesting the deep-sea sediments from Okinawa Trough harbored a plethora of different fungal communities compared with other deep-sea environments. To our knowledge, this study is the first exploration of the fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing.

Reconstructing the history of a fragmented and heavily exploited red deer population using ancient and contemporary DNA.

PubMed

Rosvold, Jørgen; Røed, Knut H; Hufthammer, Anne Karin; Andersen, Reidar; Stenøien, Hans K

2012-09-26

Red deer (Cervus elaphus) have been an important human resource for millennia, experiencing intensive human influence through habitat alterations, hunting and translocation of animals. In this study we investigate a time series of ancient and contemporary DNA from Norwegian red deer spanning about 7,000 years. Our main aim was to investigate how increasing agricultural land use, hunting pressure and possibly human mediated translocation of animals have affected the genetic diversity on a long-term scale. We obtained mtDNA (D-loop) sequences from 73 ancient specimens. These show higher genetic diversity in ancient compared to extant samples, with the highest diversity preceding the onset of agricultural intensification in the Early Iron Age. Using standard diversity indices, Bayesian skyline plot and approximate Bayesian computation, we detected a population reduction which was more prolonged than, but not as severe as, historic documents indicate. There are signs of substantial changes in haplotype frequencies primarily due to loss of haplotypes through genetic drift. There is no indication of human mediated translocations into the Norwegian population. All the Norwegian sequences show a western European origin, from which the Norwegian lineage diverged approximately 15,000 years ago. Our results provide direct insight into the effects of increasing habitat fragmentation and human hunting pressure on genetic diversity and structure of red deer populations. They also shed light on the northward post-glacial colonisation process of red deer in Europe and suggest increased precision in inferring past demographic events when including both ancient and contemporary DNA.

Short-term direct contact with soil and plant materials leads to an immediate increase in diversity of skin microbiota.

PubMed

Grönroos, Mira; Parajuli, Anirudra; Laitinen, Olli H; Roslund, Marja I; Vari, Heli K; Hyöty, Heikki; Puhakka, Riikka; Sinkkonen, Aki

2018-05-29

Immune-mediated diseases have increased during the last decades in urban environments. The hygiene hypothesis suggests that increased hygiene level and reduced contacts with natural biodiversity are related to the increase in immune-mediated diseases. We tested whether short-time contact with microbiologically diverse nature-based materials immediately change bacterial diversity on human skin. We tested direct skin contact, as two volunteers rubbed their hands with sixteen soil and plant based materials, and an exposure via fabric packets filled with moss material. Skin swabs were taken before and after both exposures. Next-generation sequencing showed that exposures increased, at least temporarily, the total diversity of skin microbiota and the diversity of Acidobacteria, Actinobacteria, Bacteroidetes, Proteobacteria and Alpha-, Beta- and Gammaproteobacteria suggesting that contact with nature-based materials modify skin microbiome and increase skin microbial diversity. Until now, approaches to cure or prevent immune system disorders using microbe-based treatments have been limited to use of a few microbial species. We propose that nature-based materials with high natural diversity, such as the materials tested here, might be more effective in modifying human skin microbiome, and eventually, in reducing immune system disorders. Future studies should investigate how long-term changes in skin microbiota are achieved and if the exposure induces beneficial changes in the immune system markers. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Sequence-Dependent Self-Assembly and Structural Diversity of Islet Amyloid Polypeptide-Derived β-Sheet Fibrils

DOE PAGES

Wang, Shih-Ting; Lin, Yiyang; Spencer, Ryan K.; ...

2017-08-03

Determining the structural origins of amyloid fibrillation is essential for understanding both the pathology of amyloidosis and the rational design of inhibitors to prevent or reverse amyloid formation. In this work, the decisive roles of peptide structures on amyloid self-assembly and morphological diversity were investigated by the design of eight amyloidogenic peptides derived from islet amyloid polypeptide. Among the segments, two distinct morphologies were highlighted in the form of twisted and planar (untwisted) ribbons with varied diameters, thicknesses, and lengths. In particular, transformation of amyloid fibrils from twisted ribbons into untwisted structures was triggered by substitution of the C-terminal serinemore » with threonine, where the side chain methyl group was responsible for the distinct morphological change. This effect was confirmed following serine substitution with alanine and valine and was ascribed to the restriction of intersheet torsional strain through the increased hydrophobic interactions and hydrogen bonding. We also studied the variation of fibril morphology (i.e., association and helicity) and peptide aggregation propensity by increasing the hydrophobicity of the peptide side group, capping the N-terminus, and extending sequence length. Lastly, we anticipate that our insights into sequence-dependent fibrillation and morphological diversity will shed light on the structural interpretation of amyloidogenesis and development of structure-specific imaging agents and aggregation inhibitors.« less

Reducing Freshwater Toxicity while Maintaining Weed Control, Profits, And Productivity: Effects of Increased Crop Rotation Diversity and Reduced Herbicide Usage.

PubMed

Hunt, Natalie D; Hill, Jason D; Liebman, Matt

2017-02-07

Increasing crop rotation diversity while reducing herbicide applications may maintain effective weed control while reducing freshwater toxicity. To test this hypothesis, we applied the model USEtox 2.0 to data from a long-term Iowa field experiment that included three crop rotation systems: a 2-year corn-soybean sequence, a 3-year corn-soybean-oat/red clover sequence, and 4-year corn-soybean-oat/alfalfa-alfalfa sequence. Corn and soybean in each rotation were managed with conventional or low-herbicide regimes. Oat, red clover, and alfalfa were not treated with herbicides. Data from 2008-2015 showed that use of the low-herbicide regime reduced freshwater toxicity loads by 81-96%, and that use of the more diverse rotations reduced toxicity and system dependence on herbicides by 25-51%. Mean weed biomass in corn and soybean was <25 kg ha -1 in all rotation × herbicide combinations except the low-herbicide 3-year rotation, which contained ∼110 kg ha -1 of weed biomass. Corn and soybean yields and net returns were as high or higher for the 3- and 4-year rotations managed with the low-herbicide regime as for the conventional-herbicide 2-year rotation. These results indicate that certain forms of cropping system diversification and alternative weed management strategies can maintain yield, profit, and weed suppression while delivering enhanced environmental performance.

High Diversity of Myocyanophage in Various Aquatic Environments Revealed by High-Throughput Sequencing of Major Capsid Protein Gene With a New Set of Primers.

PubMed

Hou, Weiguo; Wang, Shang; Briggs, Brandon R; Li, Gaoyuan; Xie, Wei; Dong, Hailiang

2018-01-01

Myocyanophages, a group of viruses infecting cyanobacteria, are abundant and play important roles in elemental cycling. Here we investigated the particle-associated viral communities retained on 0.2 μm filters and in sediment samples (representing ancient cyanophage communities) from four ocean and three lake locations, using high-throughput sequencing and a newly designed primer pair targeting a gene fragment (∼145-bp in length) encoding the cyanophage gp23 major capsid protein (MCP). Diverse viral communities were detected in all samples. The fragments of 142-, 145-, and 148-bp in length were most abundant in the amplicons, and most sequences (>92%) belonged to cyanophages. Additionally, different sequencing depths resulted in different diversity estimates of the viral community. Operational taxonomic units obtained from deep sequencing of the MCP gene covered the majority of those obtained from shallow sequencing, suggesting that deep sequencing exhibited a more complete picture of cyanophage community than shallow sequencing. Our results also revealed a wide geographic distribution of marine myocyanophages, i.e., higher dissimilarities of the myocyanophage communities corresponded with the larger distances between the sampling sites. Collectively, this study suggests that the newly designed primer pair can be effectively used to study the community and diversity of myocyanophage from different environments, and the high-throughput sequencing represents a good method to understand viral diversity.

High Diversity of Myocyanophage in Various Aquatic Environments Revealed by High-Throughput Sequencing of Major Capsid Protein Gene With a New Set of Primers

PubMed Central

Hou, Weiguo; Wang, Shang; Briggs, Brandon R.; Li, Gaoyuan; Xie, Wei; Dong, Hailiang

2018-01-01

Myocyanophages, a group of viruses infecting cyanobacteria, are abundant and play important roles in elemental cycling. Here we investigated the particle-associated viral communities retained on 0.2 μm filters and in sediment samples (representing ancient cyanophage communities) from four ocean and three lake locations, using high-throughput sequencing and a newly designed primer pair targeting a gene fragment (∼145-bp in length) encoding the cyanophage gp23 major capsid protein (MCP). Diverse viral communities were detected in all samples. The fragments of 142-, 145-, and 148-bp in length were most abundant in the amplicons, and most sequences (>92%) belonged to cyanophages. Additionally, different sequencing depths resulted in different diversity estimates of the viral community. Operational taxonomic units obtained from deep sequencing of the MCP gene covered the majority of those obtained from shallow sequencing, suggesting that deep sequencing exhibited a more complete picture of cyanophage community than shallow sequencing. Our results also revealed a wide geographic distribution of marine myocyanophages, i.e., higher dissimilarities of the myocyanophage communities corresponded with the larger distances between the sampling sites. Collectively, this study suggests that the newly designed primer pair can be effectively used to study the community and diversity of myocyanophage from different environments, and the high-throughput sequencing represents a good method to understand viral diversity.

A Reference Viral Database (RVDB) To Enhance Bioinformatics Analysis of High-Throughput Sequencing for Novel Virus Detection

PubMed Central

Goodacre, Norman; Aljanahi, Aisha; Nandakumar, Subhiksha; Mikailov, Mike

2018-01-01

ABSTRACT Detection of distantly related viruses by high-throughput sequencing (HTS) is bioinformatically challenging because of the lack of a public database containing all viral sequences, without abundant nonviral sequences, which can extend runtime and obscure viral hits. Our reference viral database (RVDB) includes all viral, virus-related, and virus-like nucleotide sequences (excluding bacterial viruses), regardless of length, and with overall reduced cellular sequences. Semantic selection criteria (SEM-I) were used to select viral sequences from GenBank, resulting in a first-generation viral database (VDB). This database was manually and computationally reviewed, resulting in refined, semantic selection criteria (SEM-R), which were applied to a new download of updated GenBank sequences to create a second-generation VDB. Viral entries in the latter were clustered at 98% by CD-HIT-EST to reduce redundancy while retaining high viral sequence diversity. The viral identity of the clustered representative sequences (creps) was confirmed by BLAST searches in NCBI databases and HMMER searches in PFAM and DFAM databases. The resulting RVDB contained a broad representation of viral families, sequence diversity, and a reduced cellular content; it includes full-length and partial sequences and endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Testing of RVDBv10.2, with an in-house HTS transcriptomic data set indicated a significantly faster run for virus detection than interrogating the entirety of the NCBI nonredundant nucleotide database, which contains all viral sequences but also nonviral sequences. RVDB is publically available for facilitating HTS analysis, particularly for novel virus detection. It is meant to be updated on a regular basis to include new viral sequences added to GenBank. IMPORTANCE To facilitate bioinformatics analysis of high-throughput sequencing (HTS) data for the detection of both known and novel viruses, we have developed a new reference viral database (RVDB) that provides a broad representation of different virus species from eukaryotes by including all viral, virus-like, and virus-related sequences (excluding bacteriophages), regardless of their size. In particular, RVDB contains endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Sequences were clustered to reduce redundancy while retaining high viral sequence diversity. A particularly useful feature of RVDB is the reduction of cellular sequences, which can enhance the run efficiency of large transcriptomic and genomic data analysis and increase the specificity of virus detection. PMID:29564396

A Reference Viral Database (RVDB) To Enhance Bioinformatics Analysis of High-Throughput Sequencing for Novel Virus Detection.

PubMed

Goodacre, Norman; Aljanahi, Aisha; Nandakumar, Subhiksha; Mikailov, Mike; Khan, Arifa S

2018-01-01

Detection of distantly related viruses by high-throughput sequencing (HTS) is bioinformatically challenging because of the lack of a public database containing all viral sequences, without abundant nonviral sequences, which can extend runtime and obscure viral hits. Our reference viral database (RVDB) includes all viral, virus-related, and virus-like nucleotide sequences (excluding bacterial viruses), regardless of length, and with overall reduced cellular sequences. Semantic selection criteria (SEM-I) were used to select viral sequences from GenBank, resulting in a first-generation viral database (VDB). This database was manually and computationally reviewed, resulting in refined, semantic selection criteria (SEM-R), which were applied to a new download of updated GenBank sequences to create a second-generation VDB. Viral entries in the latter were clustered at 98% by CD-HIT-EST to reduce redundancy while retaining high viral sequence diversity. The viral identity of the clustered representative sequences (creps) was confirmed by BLAST searches in NCBI databases and HMMER searches in PFAM and DFAM databases. The resulting RVDB contained a broad representation of viral families, sequence diversity, and a reduced cellular content; it includes full-length and partial sequences and endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Testing of RVDBv10.2, with an in-house HTS transcriptomic data set indicated a significantly faster run for virus detection than interrogating the entirety of the NCBI nonredundant nucleotide database, which contains all viral sequences but also nonviral sequences. RVDB is publically available for facilitating HTS analysis, particularly for novel virus detection. It is meant to be updated on a regular basis to include new viral sequences added to GenBank. IMPORTANCE To facilitate bioinformatics analysis of high-throughput sequencing (HTS) data for the detection of both known and novel viruses, we have developed a new reference viral database (RVDB) that provides a broad representation of different virus species from eukaryotes by including all viral, virus-like, and virus-related sequences (excluding bacteriophages), regardless of their size. In particular, RVDB contains endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Sequences were clustered to reduce redundancy while retaining high viral sequence diversity. A particularly useful feature of RVDB is the reduction of cellular sequences, which can enhance the run efficiency of large transcriptomic and genomic data analysis and increase the specificity of virus detection.

Effects of habitat fragmentation on the genetic diversity and differentiation of Dendrolimus punctatus (Lepidoptera: Lasiocampidae) in Thousand Island Lake, China, based on mitochondrial COI gene sequences.

PubMed

Lv, K; Wang, J-R; Li, T-Q; Zhou, J; Gu, J-Q; Zhou, G-X; Xu, Z-H

2018-05-10

Thousand Island Lake (TIL) is a typical fragmented landscape and an ideal model to study ecological effects of fragmentation. Partial fragments of the mitochondrial cytochrome oxidase subunit I gene of 23 island populations of Dendrolimus punctatus in TIL were sequenced, 141 haplotypes being identified. The number of haplotypes increased significantly with the increase in island area and shape index, whereas no significant correlation was detected between three island attributes (area, shape and isolation) and haplotype diversity. However, the correlation with number of haplotypes was no longer significant when the 'outlier' island JSD (the largest island) was not included. Additionally, we found no significant relationship between geographic distance and genetic distance. Geographic isolation did not obstruct the gene flow among D. punctatus populations, which might be because of the high dispersal capacity of this pine moth. Fragmentation resulted in the conversion of large and continuous habitats into isolated, small and insular patches, which was the primary effect on the genetic diversity of D. punctatus in TIL. The conclusion to emphasize from our research is that habitat fragmentation reduced the biological genetic diversity to some extent, further demonstrating the importance of habitat continuity in biodiversity protection.

Semiconductor Sequencing Reveals the Diversity of Bacterial Communities in an Amazon Reservoir Considered as a Methane Source

NASA Astrophysics Data System (ADS)

Graças, D. A.; Ramos, R. T.; Sá, P. G.; Baraúna, R. A.; Schneider, M. C.; Silva, A.

2013-05-01

The Amazon region has enormous hydro potential which is used for power generation. In fact, there are several hydroelectric power stations (HPS) already installed and many under construction or designed. It's in the Amazon which the HPS of Tucuruí, fifth largest in the world, is located. The construction of this hydroelectric dam flooded an area of 2,400 km2 of forest that decomposing, releasing greenhouse gases such as methane (CH4). Methane is the most abundant organic gas in the atmosphere and the second most important greenhouse gas. In this study, we use semicondutor sequencing to assess the bacterial diversity along a water column of 70 meters deep in the Tucuruí reservoir. One liter of water was collected every 10 meters along the water column for total DNA extraction. A fragment of approximately 150 base pairs of the 16S rRNA gene was amplified by polymerase chain reaction using universal primers. These fragments were then paralleled sequenced in Ion Torrent® platform using barcodes on the 316 chip. After the quality filters, about 237 thousands reads were obtained, representing more than 300 Mbp. For bacterial diversity analysis, we used only reads longer than 100 base pairs. The taxonomic diversity was obtained from the Ribosomal Database Project Classifier and alpha diversity analysis (diversity indices and rarefaction) was performed using the RDP pyrosequencing pipeline. Although it is recommended for data pyrosequencing, that pipeline is able to process data obtained from semiconductor sequencing once all of them are fasta files. Over 75% of the sequences were not classified in any phylum, which leads us to believe that there is a huge diversity in the bacterial environment whose function is still unclear. Among the sequences that could be classified, there is a predominance of proteobacteria in all layers, but in higher concentrations at the lower layers. Cyanobacteria accounted for about 3% in the layers of 0m and 10m, leading us to conclude that oxygen production is considerable in this layer. The oxygen produced by Cyanobacteria coupled to atmospheric oxygen provides the ideal environment for the methanotrophic bacteria oxidize methane. Indeed, methanotrophic bacteria represented approximately 10% in the upper layers. Another bacterial phylum well represented in the upper layers was Bacteroidetes, which accounted for about 3% in the layers of 0-30m. Rarefaction analyses, using a cutoff of 3%, tell us the existence of 3212, 6657, 10171, 4209, 10533, 74, 24345 and 64683 OTUs for the layers of 0, 10, 20, 30, 40, 50, 60 and 70 meters, respectively. Bacterial diversity seems to increase with depth, probably due to the large amount of organic matter deposited in the pellet. The 50 meter depth layer showed the lowest diversity due to low quality sequencing of this barcode, which hampered the analysis. The abundance of methanotrophic bacteria shows that the microbial profile of the reservoir is able to consume much of the methane produced by methanogenic archaea in the sediment and that there is a huge diversity whose function is still unknown. The use of semiconductor sequencing proved to be a robust tool to analysis of the microbial community, as an alternative to pyrosequencing.

Enrichment allows identification of diverse, rare elements in metagenomic resistome-virulome sequencing.

PubMed

Noyes, Noelle R; Weinroth, Maggie E; Parker, Jennifer K; Dean, Chris J; Lakin, Steven M; Raymond, Robert A; Rovira, Pablo; Doster, Enrique; Abdo, Zaid; Martin, Jennifer N; Jones, Kenneth L; Ruiz, Jaime; Boucher, Christina A; Belk, Keith E; Morley, Paul S

2017-10-17

Shotgun metagenomic sequencing is increasingly utilized as a tool to evaluate ecological-level dynamics of antimicrobial resistance and virulence, in conjunction with microbiome analysis. Interest in use of this method for environmental surveillance of antimicrobial resistance and pathogenic microorganisms is also increasing. In published metagenomic datasets, the total of all resistance- and virulence-related sequences accounts for < 1% of all sequenced DNA, leading to limitations in detection of low-abundance resistome-virulome elements. This study describes the extent and composition of the low-abundance portion of the resistome-virulome, using a bait-capture and enrichment system that incorporates unique molecular indices to count DNA molecules and correct for enrichment bias. The use of the bait-capture and enrichment system significantly increased on-target sequencing of the resistome-virulome, enabling detection of an additional 1441 gene accessions and revealing a low-abundance portion of the resistome-virulome that was more diverse and compositionally different than that detected by more traditional metagenomic assays. The low-abundance portion of the resistome-virulome also contained resistance genes with public health importance, such as extended-spectrum betalactamases, that were not detected using traditional shotgun metagenomic sequencing. In addition, the use of the bait-capture and enrichment system enabled identification of rare resistance gene haplotypes that were used to discriminate between sample origins. These results demonstrate that the rare resistome-virulome contains valuable and unique information that can be utilized for both surveillance and population genetic investigations of resistance. Access to the rare resistome-virulome using the bait-capture and enrichment system validated in this study can greatly advance our understanding of microbiome-resistome dynamics.

Anthropogenic disturbance equalizes diversity levels in arbuscular mycorrhizal fungal communities.

PubMed

García de León, David; Davison, John; Moora, Mari; Öpik, Maarja; Feng, Huyuan; Hiiesalu, Inga; Jairus, Teele; Koorem, Kadri; Liu, Yongjun; Phosri, Cherdchai; Sepp, Siim-Kaarel; Vasar, Martti; Zobel, Martin

2018-03-24

The arbuscular mycorrhizal (AM) symbiosis is a key plant-microbe interaction in sustainable functioning ecosystems. Increasing anthropogenic disturbance poses a threat to AM fungal communities worldwide, but there is little empirical evidence about its potential negative consequences. In this global study, we sequenced AM fungal DNA in soil samples collected from pairs of natural (undisturbed) and anthropogenic (disturbed) plots in two ecosystem types (10 naturally wooded and six naturally unwooded ecosystems). We found that ecosystem type had stronger directional effects than anthropogenic disturbance on AM fungal alpha and beta diversity. However, disturbance increased alpha and beta diversity at sites where natural diversity was low and decreased diversity at sites where natural diversity was high. Cultured AM fungal taxa were more prevalent in anthropogenic than natural plots, probably due to their efficient colonization strategies and ability to recover from disturbance. We conclude that anthropogenic disturbance does not have a consistent directional effect on AM fungal diversity; rather, disturbance equalizes levels of diversity at large scales and causes changes in community functional structure. © 2018 John Wiley & Sons Ltd.

Error correction and statistical analyses for intra-host comparisons of feline immunodeficiency virus diversity from high-throughput sequencing data.

PubMed

Liu, Yang; Chiaromonte, Francesca; Ross, Howard; Malhotra, Raunaq; Elleder, Daniel; Poss, Mary

2015-06-30

Infection with feline immunodeficiency virus (FIV) causes an immunosuppressive disease whose consequences are less severe if cats are co-infected with an attenuated FIV strain (PLV). We use virus diversity measurements, which reflect replication ability and the virus response to various conditions, to test whether diversity of virulent FIV in lymphoid tissues is altered in the presence of PLV. Our data consisted of the 3' half of the FIV genome from three tissues of animals infected with FIV alone, or with FIV and PLV, sequenced by 454 technology. Since rare variants dominate virus populations, we had to carefully distinguish sequence variation from errors due to experimental protocols and sequencing. We considered an exponential-normal convolution model used for background correction of microarray data, and modified it to formulate an error correction approach for minor allele frequencies derived from high-throughput sequencing. Similar to accounting for over-dispersion in counts, this accounts for error-inflated variability in frequencies - and quite effectively reproduces empirically observed distributions. After obtaining error-corrected minor allele frequencies, we applied ANalysis Of VAriance (ANOVA) based on a linear mixed model and found that conserved sites and transition frequencies in FIV genes differ among tissues of dual and single infected cats. Furthermore, analysis of minor allele frequencies at individual FIV genome sites revealed 242 sites significantly affected by infection status (dual vs. single) or infection status by tissue interaction. All together, our results demonstrated a decrease in FIV diversity in bone marrow in the presence of PLV. Importantly, these effects were weakened or undetectable when error correction was performed with other approaches (thresholding of minor allele frequencies; probabilistic clustering of reads). We also queried the data for cytidine deaminase activity on the viral genome, which causes an asymmetric increase in G to A substitutions, but found no evidence for this host defense strategy. Our error correction approach for minor allele frequencies (more sensitive and computationally efficient than other algorithms) and our statistical treatment of variation (ANOVA) were critical for effective use of high-throughput sequencing data in understanding viral diversity. We found that co-infection with PLV shifts FIV diversity from bone marrow to lymph node and spleen.

Dramatic Increases of Soil Microbial Functional Gene Diversity at the Treeline Ecotone of Changbai Mountain.

PubMed

Shen, Congcong; Shi, Yu; Ni, Yingying; Deng, Ye; Van Nostrand, Joy D; He, Zhili; Zhou, Jizhong; Chu, Haiyan

2016-01-01

The elevational and latitudinal diversity patterns of microbial taxa have attracted great attention in the past decade. Recently, the distribution of functional attributes has been in the spotlight. Here, we report a study profiling soil microbial communities along an elevation gradient (500-2200 m) on Changbai Mountain. Using a comprehensive functional gene microarray (GeoChip 5.0), we found that microbial functional gene richness exhibited a dramatic increase at the treeline ecotone, but the bacterial taxonomic and phylogenetic diversity based on 16S rRNA gene sequencing did not exhibit such a similar trend. However, the β-diversity (compositional dissimilarity among sites) pattern for both bacterial taxa and functional genes was similar, showing significant elevational distance-decay patterns which presented increased dissimilarity with elevation. The bacterial taxonomic diversity/structure was strongly influenced by soil pH, while the functional gene diversity/structure was significantly correlated with soil dissolved organic carbon (DOC). This finding highlights that soil DOC may be a good predictor in determining the elevational distribution of microbial functional genes. The finding of significant shifts in functional gene diversity at the treeline ecotone could also provide valuable information for predicting the responses of microbial functions to climate change.

Genomics of crop wild relatives: expanding the gene pool for crop improvement.

PubMed

Brozynska, Marta; Furtado, Agnelo; Henry, Robert J

2016-04-01

Plant breeders require access to new genetic diversity to satisfy the demands of a growing human population for more food that can be produced in a variable or changing climate and to deliver the high-quality food with nutritional and health benefits demanded by consumers. The close relatives of domesticated plants, crop wild relatives (CWRs), represent a practical gene pool for use by plant breeders. Genomics of CWR generates data that support the use of CWR to expand the genetic diversity of crop plants. Advances in DNA sequencing technology are enabling the efficient sequencing of CWR and their increased use in crop improvement. As the sequencing of genomes of major crop species is completed, attention has shifted to analysis of the wider gene pool of major crops including CWR. A combination of de novo sequencing and resequencing is required to efficiently explore useful genetic variation in CWR. Analysis of the nuclear genome, transcriptome and maternal (chloroplast and mitochondrial) genome of CWR is facilitating their use in crop improvement. Genome analysis results in discovery of useful alleles in CWR and identification of regions of the genome in which diversity has been lost in domestication bottlenecks. Targeting of high priority CWR for sequencing will maximize the contribution of genome sequencing of CWR. Coordination of global efforts to apply genomics has the potential to accelerate access to and conservation of the biodiversity essential to the sustainability of agriculture and food production. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Analysis of large 16S rRNA Illumina data sets: Impact of singleton read filtering on microbial community description.

PubMed

Auer, Lucas; Mariadassou, Mahendra; O'Donohue, Michael; Klopp, Christophe; Hernandez-Raquet, Guillermina

2017-11-01

Next-generation sequencing technologies give access to large sets of data, which are extremely useful in the study of microbial diversity based on 16S rRNA gene. However, the production of such large data sets is not only marred by technical biases and sequencing noise but also increases computation time and disc space use. To improve the accuracy of OTU predictions and overcome both computations, storage and noise issues, recent studies and tools suggested removing all single reads and low abundant OTUs, considering them as noise. Although the effect of applying an OTU abundance threshold on α- and β-diversity has been well documented, the consequences of removing single reads have been poorly studied. Here, we test the effect of singleton read filtering (SRF) on microbial community composition using in silico simulated data sets as well as sequencing data from synthetic and real communities displaying different levels of diversity and abundance profiles. Scalability to large data sets is also assessed using a complete MiSeq run. We show that SRF drastically reduces the chimera content and computational time, enabling the analysis of a complete MiSeq run in just a few minutes. Moreover, SRF accurately determines the actual community diversity: the differences in α- and β-community diversity obtained with SRF and standard procedures are much smaller than the intrinsic variability of technical and biological replicates. © 2017 John Wiley & Sons Ltd.

Strong latitudinal and vertical biogeography of Synechococcus diversity in the equatorial Pacific Ocean

NASA Astrophysics Data System (ADS)

Martiny, A.; Kent, A. G.; Mouginot, C.; Baer, S. E.; Lomas, M. W.

2016-02-01

Extensive genetic diversity has been observed within Synechococcus including the presence of multiple major clades. However, the biogeography and underlying environmental drivers of these clades remain elusive. Here, we developed a new high-throughput sequencing assay using rpoC1 as marker combined with Illumina sequencing. Using this, we identified the genetic diversity of Synechococcus from 200 samples in an eastern Pacific Ocean transect between 19˚N and 3˚S. We used a placement method to identify the phylogenetic affiliation of each sequence and detected extensive diversity including multiple previously undescribed clades. We observed clear biogeographical domains, with Clade 2 dominant in the northern part of the transect, Clade CRD peaking at the equator, and Clade 1 dominant deeper in the water column throughout the transect. This biogeography, along with physical and nutrient data, suggests that Clade 2 represents a high temperature, low macronutrient ecotype, CRD a high temperature but low iron ecotype, and at least part of Clade 1 a low-light ecotype. The shift between Clade 2 and CRD occurred at 7˚N, whereas the concentration of macronutrients was low down to 4˚N, before increasing. This biogeography indicates that Synechococcus cells experience iron stress up to 7˚N despite low concentrations of phosphate and nitrate. The overall biogeography closely matched the distribution of Prochlorococcus diversity in this region, suggesting a parallel evolution of ecotypes in these two major lineages of marine Cyanobacteria.

Elevational Patterns in Archaeal Diversity on Mt. Fuji

PubMed Central

Singh, Dharmesh; Takahashi, Koichi; Adams, Jonathan M.

2012-01-01

Little is known of how archaeal diversity and community ecology behaves along elevational gradients. We chose to study Mount Fuji of Japan as a geologically and topographically uniform mountain system, with a wide range of elevational zones. PCR-amplified soil DNA for the archaeal 16 S rRNA gene was pyrosequenced and taxonomically classified against EzTaxon-e archaeal database. At a bootstrap cut-off of 80%, most of the archaeal sequences were classified into phylum Thaumarchaeota (96%) and Euryarchaeota (3.9%), with no sequences classified into other phyla. Archaeal OTU richness and diversity on Fuji showed a pronounced ‘peak’ in the mid-elevations, around 1500 masl, within the boreal forest zone, compared to the temperate forest zone below and the alpine fell-field and desert zones above. Diversity decreased towards higher elevations followed by a subtle increase at the summit, mainly due to an increase in the relative abundance of the group I.1b of Thaumarchaeota. Archaeal diversity showed a strong positive correlation with soil NH4 +, K and NO3 − . Archaeal diversity does not parallel plant diversity, although it does roughly parallel bacterial diversity. Ecological hypotheses to explain the mid diversity bulge on Fuji include intermediate disturbance effects, and the result of mid elevations combining a mosaic of upper and lower slope environments. Our findings show clearly that archaeal soil communities are highly responsive to soil environmental gradients, in terms of both their diversity and community composition. Distinct communities of archaea specific to each elevational zone suggest that many archaea may be quite finely niche-adapted within the range of soil environments. A further interesting finding is the presence of a mesophilic component of archaea at high altitudes on a mountain that is not volcanically active. This emphasizes the importance of microclimate – in this case solar heating of the black volcanic ash surface – for the ecology of soil archaea. PMID:22970233

Deep sampling of the Palomero maize transcriptome by a high throughput strategy of pyrosequencing.

PubMed

Vega-Arreguín, Julio C; Ibarra-Laclette, Enrique; Jiménez-Moraila, Beatriz; Martínez, Octavio; Vielle-Calzada, Jean Philippe; Herrera-Estrella, Luis; Herrera-Estrella, Alfredo

2009-07-06

In-depth sequencing analysis has not been able to determine the overall complexity of transcriptional activity of a plant organ or tissue sample. In some cases, deep parallel sequencing of Expressed Sequence Tags (ESTs), although not yet optimized for the sequencing of cDNAs, has represented an efficient procedure for validating gene prediction and estimating overall gene coverage. This approach could be very valuable for complex plant genomes. In addition, little emphasis has been given to efforts aiming at an estimation of the overall transcriptional universe found in a multicellular organism at a specific developmental stage. To explore, in depth, the transcriptional diversity in an ancient maize landrace, we developed a protocol to optimize the sequencing of cDNAs and performed 4 consecutive GS20-454 pyrosequencing runs of a cDNA library obtained from 2 week-old Palomero Toluqueño maize plants. The protocol reported here allowed obtaining over 90% of informative sequences. These GS20-454 runs generated over 1.5 Million reads, representing the largest amount of sequences reported from a single plant cDNA library. A collection of 367,391 quality-filtered reads (30.09 Mb) from a single run was sufficient to identify transcripts corresponding to 34% of public maize ESTs databases; total sequences generated after 4 filtered runs increased this coverage to 50%. Comparisons of all 1.5 Million reads to the Maize Assembled Genomic Islands (MAGIs) provided evidence for the transcriptional activity of 11% of MAGIs. We estimate that 5.67% (86,069 sequences) do not align with public ESTs or annotated genes, potentially representing new maize transcripts. Following the assembly of 74.4% of the reads in 65,493 contigs, real-time PCR of selected genes confirmed a predicted correlation between the abundance of GS20-454 sequences and corresponding levels of gene expression. A protocol was developed that significantly increases the number, length and quality of cDNA reads using massive 454 parallel sequencing. We show that recurrent 454 pyrosequencing of a single cDNA sample is necessary to attain a thorough representation of the transcriptional universe present in maize, that can also be used to estimate transcript abundance of specific genes. This data suggests that the molecular and functional diversity contained in the vast native landraces remains to be explored, and that large-scale transcriptional sequencing of a presumed ancestor of the modern maize varieties represents a valuable approach to characterize the functional diversity of maize for future agricultural and evolutionary studies.

It's all relative: ranking the diversity of aquatic bacterial communities.

PubMed

Shaw, Allison K; Halpern, Aaron L; Beeson, Karen; Tran, Bao; Venter, J Craig; Martiny, Jennifer B H

2008-09-01

The study of microbial diversity patterns is hampered by the enormous diversity of microbial communities and the lack of resources to sample them exhaustively. For many questions about richness and evenness, however, one only needs to know the relative order of diversity among samples rather than total diversity. We used 16S libraries from the Global Ocean Survey to investigate the ability of 10 diversity statistics (including rarefaction, non-parametric, parametric, curve extrapolation and diversity indices) to assess the relative diversity of six aquatic bacterial communities. Overall, we found that the statistics yielded remarkably similar rankings of the samples for a given sequence similarity cut-off. This correspondence, despite the different underlying assumptions of the statistics, suggests that diversity statistics are a useful tool for ranking samples of microbial diversity. In addition, sequence similarity cut-off influenced the diversity ranking of the samples, demonstrating that diversity statistics can also be used to detect differences in phylogenetic structure among microbial communities. Finally, a subsampling analysis suggests that further sequencing from these particular clone libraries would not have substantially changed the richness rankings of the samples.

On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires.

PubMed

Silas, Sukrit; Makarova, Kira S; Shmakov, Sergey; Páez-Espino, David; Mohr, Georg; Liu, Yi; Davison, Michelle; Roux, Simon; Krishnamurthy, Siddharth R; Fu, Becky Xu Hua; Hansen, Loren L; Wang, David; Sullivan, Matthew B; Millard, Andrew; Clokie, Martha R; Bhaya, Devaki; Lambowitz, Alan M; Kyrpides, Nikos C; Koonin, Eugene V; Fire, Andrew Z

2017-07-11

Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium- Arthrospira platensis Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown. IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis , a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic. Copyright © 2017 Silas et al.

The utility of DNA metabarcoding for studying the response of arthropod diversity and composition to land-use change in the tropics

PubMed Central

Beng, Kingsly Chuo; Tomlinson, Kyle W.; Shen, Xian Hui; Surget-Groba, Yann; Hughes, Alice C.; Corlett, Richard T.; Slik, J. W. Ferry

2016-01-01

Metabarcoding potentially offers a rapid and cheap method of monitoring biodiversity, but real-world applications are few. We investigated its utility in studying patterns of litter arthropod diversity and composition in the tropics. We collected litter arthropods from 35 matched forest-plantation sites across Xishuangbanna, southwestern China. A new primer combination and the MiSeq platform were used to amplify and sequence a wide variety of litter arthropods using simulated and real-world communities. Quality filtered reads were clustered into 3,624 MOTUs at ≥97% similarity and the taxonomy of each MOTU was predicted. We compared diversity and compositional differences between forests and plantations (rubber and tea) for all MOTUs and for eight arthropod groups. We obtained ~100% detection rate after in silico sequencing six mock communities with known arthropod composition. Ordination showed that rubber, tea and forest communities formed distinct clusters. α-diversity declined significantly between forests and adjacent plantations for more arthropod groups in rubber than tea, and diversity of order Orthoptera increased significantly in tea. Turnover was higher in forests than plantations, but patterns differed among groups. Metabarcoding is useful for quantifying diversity patterns of arthropods under different land-uses and the MiSeq platform is effective for arthropod metabarcoding in the tropics. PMID:27112993

The genetic diversity of merozoite surface antigen 1 (MSA-1) among Babesia bovis detected from cattle populations in Thailand, Brazil and Ghana.

PubMed

Nagano, Daisuke; Sivakumar, Thillaiampalam; De De Macedo, Alane Caine Costa; Inpankaew, Tawin; Alhassan, Andy; Igarashi, Ikuo; Yokoyama, Naoaki

2013-11-01

In the present study, we screened blood DNA samples obtained from cattle bred in Brazil (n=164) and Ghana (n=80) for Babesia bovis using a diagnostic PCR assay and found prevalences of 14.6% and 46.3%, respectively. Subsequently, the genetic diversity of B. bovis in Thailand, Brazil and Ghana was analyzed, based on the DNA sequence of merozoite surface antigen-1 (MSA-1). In Thailand, MSA-1 sequences were relatively conserved and found in a single clade of the phylogram, while Brazilian MSA-1 sequences showed high genetic diversity and were dispersed across three different clades. In contrast, the sequences from Ghanaian samples were detected in two different clades, one of which contained only a single Ghanaian sequence. The identities among the MSA-1 sequences from Thailand, Brazil and Ghana were 99.0-100%, 57.5-99.4% and 60.3-100%, respectively, while the similarities among the deduced MSA-1 amino acid sequences within the respective countries were 98.4-100%, 59.4-99.7% and 58.7-100%, respectively. These observations suggested that the genetic diversity of B. bovis based on MSA-1 sequences was higher in Brazil and Ghana than in Thailand. The current data highlight the importance of conducting extensive studies on the genetic diversity of B. bovis before designing immune control strategies in each surveyed country.

Comprehensive phylogenetic analysis of bacterial reverse transcriptases.

PubMed

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology.

Comprehensive Phylogenetic Analysis of Bacterial Reverse Transcriptases

PubMed Central

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology. PMID:25423096

Cyberinfrastructure for Fusarium (CiF)

USDA-ARS?s Scientific Manuscript database

The rapidly increasing number of genome sequences from diverse fungal species and expanding phylogenetic data necessitate highly integrated informatics platforms to adequately support the use of these resources for studying fungal biology and evolution. The long-term goal of Cyberinfrastructure for...

Estimating Bacterial Diversity for Ecological Studies: Methods, Metrics, and Assumptions

PubMed Central

Birtel, Julia; Walser, Jean-Claude; Pichon, Samuel; Bürgmann, Helmut; Matthews, Blake

2015-01-01

Methods to estimate microbial diversity have developed rapidly in an effort to understand the distribution and diversity of microorganisms in natural environments. For bacterial communities, the 16S rRNA gene is the phylogenetic marker gene of choice, but most studies select only a specific region of the 16S rRNA to estimate bacterial diversity. Whereas biases derived from from DNA extraction, primer choice and PCR amplification are well documented, we here address how the choice of variable region can influence a wide range of standard ecological metrics, such as species richness, phylogenetic diversity, β-diversity and rank-abundance distributions. We have used Illumina paired-end sequencing to estimate the bacterial diversity of 20 natural lakes across Switzerland derived from three trimmed variable 16S rRNA regions (V3, V4, V5). Species richness, phylogenetic diversity, community composition, β-diversity, and rank-abundance distributions differed significantly between 16S rRNA regions. Overall, patterns of diversity quantified by the V3 and V5 regions were more similar to one another than those assessed by the V4 region. Similar results were obtained when analyzing the datasets with different sequence similarity thresholds used during sequences clustering and when the same analysis was used on a reference dataset of sequences from the Greengenes database. In addition we also measured species richness from the same lake samples using ARISA Fingerprinting, but did not find a strong relationship between species richness estimated by Illumina and ARISA. We conclude that the selection of 16S rRNA region significantly influences the estimation of bacterial diversity and species distributions and that caution is warranted when comparing data from different variable regions as well as when using different sequencing techniques. PMID:25915756

Overlap and diversity in antimicrobial peptide databases: compiling a non-redundant set of sequences.

PubMed

Aguilera-Mendoza, Longendri; Marrero-Ponce, Yovani; Tellez-Ibarra, Roberto; Llorente-Quesada, Monica T; Salgado, Jesús; Barigye, Stephen J; Liu, Jun

2015-08-01

The large variety of antimicrobial peptide (AMP) databases developed to date are characterized by a substantial overlap of data and similarity of sequences. Our goals are to analyze the levels of redundancy for all available AMP databases and use this information to build a new non-redundant sequence database. For this purpose, a new software tool is introduced. A comparative study of 25 AMP databases reveals the overlap and diversity among them and the internal diversity within each database. The overlap analysis shows that only one database (Peptaibol) contains exclusive data, not present in any other, whereas all sequences in the LAMP_Patent database are included in CAMP_Patent. However, the majority of databases have their own set of unique sequences, as well as some overlap with other databases. The complete set of non-duplicate sequences comprises 16 990 cases, which is almost half of the total number of reported peptides. On the other hand, the diversity analysis identifies the most and least diverse databases and proves that all databases exhibit some level of redundancy. Finally, we present a new parallel-free software, named Dover Analyzer, developed to compute the overlap and diversity between any number of databases and compile a set of non-redundant sequences. These results are useful for selecting or building a suitable representative set of AMPs, according to specific needs. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Differential sequence diversity at merozoite surface protein-1 locus of Plasmodium knowlesi from humans and macaques in Thailand.

PubMed

Putaporntip, Chaturong; Thongaree, Siriporn; Jongwutiwes, Somchai

2013-08-01

To determine the genetic diversity and potential transmission routes of Plasmodium knowlesi, we analyzed the complete nucleotide sequence of the gene encoding the merozoite surface protein-1 of this simian malaria (Pkmsp-1), an asexual blood-stage vaccine candidate, from naturally infected humans and macaques in Thailand. Analysis of Pkmsp-1 sequences from humans (n=12) and monkeys (n=12) reveals five conserved and four variable domains. Most nucleotide substitutions in conserved domains were dimorphic whereas three of four variable domains contained complex repeats with extensive sequence and size variation. Besides purifying selection in conserved domains, evidence of intragenic recombination scattering across Pkmsp-1 was detected. The number of haplotypes, haplotype diversity, nucleotide diversity and recombination sites of human-derived sequences exceeded that of monkey-derived sequences. Phylogenetic networks based on concatenated conserved sequences of Pkmsp-1 displayed a character pattern that could have arisen from sampling process or the presence of two independent routes of P. knowlesi transmission, i.e. from macaques to human and from human to humans in Thailand. Copyright © 2013 Elsevier B.V. All rights reserved.

Characterizing novel endogenous retroviruses from genetic variation inferred from short sequence reads

PubMed Central

Mourier, Tobias; Mollerup, Sarah; Vinner, Lasse; Hansen, Thomas Arn; Kjartansdóttir, Kristín Rós; Guldberg Frøslev, Tobias; Snogdal Boutrup, Torsten; Nielsen, Lars Peter; Willerslev, Eske; Hansen, Anders J.

2015-01-01

From Illumina sequencing of DNA from brain and liver tissue from the lion, Panthera leo, and tumor samples from the pike-perch, Sander lucioperca, we obtained two assembled sequence contigs with similarity to known retroviruses. Phylogenetic analyses suggest that the pike-perch retrovirus belongs to the epsilonretroviruses, and the lion retrovirus to the gammaretroviruses. To determine if these novel retroviral sequences originate from an endogenous retrovirus or from a recently integrated exogenous retrovirus, we assessed the genetic diversity of the parental sequences from which the short Illumina reads are derived. First, we showed by simulations that we can robustly infer the level of genetic diversity from short sequence reads. Second, we find that the measures of nucleotide diversity inferred from our retroviral sequences significantly exceed the level observed from Human Immunodeficiency Virus infections, prompting us to conclude that the novel retroviruses are both of endogenous origin. Through further simulations, we rule out the possibility that the observed elevated levels of nucleotide diversity are the result of co-infection with two closely related exogenous retroviruses. PMID:26493184

Analysis of genetic diversity using SNP markers in oat

USDA-ARS?s Scientific Manuscript database

A large-scale single nucleotide polymorphism (SNP) discovery was carried out in cultivated oat using Roche 454 sequencing methods. DNA sequences were generated from cDNAs originating from a panel of 20 diverse oat cultivars, and from Diversity Array Technology (DArT) genomic complexity reductions fr...

Metagenomics and the protein universe

PubMed Central

Godzik, Adam

2011-01-01

Metagenomics sequencing projects have dramatically increased our knowledge of the protein universe and provided over one-half of currently known protein sequences; they have also introduced a much broader phylogenetic diversity into the protein databases. The full analysis of metagenomic datasets is only beginning, but it has already led to the discovery of thousands of new protein families, likely representing novel functions specific to given environments. At the same time, a deeper analysis of such novel families, including experimental structure determination of some representatives, suggests that most of them represent distant homologs of already characterized protein families, and thus most of the protein diversity present in the new environments are due to functional divergence of the known protein families rather than the emergence of new ones. PMID:21497084

Emerging from the bottleneck: Benefits of the comparative approach to modern neuroscience

PubMed Central

Brenowitz, Eliot A.; Zakon, Harold H.

2015-01-01

Neuroscience historically exploited a wide diversity of animal taxa. Recently, however, research focused increasingly on a few model species. This trend accelerated with the genetic revolution, as genomic sequences and genetic tools became available for a few species, which formed a bottleneck. This coalescence on a small set of model species comes with several costs often not considered, especially in the current drive to use mice explicitly as models for human diseases. Comparative studies of strategically chosen non-model species can complement model species research and yield more rigorous studies. As genetic sequences and tools become available for many more species, we are poised to emerge from the bottleneck and once again exploit the rich biological diversity offered by comparative studies. PMID:25800324

Microbial community structure and diversity in the soil spatial profile of 5-year-old Robinia pseudoacacia 'Idaho,' determined by 454 sequencing of the 16S RNA gene.

PubMed

Chang, Yanping; Bu, Xiangpan; Niu, Weibo; Xiu, Yu; Wang, Huafang

2013-01-01

Relatively little information is available regarding the variability of microbial communities inhabiting deeper soil layers. We investigated the distribution of soil microbial communities down to 1.2 m in 5-year-old Robinia pseudoacacia 'Idaho' soil by 454 sequencing of the 16S RNA gene. The average number of sequences per sample was 12,802. The Shannon and Chao 1 indices revealed various relative microbial abundances and even distribution of microbial diversity for all evaluated sample depths. The predicted diversity in the topsoil exceeded that of the corresponding subsoil. The changes in the relative abundance of the major soil bacterial phyla showed decreasing, increasing, or no consistent trends with respect to sampling depth. Despite their novelty, members of the new candidate phyla OD1 and TM7 were widespread. Environmental variables affecting the bacterial community within the environment appeared to differ from those reported previously, especially the lack of detectable effect from pH. Overall, we found that the overall relative abundance fluctuated with the physical and chemical properties of the soil, root system, and sampling depth. Such information may facilitate forest soil management.

IMNGS: A comprehensive open resource of processed 16S rRNA microbial profiles for ecology and diversity studies.

PubMed

Lagkouvardos, Ilias; Joseph, Divya; Kapfhammer, Martin; Giritli, Sabahattin; Horn, Matthias; Haller, Dirk; Clavel, Thomas

2016-09-23

The SRA (Sequence Read Archive) serves as primary depository for massive amounts of Next Generation Sequencing data, and currently host over 100,000 16S rRNA gene amplicon-based microbial profiles from various host habitats and environments. This number is increasing rapidly and there is a dire need for approaches to utilize this pool of knowledge. Here we created IMNGS (Integrated Microbial Next Generation Sequencing), an innovative platform that uniformly and systematically screens for and processes all prokaryotic 16S rRNA gene amplicon datasets available in SRA and uses them to build sample-specific sequence databases and OTU-based profiles. Via a web interface, this integrative sequence resource can easily be queried by users. We show examples of how the approach allows testing the ecological importance of specific microorganisms in different hosts or ecosystems, and performing targeted diversity studies for selected taxonomic groups. The platform also offers a complete workflow for de novo analysis of users' own raw 16S rRNA gene amplicon datasets for the sake of comparison with existing data. IMNGS can be accessed at www.imngs.org.

Genetic diversity and connectivity in the East African giant mud crab Scylla serrata: Implications for fisheries management.

PubMed

Rumisha, Cyrus; Huyghe, Filip; Rapanoel, Diary; Mascaux, Nemo; Kochzius, Marc

2017-01-01

The giant mud crab Scylla serrata provides an important source of income and food to coastal communities in East Africa. However, increasing demand and exploitation due to the growing coastal population, export trade, and tourism industry are threatening the sustainability of the wild stock of this species. Because effective management requires a clear understanding of the connectivity among populations, this study was conducted to assess the genetic diversity and connectivity in the East African mangrove crab S. serrata. A section of 535 base pairs of the cytochrome oxidase subunit I (COI) gene and eight microsatellite loci were analysed from 230 tissue samples of giant mud crabs collected from Kenya, Tanzania, Mozambique, Madagascar, and South Africa. Microsatellite genetic diversity (He) ranged between 0.56 and 0.6. The COI sequences showed 57 different haplotypes associated with low nucleotide diversity (current nucleotide diversity = 0.29%). In addition, the current nucleotide diversity was lower than the historical nucleotide diversity, indicating overexploitation or historical bottlenecks in the recent history of the studied population. Considering that the coastal population is growing rapidly, East African countries should promote sustainable fishing practices and sustainable use of mangrove resources to protect mud crabs and other marine fauna from the increasing pressure of exploitation. While microsatellite loci did not show significant genetic differentiation (p > 0.05), COI sequences revealed significant genetic divergence between sites on the East coast of Madagascar (ECM) and sites on the West coast of Madagascar, mainland East Africa, as well as the Seychelles. Since East African countries agreed to achieve the Convention on Biological Diversity (CBD) target to protect over 10% of their marine areas by 2020, the observed pattern of connectivity and the measured genetic diversity can serve to provide useful information for designing networks of marine protected areas.

Genotypic diversity of stress response in Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus pentosus.

PubMed

Ricciardi, Annamaria; Parente, Eugenio; Guidone, Angela; Ianniello, Rocco Gerardo; Zotta, Teresa; Abu Sayem, S M; Varcamonti, Mario

2012-07-02

Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum are three closely related species which are widespread in food and non-food environments, and are important as starter bacteria or probiotics. In order to evaluate the phenotypic diversity of stress tolerance in the L. plantarum group and the ability to mount an adaptive heat shock response, the survival of exponential and stationary phase and of heat adapted exponential phase cells of six L. plantarum subsp. plantarum, one L. plantarum subsp. argentoratensis, one L. pentosus and two L. paraplantarum strains selected in a previous work upon exposure to oxidative, heat, detergent, starvation and acid stresses was compared to that of the L. plantarum WCFS1 strain. Furthermore, to evaluate the genotypic diversity in stress response genes, ten genes (encoding for chaperones DnaK, GroES and GroEL, regulators CtsR, HrcA and CcpA, ATPases/proteases ClpL, ClpP, ClpX and protease FtsH) were amplified using primers derived from the WCFS1 genome sequence and submitted to restriction with one or two endonucleases. The results were compared by univariate and multivariate statistical methods. In addition, the amplicons for hrcA and ctsR were sequenced and compared by multiple sequence alignment and polymorphism analysis. Although there was evidence of a generalized stress response in the stationary phase, with increase of oxidative, heat, and, to a lesser extent, starvation stress tolerance, and for adaptive heat stress response, with increased tolerance to heat, acid and detergent, different growth phases and adaptation patterns were found. Principal component analysis showed that while heat, acid and detergent stresses respond similarly to growth phase and adaptation, tolerance to oxidative and starvation stresses implies completely unrelated mechanisms. A dendrogram obtained using the data from multilocus restriction typing (MLRT) of stress response genes clearly separated two groups of L. plantarum strains from the other species but there was no correlation between genotypic grouping and grouping obtained on the basis of the stress response pattern, nor with the phylograms obtained from hrcA and ctsR sequences. Differences in sequence in L. plantarum strains were mostly due to single nucleotide polymorphisms with a high frequency of synonymous nucleotide changes and, while hrcA was characterized by an excess of low frequency polymorphism, very low diversity was found in ctsR sequences. Sequence alignment of hrcA allowed a correct discrimination of the strains at the species level, thus confirming the relevance of stress response genes for taxonomy. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification of Single-Copy Orthologous Genes between Physalis and Solanum lycopersicum and Analysis of Genetic Diversity in Physalis Using Molecular Markers

PubMed Central

Wei, Jingli; Hu, Xiaorong; Yang, Jingjing; Yang, Wencai

2012-01-01

The genus Physalis includes a number of commercially important edible and ornamental species. Its high nutritional value and potential medicinal properties leads to the increased commercial interest in the products of this genus worldwide. However, lack of molecular markers prevents the detailed study of genetics and phylogeny in Physalis, which limits the progress of breeding. In the present study, we compared the DNA sequences between Physalis and tomato, and attempted to analyze genetic diversity in Physalis using tomato markers. Blasting 23180 DNA sequences derived from Physalis against the International Tomato Annotation Group (ITAG) Release2.3 Predicted CDS (SL2.40) discovered 3356 single-copy orthologous genes between them. A total of 38 accessions from at least six species of Physalis were subjected to genetic diversity analysis using 97 tomato markers and 25 SSR markers derived from P. peruviana. Majority (73.2%) of tomato markers could amplify DNA fragments from at least one accession of Physalis. Diversity in Physalis at molecular level was also detected. The average Nei’s genetic distance between accessions was 0.3806 with a range of 0.2865 to 0.7091. These results indicated Physalis and tomato had similarity at both molecular marker and DNA sequence levels. Therefore, the molecular markers developed in tomato can be used in genetic study in Physalis. PMID:23166835

Identification of single-copy orthologous genes between Physalis and Solanum lycopersicum and analysis of genetic diversity in Physalis using molecular markers.

PubMed

Wei, Jingli; Hu, Xiaorong; Yang, Jingjing; Yang, Wencai

2012-01-01

The genus Physalis includes a number of commercially important edible and ornamental species. Its high nutritional value and potential medicinal properties leads to the increased commercial interest in the products of this genus worldwide. However, lack of molecular markers prevents the detailed study of genetics and phylogeny in Physalis, which limits the progress of breeding. In the present study, we compared the DNA sequences between Physalis and tomato, and attempted to analyze genetic diversity in Physalis using tomato markers. Blasting 23180 DNA sequences derived from Physalis against the International Tomato Annotation Group (ITAG) Release2.3 Predicted CDS (SL2.40) discovered 3356 single-copy orthologous genes between them. A total of 38 accessions from at least six species of Physalis were subjected to genetic diversity analysis using 97 tomato markers and 25 SSR markers derived from P. peruviana. Majority (73.2%) of tomato markers could amplify DNA fragments from at least one accession of Physalis. Diversity in Physalis at molecular level was also detected. The average Nei's genetic distance between accessions was 0.3806 with a range of 0.2865 to 0.7091. These results indicated Physalis and tomato had similarity at both molecular marker and DNA sequence levels. Therefore, the molecular markers developed in tomato can be used in genetic study in Physalis.

Long-term nutrient addition differentially alters community composition and diversity of genes that control nitrous oxide flux from salt marsh sediments

NASA Astrophysics Data System (ADS)

Kearns, Patrick J.; Angell, John H.; Feinman, Sarah G.; Bowen, Jennifer L.

2015-03-01

Enrichment of natural waters, soils, and sediments by inorganic nutrients, including nitrogen, is occurring at an increasing rate and has fundamentally altered global biogeochemical cycles. Salt marshes are critical for the removal of land-derived nitrogen before it enters coastal waters. This is accomplished via multiple microbially mediated pathways, including denitrification. Many of these pathways, however, are also a source of the greenhouse gas nitrous oxide (N2O). We used clone libraries and quantative PCR (qPCR) to examine the effect of fertilization on the diversity and abundance of two functional genes associated with denitrification and N2O production (norB and nosZ) in experimental plots at the Great Sippewissett Salt Marsh (Falmouth, MA, USA) that have been enriched with nutrients for over 40 years. Our data showed distinct nosZ and norB community structures at different nitrogen loads, especially at the highest level of fertilization. Furthermore, calculations of the Shannon Diversity Index and Chao1 Richness Estimator indicated that nosZ gene diversity and richness increased with increased nitrogen supply, however no such relationship existed with regard to richness and diversity of the norB gene. Results from qPCR demonstrated that nosZ gene abundance was an order of magnitude lower in the extra-highly fertilized plots compared to the other plots, but the abundance of norB was not affected by fertilization. The majority of sequences obtained from the marsh plots had no close cultured relatives and they were divergent from previously sequenced norB and nosZ fragments. Despite their divergence from any cultured representatives, most of the norB and nosZ sequences appeared to be from members of the Alpha- and Betaproteobacteria, suggesting that these classes are particularly important in salt marsh nitrogen cycling. Our results suggest that both norB and nosZ containing microbes are affected by fertilization and that the Great Sippewissett Marsh may harbor distinct clades of novel denitrifying microorganisms that are responsible for both the production and removal of N2O.

Transferring the Characteristics of Naturally Occurring and Biased Antibody Repertoires to Human Antibody Libraries by Trapping CDRH3 Sequences

PubMed Central

Venet, Sophie; Ravn, Ulla; Buatois, Vanessa; Gueneau, Franck; Calloud, Sébastien; Kosco-Vilbois, Marie; Fischer, Nicolas

2012-01-01

Antibody repertoires are characterized by diversity as they vary not only amongst individuals and post antigen exposure but also differ significantly between vertebrate species. Such plasticity can be exploited to generate human antibody libraries featuring hallmarks of these diverse repertoires. In this study, the focus was to capture CDRH3 sequences, as this region generally accounts for most of the interaction energy with antigen. Sequences from human as well as non-human sources were successfully integrated into human antibody libraries. Next generation sequencing of these libraries proved that the CDRH3 lengths and amino acid composition corresponded to the species of origin. Specific CDRH3 sequences, biased towards the recognition of a model antigen either by immunizing mice or by selecting with phage display, were then integrated into another set of libraries. From these antigen biased libraries, highly potent antibodies were more frequently isolated, indicating that the characteristics of an immune repertoire is transferrable via CDRH3 sequences into a human antibody library. Taken together, these data demonstrate that the properties of naturally or experimentally biased repertoires can be effectively harnessed for the generation of targeted human antibody libraries, substantially increasing the probability of isolating antibodies suitable for therapeutic and diagnostic applications. PMID:22937053

The need for high-quality whole-genome sequence databases in microbial forensics.

PubMed

Sjödin, Andreas; Broman, Tina; Melefors, Öjar; Andersson, Gunnar; Rasmusson, Birgitta; Knutsson, Rickard; Forsman, Mats

2013-09-01

Microbial forensics is an important part of a strengthened capability to respond to biocrime and bioterrorism incidents to aid in the complex task of distinguishing between natural outbreaks and deliberate acts. The goal of a microbial forensic investigation is to identify and criminally prosecute those responsible for a biological attack, and it involves a detailed analysis of the weapon--that is, the pathogen. The recent development of next-generation sequencing (NGS) technologies has greatly increased the resolution that can be achieved in microbial forensic analyses. It is now possible to identify, quickly and in an unbiased manner, previously undetectable genome differences between closely related isolates. This development is particularly relevant for the most deadly bacterial diseases that are caused by bacterial lineages with extremely low levels of genetic diversity. Whole-genome analysis of pathogens is envisaged to be increasingly essential for this purpose. In a microbial forensic context, whole-genome sequence analysis is the ultimate method for strain comparisons as it is informative during identification, characterization, and attribution--all 3 major stages of the investigation--and at all levels of microbial strain identity resolution (ie, it resolves the full spectrum from family to isolate). Given these capabilities, one bottleneck in microbial forensics investigations is the availability of high-quality reference databases of bacterial whole-genome sequences. To be of high quality, databases need to be curated and accurate in terms of sequences, metadata, and genetic diversity coverage. The development of whole-genome sequence databases will be instrumental in successfully tracing pathogens in the future.

Characterization of the bacterial biodiversity in Pico cheese (an artisanal Azorean food).

PubMed

Riquelme, Cristina; Câmara, Sandra; Dapkevicius, Maria de Lurdes N Enes; Vinuesa, Pablo; da Silva, Célia Costa Gomes; Malcata, F Xavier; Rego, Oldemiro A

2015-01-02

This work presents the first study on the bacterial communities in Pico cheese, a traditional cheese of the Azores (Portugal), made from raw cow's milk. Pyrosequencing of tagged amplicons of the V3-V4 regions of the 16S rDNA and Operational Taxonomic Unit-based (OTU-based) analysis were applied to obtain an overall idea of the microbiota in Pico cheese and to elucidate possible differences between cheese-makers (A, B and C) and maturation times. Pyrosequencing revealed a high bacterial diversity in Pico cheese. Four phyla (Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes) and 54 genera were identified. The predominant genus was Lactococcus (77% of the sequences). Sequences belonging to major cheese-borne pathogens were not found. Staphylococcus accounted for 0.5% of the sequences. Significant differences in bacterial community composition were observed between cheese-maker B and the other two units that participated in the study. However, OTU analysis identified a set of taxa (Lactococcus, Streptococcus, Acinetobacter, Enterococcus, Lactobacillus, Staphylococcus, Rothia, Pantoea and unclassified genera belonging to the Enterobacteriaceae family) that would represent the core components of artisanal Pico cheese microbiota. A diverse bacterial community was present at early maturation, with an increase in the number of phylotypes up to 2 weeks, followed by a decrease at the end of ripening. The most remarkable trend in abundance patterns throughout ripening was an increase in the number of sequences belonging to the Lactobacillus genus, with a concomitant decrease in Acinetobacter, and Stenotrophomonas. Microbial rank abundance curves showed that Pico cheese's bacterial communities are characterized by a few dominant taxa and many low-abundance, highly diverse taxa that integrate the so-called "rare biosphere". Copyright © 2014 Elsevier B.V. All rights reserved.

Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons

PubMed Central

Haas, Brian J.; Gevers, Dirk; Earl, Ashlee M.; Feldgarden, Mike; Ward, Doyle V.; Giannoukos, Georgia; Ciulla, Dawn; Tabbaa, Diana; Highlander, Sarah K.; Sodergren, Erica; Methé, Barbara; DeSantis, Todd Z.; Petrosino, Joseph F.; Knight, Rob; Birren, Bruce W.

2011-01-01

Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys. PMID:21212162

Construction of nested genetic core collections to optimize the exploitation of natural diversity in Vitis vinifera L. subsp. sativa

PubMed Central

Le Cunff, Loïc; Fournier-Level, Alexandre; Laucou, Valérie; Vezzulli, Silvia; Lacombe, Thierry; Adam-Blondon, Anne-Françoise; Boursiquot, Jean-Michel; This, Patrice

2008-01-01

Background The first high quality draft of the grape genome sequence has just been published. This is a critical step in accessing all the genes of this species and increases the chances of exploiting the natural genetic diversity through association genetics. However, our basic knowledge of the extent of allelic variation within the species is still not sufficient. Towards this goal, we constructed nested genetic core collections (G-cores) to capture the simple sequence repeat (SSR) diversity of the grape cultivated compartment (Vitis vinifera L. subsp. sativa) from the world's largest germplasm collection (Domaine de Vassal, INRA Hérault, France), containing 2262 unique genotypes. Results Sub-samples of 12, 24, 48 and 92 varieties of V. vinifera L. were selected based on their genotypes for 20 SSR markers using the M-strategy. They represent respectively 58%, 73%, 83% and 100% of total SSR diversity. The capture of allelic diversity was analyzed by sequencing three genes scattered throughout the genome on 233 individuals: 41 single nucleotide polymorphisms (SNPs) were identified using the G-92 core (one SNP for every 49 nucleotides) while only 25 were observed using a larger sample of 141 individuals selected on the basis of 50 morphological traits, thus demonstrating the reliability of the approach. Conclusion The G-12 and G-24 core-collections displayed respectively 78% and 88% of the SNPs respectively, and are therefore of great interest for SNP discovery studies. Furthermore, the nested genetic core collections satisfactorily reflected the geographic and the genetic diversity of grape, which are also of great interest for the study of gene evolution in this species. PMID:18384667

SNiPlay: a web-based tool for detection, management and analysis of SNPs. Application to grapevine diversity projects.

PubMed

Dereeper, Alexis; Nicolas, Stéphane; Le Cunff, Loïc; Bacilieri, Roberto; Doligez, Agnès; Peros, Jean-Pierre; Ruiz, Manuel; This, Patrice

2011-05-05

High-throughput re-sequencing, new genotyping technologies and the availability of reference genomes allow the extensive characterization of Single Nucleotide Polymorphisms (SNPs) and insertion/deletion events (indels) in many plant species. The rapidly increasing amount of re-sequencing and genotyping data generated by large-scale genetic diversity projects requires the development of integrated bioinformatics tools able to efficiently manage, analyze, and combine these genetic data with genome structure and external data. In this context, we developed SNiPlay, a flexible, user-friendly and integrative web-based tool dedicated to polymorphism discovery and analysis. It integrates:1) a pipeline, freely accessible through the internet, combining existing softwares with new tools to detect SNPs and to compute different types of statistical indices and graphical layouts for SNP data. From standard sequence alignments, genotyping data or Sanger sequencing traces given as input, SNiPlay detects SNPs and indels events and outputs submission files for the design of Illumina's SNP chips. Subsequently, it sends sequences and genotyping data into a series of modules in charge of various processes: physical mapping to a reference genome, annotation (genomic position, intron/exon location, synonymous/non-synonymous substitutions), SNP frequency determination in user-defined groups, haplotype reconstruction and network, linkage disequilibrium evaluation, and diversity analysis (Pi, Watterson's Theta, Tajima's D).Furthermore, the pipeline allows the use of external data (such as phenotype, geographic origin, taxa, stratification) to define groups and compare statistical indices.2) a database storing polymorphisms, genotyping data and grapevine sequences released by public and private projects. It allows the user to retrieve SNPs using various filters (such as genomic position, missing data, polymorphism type, allele frequency), to compare SNP patterns between populations, and to export genotyping data or sequences in various formats. Our experiments on grapevine genetic projects showed that SNiPlay allows geneticists to rapidly obtain advanced results in several key research areas of plant genetic diversity. Both the management and treatment of large amounts of SNP data are rendered considerably easier for end-users through automation and integration. Current developments are taking into account new advances in high-throughput technologies.SNiPlay is available at: http://sniplay.cirad.fr/.

Genotyping-by-sequencing (GBS) revealed molecular genetic diversity of Iranian wheat landraces and cultivars

USDA-ARS?s Scientific Manuscript database

Genetic diversity is an essential resource for breeders to improve new cultivars with desirable characteristics. Recently genotyping-by-sequencing (GBS), a next generation sequencing (NGS) based technology that can simplify complex genomes, has been used as a high-throughput and cost-effective molec...

Transcriptome sequencing of diverse peanut (arachis) wild species and the cultivated species reveals a wealth of untapped genetic variability

USDA-ARS?s Scientific Manuscript database

Next generation sequencing technologies and improved bioinformatics methods have provided opportunities to study sequence variability in complex polyploid transcriptomes. In this study, we used a diverse panel of twenty-two Arachis accessions representing seven Arachis hypogaea market classes, A-, B...

Global patterns in coronavirus diversity

PubMed Central

Johnson, Christine K.; Greig, Denise J.; Kramer, Sarah; Che, Xiaoyu; Wells, Heather; Hicks, Allison L.; Joly, Damien O.; Wolfe, Nathan D.; Daszak, Peter; Karesh, William; Lipkin, W. I.; Morse, Stephen S.; Mazet, Jonna A. K.

2017-01-01

Abstract Since the emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrom Coronavirus (MERS-CoV) it has become increasingly clear that bats are important reservoirs of CoVs. Despite this, only 6% of all CoV sequences in GenBank are from bats. The remaining 94% largely consist of known pathogens of public health or agricultural significance, indicating that current research effort is heavily biased towards describing known diseases rather than the ‘pre-emergent’ diversity in bats. Our study addresses this critical gap, and focuses on resource poor countries where the risk of zoonotic emergence is believed to be highest. We surveyed the diversity of CoVs in multiple host taxa from twenty countries to explore the factors driving viral diversity at a global scale. We identified sequences representing 100 discrete phylogenetic clusters, ninety-one of which were found in bats, and used ecological and epidemiologic analyses to show that patterns of CoV diversity correlate with those of bat diversity. This cements bats as the major evolutionary reservoirs and ecological drivers of CoV diversity. Co-phylogenetic reconciliation analysis was also used to show that host switching has contributed to CoV evolution, and a preliminary analysis suggests that regional variation exists in the dynamics of this process. Overall our study represents a model for exploring global viral diversity and advances our fundamental understanding of CoV biodiversity and the potential risk factors associated with zoonotic emergence. PMID:28630747

Phylogenetically Structured Differences in rRNA Gene Sequence Variation among Species of Arbuscular Mycorrhizal Fungi and Their Implications for Sequence Clustering

PubMed Central

Ekanayake, Saliya; Ruan, Yang; Schütte, Ursel M. E.; Kaonongbua, Wittaya; Fox, Geoffrey; Ye, Yuzhen; Bever, James D.

2016-01-01

ABSTRACT Arbuscular mycorrhizal (AM) fungi form mutualisms with plant roots that increase plant growth and shape plant communities. Each AM fungal cell contains a large amount of genetic diversity, but it is unclear if this diversity varies across evolutionary lineages. We found that sequence variation in the nuclear large-subunit (LSU) rRNA gene from 29 isolates representing 21 AM fungal species generally assorted into genus- and species-level clades, with the exception of species of the genera Claroideoglomus and Entrophospora. However, there were significant differences in the levels of sequence variation across the phylogeny and between genera, indicating that it is an evolutionarily constrained trait in AM fungi. These consistent patterns of sequence variation across both phylogenetic and taxonomic groups pose challenges to interpreting operational taxonomic units (OTUs) as approximations of species-level groups of AM fungi. We demonstrate that the OTUs produced by five sequence clustering methods using 97% or equivalent sequence similarity thresholds failed to match the expected species of AM fungi, although OTUs from AbundantOTU, CD-HIT-OTU, and CROP corresponded better to species than did OTUs from mothur or UPARSE. This lack of OTU-to-species correspondence resulted both from sequences of one species being split into multiple OTUs and from sequences of multiple species being lumped into the same OTU. The OTU richness therefore will not reliably correspond to the AM fungal species richness in environmental samples. Conservatively, this error can overestimate species richness by 4-fold or underestimate richness by one-half, and the direction of this error will depend on the genera represented in the sample. IMPORTANCE Arbuscular mycorrhizal (AM) fungi form important mutualisms with the roots of most plant species. Individual AM fungi are genetically diverse, but it is unclear whether the level of this diversity differs among evolutionary lineages. We found that the amount of sequence variation in an rRNA gene that is commonly used to identify AM fungal species varied significantly between evolutionary groups that correspond to different genera, with the exception of two genera that are genetically indistinguishable from each other. When we clustered groups of similar sequences into operational taxonomic units (OTUs) using five different clustering methods, these patterns of sequence variation caused the number of OTUs to either over- or underestimate the actual number of AM fungal species, depending on the genus. Our results indicate that OTU-based inferences about AM fungal species composition from environmental sequences can be improved if they take these taxonomically structured patterns of sequence variation into account. PMID:27260357

Twenty-one genome sequences from Pseudomonas species and 19 genome sequences from diverse bacteria isolated from the rhizosphere and endosphere of Populus deltoides.

PubMed

Brown, Steven D; Utturkar, Sagar M; Klingeman, Dawn M; Johnson, Courtney M; Martin, Stanton L; Land, Miriam L; Lu, Tse-Yuan S; Schadt, Christopher W; Doktycz, Mitchel J; Pelletier, Dale A

2012-11-01

To aid in the investigation of the Populus deltoides microbiome, we generated draft genome sequences for 21 Pseudomonas strains and 19 other diverse bacteria isolated from Populus deltoides roots. Genome sequences for isolates similar to Acidovorax, Bradyrhizobium, Brevibacillus, Caulobacter, Chryseobacterium, Flavobacterium, Herbaspirillum, Novosphingobium, Pantoea, Phyllobacterium, Polaromonas, Rhizobium, Sphingobium, and Variovorax were generated.

Global sequence diversity of the lactate dehydrogenase gene in Plasmodium falciparum.

PubMed

Simpalipan, Phumin; Pattaradilokrat, Sittiporn; Harnyuttanakorn, Pongchai

2018-01-09

Antigen-detecting rapid diagnostic tests (RDTs) have been recommended by the World Health Organization for use in remote areas to improve malaria case management. Lactate dehydrogenase (LDH) of Plasmodium falciparum is one of the main parasite antigens employed by various commercial RDTs. It has been hypothesized that the poor detection of LDH-based RDTs is attributed in part to the sequence diversity of the gene. To test this, the present study aimed to investigate the genetic diversity of the P. falciparum ldh gene in Thailand and to construct the map of LDH sequence diversity in P. falciparum populations worldwide. The ldh gene was sequenced for 50 P. falciparum isolates in Thailand and compared with hundreds of sequences from P. falciparum populations worldwide. Several indices of molecular variation were calculated, including the proportion of polymorphic sites, the average nucleotide diversity index (π), and the haplotype diversity index (H). Tests of positive selection and neutrality tests were performed to determine signatures of natural selection on the gene. Mean genetic distance within and between species of Plasmodium ldh was analysed to infer evolutionary relationships. Nucleotide sequences of P. falciparum ldh could be classified into 9 alleles, encoding 5 isoforms of LDH. L1a was the most common allelic type and was distributed in P. falciparum populations worldwide. Plasmodium falciparum ldh sequences were highly conserved, with haplotype and nucleotide diversity values of 0.203 and 0.0004, respectively. The extremely low genetic diversity was maintained by purifying selection, likely due to functional constraints. Phylogenetic analysis inferred the close genetic relationship of P. falciparum to malaria parasites of great apes, rather than to other human malaria parasites. This study revealed the global genetic variation of the ldh gene in P. falciparum, providing knowledge for improving detection of LDH-based RDTs and supporting the candidacy of LDH as a therapeutic drug target.

Rogue taxa phenomenon: a biological companion to simulation analysis

PubMed Central

Westover, Kristi M.; Rusinko, Joseph P.; Hoin, Jon; Neal, Matthew

2013-01-01

To provide a baseline biological comparison to simulation study predictions about the frequency of rogue taxa effects, we evaluated the frequency of a rogue taxa effect using viral data sets which differed in diversity. Using a quartet-tree framework, we measured the frequency of a rogue taxa effect in three data sets of increasing genetic variability (within viral serotype, between viral serotype, and between viral family) to test whether the rogue taxa was correlated with the mean sequence diversity of the respective data sets. We found a slight increase in the percentage of rogues as nucleotide diversity increased. Even though the number of rogues increased with diversity, the distribution of the types of rogues (friendly, crazy, or evil) did not depend on the diversity and in the case of the order-level data set the net rogue effect was slightly positive. This study, assessing frequency of the rogue taxa effect using biological data, indicated that simulation studies may over-predict the prevalence of the rogue taxa effect. Further investigations are necessary to understand which types of data sets are susceptible to a negative rogue effect and thus merit the removal of taxa from large phylogenetic reconstructions. PMID:23707704

Rogue taxa phenomenon: a biological companion to simulation analysis.

PubMed

Westover, Kristi M; Rusinko, Joseph P; Hoin, Jon; Neal, Matthew

2013-10-01

To provide a baseline biological comparison to simulation study predictions about the frequency of rogue taxa effects, we evaluated the frequency of a rogue taxa effect using viral data sets which differed in diversity. Using a quartet-tree framework, we measured the frequency of a rogue taxa effect in three data sets of increasing genetic variability (within viral serotype, between viral serotype, and between viral family) to test whether the rogue taxa was correlated with the mean sequence diversity of the respective data sets. We found a slight increase in the percentage of rogues as nucleotide diversity increased. Even though the number of rogues increased with diversity, the distribution of the types of rogues (friendly, crazy, or evil) did not depend on the diversity and in the case of the order-level data set the net rogue effect was slightly positive. This study, assessing frequency of the rogue taxa effect using biological data, indicated that simulation studies may over-predict the prevalence of the rogue taxa effect. Further investigations are necessary to understand which types of data sets are susceptible to a negative rogue effect and thus merit the removal of taxa from large phylogenetic reconstructions. Copyright © 2013 Elsevier Inc. All rights reserved.

HIV-1 sequence variation between isolates from mother-infant transmission pairs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wike, C.M.; Daniels, M.R.; Furtado, M.

1991-12-31

To examine the sequence diversity of human immunodeficiency virus type 1 (HIV-1) between known transmission sets, sequences from the V3 and V4-V5 region of the env gene from 4 mother-infant pairs were analyzed. The mean interpatient sequence variation between isolates from linked mother-infant pairs was comparable to the sequence diversity found between isolates from other close contacts. The mean intrapatient variation was significantly less in the infants` isolates then the isolates from both their mothers and other characterized intrapatient sequence sets. In addition, a distinct and characteristic difference in the glycosylation pattern preceding the V3 loop was found between eachmore » linked transmission pair. These findings indicate that selection of specific genotypic variants, which may play a role in some direct transmission sets, and the duration of infection are important factors in the degree of diversity seen between the sequence sets.« less

Whole-genome sequencing and analyses identify high genetic heterogeneity, diversity and endemicity of rotavirus genotype P[6] strains circulating in Africa.

PubMed

Nyaga, Martin M; Tan, Yi; Seheri, Mapaseka L; Halpin, Rebecca A; Akopov, Asmik; Stucker, Karla M; Fedorova, Nadia B; Shrivastava, Susmita; Duncan Steele, A; Mwenda, Jason M; Pickett, Brett E; Das, Suman R; Jeffrey Mphahlele, M

2018-05-18

Rotavirus A (RVA) exhibits a wide genotype diversity globally. Little is known about the genetic composition of genotype P[6] from Africa. This study investigated possible evolutionary mechanisms leading to genetic diversity of genotype P[6] VP4 sequences. Phylogenetic analyses on 167 P[6] VP4 full-length sequences were conducted, which included six porcine-origin sequences. Of the 167 sequences, 57 were newly acquired through whole genome sequencing as part of this study. The other 110 sequences were all publicly-available global P[6] VP4 full-length sequences downloaded from GenBank. The strength of association between the phenotypic features and the phylogeny was also determined. A number of reassortment and mixed infections of RVA genotype P[6] strains were observed in this study. Phylogenetic analyses demostrated the extensive genetic diversity that exists among human P[6] strains, porcine-like strains, their concomitant clades/subclades and estimated that P[6] VP4 gene has a higher substitution rate with the mean of 1.05E-3 substitutions/site/year. Further, the phylogenetic analyses indicated that genotype P[6] strains were endemic in Africa, characterised by an extensive genetic diversity and long-time local evolution of the viruses. This was also supported by phylogeographic clustering and G-genotype clustering of the P[6] strains when Bayesian Tip-association Significance testing (BaTS) was applied, clearly supporting that the viruses evolved locally in Africa instead of spatial mixing among different regions. Overall, the results demonstrated that multiple mechanisms such as reassortment events, various mutations and possibly interspecies transmission account for the enormous diversity of genotype P[6] strains in Africa. These findings highlight the need for continued global surveillance of rotavirus diversity. Copyright © 2018 Elsevier B.V. All rights reserved.

Microbial community profiling of fresh basil and pitfalls in taxonomic assignment of enterobacterial pathogenic species based upon 16S rRNA amplicon sequencing.

PubMed

Ceuppens, Siele; De Coninck, Dieter; Bottledoorn, Nadine; Van Nieuwerburgh, Filip; Uyttendaele, Mieke

2017-09-18

Application of 16S rRNA (gene) amplicon sequencing on food samples is increasingly applied for assessing microbial diversity but may as unintended advantage also enable simultaneous detection of any human pathogens without a priori definition. In the present study high-throughput next-generation sequencing (NGS) of the V1-V2-V3 regions of the 16S rRNA gene was applied to identify the bacteria present on fresh basil leaves. However, results were strongly impacted by variations in the bioinformatics analysis pipelines (MEGAN, SILVAngs, QIIME and MG-RAST), including the database choice (Greengenes, RDP and M5RNA) and the annotation algorithm (best hit, representative hit and lowest common ancestor). The use of pipelines with default parameters will lead to discrepancies. The estimate of microbial diversity of fresh basil using 16S rRNA (gene) amplicon sequencing is thus indicative but subject to biases. Salmonella enterica was detected at low frequencies, between 0.1% and 0.4% of bacterial sequences, corresponding with 37 to 166 reads. However, this result was dependent upon the pipeline used: Salmonella was detected by MEGAN, SILVAngs and MG-RAST, but not by QIIME. Confirmation of Salmonella sequences by real-time PCR was unsuccessful. It was shown that taxonomic resolution obtained from the short (500bp) sequence reads of the 16S rRNA gene containing the hypervariable regions V1-V3 cannot allow distinction of Salmonella with closely related enterobacterial species. In conclusion 16S amplicon sequencing, getting the status of standard method in microbial ecology studies of foods, needs expertise on both bioinformatics and microbiology for analysis of results. It is a powerful tool to estimate bacterial diversity but amenable to biases. Limitations concerning taxonomic resolution for some bacterial species or its inability to detect sub-dominant (pathogenic) species should be acknowledged in order to avoid overinterpretation of results. Copyright © 2017 Elsevier B.V. All rights reserved.

Reproducibility and quantitation of amplicon sequencing-based detection

PubMed Central

Zhou, Jizhong; Wu, Liyou; Deng, Ye; Zhi, Xiaoyang; Jiang, Yi-Huei; Tu, Qichao; Xie, Jianping; Van Nostrand, Joy D; He, Zhili; Yang, Yunfeng

2011-01-01

To determine the reproducibility and quantitation of the amplicon sequencing-based detection approach for analyzing microbial community structure, a total of 24 microbial communities from a long-term global change experimental site were examined. Genomic DNA obtained from each community was used to amplify 16S rRNA genes with two or three barcode tags as technical replicates in the presence of a small quantity (0.1% wt/wt) of genomic DNA from Shewanella oneidensis MR-1 as the control. The technical reproducibility of the amplicon sequencing-based detection approach is quite low, with an average operational taxonomic unit (OTU) overlap of 17.2%±2.3% between two technical replicates, and 8.2%±2.3% among three technical replicates, which is most likely due to problems associated with random sampling processes. Such variations in technical replicates could have substantial effects on estimating β-diversity but less on α-diversity. A high variation was also observed in the control across different samples (for example, 66.7-fold for the forward primer), suggesting that the amplicon sequencing-based detection approach could not be quantitative. In addition, various strategies were examined to improve the comparability of amplicon sequencing data, such as increasing biological replicates, and removing singleton sequences and less-representative OTUs across biological replicates. Finally, as expected, various statistical analyses with preprocessed experimental data revealed clear differences in the composition and structure of microbial communities between warming and non-warming, or between clipping and non-clipping. Taken together, these results suggest that amplicon sequencing-based detection is useful in analyzing microbial community structure even though it is not reproducible and quantitative. However, great caution should be taken in experimental design and data interpretation when the amplicon sequencing-based detection approach is used for quantitative analysis of the β-diversity of microbial communities. PMID:21346791

Evolution properties of online user preference diversity

NASA Astrophysics Data System (ADS)

Guo, Qiang; Ji, Lei; Liu, Jian-Guo; Han, Jingti

2017-02-01

Detecting the evolution properties of online user preference diversity is of significance for deeply understanding online collective behaviors. In this paper, we empirically explore the evolution patterns of online user rating preference, where the preference diversity is measured by the variation coefficient of the user rating sequence. The statistical results for four real systems show that, for movies and reviews, the user rating preference would become diverse and then get centralized finally. By introducing the empirical variation coefficient, we present a Markov model, which could regenerate the evolution properties of two online systems regarding to the stable variation coefficients. In addition, we investigate the evolution of the correlation between the user ratings and the object qualities, and find that the correlation would keep increasing as the user degree increases. This work could be helpful for understanding the anchoring bias and memory effects of the online user collective behaviors.

Nonpareil 3: Fast Estimation of Metagenomic Coverage and Sequence Diversity.

PubMed

Rodriguez-R, Luis M; Gunturu, Santosh; Tiedje, James M; Cole, James R; Konstantinidis, Konstantinos T

2018-01-01

Estimations of microbial community diversity based on metagenomic data sets are affected, often to an unknown degree, by biases derived from insufficient coverage and reference database-dependent estimations of diversity. For instance, the completeness of reference databases cannot be generally estimated since it depends on the extant diversity sampled to date, which, with the exception of a few habitats such as the human gut, remains severely undersampled. Further, estimation of the degree of coverage of a microbial community by a metagenomic data set is prohibitively time-consuming for large data sets, and coverage values may not be directly comparable between data sets obtained with different sequencing technologies. Here, we extend Nonpareil, a database-independent tool for the estimation of coverage in metagenomic data sets, to a high-performance computing implementation that scales up to hundreds of cores and includes, in addition, a k -mer-based estimation as sensitive as the original alignment-based version but about three hundred times as fast. Further, we propose a metric of sequence diversity ( N d ) derived directly from Nonpareil curves that correlates well with alpha diversity assessed by traditional metrics. We use this metric in different experiments demonstrating the correlation with the Shannon index estimated on 16S rRNA gene profiles and show that N d additionally reveals seasonal patterns in marine samples that are not captured by the Shannon index and more precise rankings of the magnitude of diversity of microbial communities in different habitats. Therefore, the new version of Nonpareil, called Nonpareil 3, advances the toolbox for metagenomic analyses of microbiomes. IMPORTANCE Estimation of the coverage provided by a metagenomic data set, i.e., what fraction of the microbial community was sampled by DNA sequencing, represents an essential first step of every culture-independent genomic study that aims to robustly assess the sequence diversity present in a sample. However, estimation of coverage remains elusive because of several technical limitations associated with high computational requirements and limiting statistical approaches to quantify diversity. Here we described Nonpareil 3, a new bioinformatics algorithm that circumvents several of these limitations and thus can facilitate culture-independent studies in clinical or environmental settings, independent of the sequencing platform employed. In addition, we present a new metric of sequence diversity based on rarefied coverage and demonstrate its use in communities from diverse ecosystems.

Diversity and stratification of archaea in a hypersaline microbial mat.

PubMed

Robertson, Charles E; Spear, John R; Harris, J Kirk; Pace, Norman R

2009-04-01

The Guerrero Negro (GN) hypersaline microbial mats have become one focus for biogeochemical studies of stratified ecosystems. The GN mats are found beneath several of a series of ponds of increasing salinity that make up a solar saltern fed from Pacific Ocean water pumped from the Laguna Ojo de Liebre near GN, Baja California Sur, Mexico. Molecular surveys of the laminated photosynthetic microbial mat below the fourth pond in the series identified an enormous diversity of bacteria in the mat, but archaea have received little attention. To determine the bulk contribution of archaeal phylotypes to the pond 4 study site, we determined the phylogenetic distribution of archaeal rRNA gene sequences in PCR libraries based on nominally universal primers. The ratios of bacterial/archaeal/eukaryotic rRNA genes, 90%/9%/1%, suggest that the archaeal contribution to the metabolic activities of the mat may be significant. To explore the distribution of archaea in the mat, sequences derived using archaeon-specific PCR primers were surveyed in 10 strata of the 6-cm-thick mat. The diversity of archaea overall was substantial albeit less than the diversity observed previously for bacteria. Archaeal diversity, mainly euryarchaeotes, was highest in the uppermost 2 to 3 mm of the mat and decreased rapidly with depth, where crenarchaeotes dominated. Only 3% of the sequences were specifically related to known organisms including methanogens. While some mat archaeal clades corresponded with known chemical gradients, others did not, which is likely explained by heretofore-unrecognized gradients. Some clades did not segregate by depth in the mat, indicating broad metabolic repertoires, undersampling, or both.

Diversity of picoeukaryotes at an oligotrophic site off the Northeastern Red Sea Coast

PubMed Central

2013-01-01

Background Picoeukaryotes are protists ≤ 3 μm composed of a wide diversity of taxonomic groups. They are an important constituent of the ocean’s microbiota and perform essential ecological roles in marine nutrient and carbon cycles. Despite their importance, the true extent of their diversity has only recently been uncovered by molecular surveys that resulted in the discovery of a substantial number of previously unknown groups. No study on picoeukaryote diversity has been conducted so far in the main Red Sea basin-a unique marine environment characterized by oligotrophic conditions, high levels of irradiance, high salinity and increased water temperature. Results We sampled surface waters off the coast of the northeastern Red Sea and analyzed the picoeukaryotic diversity using Sanger-based clone libraries of the 18S rRNA gene in order to produce high quality, nearly full-length sequences. The community captured by our approach was dominated by three main phyla, the alveolates, stramenopiles and chlorophytes; members of Radiolaria, Cercozoa and Haptophyta were also found, albeit in low abundances. Photosynthetic organisms were especially diverse and abundant in the sample, confirming the importance of picophytoplankton for primary production in the basin as well as indicating the existence of numerous ecological micro-niches for this trophic level in the upper euphotic zone. Heterotrophic organisms were mostly composed of the presumably parasitic Marine Alveolates (MALV) and the presumably bacterivorous Marine Stramenopiles (MAST) groups. A small number of sequences that did not cluster closely with known clades were also found, especially in the MALV-II group, some of which could potentially belong to novel clades. Conclusions This study provides the first snapshot of the picoeukaryotic diversity present in surface waters of the Red Sea, hence setting the stage for large-scale surveying and characterization of the eukaryotic diversity in the entire basin. Our results indicate that the picoeukaryotic community in the northern Red Sea, despite its unique physiochemical conditions (i.e. increased temperatures, increased salinity, and high UV irradiance) does not differ vastly from its counterparts in other oligotrophic marine habitats. PMID:23962380

Diversity of picoeukaryotes at an oligotrophic site off the Northeastern Red Sea Coast.

PubMed

Acosta, Francisco; Ngugi, David Kamanda; Stingl, Ulrich

2013-08-20

Picoeukaryotes are protists ≤ 3 μm composed of a wide diversity of taxonomic groups. They are an important constituent of the ocean's microbiota and perform essential ecological roles in marine nutrient and carbon cycles. Despite their importance, the true extent of their diversity has only recently been uncovered by molecular surveys that resulted in the discovery of a substantial number of previously unknown groups. No study on picoeukaryote diversity has been conducted so far in the main Red Sea basin-a unique marine environment characterized by oligotrophic conditions, high levels of irradiance, high salinity and increased water temperature. We sampled surface waters off the coast of the northeastern Red Sea and analyzed the picoeukaryotic diversity using Sanger-based clone libraries of the 18S rRNA gene in order to produce high quality, nearly full-length sequences. The community captured by our approach was dominated by three main phyla, the alveolates, stramenopiles and chlorophytes; members of Radiolaria, Cercozoa and Haptophyta were also found, albeit in low abundances. Photosynthetic organisms were especially diverse and abundant in the sample, confirming the importance of picophytoplankton for primary production in the basin as well as indicating the existence of numerous ecological micro-niches for this trophic level in the upper euphotic zone. Heterotrophic organisms were mostly composed of the presumably parasitic Marine Alveolates (MALV) and the presumably bacterivorous Marine Stramenopiles (MAST) groups. A small number of sequences that did not cluster closely with known clades were also found, especially in the MALV-II group, some of which could potentially belong to novel clades. This study provides the first snapshot of the picoeukaryotic diversity present in surface waters of the Red Sea, hence setting the stage for large-scale surveying and characterization of the eukaryotic diversity in the entire basin. Our results indicate that the picoeukaryotic community in the northern Red Sea, despite its unique physiochemical conditions (i.e. increased temperatures, increased salinity, and high UV irradiance) does not differ vastly from its counterparts in other oligotrophic marine habitats.

Out of the dark: transitional subsurface-to-surface microbial diversity in a terrestrial serpentinizing seep (Manleluag, Pangasinan, the Philippines)

PubMed Central

Woycheese, Kristin M.; Meyer-Dombard, D'Arcy R.; Cardace, Dawn; Argayosa, Anacleto M.; Arcilla, Carlo A.

2015-01-01

In the Zambales ophiolite range, terrestrial serpentinizing fluid seeps host diverse microbial assemblages. The fluids fall within the profile of Ca2+-OH−-type waters, indicative of active serpentinization, and are low in dissolved inorganic carbon (DIC) (<0.5 ppm). Influx of atmospheric carbon dioxide (CO2) affects the solubility of calcium carbonate as distance from the source increases, triggering the formation of meter-scale travertine terraces. Samples were collected at the source and along the outflow channel to determine subsurface microbial community response to surface exposure. DNA was extracted and submitted for high-throughput 16S rRNA gene sequencing on the Illumina MiSeq platform. Taxonomic assignment of the sequence data indicates that 8.1% of the total sequence reads at the source of the seep affiliate with the genus Methanobacterium. Other major classes detected at the source include anaerobic taxa such as Bacteroidetes (40.7% of total sequence reads) and Firmicutes (19.1% of total reads). Hydrogenophaga spp. increase in relative abundance as redox potential increases. At the carbonate terrace, 45% of sequence reads affiliate with Meiothermus spp. Taxonomic observations and geochemical data suggest that several putative metabolisms may be favorable, including hydrogen oxidation, H2-associated sulfur cycling, methanogenesis, methanotrophy, nitrogen fixation, ammonia oxidation, denitrification, nitrate respiration, methylotrophy, carbon monoxide respiration, and ferrous iron oxidation, based on capabilities of nearest known neighbors. Scanning electron microscopy and energy dispersive X-ray spectroscopy suggest that microbial activity produces chemical and physical traces in the precipitated carbonates forming downstream of the seep's source. These data provide context for future serpentinizing seep ecosystem studies, particularly with regards to tropical biomes. PMID:25745416

Out of the dark: transitional subsurface-to-surface microbial diversity in a terrestrial serpentinizing seep (Manleluag, Pangasinan, the Philippines).

PubMed

Woycheese, Kristin M; Meyer-Dombard, D'Arcy R; Cardace, Dawn; Argayosa, Anacleto M; Arcilla, Carlo A

2015-01-01

In the Zambales ophiolite range, terrestrial serpentinizing fluid seeps host diverse microbial assemblages. The fluids fall within the profile of Ca(2+)-OH(-)-type waters, indicative of active serpentinization, and are low in dissolved inorganic carbon (DIC) (<0.5 ppm). Influx of atmospheric carbon dioxide (CO2) affects the solubility of calcium carbonate as distance from the source increases, triggering the formation of meter-scale travertine terraces. Samples were collected at the source and along the outflow channel to determine subsurface microbial community response to surface exposure. DNA was extracted and submitted for high-throughput 16S rRNA gene sequencing on the Illumina MiSeq platform. Taxonomic assignment of the sequence data indicates that 8.1% of the total sequence reads at the source of the seep affiliate with the genus Methanobacterium. Other major classes detected at the source include anaerobic taxa such as Bacteroidetes (40.7% of total sequence reads) and Firmicutes (19.1% of total reads). Hydrogenophaga spp. increase in relative abundance as redox potential increases. At the carbonate terrace, 45% of sequence reads affiliate with Meiothermus spp. Taxonomic observations and geochemical data suggest that several putative metabolisms may be favorable, including hydrogen oxidation, H2-associated sulfur cycling, methanogenesis, methanotrophy, nitrogen fixation, ammonia oxidation, denitrification, nitrate respiration, methylotrophy, carbon monoxide respiration, and ferrous iron oxidation, based on capabilities of nearest known neighbors. Scanning electron microscopy and energy dispersive X-ray spectroscopy suggest that microbial activity produces chemical and physical traces in the precipitated carbonates forming downstream of the seep's source. These data provide context for future serpentinizing seep ecosystem studies, particularly with regards to tropical biomes.

Microbial Diversity in Deep-sea Methane Seep Sediments Presented by SSU rRNA Gene Tag Sequencing

PubMed Central

Nunoura, Takuro; Takaki, Yoshihiro; Kazama, Hiromi; Hirai, Miho; Ashi, Juichiro; Imachi, Hiroyuki; Takai, Ken

2012-01-01

Microbial community structures in methane seep sediments in the Nankai Trough were analyzed by tag-sequencing analysis for the small subunit (SSU) rRNA gene using a newly developed primer set. The dominant members of Archaea were Deep-sea Hydrothermal Vent Euryarchaeotic Group 6 (DHVEG 6), Marine Group I (MGI) and Deep Sea Archaeal Group (DSAG), and those in Bacteria were Alpha-, Gamma-, Delta- and Epsilonproteobacteria, Chloroflexi, Bacteroidetes, Planctomycetes and Acidobacteria. Diversity and richness were examined by 8,709 and 7,690 tag-sequences from sediments at 5 and 25 cm below the seafloor (cmbsf), respectively. The estimated diversity and richness in the methane seep sediment are as high as those in soil and deep-sea hydrothermal environments, although the tag-sequences obtained in this study were not sufficient to show whole microbial diversity in this analysis. We also compared the diversity and richness of each taxon/division between the sediments from the two depths, and found that the diversity and richness of some taxa/divisions varied significantly along with the depth. PMID:22510646

Dramatic Increases of Soil Microbial Functional Gene Diversity at the Treeline Ecotone of Changbai Mountain

PubMed Central

Shen, Congcong; Shi, Yu; Ni, Yingying; Deng, Ye; Van Nostrand, Joy D.; He, Zhili; Zhou, Jizhong; Chu, Haiyan

2016-01-01

The elevational and latitudinal diversity patterns of microbial taxa have attracted great attention in the past decade. Recently, the distribution of functional attributes has been in the spotlight. Here, we report a study profiling soil microbial communities along an elevation gradient (500–2200 m) on Changbai Mountain. Using a comprehensive functional gene microarray (GeoChip 5.0), we found that microbial functional gene richness exhibited a dramatic increase at the treeline ecotone, but the bacterial taxonomic and phylogenetic diversity based on 16S rRNA gene sequencing did not exhibit such a similar trend. However, the β-diversity (compositional dissimilarity among sites) pattern for both bacterial taxa and functional genes was similar, showing significant elevational distance-decay patterns which presented increased dissimilarity with elevation. The bacterial taxonomic diversity/structure was strongly influenced by soil pH, while the functional gene diversity/structure was significantly correlated with soil dissolved organic carbon (DOC). This finding highlights that soil DOC may be a good predictor in determining the elevational distribution of microbial functional genes. The finding of significant shifts in functional gene diversity at the treeline ecotone could also provide valuable information for predicting the responses of microbial functions to climate change. PMID:27524983

Genetic Diversity of Bacterial Communities and Gene Transfer Agents in Northern South China Sea

PubMed Central

Sun, Fu-Lin; Wang, You-Shao; Wu, Mei-Lin; Jiang, Zhao-Yu; Sun, Cui-Ci; Cheng, Hao

2014-01-01

Pyrosequencing of the 16S ribosomal RNA gene (rDNA) amplicons was performed to investigate the unique distribution of bacterial communities in northern South China Sea (nSCS) and evaluate community structure and spatial differences of bacterial diversity. Cyanobacteria, Proteobacteria, Actinobacteria, and Bacteroidetes constitute the majority of bacteria. The taxonomic description of bacterial communities revealed that more Chroococcales, SAR11 clade, Acidimicrobiales, Rhodobacterales, and Flavobacteriales are present in the nSCS waters than other bacterial groups. Rhodobacterales were less abundant in tropical water (nSCS) than in temperate and cold waters. Furthermore, the diversity of Rhodobacterales based on the gene transfer agent (GTA) major capsid gene (g5) was investigated. Four g5 gene clone libraries were constructed from samples representing different regions and yielded diverse sequences. Fourteen g5 clusters could be identified among 197 nSCS clones. These clusters were also related to known g5 sequences derived from genome-sequenced Rhodobacterales. The composition of g5 sequences in surface water varied with the g5 sequences in the sampling sites; this result indicated that the Rhodobacterales population could be highly diverse in nSCS. Phylogenetic tree analysis result indicated distinguishable diversity patterns among tropical (nSCS), temperate, and cold waters, thereby supporting the niche adaptation of specific Rhodobacterales members in unique environments. PMID:25364820

Phylogenetic diversity and biogeography of the Mamiellophyceae lineage of eukaryotic phytoplankton across the oceans.

PubMed

Monier, Adam; Worden, Alexandra Z; Richards, Thomas A

2016-08-01

High-throughput diversity amplicon sequencing of marine microbial samples has revealed that members of the Mamiellophyceae lineage are successful phytoplankton in many oceanic habitats. Indeed, these eukaryotic green algae can dominate the picoplanktonic biomass, however, given the broad expanses of the oceans, their geographical distributions and the phylogenetic diversity of some groups remain poorly characterized. As these algae play a foundational role in marine food webs, it is crucial to assess their global distribution in order to better predict potential changes in abundance and community structure. To this end, we analyzed the V9-18S small subunit rDNA sequences deposited from the Tara Oceans expedition to evaluate the diversity and biogeography of these phytoplankton. Our results show that the phylogenetic composition of Mamiellophyceae communities is in part determined by geographical provenance, and do not appear to be influenced - in the samples recovered - by water depth, at least at the resolution possible with the V9-18S. Phylogenetic classification of Mamiellophyceae sequences revealed that the Dolichomastigales order encompasses more sequence diversity than other orders in this lineage. These results indicate that a large fraction of the Mamiellophyceae diversity has been hitherto overlooked, likely because of a combination of size fraction, sequencing and geographical limitations. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Growth and splitting of neural sequences in songbird vocal development

PubMed Central

Okubo, Tatsuo S.; Mackevicius, Emily L.; Payne, Hannah L.; Lynch, Galen F.; Fee, Michale S.

2015-01-01

Neural sequences are a fundamental feature of brain dynamics underlying diverse behaviors, but the mechanisms by which they develop during learning remain unknown. Songbirds learn vocalizations composed of syllables; in adult birds, each syllable is produced by a different sequence of action potential bursts in the premotor cortical area HVC. Here we carried out recordings of large populations of HVC neurons in singing juvenile birds throughout learning to examine the emergence of neural sequences. Early in vocal development, HVC neurons begin producing rhythmic bursts, temporally locked to a ‘prototype’ syllable. Different neurons are active at different latencies relative to syllable onset to form a continuous sequence. Through development, as new syllables emerge from the prototype syllable, initially highly overlapping burst sequences become increasingly distinct. We propose a mechanistic model in which multiple neural sequences can emerge from the growth and splitting of a common precursor sequence. PMID:26618871

New Insight Into the Diversity of SemiSWEET Sugar Transporters and the Homologs in Prokaryotes

PubMed Central

Jia, Baolei; Hao, Lujiang; Xuan, Yuan Hu; Jeon, Che Ok

2018-01-01

Sugars will eventually be exported transporters (SWEETs) and SemiSWEETs represent a family of sugar transporters in eukaryotes and prokaryotes, respectively. SWEETs contain seven transmembrane helices (TMHs), while SemiSWEETs contain three. The functions of SemiSWEETs are less studied. In this perspective article, we analyzed the diversity and conservation of SemiSWEETs and further proposed the possible functions. 1,922 SemiSWEET homologs were retrieved from the UniProt database, which is not proportional to the sequenced prokaryotic genomes. However, these proteins are very diverse in sequences and can be classified into 19 clusters when >50% sequence identity is required. Moreover, a gene context analysis indicated that several SemiSWEETs are located in the operons that are related to diverse carbohydrate metabolism. Several proteins with seven TMHs can be found in bacteria, and sequence alignment suggested that these proteins in bacteria may be formed by the duplication and fusion. Multiple sequence alignments showed that the amino acids for sugar translocation are still conserved and coevolved, although the sequences show diversity. Among them, the functions of a few amino acids are still not clear. These findings highlight the challenges that exist in SemiSWEETs and provide future researchers the foundation to explore these uncharted areas. PMID:29872447

New Insight Into the Diversity of SemiSWEET Sugar Transporters and the Homologs in Prokaryotes.

PubMed

Jia, Baolei; Hao, Lujiang; Xuan, Yuan Hu; Jeon, Che Ok

2018-01-01

Sugars will eventually be exported transporters (SWEETs) and SemiSWEETs represent a family of sugar transporters in eukaryotes and prokaryotes, respectively. SWEETs contain seven transmembrane helices (TMHs), while SemiSWEETs contain three. The functions of SemiSWEETs are less studied. In this perspective article, we analyzed the diversity and conservation of SemiSWEETs and further proposed the possible functions. 1,922 SemiSWEET homologs were retrieved from the UniProt database, which is not proportional to the sequenced prokaryotic genomes. However, these proteins are very diverse in sequences and can be classified into 19 clusters when >50% sequence identity is required. Moreover, a gene context analysis indicated that several SemiSWEETs are located in the operons that are related to diverse carbohydrate metabolism. Several proteins with seven TMHs can be found in bacteria, and sequence alignment suggested that these proteins in bacteria may be formed by the duplication and fusion. Multiple sequence alignments showed that the amino acids for sugar translocation are still conserved and coevolved, although the sequences show diversity. Among them, the functions of a few amino acids are still not clear. These findings highlight the challenges that exist in SemiSWEETs and provide future researchers the foundation to explore these uncharted areas.

Comparing Sanger sequencing and high-throughput metabarcoding for inferring photobiont diversity in lichens.

PubMed

Paul, Fiona; Otte, Jürgen; Schmitt, Imke; Dal Grande, Francesco

2018-06-05

The implementation of HTS (high-throughput sequencing) approaches is rapidly changing our understanding of the lichen symbiosis, by uncovering high bacterial and fungal diversity, which is often host-specific. Recently, HTS methods revealed the presence of multiple photobionts inside a single thallus in several lichen species. This differs from Sanger technology, which typically yields a single, unambiguous algal sequence per individual. Here we compared HTS and Sanger methods for estimating the diversity of green algal symbionts within lichen thalli using 240 lichen individuals belonging to two species of lichen-forming fungi. According to HTS data, Sanger technology consistently yielded the most abundant photobiont sequence in the sample. However, if the second most abundant photobiont exceeded 30% of the total HTS reads in a sample, Sanger sequencing generally failed. Our results suggest that most lichen individuals in the two analyzed species, Lasallia hispanica and L. pustulata, indeed contain a single, predominant green algal photobiont. We conclude that Sanger sequencing is a valid approach to detect the dominant photobionts in lichen individuals and populations. We discuss which research areas in lichen ecology and evolution will continue to benefit from Sanger sequencing, and which areas will profit from HTS approaches to assessing symbiont diversity.

Twenty-One Genome Sequences from Pseudomonas Species and 19 Genome Sequences from Diverse Bacteria Isolated from the Rhizosphere and Endosphere of Populus deltoides

PubMed Central

Utturkar, Sagar M.; Klingeman, Dawn M.; Johnson, Courtney M.; Martin, Stanton L.; Land, Miriam L.; Lu, Tse-Yuan S.; Schadt, Christopher W.; Doktycz, Mitchel J.

2012-01-01

To aid in the investigation of the Populus deltoides microbiome, we generated draft genome sequences for 21 Pseudomonas strains and 19 other diverse bacteria isolated from Populus deltoides roots. Genome sequences for isolates similar to Acidovorax, Bradyrhizobium, Brevibacillus, Caulobacter, Chryseobacterium, Flavobacterium, Herbaspirillum, Novosphingobium, Pantoea, Phyllobacterium, Polaromonas, Rhizobium, Sphingobium, and Variovorax were generated. PMID:23045501

Twenty-One Genome Sequences from Pseudomonas Species and 19 Genome Sequences from Diverse Bacteria Isolated from the Rhizosphere and Endosphere of Populus deltoides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Steven D; Utturkar, Sagar M; Klingeman, Dawn Marie

To aid in the investigation of the Populus deltoides microbiome we generated draft genome sequences for twenty one Pseudomonas and twenty one other diverse bacteria isolated from Populus deltoides roots. Genome sequences for isolates similar to Acidovorax, Bradyrhizobium, Brevibacillus, Burkholderia, Caulobacter, Chryseobacterium, Flavobacterium, Herbaspirillum, Novosphingobium, Pantoea, Phyllobacterium, Polaromonas, Rhizobium, Sphingobium and Variovorax were generated.

[Research on bacteria microecology in root rot rhizosphere soil of Coptis chinensis produced in Shizhu city].

PubMed

Song, Xu-Hong; Wang, Yu; Li, Long-Yun; Tan, Jun

2017-04-01

Illumina Hiseq 2500 high-throughput sequencing platform was used to study the bacteria richness and diversity, the soil enzyme activities, nutrients in unplanted soil, root-rot and healthy rhizophere soil of Coptis chinensis for deeply discussing the mechanism of the root-rot of C. chinensis. The high-throughput sequencing result showed that the artificial cultivation effected the bacteria community richness and diversity. The bacteria community richness in healthy and diseased rhizosphere soil showed significant lower than that of in unplanted soil (P<0.05) and declined bacteria diversity. The bacteria community richness in root-rot rhizosphere soil increased significantly than that of health and unplanted soil and the diversity was lower significant than that of unplanted soil (P<0.05). The results of soil nutrients and enzyme activities detected that the pH value, available phosphorus and urease activity decreased and the sucrase activity increased significantly (P<0.05). The content of organic carbon and alkaline hydrolysis nitrogen the catalase and urease activity in root rot soil samples was significantly lower than that of healthy soil samples (P<0.05). However, the contents of available phosphorus and available potassium were significantly in root-rot sample higher than that of healthy soil samples (P<0.05). Comprehensive analysis showed that the artificial cultivation declined the bacteria community richness and diversity. The bacteria community richness decreased significantly and the decreased diversity may be the cause of the root-rot. Meanwhile, the decrease of carbon and the catalase activity may be another cause of the root-rot in C. chinensis produced in Shizhu city, Chongqing province. Copyright© by the Chinese Pharmaceutical Association.

Genetic diversity of Clostridium perfringens type A isolates from animals, food poisoning outbreaks and sludge

PubMed Central

Johansson, Anders; Aspan, Anna; Bagge, Elisabeth; Båverud, Viveca; Engström, Björn E; Johansson, Karl-Erik

2006-01-01

Background Clostridium perfringens, a serious pathogen, causes enteric diseases in domestic animals and food poisoning in humans. The epidemiological relationship between C. perfringens isolates from the same source has previously been investigated chiefly by pulsed-field gel electrophoresis (PFGE). In this study the genetic diversity of C. perfringens isolated from various animals, from food poisoning outbreaks and from sludge was investigated. Results We used PFGE to examine the genetic diversity of 95 C. perfringens type A isolates from eight different sources. The isolates were also examined for the presence of the beta2 toxin gene (cpb2) and the enterotoxin gene (cpe). The cpb2 gene from the 28 cpb2-positive isolates was also partially sequenced (519 bp, corresponding to positions 188 to 706 in the consensus cpb2 sequence). The results of PFGE revealed a wide genetic diversity among the C. perfringens type A isolates. The genetic relatedness of the isolates ranged from 58 to 100% and 56 distinct PFGE types were identified. Almost all clusters with similar patterns comprised isolates with a known epidemiological correlation. Most of the isolates from pig, horse and sheep carried the cpb2 gene. All isolates originating from food poisoning outbreaks carried the cpe gene and three of these also carried cpb2. Two evolutionary different populations were identified by sequence analysis of the partially sequenced cpb2 genes from our study and cpb2 sequences previously deposited in GenBank. Conclusion As revealed by PFGE, there was a wide genetic diversity among C. perfringens isolates from different sources. Epidemiologically related isolates showed a high genetic similarity, as expected, while isolates with no obvious epidemiological relationship expressed a lesser degree of genetic similarity. The wide diversity revealed by PFGE was not reflected in the 16S rRNA sequences, which had a considerable degree of sequence similarity. Sequence comparison of the partially sequenced cpb2 gene revealed two genetically different populations. This is to our knowledge the first study in which the genetic diversity of C. perfringens isolates both from different animals species, from food poisoning outbreaks and from sludge has been investigated. PMID:16737528

The origins and impact of primate segmental duplications.

PubMed

Marques-Bonet, Tomas; Girirajan, Santhosh; Eichler, Evan E

2009-10-01

Duplicated sequences are substrates for the emergence of new genes and are an important source of genetic instability associated with rare and common diseases. Analyses of primate genomes have shown an increase in the proportion of interspersed segmental duplications (SDs) within the genomes of humans and great apes. This contrasts with other mammalian genomes that seem to have their recently duplicated sequences organized in a tandem configuration. In this review, we focus on the mechanistic origin and impact of this difference with respect to evolution, genetic diversity and primate phenotype. Although many genomes will be sequenced in the future, resolution of this aspect of genomic architecture still requires high quality sequences and detailed analyses.

Low DNA Sequence Diversity of the Intergenic Spacer 1 Region in the Human Skin Commensal Fungi Malassezia sympodialis and M. dermatis Isolated from Patients with Malassezia-Associated Skin Diseases and Healthy Subjects.

PubMed

Cho, Otomi; Sugita, Takashi

2016-12-01

As DNA sequences of the intergenic spacer (IGS) region in the rRNA gene show remarkable intraspecies diversity compared with the small subunit, large subunit, and internal transcribed spacer region, the IGS region has been used as an epidemiological tool in studies on Malassezia globosa and M. restricta, which are responsible for the exacerbation of atopic dermatitis (AD) and seborrheic dermatitis (SD). However, the IGS regions of M. sympodialis and M. dermatis obtained from the skin of patients with AD and SD, as well as healthy subjects, lacked sequence diversity. Of the 105 M. sympodialis strains and the 40 M. dermatis strains, the sequences of 103 (98.1 %) and 39 (97.5 %), respectively, were identical. Thus, given the lack of intraspecies diversity in the IGS regions of M. sympodialis and M. dermatis, studies of the diversity of these species should be performed using appropriate genes and not the IGS.

Endophytic bacterial diversity in grapevine (Vitis vinifera L.) leaves described by 16S rRNA gene sequence analysis and length heterogeneity-PCR.

PubMed

Bulgari, Daniela; Casati, Paola; Brusetti, Lorenzo; Quaglino, Fabio; Brasca, Milena; Daffonchio, Daniele; Bianco, Piero Attilio

2009-08-01

Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. In order to identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Sequence analysis of 16S rRNA gene libraries underscored five diverse Operational Taxonomic Units (OTUs), showing best sequence matches with gamma-Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Bacteria isolation through cultivation revealed the presence of six OTUs, showing best sequence matches with Actinobacteria, genus Curtobacterium, and with Firmicutes genera Bacillus and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Analysis of healthy and phytoplasma-infected grapevine plants showed that LH-PCR could be a useful complementary tool for examining the diversity of bacterial endophytes especially for diversity survey on a large number of samples.

Carnivore-specific SINEs (Can-SINEs): distribution, evolution, and genomic impact.

PubMed

Walters-Conte, Kathryn B; Johnson, Diana L E; Allard, Marc W; Pecon-Slattery, Jill

2011-01-01

Short interspersed nuclear elements (SINEs) are a type of class 1 transposable element (retrotransposon) with features that allow investigators to resolve evolutionary relationships between populations and species while providing insight into genome composition and function. Characterization of a Carnivora-specific SINE family, Can-SINEs, has, has aided comparative genomic studies by providing rare genomic changes, and neutral sequence variants often needed to resolve difficult evolutionary questions. In addition, Can-SINEs constitute a significant source of functional diversity with Carnivora. Publication of the whole-genome sequence of domestic dog, domestic cat, and giant panda serves as a valuable resource in comparative genomic inferences gleaned from Can-SINEs. In anticipation of forthcoming studies bolstered by new genomic data, this review describes the discovery and characterization of Can-SINE motifs as well as describes composition, distribution, and effect on genome function. As the contribution of noncoding sequences to genomic diversity becomes more apparent, SINEs and other transposable elements will play an increasingly large role in mammalian comparative genomics.

Carnivore-Specific SINEs (Can-SINEs): Distribution, Evolution, and Genomic Impact

PubMed Central

Johnson, Diana L.E.; Allard, Marc W.; Pecon-Slattery, Jill

2011-01-01

Short interspersed nuclear elements (SINEs) are a type of class 1 transposable element (retrotransposon) with features that allow investigators to resolve evolutionary relationships between populations and species while providing insight into genome composition and function. Characterization of a Carnivora-specific SINE family, Can-SINEs, has, has aided comparative genomic studies by providing rare genomic changes, and neutral sequence variants often needed to resolve difficult evolutionary questions. In addition, Can-SINEs constitute a significant source of functional diversity with Carnivora. Publication of the whole-genome sequence of domestic dog, domestic cat, and giant panda serves as a valuable resource in comparative genomic inferences gleaned from Can-SINEs. In anticipation of forthcoming studies bolstered by new genomic data, this review describes the discovery and characterization of Can-SINE motifs as well as describes composition, distribution, and effect on genome function. As the contribution of noncoding sequences to genomic diversity becomes more apparent, SINEs and other transposable elements will play an increasingly large role in mammalian comparative genomics. PMID:21846743

1,003 reference genomes of bacterial and archaeal isolates expand coverage of the tree of life

DOE PAGES

Mukherjee, Supratim; Seshadri, Rekha; Varghese, Neha J.; ...

2017-06-12

We present 1,003 reference genomes that were sequenced as part of the Genomic Encyclopedia of Bacteria and Archaea (GEBA) initiative, selected to maximize sequence coverage of phylogenetic space. These genomes double the number of existing type strains and expand their overall phylogenetic diversity by 25%. Comparative analyses with previously available finished and draft genomes reveal a 10.5% increase in novel protein families as a function of phylogenetic diversity. The GEBA genomes recruit 25 million previously unassigned metagenomic proteins from 4,650 samples, improving their phylogenetic and functional interpretation. We identify numerous biosynthetic clusters and experimentally validate a divergent phenazine cluster withmore » potential new chemical structure and antimicrobial activity. This Resource is the largest single release of reference genomes to date. Bacterial and archaeal isolate sequence space is still far from saturated, and future endeavors in this direction will continue to be a valuable resource for scientific discovery.« less

Molecular diversity of arbuscular mycorrhizal fungi in relation to soil chemical properties and heavy metal contamination.

PubMed

Zarei, Mehdi; Hempel, Stefan; Wubet, Tesfaye; Schäfer, Tina; Savaghebi, Gholamreza; Jouzani, Gholamreza Salehi; Nekouei, Mojtaba Khayam; Buscot, François

2010-08-01

Abundance and diversity of arbuscular mycorrhizal fungi (AMF) associated with dominant plant species were studied along a transect from highly lead (Pb) and zinc (Zn) polluted to non-polluted soil at the Anguran open pit mine in Iran. Using an established primer set for AMF in the internal transcribed spacer (ITS) region of rDNA, nine different AMF sequence types were distinguished after phylogenetic analyses, showing remarkable differences in their distribution patterns along the transect. With decreasing Pb and Zn concentration, the number of AMF sequence types increased, however one sequence type was only found in the highly contaminated area. Multivariate statistical analysis revealed that further factors than HM soil concentration affect the AMF community at contaminated sites. Specifically, the soils' calcium carbonate equivalent and available P proved to be of importance, which illustrates that field studies on AMF distribution should also consider important environmental factors and their possible interactions. Copyright 2010 Elsevier Ltd. All rights reserved.

1,003 reference genomes of bacterial and archaeal isolates expand coverage of the tree of life

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mukherjee, Supratim; Seshadri, Rekha; Varghese, Neha J.

We present 1,003 reference genomes that were sequenced as part of the Genomic Encyclopedia of Bacteria and Archaea (GEBA) initiative, selected to maximize sequence coverage of phylogenetic space. These genomes double the number of existing type strains and expand their overall phylogenetic diversity by 25%. Comparative analyses with previously available finished and draft genomes reveal a 10.5% increase in novel protein families as a function of phylogenetic diversity. The GEBA genomes recruit 25 million previously unassigned metagenomic proteins from 4,650 samples, improving their phylogenetic and functional interpretation. We identify numerous biosynthetic clusters and experimentally validate a divergent phenazine cluster withmore » potential new chemical structure and antimicrobial activity. This Resource is the largest single release of reference genomes to date. Bacterial and archaeal isolate sequence space is still far from saturated, and future endeavors in this direction will continue to be a valuable resource for scientific discovery.« less

Functional Genotyping of Sulfurospirillum spp. in Mixed Cultures Allowed the Identification of a New Tetrachloroethene Reductive Dehalogenase

PubMed Central

Buttet, Géraldine F.; Holliger, Christof

2013-01-01

Reductive dehalogenases are the key enzymes involved in the anaerobic respiration of organohalides such as the widespread groundwater pollutant tetrachloroethene. The increasing number of available bacterial genomes and metagenomes gives access to hundreds of new putative reductive dehalogenase genes that display a high level of sequence diversity and for which substrate prediction remains very challenging. In this study, we present the development of a functional genotyping method targeting the diverse reductive dehalogenases present in Sulfurospirillum spp., which allowed us to unambiguously identify a new reductive dehalogenase from our tetrachloroethene-dechlorinating SL2 bacterial consortia. The new enzyme, named PceATCE, shows 92% sequence identity with the well-characterized PceA enzyme of Sulfurospirillum multivorans, but in contrast to the latter, it is restricted to tetrachloroethene as a substrate. Its apparent higher dechlorinating activity with tetrachloroethene likely allowed its selection and maintenance in the bacterial consortia among other enzymes showing broader substrate ranges. The sequence-substrate relationships within tetrachloroethene reductive dehalogenases are also discussed. PMID:23995945

Activity and diversity of methane-oxidizing bacteria along a Norwegian sub-Arctic glacier forefield.

PubMed

Mateos-Rivera, Alejandro; Øvreås, Lise; Wilson, Bryan; Yde, Jacob C; Finster, Kai W

2018-05-01

Methane (CH4) is one of the most abundant greenhouse gases in the atmosphere and identification of its sources and sinks is crucial for the reliability of climate model outputs. Although CH4 production and consumption rates have been reported from a broad spectrum of environments, data obtained from glacier forefields are restricted to a few locations. We report the activities of methanotrophic communities and their diversity along a chronosequence in front of a sub-Arctic glacier using high-throughput sequencing and gas flux measurements. CH4 oxidation rates were measured in the field throughout the growing season during three sampling times at eight different sampling points in combination with laboratory incubation experiments. The overall results showed that the methanotrophic community had similar trends of increased CH4 consumption and increased abundance as a function of soil development and time of year. Sequencing results revealed that the methanotrophic community was dominated by a few OTUs and that a short-term increase in CH4 concentration, as performed in the field measurements, altered slightly the relative abundance of the OTUs.

Estimation of pea (Pisum sativum L.) microsatellite mutation rate based on pedigree and single-seed descent analyses.

PubMed

Cieslarová, Jaroslava; Hanáček, Pavel; Fialová, Eva; Hýbl, Miroslav; Smýkal, Petr

2011-11-01

Microsatellites, or simple sequence repeats (SSRs) are widespread class of repetitive DNA sequences, used in population genetics, genetic diversity and mapping studies. In spite of the SSR utility, the genetic and evolutionary mechanisms are not fully understood. We have investigated three microsatellite loci with different position in the pea (Pisum sativum L.) genome, the A9 locus residing in LTR region of abundant retrotransposon, AD270 as intergenic and AF016458 located in 5'untranslated region of expressed gene. Comparative analysis of a 35 pair samples from seven pea varieties propagated by single-seed descent for ten generations, revealed single 4 bp mutation in 10th generation sample at AD270 locus corresponding to stepwise increase in one additional ATCT repeat unit. The estimated mutation rate was 4.76 × 10(-3) per locus per generation, with a 95% confidence interval of 1.2 × 10(-4) to 2.7 × 10(-2). The comparison of cv. Bohatýr accessions retrieved from different collections, showed intra-, inter-accession variation and differences in flanking and repeat sequences. Fragment size and sequence alternations were also found in long term in vitro organogenic culture, established at 1983, indicative of somatic mutation process. The evidence of homoplasy was detected across of unrelated pea genotypes, which adversaly affects the reliability of diversity estimates not only for diverse germplasm but also highly bred material. The findings of this study have important implications for Pisum phylogeny studies, variety identification and registration process in pea breeding where mutation rate influences the genetic diversity and the effective population size estimates.

Fungal diversity in deep-sea sediments associated with asphalt seeps at the Sao Paulo Plateau

NASA Astrophysics Data System (ADS)

Nagano, Yuriko; Miura, Toshiko; Nishi, Shinro; Lima, Andre O.; Nakayama, Cristina; Pellizari, Vivian H.; Fujikura, Katsunori

2017-12-01

We investigated the fungal diversity in a total of 20 deep-sea sediment samples (of which 14 samples were associated with natural asphalt seeps and 6 samples were not associated) collected from two different sites at the Sao Paulo Plateau off Brazil by Ion Torrent PGM targeting ITS region of ribosomal RNA. Our results suggest that diverse fungi (113 operational taxonomic units (OTUs) based on clustering at 97% sequence similarity assigned into 9 classes and 31 genus) are present in deep-sea sediment samples collected at the Sao Paulo Plateau, dominated by Ascomycota (74.3%), followed by Basidiomycota (11.5%), unidentified fungi (7.1%), and sequences with no affiliation to any organisms in the public database (7.1%). However, it was revealed that only three species, namely Penicillium sp., Cadophora malorum and Rhodosporidium diobovatum, were dominant, with the majority of OTUs remaining a minor community. Unexpectedly, there was no significant difference in major fungal community structure between the asphalt seep and non-asphalt seep sites, despite the presence of mass hydrocarbon deposits and the high amount of macro organisms surrounding the asphalt seeps. However, there were some differences in the minor fungal communities, with possible asphalt degrading fungi present specifically in the asphalt seep sites. In contrast, some differences were found between the two different sampling sites. Classification of OTUs revealed that only 47 (41.6%) fungal OTUs exhibited >97% sequence similarity, in comparison with pre-existing ITS sequences in public databases, indicating that a majority of deep-sea inhabiting fungal taxa still remain undescribed. Although our knowledge on fungi and their role in deep-sea environments is still limited and scarce, this study increases our understanding of fungal diversity and community structure in deep-sea environments.

3D: diversity, dynamics, differential testing - a proposed pipeline for analysis of next-generation sequencing T cell repertoire data.

PubMed

Zhang, Li; Cham, Jason; Paciorek, Alan; Trager, James; Sheikh, Nadeem; Fong, Lawrence

2017-02-27

Cancer immunotherapy has demonstrated significant clinical activity in different cancers. T cells represent a crucial component of the adaptive immune system and are thought to mediate anti-tumoral immunity. Antigen-specific recognition by T cells is via the T cell receptor (TCR) which is unique for each T cell. Next generation sequencing (NGS) of the TCRs can be used as a platform to profile the T cell repertoire. Though there are a number of software tools available for processing repertoire data by mapping antigen receptor segments to sequencing reads and assembling the clonotypes, most of them are not designed to track and examine the dynamic nature of the TCR repertoire across multiple time points or between different biologic compartments (e.g., blood and tissue samples) in a clinical context. We integrated different diversity measures to assess the T cell repertoire diversity and examined the robustness of the diversity indices. Among those tested, Clonality was identified for its robustness as a key metric for study design and the first choice to measure TCR repertoire diversity. To evaluate the dynamic nature of T cell clonotypes across time, we utilized several binary similarity measures (such as Baroni-Urbani and Buser overlap index), relative clonality and Morisita's overlap index, as well as the intraclass correlation coefficient, and performed fold change analysis, which was further extended to investigate the transition of clonotypes among different biological compartments. Furthermore, the application of differential testing enabled the detection of clonotypes which were significantly changed across time. By applying the proposed "3D" analysis pipeline to the real example of prostate cancer subjects who received sipuleucel-T, an FDA-approved immunotherapy, we were able to detect changes in TCR sequence frequency and diversity thus demonstrating that sipuleucel-T treatment affected TCR repertoire in blood and in prostate tissue. We also found that the increase in common TCR sequences between tissue and blood after sipuleucel-T treatment supported the hypothesis that treatment-induced T cell migrated into the prostate tissue. In addition, a second example of prostate cancer subjects treated with Ipilimumab and granulocyte macrophage colony stimulating factor (GM-CSF) was presented in the supplementary documents to further illustrate assessing the treatment-associated change in a clinical context by the proposed workflow. Our paper provides guidance to study the diversity and dynamics of NGS-based TCR repertoire profiling in a clinical context to ensure consistency and reproducibility of post-analysis. This analysis pipeline will provide an initial workflow for TCR sequencing data with serial time points and for comparing T cells in multiple compartments for a clinical study.

High Epstein-Barr Virus Load and Genomic Diversity Are Associated with Generation of gp350-Specific Neutralizing Antibodies following Acute Infectious Mononucleosis

PubMed Central

Weiss, Eric R.; Alter, Galit; Ogembo, Javier Gordon; Henderson, Jennifer L.; Tabak, Barbara; Bakiş, Yasin; Somasundaran, Mohan; Garber, Manuel; Selin, Liisa

2016-01-01

ABSTRACT The Epstein-Barr virus (EBV) gp350 glycoprotein interacts with the cellular receptor to mediate viral entry and is thought to be the major target for neutralizing antibodies. To better understand the role of EBV-specific antibodies in the control of viral replication and the evolution of sequence diversity, we measured EBV gp350-specific antibody responses and sequenced the gp350 gene in samples obtained from individuals experiencing primary EBV infection (acute infectious mononucleosis [AIM]) and again 6 months later (during convalescence [CONV]). EBV gp350-specific IgG was detected in the sera of 17 (71%) of 24 individuals at the time of AIM and all 24 (100%) individuals during CONV; binding antibody titers increased from AIM through CONV, reaching levels equivalent to those in age-matched, chronically infected individuals. Antibody-dependent cell-mediated phagocytosis (ADCP) was rarely detected during AIM (4 of 24 individuals; 17%) but was commonly detected during CONV (19 of 24 individuals; 79%). The majority (83%) of samples taken during AIM neutralized infection of primary B cells; all samples obtained at 6 months postdiagnosis neutralized EBV infection of cultured and primary target cells. Deep sequencing revealed interpatient gp350 sequence variation but conservation of the CR2-binding site. The levels of gp350-specific neutralizing activity directly correlated with higher peripheral blood EBV DNA levels during AIM and a greater evolution of diversity in gp350 nucleotide sequences from AIM to CONV. In summary, we conclude that the viral load and EBV gp350 diversity during early infection are associated with the development of neutralizing antibody responses following AIM. IMPORTANCE Antibodies against viral surface proteins can blunt the spread of viral infection by coating viral particles, mediating uptake by immune cells, or blocking interaction with host cell receptors, making them a desirable component of a sterilizing vaccine. The EBV surface protein gp350 is a major target for antibodies. We report the detection of EBV gp350-specific antibodies capable of neutralizing EBV infection in vitro. The majority of gp350-directed vaccines focus on glycoproteins from lab-adapted strains, which may poorly reflect primary viral envelope diversity. We report some of the first primary gp350 sequences, noting that the gp350 host receptor binding site is remarkably stable across patients and time. However, changes in overall gene diversity were detectable during infection. Patients with higher peripheral blood viral loads in primary infection and greater changes in viral diversity generated more efficient antibodies. Our findings provide insight into the generation of functional antibodies, necessary for vaccine development. PMID:27733645

High Epstein-Barr Virus Load and Genomic Diversity Are Associated with Generation of gp350-Specific Neutralizing Antibodies following Acute Infectious Mononucleosis.

PubMed

Weiss, Eric R; Alter, Galit; Ogembo, Javier Gordon; Henderson, Jennifer L; Tabak, Barbara; Bakiş, Yasin; Somasundaran, Mohan; Garber, Manuel; Selin, Liisa; Luzuriaga, Katherine

2017-01-01

The Epstein-Barr virus (EBV) gp350 glycoprotein interacts with the cellular receptor to mediate viral entry and is thought to be the major target for neutralizing antibodies. To better understand the role of EBV-specific antibodies in the control of viral replication and the evolution of sequence diversity, we measured EBV gp350-specific antibody responses and sequenced the gp350 gene in samples obtained from individuals experiencing primary EBV infection (acute infectious mononucleosis [AIM]) and again 6 months later (during convalescence [CONV]). EBV gp350-specific IgG was detected in the sera of 17 (71%) of 24 individuals at the time of AIM and all 24 (100%) individuals during CONV; binding antibody titers increased from AIM through CONV, reaching levels equivalent to those in age-matched, chronically infected individuals. Antibody-dependent cell-mediated phagocytosis (ADCP) was rarely detected during AIM (4 of 24 individuals; 17%) but was commonly detected during CONV (19 of 24 individuals; 79%). The majority (83%) of samples taken during AIM neutralized infection of primary B cells; all samples obtained at 6 months postdiagnosis neutralized EBV infection of cultured and primary target cells. Deep sequencing revealed interpatient gp350 sequence variation but conservation of the CR2-binding site. The levels of gp350-specific neutralizing activity directly correlated with higher peripheral blood EBV DNA levels during AIM and a greater evolution of diversity in gp350 nucleotide sequences from AIM to CONV. In summary, we conclude that the viral load and EBV gp350 diversity during early infection are associated with the development of neutralizing antibody responses following AIM. Antibodies against viral surface proteins can blunt the spread of viral infection by coating viral particles, mediating uptake by immune cells, or blocking interaction with host cell receptors, making them a desirable component of a sterilizing vaccine. The EBV surface protein gp350 is a major target for antibodies. We report the detection of EBV gp350-specific antibodies capable of neutralizing EBV infection in vitro The majority of gp350-directed vaccines focus on glycoproteins from lab-adapted strains, which may poorly reflect primary viral envelope diversity. We report some of the first primary gp350 sequences, noting that the gp350 host receptor binding site is remarkably stable across patients and time. However, changes in overall gene diversity were detectable during infection. Patients with higher peripheral blood viral loads in primary infection and greater changes in viral diversity generated more efficient antibodies. Our findings provide insight into the generation of functional antibodies, necessary for vaccine development. Copyright © 2016 American Society for Microbiology.

Mountain gorilla genomes reveal the impact of long-term population decline and inbreeding

PubMed Central

Ayub, Qasim; Szpak, Michal; Frandsen, Peter; Chen, Yuan; Yngvadottir, Bryndis; Cooper, David N.; de Manuel, Marc; Hernandez-Rodriguez, Jessica; Lobon, Irene; Siegismund, Hans R.; Pagani, Luca; Quail, Michael A.; Hvilsom, Christina; Mudakikwa, Antoine; Eichler, Evan E.; Cranfield, Michael R.; Marques-Bonet, Tomas; Tyler-Smith, Chris; Scally, Aylwyn

2015-01-01

Mountain gorillas are an endangered great ape subspecies and a prominent focus for conservation, yet we know little about their genomic diversity and evolutionary past. We sequenced whole genomes from multiple wild individuals and compared the genomes of all four Gorilla subspecies. We found that the two eastern subspecies have experienced a prolonged population decline over the past 100,000 years, resulting in very low genetic diversity and an increased overall burden of deleterious variation. A further recent decline in the mountain gorilla population has led to extensive inbreeding, such that individuals are typically homozygous at 34% of their sequence, leading to the purging of severely deleterious recessive mutations from the population. We discuss the causes of their decline and the consequences for their future survival. PMID:25859046

The domestication of the probiotic bacterium Lactobacillus acidophilus

PubMed Central

Bull, Matthew J.; Jolley, Keith A.; Bray, James E.; Aerts, Maarten; Vandamme, Peter; Maiden, Martin C. J.; Marchesi, Julian R.; Mahenthiralingam, Eshwar

2014-01-01

Lactobacillus acidophilus is a Gram-positive lactic acid bacterium that has had widespread historical use in the dairy industry and more recently as a probiotic. Although L. acidophilus has been designated as safe for human consumption, increasing commercial regulation and clinical demands for probiotic validation has resulted in a need to understand its genetic diversity. By drawing on large, well-characterised collections of lactic acid bacteria, we examined L. acidophilus isolates spanning 92 years and including multiple strains in current commercial use. Analysis of the whole genome sequence data set (34 isolate genomes) demonstrated L. acidophilus was a low diversity, monophyletic species with commercial isolates essentially identical at the sequence level. Our results indicate that commercial use has domesticated L. acidophilus with genetically stable, invariant strains being consumed globally by the human population. PMID:25425319

The domestication of the probiotic bacterium Lactobacillus acidophilus.

PubMed

Bull, Matthew J; Jolley, Keith A; Bray, James E; Aerts, Maarten; Vandamme, Peter; Maiden, Martin C J; Marchesi, Julian R; Mahenthiralingam, Eshwar

2014-11-26

Lactobacillus acidophilus is a Gram-positive lactic acid bacterium that has had widespread historical use in the dairy industry and more recently as a probiotic. Although L. acidophilus has been designated as safe for human consumption, increasing commercial regulation and clinical demands for probiotic validation has resulted in a need to understand its genetic diversity. By drawing on large, well-characterised collections of lactic acid bacteria, we examined L. acidophilus isolates spanning 92 years and including multiple strains in current commercial use. Analysis of the whole genome sequence data set (34 isolate genomes) demonstrated L. acidophilus was a low diversity, monophyletic species with commercial isolates essentially identical at the sequence level. Our results indicate that commercial use has domesticated L. acidophilus with genetically stable, invariant strains being consumed globally by the human population.

Generation of diversity in Streptococcus mutans genes demonstrated by MLST.

PubMed

Do, Thuy; Gilbert, Steven C; Clark, Douglas; Ali, Farida; Fatturi Parolo, Clarissa C; Maltz, Marisa; Russell, Roy R; Holbrook, Peter; Wade, William G; Beighton, David

2010-02-05

Streptococcus mutans, consisting of serotypes c, e, f and k, is an oral aciduric organism associated with the initiation and progression of dental caries. A total of 135 independent Streptococcus mutans strains from caries-free and caries-active subjects isolated from various geographical locations were examined in two versions of an MLST scheme consisting of either 6 housekeeping genes [accC (acetyl-CoA carboxylase biotin carboxylase subunit), gki (glucokinase), lepA (GTP-binding protein), recP (transketolase), sodA (superoxide dismutase), and tyrS (tyrosyl-tRNA synthetase)] or the housekeeping genes supplemented with 2 extracellular putative virulence genes [gtfB (glucosyltransferase B) and spaP (surface protein antigen I/II)] to increase sequence type diversity. The number of alleles found varied between 20 (lepA) and 37 (spaP). Overall, 121 sequence types (STs) were defined using the housekeeping genes alone and 122 with all genes. However pi, nucleotide diversity per site, was low for all loci being in the range 0.019-0.007. The virulence genes exhibited the greatest nucleotide diversity and the recombination/mutation ratio was 0.67 [95% confidence interval 0.3-1.15] compared to 8.3 [95% confidence interval 5.0-14.5] for the 6 concatenated housekeeping genes alone. The ML trees generated for individual MLST loci were significantly incongruent and not significantly different from random trees. Analysis using ClonalFrame indicated that the majority of isolates were singletons and no evidence for a clonal structure or evidence to support serotype c strains as the ancestral S. mutans strain was apparent. There was also no evidence of a geographical distribution of individual isolates or that particular isolate clusters were associated with caries. The overall low sequence diversity suggests that S. mutans is a newly emerged species which has not accumulated large numbers of mutations but those that have occurred have been shuffled as a consequence of intra-species recombination generating genotypes which can be readily distinguished by sequence analysis.

Shifts in taxonomic and functional microbial diversity with agriculture: How fragile is the Brazilian Cerrado?

PubMed

Souza, Renata Carolini; Mendes, Iêda Carvalho; Reis-Junior, Fábio Bueno; Carvalho, Fabíola Marques; Nogueira, Marco Antonio; Vasconcelos, Ana Tereza Ribeiro; Vicente, Vânia Aparecida; Hungria, Mariangela

2016-03-16

The Cerrado--an edaphic type of savannah--comprises the second largest biome of the Brazilian territory and is the main area for grain production in the country, but information about the impact of land conversion to agriculture on microbial diversity is still scarce. We used a shotgun metagenomic approach to compare undisturbed (native) soil and soils cropped for 23 years with soybean/maize under conservation tillage--"no-till" (NT)--and conventional tillage (CT) systems in the Cerrado biome. Soil management and fertilizer inputs with the introduction of agriculture improved chemical properties, but decreased soil macroporosity and microbial biomass of carbon and nitrogen. Principal coordinates analyses confirmed different taxonomic and functional profiles for each treatment. There was predominance of the Bacteria domain, especially the phylum Proteobacteria, with higher numbers of sequences in the NT and CT treatments; Archaea and Viruses also had lower numbers of sequences in the undisturbed soil. Within the Alphaproteobacteria, there was dominance of Rhizobiales and of the genus Bradyrhizobium in the NT and CT systems, attributed to massive inoculation of soybean, and also of Burkholderiales. In contrast, Rhizobium, Azospirillum, Xanthomonas, Pseudomonas and Acidobacterium predominated in the native Cerrado. More Eukaryota, especially of the phylum Ascomycota were detected in the NT. The functional analysis revealed lower numbers of sequences in the five dominant categories for the CT system, whereas the undisturbed Cerrado presented higher abundance. High impact of agriculture in taxonomic and functional microbial diversity in the biome Cerrado was confirmed. Functional diversity was not necessarily associated with taxonomic diversity, as the less conservationist treatment (CT) presented increased taxonomic sequences and reduced functional profiles, indicating a strategy to try to maintain soil functioning by favoring taxa that are probably not the most efficient for some functions. Our results highlight that underneath the rustic appearance of the Cerrado vegetation there is a fragile soil microbial community.

Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies.

PubMed

Harris, Katherine E; Aldred, Shelley Force; Davison, Laura M; Ogana, Heather Anne N; Boudreau, Andrew; Brüggemann, Marianne; Osborn, Michael; Ma, Biao; Buelow, Benjamin; Clarke, Starlynn C; Dang, Kevin H; Iyer, Suhasini; Jorgensen, Brett; Pham, Duy T; Pratap, Payal P; Rangaswamy, Udaya S; Schellenberger, Ute; van Schooten, Wim C; Ugamraj, Harshad S; Vafa, Omid; Buelow, Roland; Trinklein, Nathan D

2018-01-01

We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

Diversity and phylogenetic analyses of bacteria from a shallow-water hydrothermal vent in Milos island (Greece).

PubMed

Giovannelli, Donato; d'Errico, Giuseppe; Manini, Elena; Yakimov, Michail; Vetriani, Costantino

2013-01-01

Studies of shallow-water hydrothermal vents have been lagging behind their deep-sea counterparts. Hence, the importance of these systems and their contribution to the local and regional diversity and biogeochemistry is unclear. This study analyzes the bacterial community along a transect at the shallow-water hydrothermal vent system of Milos island, Greece. The abundance and biomass of the prokaryotic community is comparable to areas not affected by hydrothermal activity and was, on average, 1.34 × 10(8) cells g(-1). The abundance, biomass and diversity of the prokaryotic community increased with the distance from the center of the vent and appeared to be controlled by the temperature gradient rather than the trophic conditions. The retrieved 16S rRNA gene fragments matched sequences from a variety of geothermal environments, although the average similarity was low (94%), revealing previously undiscovered taxa. Epsilonproteobacteria constituted the majority of the population along the transect, with an average contribution to the total diversity of 60%. The larger cluster of 16S rRNA gene sequences was related to chemolithoautotrophic Sulfurovum spp., an Epsilonproteobacterium so far detected only at deep-sea hydrothermal vents. The presence of previously unknown lineages of Epsilonproteobacteria could be related to the abundance of organic matter in these systems, which may support alternative metabolic strategies to chemolithoautotrophy. The relative contribution of Gammaproteobacteria to the Milos microbial community increased along the transect as the distance from the center of the vent increased. Further attempts to isolate key species from these ecosystems will be critical to shed light on their evolution and ecology.

Diversity and phylogenetic analyses of bacteria from a shallow-water hydrothermal vent in Milos island (Greece)

PubMed Central

Giovannelli, Donato; d'Errico, Giuseppe; Manini, Elena; Yakimov, Michail; Vetriani, Costantino

2013-01-01

Studies of shallow-water hydrothermal vents have been lagging behind their deep-sea counterparts. Hence, the importance of these systems and their contribution to the local and regional diversity and biogeochemistry is unclear. This study analyzes the bacterial community along a transect at the shallow-water hydrothermal vent system of Milos island, Greece. The abundance and biomass of the prokaryotic community is comparable to areas not affected by hydrothermal activity and was, on average, 1.34 × 108 cells g−1. The abundance, biomass and diversity of the prokaryotic community increased with the distance from the center of the vent and appeared to be controlled by the temperature gradient rather than the trophic conditions. The retrieved 16S rRNA gene fragments matched sequences from a variety of geothermal environments, although the average similarity was low (94%), revealing previously undiscovered taxa. Epsilonproteobacteria constituted the majority of the population along the transect, with an average contribution to the total diversity of 60%. The larger cluster of 16S rRNA gene sequences was related to chemolithoautotrophic Sulfurovum spp., an Epsilonproteobacterium so far detected only at deep-sea hydrothermal vents. The presence of previously unknown lineages of Epsilonproteobacteria could be related to the abundance of organic matter in these systems, which may support alternative metabolic strategies to chemolithoautotrophy. The relative contribution of Gammaproteobacteria to the Milos microbial community increased along the transect as the distance from the center of the vent increased. Further attempts to isolate key species from these ecosystems will be critical to shed light on their evolution and ecology. PMID:23847607

Restructuring of the Aquatic Bacterial Community by Hydric Dynamics Associated with Superstorm Sandy

PubMed Central

Ulrich, Nikea; Rosenberger, Abigail; Brislawn, Colin; Wright, Justin; Kessler, Collin; Toole, David; Solomon, Caroline; Strutt, Steven; McClure, Erin

2016-01-01

ABSTRACT Bacterial community composition and longitudinal fluctuations were monitored in a riverine system during and after Superstorm Sandy to better characterize inter- and intracommunity responses associated with the disturbance associated with a 100-year storm event. High-throughput sequencing of the 16S rRNA gene was used to assess microbial community structure within water samples from Muddy Creek Run, a second-order stream in Huntingdon, PA, at 12 different time points during the storm event (29 October to 3 November 2012) and under seasonally matched baseline conditions. High-throughput sequencing of the 16S rRNA gene was used to track changes in bacterial community structure and divergence during and after Superstorm Sandy. Bacterial community dynamics were correlated to measured physicochemical parameters and fecal indicator bacteria (FIB) concentrations. Bioinformatics analyses of 2.1 million 16S rRNA gene sequences revealed a significant increase in bacterial diversity in samples taken during peak discharge of the storm. Beta-diversity analyses revealed longitudinal shifts in the bacterial community structure. Successional changes were observed, in which Betaproteobacteria and Gammaproteobacteria decreased in 16S rRNA gene relative abundance, while the relative abundance of members of the Firmicutes increased. Furthermore, 16S rRNA gene sequences matching pathogenic bacteria, including strains of Legionella, Campylobacter, Arcobacter, and Helicobacter, as well as bacteria of fecal origin (e.g., Bacteroides), exhibited an increase in abundance after peak discharge of the storm. This study revealed a significant restructuring of in-stream bacterial community structure associated with hydric dynamics of a storm event. IMPORTANCE In order to better understand the microbial risks associated with freshwater environments during a storm event, a more comprehensive understanding of the variations in aquatic bacterial diversity is warranted. This study investigated the bacterial communities during and after Superstorm Sandy to provide fine time point resolution of dynamic changes in bacterial composition. This study adds to the current literature by revealing the variation in bacterial community structure during the course of a storm. This study employed high-throughput DNA sequencing, which generated a deep analysis of inter- and intracommunity responses during a significant storm event. This study has highlighted the utility of applying high-throughput sequencing for water quality monitoring purposes, as this approach enabled a more comprehensive investigation of the bacterial community structure. Altogether, these data suggest a drastic restructuring of the stream bacterial community during a storm event and highlight the potential of high-throughput sequencing approaches for assessing the microbiological quality of our environment. PMID:27060115

Large-scale whole-genome sequencing of the Icelandic population.

PubMed

Gudbjartsson, Daniel F; Helgason, Hannes; Gudjonsson, Sigurjon A; Zink, Florian; Oddson, Asmundur; Gylfason, Arnaldur; Besenbacher, Soren; Magnusson, Gisli; Halldorsson, Bjarni V; Hjartarson, Eirikur; Sigurdsson, Gunnar Th; Stacey, Simon N; Frigge, Michael L; Holm, Hilma; Saemundsdottir, Jona; Helgadottir, Hafdis Th; Johannsdottir, Hrefna; Sigfusson, Gunnlaugur; Thorgeirsson, Gudmundur; Sverrisson, Jon Th; Gretarsdottir, Solveig; Walters, G Bragi; Rafnar, Thorunn; Thjodleifsson, Bjarni; Bjornsson, Einar S; Olafsson, Sigurdur; Thorarinsdottir, Hildur; Steingrimsdottir, Thora; Gudmundsdottir, Thora S; Theodors, Asgeir; Jonasson, Jon G; Sigurdsson, Asgeir; Bjornsdottir, Gyda; Jonsson, Jon J; Thorarensen, Olafur; Ludvigsson, Petur; Gudbjartsson, Hakon; Eyjolfsson, Gudmundur I; Sigurdardottir, Olof; Olafsson, Isleifur; Arnar, David O; Magnusson, Olafur Th; Kong, Augustine; Masson, Gisli; Thorsteinsdottir, Unnur; Helgason, Agnar; Sulem, Patrick; Stefansson, Kari

2015-05-01

Here we describe the insights gained from sequencing the whole genomes of 2,636 Icelanders to a median depth of 20×. We found 20 million SNPs and 1.5 million insertions-deletions (indels). We describe the density and frequency spectra of sequence variants in relation to their functional annotation, gene position, pathway and conservation score. We demonstrate an excess of homozygosity and rare protein-coding variants in Iceland. We imputed these variants into 104,220 individuals down to a minor allele frequency of 0.1% and found a recessive frameshift mutation in MYL4 that causes early-onset atrial fibrillation, several mutations in ABCB4 that increase risk of liver diseases and an intronic variant in GNAS associating with increased thyroid-stimulating hormone levels when maternally inherited. These data provide a study design that can be used to determine how variation in the sequence of the human genome gives rise to human diversity.

Metagenomic applications in environmental monitoring and bioremediation.

PubMed

Techtmann, Stephen M; Hazen, Terry C

2016-10-01

With the rapid advances in sequencing technology, the cost of sequencing has dramatically dropped and the scale of sequencing projects has increased accordingly. This has provided the opportunity for the routine use of sequencing techniques in the monitoring of environmental microbes. While metagenomic applications have been routinely applied to better understand the ecology and diversity of microbes, their use in environmental monitoring and bioremediation is increasingly common. In this review we seek to provide an overview of some of the metagenomic techniques used in environmental systems biology, addressing their application and limitation. We will also provide several recent examples of the application of metagenomics to bioremediation. We discuss examples where microbial communities have been used to predict the presence and extent of contamination, examples of how metagenomics can be used to characterize the process of natural attenuation by unculturable microbes, as well as examples detailing the use of metagenomics to understand the impact of biostimulation on microbial communities.

Microbial Diversity and Host-specific Sequences of Canadian Goose Feces

EPA Science Inventory

Methods to assess the impact of goose fecal contamination are needed as the result of the increasing number of Canada goose (Branta canadensis) nearby North American inland waters. However, there is little information on goose fecal microbial communities, which is important for t...

Sequence Diversity Diagram for comparative analysis of multiple sequence alignments.

PubMed

Sakai, Ryo; Aerts, Jan

2014-01-01

The sequence logo is a graphical representation of a set of aligned sequences, commonly used to depict conservation of amino acid or nucleotide sequences. Although it effectively communicates the amount of information present at every position, this visual representation falls short when the domain task is to compare between two or more sets of aligned sequences. We present a new visual presentation called a Sequence Diversity Diagram and validate our design choices with a case study. Our software was developed using the open-source program called Processing. It loads multiple sequence alignment FASTA files and a configuration file, which can be modified as needed to change the visualization. The redesigned figure improves on the visual comparison of two or more sets, and it additionally encodes information on sequential position conservation. In our case study of the adenylate kinase lid domain, the Sequence Diversity Diagram reveals unexpected patterns and new insights, for example the identification of subgroups within the protein subfamily. Our future work will integrate this visual encoding into interactive visualization tools to support higher level data exploration tasks.

Improved serial analysis of V1 ribosomal sequence tags (SARST-V1) provides a rapid, comprehensive, sequence-based characterization of bacterial diversity and community composition.

PubMed

Yu, Zhongtang; Yu, Marie; Morrison, Mark

2006-04-01

Serial analysis of ribosomal sequence tags (SARST) is a recently developed technology that can generate large 16S rRNA gene (rrs) sequence data sets from microbiomes, but there are numerous enzymatic and purification steps required to construct the ribosomal sequence tag (RST) clone libraries. We report here an improved SARST method, which still targets the V1 hypervariable region of rrs genes, but reduces the number of enzymes, oligonucleotides, reagents, and technical steps needed to produce the RST clone libraries. The new method, hereafter referred to as SARST-V1, was used to examine the eubacterial diversity present in community DNA recovered from the microbiome resident in the ovine rumen. The 190 sequenced clones contained 1055 RSTs and no less than 236 unique phylotypes (based on > or = 95% sequence identity) that were assigned to eight different eubacterial phyla. Rarefaction and monomolecular curve analyses predicted that the complete RST clone library contains 99% of the 353 unique phylotypes predicted to exist in this microbiome. When compared with ribosomal intergenic spacer analysis (RISA) of the same community DNA sample, as well as a compilation of nine previously published conventional rrs clone libraries prepared from the same type of samples, the RST clone library provided a more comprehensive characterization of the eubacterial diversity present in rumen microbiomes. As such, SARST-V1 should be a useful tool applicable to comprehensive examination of diversity and composition in microbiomes and offers an affordable, sequence-based method for diversity analysis.

A national study of the molecular epidemiology of HIV-1 in Australia 2005-2012.

PubMed

Castley, Alison; Sawleshwarkar, Shailendra; Varma, Rick; Herring, Belinda; Thapa, Kiran; Dwyer, Dominic; Chibo, Doris; Nguyen, Nam; Hawke, Karen; Ratcliff, Rodney; Garsia, Roger; Kelleher, Anthony; Nolan, David

2017-01-01

Rates of new HIV-1 diagnoses are increasing in Australia, with evidence of an increasing proportion of non-B HIV-1 subtypes reflecting a growing impact of migration and travel. The present study aims to define HIV-1 subtype diversity patterns and investigate possible HIV-1 transmission networks within Australia. The Australian Molecular Epidemiology Network (AMEN) HIV collaborating sites in Western Australia, South Australia, Victoria, Queensland and western Sydney (New South Wales), provided baseline HIV-1 partial pol sequence, age and gender information for 4,873 patients who had genotypes performed during 2005-2012. HIV-1 phylogenetic analyses utilised MEGA V6, with a stringent classification of transmission pairs or clusters (bootstrap ≥98%, genetic distance ≤1.5% from at least one other sequence in the cluster). HIV-1 subtype B represented 74.5% of the 4,873 sequences (WA 59%, SA 68.4%, w-Syd 73.8%, Vic 75.6%, Qld 82.1%), with similar proportion of transmission pairs and clusters found in the B and non-B cohorts (23% vs 24.5% of sequences, p = 0.3). Significantly more subtype B clusters were comprised of ≥3 sequences compared with non-B clusters (45.0% vs 24.0%, p = 0.021) and significantly more subtype B pairs and clusters were male-only (88% compared to 53% CRF01_AE and 17% subtype C clusters). Factors associated with being in a cluster of any size included; being sequenced in a more recent time period (p<0.001), being younger (p<0.001), being male (p = 0.023) and having a B subtype (p = 0.02). Being in a larger cluster (>3) was associated with being sequenced in a more recent time period (p = 0.05) and being male (p = 0.008). This nationwide HIV-1 study of 4,873 patient sequences highlights the increased diversity of HIV-1 subtypes within the Australian epidemic, as well as differences in transmission networks associated with these HIV-1 subtypes. These findings provide epidemiological insights not readily available using standard surveillance methods and can inform the development of effective public health strategies in the current paradigm of HIV prevention in Australia.

Phylogenetic analysis of a spontaneous cocoa bean fermentation metagenome reveals new insights into its bacterial and fungal community diversity.

PubMed

Illeghems, Koen; De Vuyst, Luc; Papalexandratou, Zoi; Weckx, Stefan

2012-01-01

This is the first report on the phylogenetic analysis of the community diversity of a single spontaneous cocoa bean box fermentation sample through a metagenomic approach involving 454 pyrosequencing. Several sequence-based and composition-based taxonomic profiling tools were used and evaluated to avoid software-dependent results and their outcome was validated by comparison with previously obtained culture-dependent and culture-independent data. Overall, this approach revealed a wider bacterial (mainly γ-Proteobacteria) and fungal diversity than previously found. Further, the use of a combination of different classification methods, in a software-independent way, helped to understand the actual composition of the microbial ecosystem under study. In addition, bacteriophage-related sequences were found. The bacterial diversity depended partially on the methods used, as composition-based methods predicted a wider diversity than sequence-based methods, and as classification methods based solely on phylogenetic marker genes predicted a more restricted diversity compared with methods that took all reads into account. The metagenomic sequencing analysis identified Hanseniaspora uvarum, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus as the prevailing species. Also, the presence of occasional members of the cocoa bean fermentation process was revealed (such as Erwinia tasmaniensis, Lactobacillus brevis, Lactobacillus casei, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc mesenteroides, and Oenococcus oeni). Furthermore, the sequence reads associated with viral communities were of a restricted diversity, dominated by Myoviridae and Siphoviridae, and reflecting Lactobacillus as the dominant host. To conclude, an accurate overview of all members of a cocoa bean fermentation process sample was revealed, indicating the superiority of metagenomic sequencing over previously used techniques.

Automated design evolution of stereochemically randomized protein foldamers

NASA Astrophysics Data System (ADS)

Ranbhor, Ranjit; Kumar, Anil; Patel, Kirti; Ramakrishnan, Vibin; Durani, Susheel

2018-05-01

Diversification of chain stereochemistry opens up the possibilities of an ‘in principle’ increase in the design space of proteins. This huge increase in the sequence and consequent structural variation is aimed at the generation of smart materials. To diversify protein structure stereochemically, we introduced L- and D-α-amino acids as the design alphabet. With a sequence design algorithm, we explored the usage of specific variables such as chirality and the sequence of this alphabet in independent steps. With molecular dynamics, we folded stereochemically diverse homopolypeptides and evaluated their ‘fitness’ for possible design as protein-like foldamers. We propose a fitness function to prune the most optimal fold among 1000 structures simulated with an automated repetitive simulated annealing molecular dynamics (AR-SAMD) approach. The highly scored poly-leucine fold with sequence lengths of 24 and 30 amino acids were later sequence-optimized using a Dead End Elimination cum Monte Carlo based optimization tool. This paper demonstrates a novel approach for the de novo design of protein-like foldamers.

Vertical zonation of soil fungal community structure in a Korean pine forest on Changbai Mountain, China.

PubMed

Ping, Yuan; Han, Dongxue; Wang, Ning; Hu, Yanbo; Mu, Liqiang; Feng, Fujuan

2017-01-01

Changbai Mountain, with intact montane vertical vegetation belts, is located at a sensitive area of global climate change and a central distribution area of Korean pine forest. Broad-leaved Korean pine mixed forest (Pinus koraiensis as an edificator) is the most representative zonal climax vegetation in the humid region of northeastern China; their vertical zonation is the most intact and representative on Changbai Mountain. In this study, we analyzed the composition and diversity of soil fungal communities in the Korean pine forest on Changbai Mountain at elevations ranging from 699 to 1177 m using Illumina High-throughput sequencing. We obtained a total 186,663 optimized sequences, with an average length of 268.81 bp. We found soil fungal diversity index was decreased with increasing elevation from 699 to 937 m and began to rise after reaching 1044 m; the richness and evenness indices were decreased with an increase in elevation. Soil fungal compositions at the phylum, class and genus levels varied significantly at different elevations, but with the same dominant fungi. Beta-diversity analysis indicated that the similarity of fungal communities decreased with an increased vertical distance between the sample plots, showing a distance-decay relationship. Variation partition analysis showed that geographic distance (mainly elevation gradient) only explained 20.53 % of the total variation of fungal community structure, while soil physicochemical factors explained 69.78 %.

Hydrocarbon biodegradation by Arctic sea-ice and sub-ice microbial communities during microcosm experiments, Northwest Passage (Nunavut, Canada).

PubMed

Garneau, Marie-Ève; Michel, Christine; Meisterhans, Guillaume; Fortin, Nathalie; King, Thomas L; Greer, Charles W; Lee, Kenneth

2016-10-01

The increasing accessibility to navigation and offshore oil exploration brings risks of hydrocarbon releases in Arctic waters. Bioremediation of hydrocarbons is a promising mitigation strategy but challenges remain, particularly due to low microbial metabolic rates in cold, ice-covered seas. Hydrocarbon degradation potential of ice-associated microbes collected from the Northwest Passage was investigated. Microcosm incubations were run for 15 days at -1.7°C with and without oil to determine the effects of hydrocarbon exposure on microbial abundance, diversity and activity, and to estimate component-specific hydrocarbon loss. Diversity was assessed with automated ribosomal intergenic spacer analysis and Ion Torrent 16S rRNA gene sequencing. Bacterial activity was measured by (3)H-leucine uptake rates. After incubation, sub-ice and sea-ice communities degraded 94% and 48% of the initial hydrocarbons, respectively. Hydrocarbon exposure changed the composition of sea-ice and sub-ice communities; in sea-ice microcosms, Bacteroidetes (mainly Polaribacter) dominated whereas in sub-ice microcosms, the contribution of Epsilonproteobacteria increased, and that of Alphaproteobacteria and Bacteroidetes decreased. Sequencing data revealed a decline in diversity and increases in Colwellia and Moritella in oil-treated microcosms. Low concentration of dissolved organic matter (DOM) in sub-ice seawater may explain higher hydrocarbon degradation when compared to sea ice, where DOM was abundant and composed of labile exopolysaccharides. © Fisheries and Oceans Canada [2016].

Genome and Transcriptome Sequencing of the Ostreid herpesvirus 1 From Tomales Bay, California

NASA Astrophysics Data System (ADS)

Burge, C. A.; Langevin, S.; Closek, C. J.; Roberts, S. B.; Friedman, C. S.

2016-02-01

Mass mortalities of larval and seed bivalve molluscs attributed to the Ostreid herpesvirus 1 (OsHV-1) occur globally. OsHV-1 was fully sequenced and characterized as a member of the Family Malacoherpesviridae. Multiple strains of OsHV-1 exist and may vary in virulence, i.e. OsHV-1 µvar. For most global variants of OsHV-1, sequence data is limited to PCR-based sequencing of segments, including two recent genomes. In the United States, OsHV-1 is limited to detection in adjacent embayments in California, Tomales and Drakes bays. Limited DNA sequence data of OsHV-1 infecting oysters in Tomales Bay indicates the virus detected in Tomales Bay is similar but not identical to any one global variant of OsHV-1. In order to better understand both strain variation and virulence of OsHV-1 infecting oysters in Tomales Bay, we used genomic and transcriptomic sequencing. Meta-genomic sequencing (Illumina MiSeq) was conducted from infected oysters (n=4 per year) collected in 2003, 2007, and 2014, where full OsHV-1 genome sequences and low overall microbial diversity were achieved from highly infected oysters. Increased microbial diversity was detected in three of four samples sequenced from 2003, where qPCR based genome copy numbers of OsHV-1 were lower. Expression analysis (SOLiD RNA sequencing) of OsHV-1 genes expressed in oyster larvae at 24 hours post exposure revealed a nearly complete transcriptome, with several highly expressed genes, which are similar to recent transcriptomic analyses of other OsHV-1 variants. Taken together, our results indicate that genome and transcriptome sequencing may be powerful tools in understanding both strain variation and virulence of non-culturable marine viruses.

Genotype diversity of hepatitis C virus (HCV) in HCV-associated liver disease patients in Indonesia.

PubMed

Utama, Andi; Tania, Navessa Padma; Dhenni, Rama; Gani, Rino Alvani; Hasan, Irsan; Sanityoso, Andri; Lelosutan, Syafruddin A R; Martamala, Ruswhandi; Lesmana, Laurentius Adrianus; Sulaiman, Ali; Tai, Susan

2010-09-01

Hepatitis C virus (HCV) genotype distribution in Indonesia has been reported. However, the identification of HCV genotype was based on 5'-UTR or NS5B sequence. This study was aimed to observe HCV core sequence variation among HCV-associated liver disease patients in Jakarta, and to analyse the HCV genotype diversity based on the core sequence. Sixty-eight chronic hepatitis (CH), 48 liver cirrhosis (LC) and 34 hepatocellular carcinoma (HCC) were included in this study. HCV core variation was analysed by direct sequencing. Alignment of HCV core sequences demonstrated that the core sequence was relatively varied among the genotype. Indeed, 237 bases of the core sequence could classify the HCV subtype; however, 236 bases failed to differentiate several subtypes. Based on 237 bases of the core sequences, the HCV strains were classified into genotypes 1 (subtypes 1a, 1b and 1c), 2 (subtypes 2a, 2e and 2f) and 3 (subtypes 3a and 3k). The HCV 1b (47.3%) was the most prevalent, followed by subtypes 1c (18.7%), 3k (10.7%), 2a (10.0%), 1a (6.7%), 2e (5.3%), 2f (0.7%) and 3a (0.7%). HCV 1b was the most common in all patients, and the prevalence increased with the severity of liver disease (36.8% in CH, 54.2% in LC and 58.8% in HCC). These results were similar to a previous report based on NS5B sequence analysis. Hepatitis C virus core sequence (237 bases) could identify the HCV subtype and the prevalence of HCV subtype based on core sequence was similar to those based on the NS5B region.

Effect of the natural arsenic gradient on the diversity and arsenic resistance of bacterial communities of the sediments of Camarones River (Atacama Desert, Chile).

PubMed

Leon, Carla G; Moraga, Ruben; Valenzuela, Cristian; Gugliandolo, Concetta; Lo Giudice, Angelina; Papale, Maria; Vilo, Claudia; Dong, Qunfeng; Smith, Carlos T; Rossello-Mora, Ramon; Yañez, Jorge; Campos, Victor L

2018-01-01

Arsenic (As), a highly toxic metalloid, naturally present in Camarones River (Atacama Desert, Chile) is a great health concern for the local population and authorities. In this study, the taxonomic and functional characterization of bacterial communities associated to metal-rich sediments from three sites of the river (sites M1, M2 and M3), showing different arsenic concentrations, were evaluated using a combination of approaches. Diversity of bacterial communities was evaluated by Illumina sequencing. Strains resistant to arsenic concentrations varying from 0.5 to 100 mM arsenite or arsenate were isolated and the presence of genes coding for enzymes involved in arsenic oxidation (aio) or reduction (arsC) investigated. Bacterial communities showed a moderate diversity which increased as arsenic concentrations decreased along the river. Sequences of the dominant taxonomic groups (abundances ≥1%) present in all three sites were affiliated to Proteobacteria (range 40.3-47.2%), Firmicutes (8.4-24.8%), Acidobacteria (10.4-17.1%), Actinobacteria (5.4-8.1%), Chloroflexi (3.9-7.5%), Planctomycetes (1.2-5.3%), Gemmatimonadetes (1.2-1.5%), and Nitrospirae (1.1-1.2%). Bacterial communities from sites M2 and M3 showed no significant differences in diversity between each other (p = 0.9753) but they were significantly more diverse than M1 (p<0.001 and p<0.001, respectively). Sequences affiliated with Proteobacteria, Firmicutes, Acidobacteria, Chloroflexi and Actinobacteria at M1 accounted for more than 89% of the total classified bacterial sequences present but these phyla were present in lesser proportions in M2 and M3 sites. Strains isolated from the sediment of sample M1, having the greatest arsenic concentration (498 mg kg-1), showed the largest percentages of arsenic oxidation and reduction. Genes aio were more frequently detected in isolates from M1 (54%), whereas arsC genes were present in almost all isolates from all three sediments, suggesting that bacterial communities play an important role in the arsenic biogeochemical cycle and detoxification of arsenical compounds. Overall, results provide further knowledge on the microbial diversity of arsenic contaminated fresh-water sediments.

Low Diversity in the Mitogenome of Sperm Whales Revealed by Next-Generation Sequencing

PubMed Central

Alexander, Alana; Steel, Debbie; Slikas, Beth; Hoekzema, Kendra; Carraher, Colm; Parks, Matthew; Cronn, Richard; Baker, C. Scott

2013-01-01

Large population sizes and global distributions generally associate with high mitochondrial DNA control region (CR) diversity. The sperm whale (Physeter macrocephalus) is an exception, showing low CR diversity relative to other cetaceans; however, diversity levels throughout the remainder of the sperm whale mitogenome are unknown. We sequenced 20 mitogenomes from 17 sperm whales representative of worldwide diversity using Next Generation Sequencing (NGS) technologies (Illumina GAIIx, Roche 454 GS Junior). Resequencing of three individuals with both NGS platforms and partial Sanger sequencing showed low discrepancy rates (454-Illumina: 0.0071%; Sanger-Illumina: 0.0034%; and Sanger-454: 0.0023%) confirming suitability of both NGS platforms for investigating low mitogenomic diversity. Using the 17 sperm whale mitogenomes in a phylogenetic reconstruction with 41 other species, including 11 new dolphin mitogenomes, we tested two hypotheses for the low CR diversity. First, the hypothesis that CR-specific constraints have reduced diversity solely in the CR was rejected as diversity was low throughout the mitogenome, not just in the CR (overall diversity π = 0.096%; protein-coding 3rd codon = 0.22%; CR = 0.35%), and CR phylogenetic signal was congruent with protein-coding regions. Second, the hypothesis that slow substitution rates reduced diversity throughout the sperm whale mitogenome was rejected as sperm whales had significantly higher rates of CR evolution and no evidence of slow coding region evolution relative to other cetaceans. The estimated time to most recent common ancestor for sperm whale mitogenomes was 72,800 to 137,400 years ago (95% highest probability density interval), consistent with previous hypotheses of a bottleneck or selective sweep as likely causes of low mitogenome diversity. PMID:23254394

Low diversity in the mitogenome of sperm whales revealed by next-generation sequencing.

PubMed

Alexander, Alana; Steel, Debbie; Slikas, Beth; Hoekzema, Kendra; Carraher, Colm; Parks, Matthew; Cronn, Richard; Baker, C Scott

2013-01-01

Large population sizes and global distributions generally associate with high mitochondrial DNA control region (CR) diversity. The sperm whale (Physeter macrocephalus) is an exception, showing low CR diversity relative to other cetaceans; however, diversity levels throughout the remainder of the sperm whale mitogenome are unknown. We sequenced 20 mitogenomes from 17 sperm whales representative of worldwide diversity using Next Generation Sequencing (NGS) technologies (Illumina GAIIx, Roche 454 GS Junior). Resequencing of three individuals with both NGS platforms and partial Sanger sequencing showed low discrepancy rates (454-Illumina: 0.0071%; Sanger-Illumina: 0.0034%; and Sanger-454: 0.0023%) confirming suitability of both NGS platforms for investigating low mitogenomic diversity. Using the 17 sperm whale mitogenomes in a phylogenetic reconstruction with 41 other species, including 11 new dolphin mitogenomes, we tested two hypotheses for the low CR diversity. First, the hypothesis that CR-specific constraints have reduced diversity solely in the CR was rejected as diversity was low throughout the mitogenome, not just in the CR (overall diversity π = 0.096%; protein-coding 3rd codon = 0.22%; CR = 0.35%), and CR phylogenetic signal was congruent with protein-coding regions. Second, the hypothesis that slow substitution rates reduced diversity throughout the sperm whale mitogenome was rejected as sperm whales had significantly higher rates of CR evolution and no evidence of slow coding region evolution relative to other cetaceans. The estimated time to most recent common ancestor for sperm whale mitogenomes was 72,800 to 137,400 years ago (95% highest probability density interval), consistent with previous hypotheses of a bottleneck or selective sweep as likely causes of low mitogenome diversity.

High-throughput sequencing reveals unprecedented diversities of Aspergillus species in outdoor air.

PubMed

Lee, S; An, C; Xu, S; Lee, S; Yamamoto, N

2016-09-01

This study used the Illumina MiSeq to analyse compositions and diversities of Aspergillus species in outdoor air. The seasonal air samplings were performed at two locations in Seoul, South Korea. The results showed the relative abundances of all Aspergillus species combined ranging from 0·20 to 18% and from 0·19 to 21% based on the number of the internal transcribed spacer 1 (ITS1) and β-tubulin (BenA) gene sequences respectively. Aspergillus fumigatus was the most dominant species with the mean relative abundances of 1·2 and 5·5% based on the number of the ITS1 and BenA sequences respectively. A total of 29 Aspergillus species were detected and identified down to the species rank, among which nine species were known opportunistic pathogens. Remarkably, eight of the nine pathogenic species were detected by either one of the two markers, suggesting the need of using multiple markers and/or primer pairs when the assessments are made based on the high-throughput sequencing. Due to diversity of species within the genus Aspergillus, the high-throughput sequencing was useful to characterize their compositions and diversities in outdoor air, which are thought to be difficult to be accurately characterized by conventional culture and/or Sanger sequencing-based techniques. Aspergillus is a diverse genus of fungi with more than 300 species reported in literature. Aspergillus is important since some species are known allergens and opportunistic human pathogens. Traditionally, growth-dependent methods have been used to detect Aspergillus species in air. However, these methods are limited in the number of isolates that can be analysed for their identities, resulting in inaccurate characterizations of Aspergillus diversities. This study used the high-throughput sequencing to explore Aspergillus diversities in outdoor, which are thought to be difficult to be accurately characterized by traditional growth-dependent techniques. © 2016 The Society for Applied Microbiology.

Assessing Species Diversity Using Metavirome Data: Methods and Challenges.

PubMed

Herath, Damayanthi; Jayasundara, Duleepa; Ackland, David; Saeed, Isaam; Tang, Sen-Lin; Halgamuge, Saman

2017-01-01

Assessing biodiversity is an important step in the study of microbial ecology associated with a given environment. Multiple indices have been used to quantify species diversity, which is a key biodiversity measure. Measuring species diversity of viruses in different environments remains a challenge relative to measuring the diversity of other microbial communities. Metagenomics has played an important role in elucidating viral diversity by conducting metavirome studies; however, metavirome data are of high complexity requiring robust data preprocessing and analysis methods. In this review, existing bioinformatics methods for measuring species diversity using metavirome data are categorised broadly as either sequence similarity-dependent methods or sequence similarity-independent methods. The former includes a comparison of DNA fragments or assemblies generated in the experiment against reference databases for quantifying species diversity, whereas estimates from the latter are independent of the knowledge of existing sequence data. Current methods and tools are discussed in detail, including their applications and limitations. Drawbacks of the state-of-the-art method are demonstrated through results from a simulation. In addition, alternative approaches are proposed to overcome the challenges in estimating species diversity measures using metavirome data.

Investigation of the bottleneck leading to the domestication of maize

PubMed Central

Eyre-Walker, Adam; Gaut, Rebecca L.; Hilton, Holly; Feldman, Dawn L.; Gaut, Brandon S.

1998-01-01

Maize (Zea mays ssp. mays) is genetically diverse, yet it is also morphologically distinct from its wild relatives. These two observations are somewhat contradictory: the first observation is consistent with a large historical population size for maize, but the latter observation is consistent with strong, diversity-limiting selection during maize domestication. In this study, we sampled sequence diversity, coupled with simulations of the coalescent process, to study the dynamics of a population bottleneck during the domestication of maize. To do this, we determined the DNA sequence of a 1,400-bp region of the Adh1 locus from 19 individuals representing maize, its presumed progenitor (Z. mays ssp. parviglumis), and a more distant relative (Zea luxurians). The sequence data were used to guide coalescent simulations of population bottlenecks associated with domestication. Our study confirms high genetic diversity in maize—maize contains 75% of the variation found in its progenitor and is more diverse than its wild relative, Z. luxurians—but it also suggests that sequence diversity in maize can be explained by a bottleneck of short duration and very small size. For example, the breadth of genetic diversity in maize is consistent with a founding population of only 20 individuals when the domestication event is 10 generations in length. PMID:9539756

Comparative immunogenomics of molluscs.

PubMed

Schultz, Jonathan H; Adema, Coen M

2017-10-01

Comparative immunology, studying both vertebrates and invertebrates, provided the earliest descriptions of phagocytosis as a general immune mechanism. However, the large scale of animal diversity challenges all-inclusive investigations and the field of immunology has developed by mostly emphasizing study of a few vertebrate species. In addressing the lack of comprehensive understanding of animal immunity, especially that of invertebrates, comparative immunology helps toward management of invertebrates that are food sources, agricultural pests, pathogens, or transmit diseases, and helps interpret the evolution of animal immunity. Initial studies showed that the Mollusca (second largest animal phylum), and invertebrates in general, possess innate defenses but lack the lymphocytic immune system that characterizes vertebrate immunology. Recognizing the reality of both common and taxon-specific immune features, and applying up-to-date cell and molecular research capabilities, in-depth studies of a select number of bivalve and gastropod species continue to reveal novel aspects of molluscan immunity. The genomics era heralded a new stage of comparative immunology; large-scale efforts yielded an initial set of full molluscan genome sequences that is available for analyses of full complements of immune genes and regulatory sequences. Next-generation sequencing (NGS), due to lower cost and effort required, allows individual researchers to generate large sequence datasets for growing numbers of molluscs. RNAseq provides expression profiles that enable discovery of immune genes and genome sequences reveal distribution and diversity of immune factors across molluscan phylogeny. Although computational de novo sequence assembly will benefit from continued development and automated annotation may require some experimental validation, NGS is a powerful tool for comparative immunology, especially increasing coverage of the extensive molluscan diversity. To date, immunogenomics revealed new levels of complexity of molluscan defense by indicating sequence heterogeneity in individual snails and bivalves, and members of expanded immune gene families are expressed differentially to generate pathogen-specific defense responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genetic Diversity and Molecular Evolution of Chinese Waxy Maize Germplasm

PubMed Central

Zheng, Hongjian; Wang, Hui; Yang, Hua; Wu, Jinhong; Shi, Biao; Cai, Run; Xu, Yunbi; Wu, Aizhong; Luo, Lijun

2013-01-01

Waxy maize (Zea mays L. var. certaina Kulesh), with many excellent characters in terms of starch composition and economic value, has grown in China for a long history and its production has increased dramatically in recent decades. However, the evolution and origin of waxy maize still remains unclear. We studied the genetic diversity of Chinese waxy maize including typical landraces and inbred lines by SSR analysis and the results showed a wide genetic diversity in the Chinese waxy maize germplasm. We analyzed the origin and evolution of waxy maize by sequencing 108 samples, and downloading 52 sequences from GenBank for the waxy locus in a number of accessions from genus Zea. A sharp reduction of nucleotide diversity and significant neutrality tests (Tajima’s D and Fu and Li’s F*) were observed at the waxy locus in Chinese waxy maize but not in nonglutinous maize. Phylogenetic analysis indicated that Chinese waxy maize originated from the cultivated flint maize and most of the modern waxy maize inbred lines showed a distinct independent origin and evolution process compared with the germplasm from Southwest China. The results indicated that an agronomic trait can be quickly improved to meet production demand by selection. PMID:23818949

Phylogenetic analyses and nitrate-reducing activity of fungal cultures isolated from the permanent, oceanic oxygen minimum zone of the Arabian Sea.

PubMed

Manohar, Cathrine Sumathi; Menezes, Larissa Danielle; Ramasamy, Kesava Priyan; Meena, Ram M

2015-03-01

Reports on the active role of fungi as denitrifiers in terrestrial ecosystems have stimulated an interest in the study of the role of fungi in oxygen-deficient marine systems. In this study, the culturable diversity of fungi was investigated from 4 stations within the permanent, oceanic, oxygen minimum zone of the Arabian Sea. The isolated cultures grouped within the 2 major fungal phyla Ascomycota and Basidiomycota; diversity estimates in the stations sampled indicated that the diversity of the oxygen-depleted environments is less than that of mangrove regions and deep-sea habitats. Phylogenetic analyses of 18S rRNA sequences revealed a few divergent isolates that clustered with environmental sequences previously obtained by others. This is significant, as these isolates represent phylotypes that so far were known only from metagenomic studies and are of phylogenetic importance. Nitrate reduction activity, the first step in the denitrification process, was recorded for isolates under simulated anoxic, deep-sea conditions showing ecological significance of fungi in the oxygen-depleted habitats. This report increases our understanding of fungal diversity in unique, poorly studied habitats and underlines the importance of fungi in the oxygen-depleted environments.

IMGD: an integrated platform supporting comparative genomics and phylogenetics of insect mitochondrial genomes

PubMed Central

Lee, Wonhoon; Park, Jongsun; Choi, Jaeyoung; Jung, Kyongyong; Park, Bongsoo; Kim, Donghan; Lee, Jaeyoung; Ahn, Kyohun; Song, Wonho; Kang, Seogchan; Lee, Yong-Hwan; Lee, Seunghwan

2009-01-01

Background Sequences and organization of the mitochondrial genome have been used as markers to investigate evolutionary history and relationships in many taxonomic groups. The rapidly increasing mitochondrial genome sequences from diverse insects provide ample opportunities to explore various global evolutionary questions in the superclass Hexapoda. To adequately support such questions, it is imperative to establish an informatics platform that facilitates the retrieval and utilization of available mitochondrial genome sequence data. Results The Insect Mitochondrial Genome Database (IMGD) is a new integrated platform that archives the mitochondrial genome sequences from 25,747 hexapod species, including 112 completely sequenced and 20 nearly completed genomes and 113,985 partially sequenced mitochondrial genomes. The Species-driven User Interface (SUI) of IMGD supports data retrieval and diverse analyses at multi-taxon levels. The Phyloviewer implemented in IMGD provides three methods for drawing phylogenetic trees and displays the resulting trees on the web. The SNP database incorporated to IMGD presents the distribution of SNPs and INDELs in the mitochondrial genomes of multiple isolates within eight species. A newly developed comparative SNU Genome Browser supports the graphical presentation and interactive interface for the identified SNPs/INDELs. Conclusion The IMGD provides a solid foundation for the comparative mitochondrial genomics and phylogenetics of insects. All data and functions described here are available at the web site . PMID:19351385

Evolutionary dynamics of Hepatitis C virus in a chronic HIV co-infected patient and its correlation with the immune status.

PubMed

Culasso, Andrés Carlos Alberto; Monzani, María Cecilia; Baré, Patricia; Campos, Rodolfo Hector

2018-05-04

The HCV evolutionary dynamics play a key role in the infection onset, maintenance of chronicity, pathogenicity, and drug resistance variants fixation, and are thought to be one of the main caveats in the development of an effective vaccine. Previous studies in HCV/HIV co-infected patients suggest that a decline in the immune status is related with increases in the HCV intra-host genetic diversity. However, these findings are based on single point sequence diversity measures or coalescence analyses in several virus-host interactions. In this work, we describe the molecular evolution of HCV-E2 region in a single HIV-co-infected patient with two clearly defined immune conditions. The phylogenetic analysis of the HCV-1a sequences from the studied patient showed that he was co-infected with three different viral lineages. These lineages were not evenly detected throughout time. The sequence diversity and coalescence analyses of these lineages suggested the action of different evolutionary patterns in different immune conditions: a slow rate, drift-like process in an immunocompromised condition (low levels of CD4+ T lymphocytes); and a fast rate, variant-switch process in an immunocompetent condition (high levels of CD4+ T lymphocytes). Copyright © 2017. Published by Elsevier B.V.

Marine Fungi: Their Ecology and Molecular Diversity

NASA Astrophysics Data System (ADS)

Richards, Thomas A.; Jones, Meredith D. M.; Leonard, Guy; Bass, David

2012-01-01

Fungi appear to be rare in marine environments. There are relatively few marine isolates in culture, and fungal small subunit ribosomal DNA (SSU rDNA) sequences are rarely recovered in marine clone library experiments (i.e., culture-independent sequence surveys of eukaryotic microbial diversity from environmental DNA samples). To explore the diversity of marine fungi, we took a broad selection of SSU rDNA data sets and calculated a summary phylogeny. Bringing these data together identified a diverse collection of marine fungi, including sequences branching close to chytrids (flagellated fungi), filamentous hypha-forming fungi, and multicellular fungi. However, the majority of the sequences branched with ascomycete and basidiomycete yeasts. We discuss evidence for 36 novel marine lineages, the majority and most divergent of which branch with the chytrids. We then investigate what these data mean for the evolutionary history of the Fungi and specifically marine-terrestrial transitions. Finally, we discuss the roles of fungi in marine ecosystems.

Variation in the hindgut microbial communities of the Florida manatee, Trichechus manatus latirostris over winter in Crystal River, Florida

USGS Publications Warehouse

Merson, Samuel D.; Ouwerkerk, Diane; Gulino, Lisa-Maree; Klieve, Athol; Bonde, Robert K.; Burgess, Elizabeth A.; Lanyon, Janet M.

2014-01-01

The Florida manatee, Trichechus manatus latirostris, is a hindgut-fermenting herbivore. In winter, manatees migrate to warm water overwintering sites where they undergo dietary shifts and may suffer from cold-induced stress. Given these seasonally induced changes in diet, the present study aimed to examine variation in the hindgut bacterial communities of wild manatees overwintering at Crystal River, west Florida. Faeces were sampled from 36 manatees of known sex and body size in early winter when manatees were newly arrived and then in mid-winter and late winter when diet had probably changed and environmental stress may have increased. Concentrations of faecal cortisol metabolite, an indicator of a stress response, were measured by enzyme immunoassay. Using 454-pyrosequencing, 2027 bacterial operational taxonomic units were identified in manatee faeces following amplicon pyrosequencing of the 16S rRNA gene V3/V4 region. Classified sequences were assigned to eight previously described bacterial phyla; only 0.36% of sequences could not be classified to phylum level. Five core phyla were identified in all samples. The majority (96.8%) of sequences were classified as Firmicutes (77.3 ± 11.1% of total sequences) or Bacteroidetes (19.5 ± 10.6%). Alpha-diversity measures trended towards higher diversity of hindgut microbiota in manatees in mid-winter compared to early and late winter. Beta-diversity measures, analysed through permanova, also indicated significant differences in bacterial communities based on the season.

Diversity of virus-host systems in hypersaline Lake Retba, Senegal.

PubMed

Sime-Ngando, Télesphore; Lucas, Soizick; Robin, Agnès; Tucker, Kimberly Pause; Colombet, Jonathan; Bettarel, Yvan; Desmond, Elie; Gribaldo, Simonetta; Forterre, Patrick; Breitbart, Mya; Prangishvili, David

2011-08-01

Remarkable morphological diversity of virus-like particles was observed by transmission electron microscopy in a hypersaline water sample from Lake Retba, Senegal. The majority of particles morphologically resembled hyperthermophilic archaeal DNA viruses isolated from extreme geothermal environments. Some hypersaline viral morphotypes have not been previously observed in nature, and less than 1% of observed particles had a head-and-tail morphology, which is typical for bacterial DNA viruses. Culture-independent analysis of the microbial diversity in the sample suggested the dominance of extremely halophilic archaea. Few of the 16S sequences corresponded to known archeal genera (Haloquadratum, Halorubrum and Natronomonas), whereas the majority represented novel archaeal clades. Three sequences corresponded to a new basal lineage of the haloarchaea. Bacteria belonged to four major phyla, consistent with the known diversity in saline environments. Metagenomic sequencing of DNA from the purified virus-like particles revealed very few similarities to the NCBI non-redundant database at either the nucleotide or amino acid level. Some of the identifiable virus sequences were most similar to previously described haloarchaeal viruses, but no sequence similarities were found to archaeal viruses from extreme geothermal environments. A large proportion of the sequences had similarity to previously sequenced viral metagenomes from solar salterns. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

Novel Method for High-Throughput Full-Length IGHV-D-J Sequencing of the Immune Repertoire from Bulk B-Cells with Single-Cell Resolution.

PubMed

Vergani, Stefano; Korsunsky, Ilya; Mazzarello, Andrea Nicola; Ferrer, Gerardo; Chiorazzi, Nicholas; Bagnara, Davide

2017-01-01

Efficient and accurate high-throughput DNA sequencing of the adaptive immune receptor repertoire (AIRR) is necessary to study immune diversity in healthy subjects and disease-related conditions. The high complexity and diversity of the AIRR coupled with the limited amount of starting material, which can compromise identification of the full biological diversity makes such sequencing particularly challenging. AIRR sequencing protocols often fail to fully capture the sampled AIRR diversity, especially for samples containing restricted numbers of B lymphocytes. Here, we describe a library preparation method for immunoglobulin sequencing that results in an exhaustive full-length repertoire where virtually every sampled B-cell is sequenced. This maximizes the likelihood of identifying and quantifying the entire IGHV-D-J repertoire of a sample, including the detection of rearrangements present in only one cell in the starting population. The methodology establishes the importance of circumventing genetic material dilution in the preamplification phases and incorporates the use of certain described concepts: (1) balancing the starting material amount and depth of sequencing, (2) avoiding IGHV gene-specific amplification, and (3) using Unique Molecular Identifier. Together, this methodology is highly efficient, in particular for detecting rare rearrangements in the sampled population and when only a limited amount of starting material is available.

Diverse molecular signatures for ribosomally ‘active’ Perkinsea in marine sediments

PubMed Central

2014-01-01

Background Perkinsea are a parasitic lineage within the eukaryotic superphylum Alveolata. Recent studies making use of environmental small sub-unit ribosomal RNA gene (SSU rDNA) sequencing methodologies have detected a significant diversity and abundance of Perkinsea-like phylotypes in freshwater environments. In contrast only a few Perkinsea environmental sequences have been retrieved from marine samples and only two groups of Perkinsea have been cultured and morphologically described and these are parasites of marine molluscs or marine protists. These two marine groups form separate and distantly related phylogenetic clusters, composed of closely related lineages on SSU rDNA trees. Here, we test the hypothesis that Perkinsea are a hitherto under-sampled group in marine environments. Using 454 diversity ‘tag’ sequencing we investigate the diversity and distribution of these protists in marine sediments and water column samples taken from the Deep Chlorophyll Maximum (DCM) and sub-surface using both DNA and RNA as the source template and sampling four European offshore locations. Results We detected the presence of 265 sequences branching with known Perkinsea, the majority of them recovered from marine sediments. Moreover, 27% of these sequences were sampled from RNA derived cDNA libraries. Phylogenetic analyses classify a large proportion of these sequences into 38 cluster groups (including 30 novel marine cluster groups), which share less than 97% sequence similarity suggesting this diversity encompasses a range of biologically and ecologically distinct organisms. Conclusions These results demonstrate that the Perkinsea lineage is considerably more diverse than previously detected in marine environments. This wide diversity of Perkinsea-like protists is largely retrieved in marine sediment with a significant proportion detected in RNA derived libraries suggesting this diversity represents ribosomally ‘active’ and intact cells. Given the phylogenetic range of hosts infected by known Perkinsea parasites, these data suggest that Perkinsea either play a significant but hitherto unrecognized role as parasites in marine sediments and/or members of this group are present in the marine sediment possibly as part of the ‘seed bank’ microbial community. PMID:24779375

Genetic Structure and Potential Environmental Determinants of Local Genetic Diversity in Japanese Honeybees (Apis cerana japonica)

PubMed Central

Nagamitsu, Teruyoshi; Yasuda, Mika; Saito-Morooka, Fuki; Inoue, Maki N.; Nishiyama, Mio; Goka, Koichi; Sugiura, Shinji; Maeto, Kaoru; Okabe, Kimiko; Taki, Hisatomo

2016-01-01

Declines in honeybee populations have been a recent concern. Although causes of the declines remain unclear, environmental factors may be responsible. We focused on the potential environmental determinants of local populations of wild honeybees, Apis cerana japonica, in Japan. This subspecies has little genetic variation in terms of its mitochondrial DNA sequences, and genetic variations at nuclear loci are as yet unknown. We estimated the genetic structure and environmental determinants of local genetic diversity in nuclear microsatellite genotypes of fathers and mothers, inferred from workers collected at 139 sites. The genotypes of fathers and mothers showed weak isolation by distance and negligible genetic structure. The local genetic diversity was high in central Japan, decreasing toward the peripheries, and depended on the climate and land use characteristics of the sites. The local genetic diversity decreased as the annual precipitation increased, and increased as the proportion of urban and paddy field areas increased. Positive effects of natural forest area, which have also been observed in terms of forager abundance in farms, were not detected with respect to the local genetic diversity. The findings suggest that A. cerana japonica forms a single population connected by gene flow in its main distributional range, and that climate and landscape properties potentially affect its local genetic diversity. PMID:27898704

Deep sequencing of the Trypanosoma cruzi GP63 surface proteases reveals diversity and diversifying selection among chronic and congenital Chagas disease patients.

PubMed

Llewellyn, Martin S; Messenger, Louisa A; Luquetti, Alejandro O; Garcia, Lineth; Torrico, Faustino; Tavares, Suelene B N; Cheaib, Bachar; Derome, Nicolas; Delepine, Marc; Baulard, Céline; Deleuze, Jean-Francois; Sauer, Sascha; Miles, Michael A

2015-04-01

Chagas disease results from infection with the diploid protozoan parasite Trypanosoma cruzi. T. cruzi is highly genetically diverse, and multiclonal infections in individual hosts are common, but little studied. In this study, we explore T. cruzi infection multiclonality in the context of age, sex and clinical profile among a cohort of chronic patients, as well as paired congenital cases from Cochabamba, Bolivia and Goias, Brazil using amplicon deep sequencing technology. A 450bp fragment of the trypomastigote TcGP63I surface protease gene was amplified and sequenced across 70 chronic and 22 congenital cases on the Illumina MiSeq platform. In addition, a second, mitochondrial target--ND5--was sequenced across the same cohort of cases. Several million reads were generated, and sequencing read depths were normalized within patient cohorts (Goias chronic, n = 43, Goias congenital n = 2, Bolivia chronic, n = 27; Bolivia congenital, n = 20), Among chronic cases, analyses of variance indicated no clear correlation between intra-host sequence diversity and age, sex or symptoms, while principal coordinate analyses showed no clustering by symptoms between patients. Between congenital pairs, we found evidence for the transmission of multiple sequence types from mother to infant, as well as widespread instances of novel genotypes in infants. Finally, non-synonymous to synonymous (dn:ds) nucleotide substitution ratios among sequences of TcGP63Ia and TcGP63Ib subfamilies within each cohort provided powerful evidence of strong diversifying selection at this locus. Our results shed light on the diversity of parasite DTUs within each patient, as well as the extent to which parasite strains pass between mother and foetus in congenital cases. Although we were unable to find any evidence that parasite diversity accumulates with age in our study cohorts, putative diversifying selection within members of the TcGP63I gene family suggests a link between genetic diversity within this gene family and survival in the mammalian host.

Diversity and phylogenetic relationships among Bartonella strains from Thai bats.

PubMed

McKee, Clifton D; Kosoy, Michael Y; Bai, Ying; Osikowicz, Lynn M; Franka, Richard; Gilbert, Amy T; Boonmar, Sumalee; Rupprecht, Charles E; Peruski, Leonard F

2017-01-01

Bartonellae are phylogenetically diverse, intracellular bacteria commonly found in mammals. Previous studies have demonstrated that bats have a high prevalence and diversity of Bartonella infections globally. Isolates (n = 42) were obtained from five bat species in four provinces of Thailand and analyzed using sequences of the citrate synthase gene (gltA). Sequences clustered into seven distinct genogroups; four of these genogroups displayed similarity with Bartonella spp. sequences from other bats in Southeast Asia, Africa, and Eastern Europe. Thirty of the isolates representing these seven genogroups were further characterized by sequencing four additional loci (ftsZ, nuoG, rpoB, and ITS) to clarify their evolutionary relationships with other Bartonella species and to assess patterns of diversity among strains. Among the seven genogroups, there were differences in the number of sequence variants, ranging from 1-5, and the amount of nucleotide divergence, ranging from 0.035-3.9%. Overall, these seven genogroups meet the criteria for distinction as novel Bartonella species, with sequence divergence among genogroups ranging from 6.4-15.8%. Evidence of intra- and intercontinental phylogenetic relationships and instances of homologous recombination among Bartonella genogroups in related bat species were found in Thai bats.

A-to-I RNA Editing Contributes to Proteomic Diversity in Cancer.

PubMed

Peng, Xinxin; Xu, Xiaoyan; Wang, Yumeng; Hawke, David H; Yu, Shuangxing; Han, Leng; Zhou, Zhicheng; Mojumdar, Kamalika; Jeong, Kang Jin; Labrie, Marilyne; Tsang, Yiu Huen; Zhang, Minying; Lu, Yiling; Hwu, Patrick; Scott, Kenneth L; Liang, Han; Mills, Gordon B

2018-05-14

Adenosine (A) to inosine (I) RNA editing introduces many nucleotide changes in cancer transcriptomes. However, due to the complexity of post-transcriptional regulation, the contribution of RNA editing to proteomic diversity in human cancers remains unclear. Here, we performed an integrated analysis of TCGA genomic data and CPTAC proteomic data. Despite limited site diversity, we demonstrate that A-to-I RNA editing contributes to proteomic diversity in breast cancer through changes in amino acid sequences. We validate the presence of editing events at both RNA and protein levels. The edited COPA protein increases proliferation, migration, and invasion of cancer cells in vitro. Our study suggests an important contribution of A-to-I RNA editing to protein diversity in cancer and highlights its translational potential. Copyright © 2018 Elsevier Inc. All rights reserved.

Combining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology.

PubMed

Tanouchi, Yu; Covert, Markus W

2017-09-19

During its lysogenic life cycle, the phage genome is integrated into the host chromosome by site-specific recombination. In this report, we analyze lambda phage integration into noncanonical sites using next-generation sequencing and show that it generates significant genetic diversity by targeting over 300 unique sites in the host Escherichia coli genome. Moreover, these integration events can have important phenotypic consequences for the host, including changes in cell motility and increased antibiotic resistance. Importantly, the new technologies that we developed to enable this study-sequencing secondary sites using next-generation sequencing and then selecting relevant lysogens using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based selection-are broadly applicable to other phage-bacterium systems. IMPORTANCE Bacteriophages play an important role in bacterial evolution through lysogeny, where the phage genome is integrated into the host chromosome. While phage integration generally occurs at a specific site in the host chromosome, it is also known to occur at other, so-called secondary sites. In this study, we developed a new experimental technology to comprehensively study secondary integration sites and discovered that phage can integrate into over 300 unique sites in the host genome, resulting in significant genetic diversity in bacteria. We further developed an assay to examine the phenotypic consequence of such diverse integration events and found that phage integration can cause changes in evolutionarily relevant traits such as bacterial motility and increases in antibiotic resistance. Importantly, our method is readily applicable to other phage-bacterium systems. Copyright © 2017 Tanouchi and Covert.

Assessing the utility of eDNA as a tool to survey reef-fish communities in the Red Sea

NASA Astrophysics Data System (ADS)

DiBattista, Joseph D.; Coker, Darren J.; Sinclair-Taylor, Tane H.; Stat, Michael; Berumen, Michael L.; Bunce, Michael

2017-12-01

Relatively small volumes of water may contain sufficient environmental DNA (eDNA) to detect target aquatic organisms via genetic sequencing. We therefore assessed the utility of eDNA to document the diversity of coral reef fishes in the central Red Sea. DNA from seawater samples was extracted, amplified using fish-specific 16S mitochondrial DNA primers, and sequenced using a metabarcoding workflow. DNA sequences were assigned to taxa using available genetic repositories or custom genetic databases generated from reference fishes. Our approach revealed a diversity of conspicuous, cryptobenthic, and commercially relevant reef fish at the genus level, with select genera in the family Labridae over-represented. Our approach, however, failed to capture a significant fraction of the fish fauna known to inhabit the Red Sea, which we attribute to limited spatial sampling, amplification stochasticity, and an apparent lack of sequencing depth. Given an increase in fish species descriptions, completeness of taxonomic checklists, and improvement in species-level assignment with custom genetic databases as shown here, we suggest that the Red Sea region may be ideal for further testing of the eDNA approach.

Exploring the Genomic Traits of Non-toxigenic Vibrio parahaemolyticus Strains Isolated in Southern Chile

PubMed Central

Castillo, Daniel; Pérez-Reytor, Diliana; Plaza, Nicolás; Ramírez-Araya, Sebastián; Blondel, Carlos J.; Corsini, Gino; Bastías, Roberto; Loyola, David E.; Jaña, Víctor; Pavez, Leonardo; García, Katherine

2018-01-01

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis worldwide. As reported in other countries, after the rise and fall of the pandemic strain in Chile, other post-pandemic strains have been associated with clinical cases, including strains lacking the major toxins TDH and TRH. Since the presence or absence of tdh and trh genes has been used for diagnostic purposes and as a proxy of the virulence of V. parahaemolyticus isolates, the understanding of virulence in V. parahaemolyticus strains lacking toxins is essential to detect these strains present in water and marine products to avoid possible food-borne infection. In this study, we characterized the genome of four environmental and two clinical non-toxigenic strains (tdh-, trh-, and T3SS2-). Using whole-genome sequencing, phylogenetic, and comparative genome analysis, we identified the core and pan-genome of V. parahaemolyticus of strains of southern Chile. The phylogenetic tree based on the core genome showed low genetic diversity but the analysis of the pan-genome revealed that all strains harbored genomic islands carrying diverse virulence and fitness factors or prophage-like elements that encode toxins like Zot and RTX. Interestingly, the three strains carrying Zot-like toxin have a different sequence, although the alignment showed some conserved areas with the zot sequence found in V. cholerae. In addition, we identified an unexpected diversity in the genetic architecture of the T3SS1 gene cluster and the presence of the T3SS2 gene cluster in a non-pandemic environmental strain. Our study sheds light on the diversity of V. parahaemolyticus strains from the southern Pacific which increases our current knowledge regarding the global diversity of this organism. PMID:29472910

Exploring bacterial diversity in hospital environments by GS-FLX Titanium pyrosequencing.

PubMed

Poza, Margarita; Gayoso, Carmen; Gómez, Manuel J; Rumbo-Feal, Soraya; Tomás, María; Aranda, Jesús; Fernández, Ana; Bou, Germán

2012-01-01

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.

Expansion of the Preimmune Antibody Repertoire by Junctional Diversity in Bos taurus

PubMed Central

Liljavirta, Jenni; Niku, Mikael; Pessa-Morikawa, Tiina; Ekman, Anna; Iivanainen, Antti

2014-01-01

Cattle have a limited range of immunoglobulin genes which are further diversified by antigen independent somatic hypermutation in fetuses. Junctional diversity generated during somatic recombination contributes to antibody diversity but its relative significance has not been comprehensively studied. We have investigated the importance of terminal deoxynucleotidyl transferase (TdT) -mediated junctional diversity to the bovine immunoglobulin repertoire. We also searched for new bovine heavy chain diversity (IGHD) genes as the information of the germline sequences is essential to define the junctional boundaries between gene segments. New heavy chain variable genes (IGHV) were explored to address the gene usage in the fetal recombinations. Our bioinformatics search revealed five new IGHD genes, which included the longest IGHD reported so far, 154 bp. By genomic sequencing we found 26 new IGHV sequences that represent potentially new IGHV genes or allelic variants. Sequence analysis of immunoglobulin heavy chain cDNA libraries of fetal bone marrow, ileum and spleen showed 0 to 36 nontemplated N-nucleotide additions between variable, diversity and joining genes. A maximum of 8 N nucleotides were also identified in the light chains. The junctional base profile was biased towards A and T nucleotide additions (64% in heavy chain VD, 52% in heavy chain DJ and 61% in light chain VJ junctions) in contrast to the high G/C content which is usually observed in mice. Sequence analysis also revealed extensive exonuclease activity, providing additional diversity. B-lymphocyte specific TdT expression was detected in bovine fetal bone marrow by reverse transcription-qPCR and immunofluorescence. These results suggest that TdT-mediated junctional diversity and exonuclease activity contribute significantly to the size of the cattle preimmune antibody repertoire already in the fetal period. PMID:24926997

Captivity results in disparate loss of gut microbial diversity in closely related hosts

PubMed Central

Kohl, Kevin D.; Skopec, Michele M.; Dearing, M. Denise

2014-01-01

The gastrointestinal tracts of animals contain diverse communities of microbes that provide a number of services to their hosts. There is recent concern that these communities may be lost as animals enter captive breeding programmes, due to changes in diet and/or exposure to environmental sources. However, empirical evidence documenting the effects of captivity and captive birth on gut communities is lacking. We conducted three studies to advance our knowledge in this area. First, we compared changes in microbial diversity of the gut communities of two species of woodrats (Neotoma albigula, a dietary generalist, and Neotoma stephensi, which specializes on juniper) before and after 6–9 months in captivity. Second, we investigated whether reintroduction of the natural diet of N. stephensi could restore microbial diversity. Third, we compared the microbial communities between offspring born in captivity and their mothers. We found that the dietary specialist, N. stephensi, lost a greater proportion of its native gut microbiota and overall diversity in response to captivity compared with N. albigula. Addition of the natural diet increased the proportion of the original microbiota but did not restore overall diversity in N. stephensi. Offspring of N. albigula more closely resembled their mothers compared with offspring–mother pairs of N. stephensi. This research suggests that the microbiota of dietary specialists may be more susceptible to captivity. Furthermore, this work highlights the need for further studies investigating the mechanisms underlying how loss of microbial diversity may vary between hosts and what an acceptable level of diversity loss may be to a host. This knowledge will aid conservation biologists in designing captive breeding programmes effective at maintaining microbial diversity. Sequence Accession Numbers: NCBI's Sequence Read Archive (SRA) – SRP033616 PMID:27293630

Population genetic structure and demographic history of the black fly vector, Simulium nodosum in Thailand.

PubMed

Chaiyasan, P; Pramual, P

2016-09-01

An understanding of the genetic structure and diversity of vector species is crucial for effective control and management. In this study, mitochondrial DNA sequences were used to examine the genetic structure, diversity and demographic history of a black fly vector, Simulium nodosum Puri (Diptera: Simuliidae), in Thailand. A total of 145 sequences were obtained from 10 sampling locations collected across geographical ranges in the country. Low genetic diversity was found in populations of S. nodosum that could be explained by the recent population history of this species. Demographic history analysis revealed a signature of demographic expansion dating back to only 2600-5200 years ago. Recent population expansion in S. nodosum possibly followed an increase in agriculture that enabled its hosts', humans and domestic animals, densities to increase. Alternatively, the Thai populations could be a derivative of an older expansion event in the more northern populations. Mitochondrial DNA genealogy revealed no genetically divergent lineages, which agrees with the previous cytogenetic study. Genetic structure analyses found that only 27% of the pairwise comparisons were significantly different. The most likely explanation for the pattern of genetic structuring is the effect of genetic drift because of recent colonization. © 2016 The Royal Entomological Society.

Bacterial diversity of surface sand samples from the Gobi and Taklamaken deserts.

PubMed

An, Shu; Couteau, Cécile; Luo, Fan; Neveu, Julie; DuBow, Michael S

2013-11-01

Arid regions represent nearly 30 % of the Earth's terrestrial surface, but their microbial biodiversity is not yet well characterized. The surface sands of deserts, a subset of arid regions, are generally subjected to large temperature fluctuations plus high UV light exposure and are low in organic matter. We examined surface sand samples from the Taklamaken (China, three samples) and Gobi (Mongolia, two samples) deserts, using pyrosequencing of PCR-amplified 16S V1/V2 rDNA sequences from total extracted DNA in order to gain an assessment of the bacterial population diversity. In total, 4,088 OTUs (using ≥97 % sequence similarity levels), with Chao1 estimates varying from 1,172 to 2,425 OTUs per sample, were discernable. These could be grouped into 102 families belonging to 15 phyla, with OTUs belonging to the Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria phyla being the most abundant. The bacterial population composition was statistically different among the samples, though members from 30 genera were found to be common among the five samples. An increase in phylotype numbers with increasing C/N ratio was noted, suggesting a possible role in the bacterial richness of these desert sand environments. Our results imply an unexpectedly large bacterial diversity residing in the harsh environment of these two Asian deserts, worthy of further investigation.

Elevational diversity and distribution of ammonia-oxidizing archaea community in meadow soils on the Tibetan Plateau.

PubMed

Zhao, Kang; Kong, Weidong; Khan, Ajmal; Liu, Jinbo; Guo, Guangxia; Muhanmmad, Said; Zhang, Xianzhou; Dong, Xiaobin

2017-09-01

Unraveling elevational diversity patterns of plants and animals has long been attracting scientific interests. However, whether soil microorganisms exhibit similar elevational patterns remains largely less explored, especially for functional microbial communities, such as ammonia oxidizers. Here, we investigated the diversity and distribution pattern of ammonia-oxidizing archaea (AOA) in meadow soils along an elevation gradient from 4400 m to the grassline at 5100 m on the Tibetan Plateau using terminal restriction fragment length polymorphism (T-RFLP) and sequencing methods by targeting amoA gene. Increasing elevations led to lower soil temperature and pH, but higher nutrients and water content. The results showed that AOA diversity and evenness monotonically increased with elevation, while richness was relatively stable. The increase of diversity and evenness was attributed to the growth inhibition of warm-adapted AOA phylotypes by lower temperature and the growth facilitation of cold-adapted AOA phylotypes by richer nutrients at higher elevations. Low temperature thus played an important role in the AOA growth and niche separation. The AOA community variation was explained by the combined effect of all soil properties (32.6%), and 8.1% of the total variation was individually explained by soil pH. The total AOA abundance decreased, whereas soil potential nitrification rate (PNR) increased with increasing elevations. Soil PNR positively correlated with the abundance of cold-adapted AOA phylotypes. Our findings suggest that low temperature plays an important role in AOA elevational diversity pattern and niche separation, rising the negative effects of warming on AOA diversity and soil nitrification process in the Tibetan region.

Uncultivated Microbial Eukaryotic Diversity: A Method to Link ssu rRNA Gene Sequences with Morphology

PubMed Central

Hirst, Marissa B.; Kita, Kelley N.; Dawson, Scott C.

2011-01-01

Protists have traditionally been identified by cultivation and classified taxonomically based on their cellular morphologies and behavior. In the past decade, however, many novel protist taxa have been identified using cultivation independent ssu rRNA sequence surveys. New rRNA “phylotypes” from uncultivated eukaryotes have no connection to the wealth of prior morphological descriptions of protists. To link phylogenetically informative sequences with taxonomically informative morphological descriptions, we demonstrate several methods for combining whole cell rRNA-targeted fluorescent in situ hybridization (FISH) with cytoskeletal or organellar immunostaining. Either eukaryote or ciliate-specific ssu rRNA probes were combined with an anti-α-tubulin antibody or phalloidin, a common actin stain, to define cytoskeletal features of uncultivated protists in several environmental samples. The eukaryote ssu rRNA probe was also combined with Mitotracker® or a hydrogenosomal-specific anti-Hsp70 antibody to localize mitochondria and hydrogenosomes, respectively, in uncultivated protists from different environments. Using rRNA probes in combination with immunostaining, we linked ssu rRNA phylotypes with microtubule structure to describe flagellate and ciliate morphology in three diverse environments, and linked Naegleria spp. to their amoeboid morphology using actin staining in hay infusion samples. We also linked uncultivated ciliates to morphologically similar Colpoda-like ciliates using tubulin immunostaining with a ciliate-specific rRNA probe. Combining rRNA-targeted FISH with cytoskeletal immunostaining or stains targeting specific organelles provides a fast, efficient, high throughput method for linking genetic sequences with morphological features in uncultivated protists. When linked to phylotype, morphological descriptions of protists can both complement and vet the increasing number of sequences from uncultivated protists, including those of novel lineages, identified in diverse environments. PMID:22174774

Reduced representation approaches to interrogate genome diversity in large repetitive plant genomes.

PubMed

Hirsch, Cory D; Evans, Joseph; Buell, C Robin; Hirsch, Candice N

2014-07-01

Technology and software improvements in the last decade now provide methodologies to access the genome sequence of not only a single accession, but also multiple accessions of plant species. This provides a means to interrogate species diversity at the genome level. Ample diversity among accessions in a collection of species can be found, including single-nucleotide polymorphisms, insertions and deletions, copy number variation and presence/absence variation. For species with small, non-repetitive rich genomes, re-sequencing of query accessions is robust, highly informative, and economically feasible. However, for species with moderate to large sized repetitive-rich genomes, technical and economic barriers prevent en masse genome re-sequencing of accessions. Multiple approaches to access a focused subset of loci in species with larger genomes have been developed, including reduced representation sequencing, exome capture and transcriptome sequencing. Collectively, these approaches have enabled interrogation of diversity on a genome scale for large plant genomes, including crop species important to worldwide food security. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Diversity of Functionally Permissive Sequences in the Receptor-Binding Site of Influenza Hemagglutinin.

PubMed

Wu, Nicholas C; Xie, Jia; Zheng, Tianqing; Nycholat, Corwin M; Grande, Geramie; Paulson, James C; Lerner, Richard A; Wilson, Ian A

2017-06-14

Influenza A virus hemagglutinin (HA) initiates viral entry by engaging host receptor sialylated glycans via its receptor-binding site (RBS). The amino acid sequence of the RBS naturally varies across avian and human influenza virus subtypes and is also evolvable. However, functional sequence diversity in the RBS has not been fully explored. Here, we performed a large-scale mutational analysis of the RBS of A/WSN/33 (H1N1) and A/Hong Kong/1/1968 (H3N2) HAs. Many replication-competent mutants not yet observed in nature were identified, including some that could escape from an RBS-targeted broadly neutralizing antibody. This functional sequence diversity is made possible by pervasive epistasis in the RBS 220-loop and can be buffered by avidity in viral receptor binding. Overall, our study reveals that the HA RBS can accommodate a much greater range of sequence diversity than previously thought, which has significant implications for the complex evolutionary interrelationships between receptor specificity and immune escape. Copyright © 2017 Elsevier Inc. All rights reserved.

Single haplotype assembly of the human genome from a hydatidiform mole.

PubMed

Steinberg, Karyn Meltz; Schneider, Valerie A; Graves-Lindsay, Tina A; Fulton, Robert S; Agarwala, Richa; Huddleston, John; Shiryev, Sergey A; Morgulis, Aleksandr; Surti, Urvashi; Warren, Wesley C; Church, Deanna M; Eichler, Evan E; Wilson, Richard K

2014-12-01

A complete reference assembly is essential for accurately interpreting individual genomes and associating variation with phenotypes. While the current human reference genome sequence is of very high quality, gaps and misassemblies remain due to biological and technical complexities. Large repetitive sequences and complex allelic diversity are the two main drivers of assembly error. Although increasing the length of sequence reads and library fragments can improve assembly, even the longest available reads do not resolve all regions. In order to overcome the issue of allelic diversity, we used genomic DNA from an essentially haploid hydatidiform mole, CHM1. We utilized several resources from this DNA including a set of end-sequenced and indexed BAC clones and 100× Illumina whole-genome shotgun (WGS) sequence coverage. We used the WGS sequence and the GRCh37 reference assembly to create an assembly of the CHM1 genome. We subsequently incorporated 382 finished BAC clone sequences to generate a draft assembly, CHM1_1.1 (NCBI AssemblyDB GCA_000306695.2). Analysis of gene, repetitive element, and segmental duplication content show this assembly to be of excellent quality and contiguity. However, comparison to assembly-independent resources, such as BAC clone end sequences and PacBio long reads, indicate misassembled regions. Most of these regions are enriched for structural variation and segmental duplication, and can be resolved in the future. This publicly available assembly will be integrated into the Genome Reference Consortium curation framework for further improvement, with the ultimate goal being a completely finished gap-free assembly. © 2014 Steinberg et al.; Published by Cold Spring Harbor Laboratory Press.

Single haplotype assembly of the human genome from a hydatidiform mole

PubMed Central

Steinberg, Karyn Meltz; Schneider, Valerie A.; Graves-Lindsay, Tina A.; Fulton, Robert S.; Agarwala, Richa; Huddleston, John; Shiryev, Sergey A.; Morgulis, Aleksandr; Surti, Urvashi; Warren, Wesley C.; Church, Deanna M.; Eichler, Evan E.; Wilson, Richard K.

2014-01-01

A complete reference assembly is essential for accurately interpreting individual genomes and associating variation with phenotypes. While the current human reference genome sequence is of very high quality, gaps and misassemblies remain due to biological and technical complexities. Large repetitive sequences and complex allelic diversity are the two main drivers of assembly error. Although increasing the length of sequence reads and library fragments can improve assembly, even the longest available reads do not resolve all regions. In order to overcome the issue of allelic diversity, we used genomic DNA from an essentially haploid hydatidiform mole, CHM1. We utilized several resources from this DNA including a set of end-sequenced and indexed BAC clones and 100× Illumina whole-genome shotgun (WGS) sequence coverage. We used the WGS sequence and the GRCh37 reference assembly to create an assembly of the CHM1 genome. We subsequently incorporated 382 finished BAC clone sequences to generate a draft assembly, CHM1_1.1 (NCBI AssemblyDB GCA_000306695.2). Analysis of gene, repetitive element, and segmental duplication content show this assembly to be of excellent quality and contiguity. However, comparison to assembly-independent resources, such as BAC clone end sequences and PacBio long reads, indicate misassembled regions. Most of these regions are enriched for structural variation and segmental duplication, and can be resolved in the future. This publicly available assembly will be integrated into the Genome Reference Consortium curation framework for further improvement, with the ultimate goal being a completely finished gap-free assembly. PMID:25373144

Genomic library screening for viruses from the human dental plaque revealed pathogen-specific lytic phage sequences.

PubMed

Al-Jarbou, Ahmed Nasser

2012-01-01

Bacterial pathogenesis presents an astounding arsenal of virulence factors that allow them to conquer many different niches throughout the course of infection. Principally fascinating is the fact that some bacterial species are able to induce different diseases by expression of different combinations of virulence factors. Nevertheless, studies aiming at screening for the presence of bacteriophages in humans have been limited. Such screening procedures would eventually lead to identification of phage-encoded properties that impart increased bacterial fitness and/or virulence in a particular niche, and hence, would potentially be used to reverse the course of bacterial infections. As the human oral cavity represents a rich and dynamic ecosystem for several upper respiratory tract pathogens. However, little is known about virus diversity in human dental plaque which is an important reservoir. We applied the culture-independent approach to characterize virus diversity in human dental plaque making a library from a virus DNA fraction amplified using a multiple displacement method and sequenced 80 clones. The resulting sequence showed 44% significant identities to GenBank databases by TBLASTX analysis. TBLAST homology comparisons showed that 66% was viral; 18% eukarya; 10% bacterial; 6% mobile elements. These sequences were sorted into 6 contigs and 45 single sequences in which 4 contigs and a single sequence showed significant identity to a small region of a putative prophage in the Corynebacterium diphtheria genome. These findings interestingly highlight the uniqueness of over half of the sequences, whilst the dominance of a pathogen-specific prophage sequences imply their role in virulence.

Mountain gorilla genomes reveal the impact of long-term population decline and inbreeding.

PubMed

Xue, Yali; Prado-Martinez, Javier; Sudmant, Peter H; Narasimhan, Vagheesh; Ayub, Qasim; Szpak, Michal; Frandsen, Peter; Chen, Yuan; Yngvadottir, Bryndis; Cooper, David N; de Manuel, Marc; Hernandez-Rodriguez, Jessica; Lobon, Irene; Siegismund, Hans R; Pagani, Luca; Quail, Michael A; Hvilsom, Christina; Mudakikwa, Antoine; Eichler, Evan E; Cranfield, Michael R; Marques-Bonet, Tomas; Tyler-Smith, Chris; Scally, Aylwyn

2015-04-10

Mountain gorillas are an endangered great ape subspecies and a prominent focus for conservation, yet we know little about their genomic diversity and evolutionary past. We sequenced whole genomes from multiple wild individuals and compared the genomes of all four Gorilla subspecies. We found that the two eastern subspecies have experienced a prolonged population decline over the past 100,000 years, resulting in very low genetic diversity and an increased overall burden of deleterious variation. A further recent decline in the mountain gorilla population has led to extensive inbreeding, such that individuals are typically homozygous at 34% of their sequence, leading to the purging of severely deleterious recessive mutations from the population. We discuss the causes of their decline and the consequences for their future survival. Copyright © 2015, American Association for the Advancement of Science.

Emerging from the bottleneck: benefits of the comparative approach to modern neuroscience.

PubMed

Brenowitz, Eliot A; Zakon, Harold H

2015-05-01

Neuroscience has historically exploited a wide diversity of animal taxa. Recently, however, research has focused increasingly on a few model species. This trend has accelerated with the genetic revolution, as genomic sequences and genetic tools became available for a few species, which formed a bottleneck. This coalescence on a small set of model species comes with several costs that are often not considered, especially in the current drive to use mice explicitly as models for human diseases. Comparative studies of strategically chosen non-model species can complement model species research and yield more rigorous studies. As genetic sequences and tools become available for many more species, we are poised to emerge from the bottleneck and once again exploit the rich biological diversity offered by comparative studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Significant variance in genetic diversity among populations of Schistosoma haematobium detected using microsatellite DNA loci from a genome-wide database.

PubMed

Glenn, Travis C; Lance, Stacey L; McKee, Anna M; Webster, Bonnie L; Emery, Aidan M; Zerlotini, Adhemar; Oliveira, Guilherme; Rollinson, David; Faircloth, Brant C

2013-10-17

Urogenital schistosomiasis caused by Schistosoma haematobium is widely distributed across Africa and is increasingly being targeted for control. Genome sequences and population genetic parameters can give insight into the potential for population- or species-level drug resistance. Microsatellite DNA loci are genetic markers in wide use by Schistosoma researchers, but there are few primers available for S. haematobium. We sequenced 1,058,114 random DNA fragments from clonal cercariae collected from a snail infected with a single Schistosoma haematobium miracidium. We assembled and aligned the S. haematobium sequences to the genomes of S. mansoni and S. japonicum, identifying microsatellite DNA loci across all three species and designing primers to amplify the loci in S. haematobium. To validate our primers, we screened 32 randomly selected primer pairs with population samples of S. haematobium. We designed >13,790 primer pairs to amplify unique microsatellite loci in S. haematobium, (available at http://www.cebio.org/projetos/schistosoma-haematobium-genome). The three Schistosoma genomes contained similar overall frequencies of microsatellites, but the frequency and length distributions of specific motifs differed among species. We identified 15 primer pairs that amplified consistently and were easily scored. We genotyped these 15 loci in S. haematobium individuals from six locations: Zanzibar had the highest levels of diversity; Malawi, Mauritius, Nigeria, and Senegal were nearly as diverse; but the sample from South Africa was much less diverse. About half of the primers in the database of Schistosoma haematobium microsatellite DNA loci should yield amplifiable and easily scored polymorphic markers, thus providing thousands of potential markers. Sequence conservation among S. haematobium, S. japonicum, and S. mansoni is relatively high, thus it should now be possible to identify markers that are universal among Schistosoma species (i.e., using DNA sequences conserved among species), as well as other markers that are specific to species or species-groups (i.e., using DNA sequences that differ among species). Full genome-sequencing of additional species and specimens of S. haematobium, S. japonicum, and S. mansoni is desirable to better characterize differences within and among these species, to develop additional genetic markers, and to examine genes as well as conserved non-coding elements associated with drug resistance.

A comparison of sequencing platforms and bioinformatics pipelines for compositional analysis of the gut microbiome.

PubMed

Allali, Imane; Arnold, Jason W; Roach, Jeffrey; Cadenas, Maria Belen; Butz, Natasha; Hassan, Hosni M; Koci, Matthew; Ballou, Anne; Mendoza, Mary; Ali, Rizwana; Azcarate-Peril, M Andrea

2017-09-13

Advancements in Next Generation Sequencing (NGS) technologies regarding throughput, read length and accuracy had a major impact on microbiome research by significantly improving 16S rRNA amplicon sequencing. As rapid improvements in sequencing platforms and new data analysis pipelines are introduced, it is essential to evaluate their capabilities in specific applications. The aim of this study was to assess whether the same project-specific biological conclusions regarding microbiome composition could be reached using different sequencing platforms and bioinformatics pipelines. Chicken cecum microbiome was analyzed by 16S rRNA amplicon sequencing using Illumina MiSeq, Ion Torrent PGM, and Roche 454 GS FLX Titanium platforms, with standard and modified protocols for library preparation. We labeled the bioinformatics pipelines included in our analysis QIIME1 and QIIME2 (de novo OTU picking [not to be confused with QIIME version 2 commonly referred to as QIIME2]), QIIME3 and QIIME4 (open reference OTU picking), UPARSE1 and UPARSE2 (each pair differs only in the use of chimera depletion methods), and DADA2 (for Illumina data only). GS FLX+ yielded the longest reads and highest quality scores, while MiSeq generated the largest number of reads after quality filtering. Declines in quality scores were observed starting at bases 150-199 for GS FLX+ and bases 90-99 for MiSeq. Scores were stable for PGM-generated data. Overall microbiome compositional profiles were comparable between platforms; however, average relative abundance of specific taxa varied depending on sequencing platform, library preparation method, and bioinformatics analysis. Specifically, QIIME with de novo OTU picking yielded the highest number of unique species and alpha diversity was reduced with UPARSE and DADA2 compared to QIIME. The three platforms compared in this study were capable of discriminating samples by treatment, despite differences in diversity and abundance, leading to similar biological conclusions. Our results demonstrate that while there were differences in depth of coverage and phylogenetic diversity, all workflows revealed comparable treatment effects on microbial diversity. To increase reproducibility and reliability and to retain consistency between similar studies, it is important to consider the impact on data quality and relative abundance of taxa when selecting NGS platforms and analysis tools for microbiome studies.

Nitrous Oxide Reductase (nosZ) Gene Fragments Differ between Native and Cultivated Michigan Soils

PubMed Central

Stres, Blaž; Mahne, Ivan; Avguštin, Gorazd; Tiedje, James M.

2004-01-01

The effect of standard agricultural management on the genetic heterogeneity of nitrous oxide reductase (nosZ) fragments from denitrifying prokaryotes in native and cultivated soil was explored. Thirty-six soil cores were composited from each of the two soil management conditions. nosZ gene fragments were amplified from triplicate samples, and PCR products were cloned and screened by restriction fragment length polymorphism (RFLP). The total nosZ RFLP profiles increased in similarity with soil sample size until triplicate 3-g samples produced visually identical RFLP profiles for each treatment. Large differences in total nosZ profiles were observed between the native and cultivated soils. The fragments representing major groups of clones encountered at least twice and four randomly selected clones with unique RFLP patterns were sequenced to verify nosZ identity. The sequence diversity of nosZ clones from the cultivated field was higher, and only eight patterns were found in clone libraries from both soils among the 182 distinct nosZ RFLP patterns identified from the two soils. A group of clones that comprised 32% of all clones dominated the gene library of native soil, whereas many minor groups were observed in the gene library of cultivated soil. The 95% confidence intervals of the Chao1 nonparametric richness estimator for nosZ RFLP data did not overlap, indicating that the levels of species richness are significantly different in the two soils, the cultivated soil having higher diversity. Phylogenetic analysis of deduced amino acid sequences grouped the majority of nosZ clones into an interleaved Michigan soil cluster whose cultured members are α-Proteobacteria. Only four nosZ sequences from cultivated soil and one from the native soil were related to sequences found in γ-Proteobacteria. Sequences from the native field formed a distinct, closely related cluster (Dmean = 0.16) containing 91.6% of the native clones. Clones from the cultivated field were more distantly related to each other (Dmean = 0.26), and 65% were found outside of the cluster from the native soil, further indicating a difference in the two communities. Overall, there appears to be a relationship between use and richness, diversity, and the phylogenetic position of nosZ sequences, indicating that agricultural use of soil caused a shift to a more diverse denitrifying community. PMID:14711656

The Hidden Diversity of Flagellated Protists in Soil.

PubMed

Venter, Paul Christiaan; Nitsche, Frank; Arndt, Hartmut

2018-07-01

Protists are among the most diverse and abundant eukaryotes in soil. However, gaps between described and sequenced protist morphospecies still present a pending problem when surveying environmental samples for known species using molecular methods. The number of sequences in the molecular PR 2 database (∼130,000) is limited compared to the species richness expected (>1 million protist species) - limiting the recovery rate. This is important, since high throughput sequencing (HTS) methods are used to find associative patterns between functional traits, taxa and environmental parameters. We performed HTS to survey soil flagellates in 150 grasslands of central Europe, and tested the recovery rate of ten previously isolated and cultivated cercomonad species, among locally found diversity. We recovered sequences for reference soil flagellate species, but also a great number of their phylogenetically evaluated genetic variants, among rare and dominant taxa with presumably own biogeography. This was recorded among dominant (cercozoans, Sandona), rare (apusozoans) and a large hidden diversity of predominantly aquatic protists in soil (choanoflagellates, bicosoecids) often forming novel clades associated with uncultured environmental sequences. Evaluating the reads, instead of the OTUs that individual reads are usually clustered into, we discovered that much of this hidden diversity may be lost due to clustering. Copyright © 2018 Elsevier GmbH. All rights reserved.

Genome skimming: A rapid approach to gaining diverse biological insights into multicellular pathogens

USDA-ARS?s Scientific Manuscript database

New genome sequence information can now be generated very quickly and cheaply for virtually any organism. The dive into genomics is increasingly tempting to scientists studying plant pathogens and other eukaryotic species without reference genomes. The ease of data collection, however, is tempered ...

Identification of Active Bacterial Communities in Drinking Water Using 16S rRNA-Based Sequence Analyses

EPA Science Inventory

DNA-based methods have considerably increased our understanding of the bacterial diversity of water distribution systems (WDS). However, as DNA may persist after cell death, the use of DNA-based methods cannot be used to describe metabolically-active microbes. In contrast, intra...

High-Throughput Sequencing of 16S rRNA Gene Amplicons: Effects of Extraction Procedure, Primer Length and Annealing Temperature

PubMed Central

Sergeant, Martin J.; Constantinidou, Chrystala; Cogan, Tristan; Penn, Charles W.; Pallen, Mark J.

2012-01-01

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers. PMID:22666455

High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature.

PubMed

Sergeant, Martin J; Constantinidou, Chrystala; Cogan, Tristan; Penn, Charles W; Pallen, Mark J

2012-01-01

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.

Rapid evolution of the env gene leader sequence in cats naturally infected with feline immunodeficiency virus

PubMed Central

Hughes, Joseph; Biek, Roman; Litster, Annette; Willett, Brian J.; Hosie, Margaret J.

2015-01-01

Analysing the evolution of feline immunodeficiency virus (FIV) at the intra-host level is important in order to address whether the diversity and composition of viral quasispecies affect disease progression. We examined the intra-host diversity and the evolutionary rates of the entire env and structural fragments of the env sequences obtained from sequential blood samples in 43 naturally infected domestic cats that displayed different clinical outcomes. We observed in the majority of cats that FIV env showed very low levels of intra-host diversity. We estimated that env evolved at a rate of 1.16×10−3 substitutions per site per year and demonstrated that recombinant sequences evolved faster than non-recombinant sequences. It was evident that the V3–V5 fragment of FIV env displayed higher evolutionary rates in healthy cats than in those with terminal illness. Our study provided the first evidence that the leader sequence of env, rather than the V3–V5 sequence, had the highest intra-host diversity and the highest evolutionary rate of all env fragments, consistent with this region being under a strong selective pressure for genetic variation. Overall, FIV env displayed relatively low intra-host diversity and evolved slowly in naturally infected cats. The maximum evolutionary rate was observed in the leader sequence of env. Although genetic stability is not necessarily a prerequisite for clinical stability, the higher genetic stability of FIV compared with human immunodeficiency virus might explain why many naturally infected cats do not progress rapidly to AIDS. PMID:25535323

Analysis of Facultative Lithotroph Distribution and Diversity on Volcanic Deposits by Use of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase†

PubMed Central

Nanba, K.; King, G. M.; Dunfield, K.

2004-01-01

A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass. PMID:15066819

Analysis of facultative lithotroph distribution and diversity on volcanic deposits by use of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase.

PubMed

Nanba, K; King, G M; Dunfield, K

2004-04-01

A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass.

Genetic Diversity among Clostridium botulinum Strains Harboring bont/A2 and bont/A3 Genes

PubMed Central

Raphael, Brian H.; Joseph, Lavin A.; Meno, Sarah R.; Fernández, Rafael A.; Maslanka, Susan E.

2012-01-01

Clostridium botulinum type A strains are known to be genetically diverse and widespread throughout the world. Genetic diversity studies have focused mainly on strains harboring one type A botulinum toxin gene, bont/A1, although all reported bont/A gene variants have been associated with botulism cases. Our study provides insight into the genetic diversity of C. botulinum type A strains, which contain bont/A2 (n = 42) and bont/A3 (n = 4) genes, isolated from diverse samples and geographic origins. Genetic diversity was assessed by using bont nucleotide sequencing, content analysis of the bont gene clusters, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sequences of bont genes obtained in this study showed 99.9 to 100% identity with other bont/A2 or bont/A3 gene sequences available in public databases. The neurotoxin gene clusters of the subtype A2 and A3 strains analyzed in this study were similar in gene content. C. botulinum strains harboring bont/A2 and bont/A3 genes were divided into six and two MLST profiles, respectively. Four groups of strains shared a similarity of at least 95% by PFGE; the largest group included 21 out of 46 strains. The strains analyzed in this study showed relatively limited genetic diversity using either MLST or PFGE. PMID:23042179

Theileria parva antigens recognized by CD8+ T cells show varying degrees of diversity in buffalo-derived infected cell lines.

PubMed

Sitt, Tatjana; Pelle, Roger; Chepkwony, Maurine; Morrison, W Ivan; Toye, Philip

2018-05-06

The extent of sequence diversity among the genes encoding 10 antigens (Tp1-10) known to be recognized by CD8+ T lymphocytes from cattle immune to Theileria parva was analysed. The sequences were derived from parasites in 23 buffalo-derived cell lines, three cattle-derived isolates and one cloned cell line obtained from a buffalo-derived stabilate. The results revealed substantial variation among the antigens through sequence diversity. The greatest nucleotide and amino acid diversity were observed in Tp1, Tp2 and Tp9. Tp5 and Tp7 showed the least amount of allelic diversity, and Tp5, Tp6 and Tp7 had the lowest levels of protein diversity. Tp6 was the most conserved protein; only a single non-synonymous substitution was found in all obtained sequences. The ratio of non-synonymous: synonymous substitutions varied from 0.84 (Tp1) to 0.04 (Tp6). Apart from Tp2 and Tp9, we observed no variation in the other defined CD8+ T cell epitopes (Tp4, 5, 7 and 8), indicating that epitope variation is not a universal feature of T. parva antigens. In addition to providing markers that can be used to examine the diversity in T. parva populations, the results highlight the potential for using conserved antigens to develop vaccines that provide broad protection against T. parva.

Genetic variations in merozoite surface antigen genes of Babesia bovis detected in Vietnamese cattle and water buffaloes.

PubMed

Yokoyama, Naoaki; Sivakumar, Thillaiampalam; Tuvshintulga, Bumduuren; Hayashida, Kyoko; Igarashi, Ikuo; Inoue, Noboru; Long, Phung Thang; Lan, Dinh Thi Bich

2015-03-01

The genes that encode merozoite surface antigens (MSAs) in Babesia bovis are genetically diverse. In this study, we analyzed the genetic diversity of B. bovis MSA-1, MSA-2b, and MSA-2c genes in Vietnamese cattle and water buffaloes. Blood DNA samples from 258 cattle and 49 water buffaloes reared in the Thua Thien Hue province of Vietnam were screened with a B. bovis-specific diagnostic PCR assay. The B. bovis-positive DNA samples (23 cattle and 16 water buffaloes) were then subjected to PCR assays to amplify the MSA-1, MSA-2b, and MSA-2c genes. Sequencing analyses showed that the Vietnamese MSA-1 and MSA-2b sequences are genetically diverse, whereas MSA-2c is relatively conserved. The nucleotide identity values for these MSA gene sequences were similar in the cattle and water buffaloes. Consistent with the sequencing data, the Vietnamese MSA-1 and MSA-2b sequences were dispersed across several clades in the corresponding phylogenetic trees, whereas the MSA-2c sequences occurred in a single clade. Cattle- and water-buffalo-derived sequences also often clustered together on the phylogenetic trees. The Vietnamese MSA-1, MSA-2b, and MSA-2c sequences were then screened for recombination with automated methods. Of the seven recombination events detected, five and two were associated with the MSA-2b and MSA-2c recombinant sequences, respectively, whereas no MSA-1 recombinants were detected among the sequences analyzed. Recombination between the sequences derived from cattle and water buffaloes was very common, and the resultant recombinant sequences were found in both host animals. These data indicate that the genetic diversity of the MSA sequences does not differ between cattle and water buffaloes in Vietnam. They also suggest that recombination between the B. bovis MSA sequences in both cattle and water buffaloes might contribute to the genetic variation in these genes in Vietnam. Copyright © 2015 Elsevier B.V. All rights reserved.

Low Diversity Cryptococcus neoformans Variety grubii Multilocus Sequence Types from Thailand Are Consistent with an Ancestral African Origin

PubMed Central

Simwami, Sitali P.; Khayhan, Kantarawee; Henk, Daniel A.; Aanensen, David M.; Boekhout, Teun; Hagen, Ferry; Brouwer, Annemarie E.; Harrison, Thomas S.; Donnelly, Christl A.; Fisher, Matthew C.

2011-01-01

The global burden of HIV-associated cryptococcal meningitis is estimated at nearly one million cases per year, causing up to a third of all AIDS-related deaths. Molecular epidemiology constitutes the main methodology for understanding the factors underpinning the emergence of this understudied, yet increasingly important, group of pathogenic fungi. Cryptococcus species are notable in the degree that virulence differs amongst lineages, and highly-virulent emerging lineages are changing patterns of human disease both temporally and spatially. Cryptococcus neoformans variety grubii (Cng, serotype A) constitutes the most ubiquitous cause of cryptococcal meningitis worldwide, however patterns of molecular diversity are understudied across some regions experiencing significant burdens of disease. We compared 183 clinical and environmental isolates of Cng from one such region, Thailand, Southeast Asia, against a global MLST database of 77 Cng isolates. Population genetic analyses showed that Thailand isolates from 11 provinces were highly homogenous, consisting of the same genetic background (globally known as VNI) and exhibiting only ten nearly identical sequence types (STs), with three (STs 44, 45 and 46) dominating our sample. This population contains significantly less diversity when compared against the global population of Cng, specifically Africa. Genetic diversity in Cng was significantly subdivided at the continental level with nearly half (47%) of the global STs unique to a genetically diverse and recombining population in Botswana. These patterns of diversity, when combined with evidence from haplotypic networks and coalescent analyses of global populations, are highly suggestive of an expansion of the Cng VNI clade out of Africa, leading to a limited number of genotypes founding the Asian populations. Divergence time testing estimates the time to the most common ancestor between the African and Asian populations to be 6,920 years ago (95% HPD 122.96 - 27,177.76). Further high-density sampling of global Cng STs is now necessary to resolve the temporal sequence underlying the global emergence of this human pathogen. PMID:21573144

Low diversity Cryptococcus neoformans variety grubii multilocus sequence types from Thailand are consistent with an ancestral African origin.

PubMed

Simwami, Sitali P; Khayhan, Kantarawee; Henk, Daniel A; Aanensen, David M; Boekhout, Teun; Hagen, Ferry; Brouwer, Annemarie E; Harrison, Thomas S; Donnelly, Christl A; Fisher, Matthew C

2011-04-01

The global burden of HIV-associated cryptococcal meningitis is estimated at nearly one million cases per year, causing up to a third of all AIDS-related deaths. Molecular epidemiology constitutes the main methodology for understanding the factors underpinning the emergence of this understudied, yet increasingly important, group of pathogenic fungi. Cryptococcus species are notable in the degree that virulence differs amongst lineages, and highly-virulent emerging lineages are changing patterns of human disease both temporally and spatially. Cryptococcus neoformans variety grubii (Cng, serotype A) constitutes the most ubiquitous cause of cryptococcal meningitis worldwide, however patterns of molecular diversity are understudied across some regions experiencing significant burdens of disease. We compared 183 clinical and environmental isolates of Cng from one such region, Thailand, Southeast Asia, against a global MLST database of 77 Cng isolates. Population genetic analyses showed that Thailand isolates from 11 provinces were highly homogenous, consisting of the same genetic background (globally known as VNI) and exhibiting only ten nearly identical sequence types (STs), with three (STs 44, 45 and 46) dominating our sample. This population contains significantly less diversity when compared against the global population of Cng, specifically Africa. Genetic diversity in Cng was significantly subdivided at the continental level with nearly half (47%) of the global STs unique to a genetically diverse and recombining population in Botswana. These patterns of diversity, when combined with evidence from haplotypic networks and coalescent analyses of global populations, are highly suggestive of an expansion of the Cng VNI clade out of Africa, leading to a limited number of genotypes founding the Asian populations. Divergence time testing estimates the time to the most common ancestor between the African and Asian populations to be 6,920 years ago (95% HPD 122.96 - 27,177.76). Further high-density sampling of global Cng STs is now necessary to resolve the temporal sequence underlying the global emergence of this human pathogen.

Visualization of Genome Diversity in German Shepherd Dogs.

PubMed

Mortlock, Sally-Anne; Booth, Rachel; Mazrier, Hamutal; Khatkar, Mehar S; Williamson, Peter

2015-01-01

A loss of genetic diversity may lead to increased disease risks in subpopulations of dogs. The canine breed structure has contributed to relatively small effective population size in many breeds and can limit the options for selective breeding strategies to maintain diversity. With the completion of the canine genome sequencing project, and the subsequent reduction in the cost of genotyping on a genomic scale, evaluating diversity in dogs has become much more accurate and accessible. This provides a potential tool for advising dog breeders and developing breeding programs within a breed. A challenge in doing this is to present complex relationship data in a form that can be readily utilized. Here, we demonstrate the use of a pipeline, known as NetView, to visualize the network of relationships in a subpopulation of German Shepherd Dogs.

Foliar fungi of Betula pendula: impact of tree species mixtures and assessment methods

PubMed Central

Nguyen, Diem; Boberg, Johanna; Cleary, Michelle; Bruelheide, Helge; Hönig, Lydia; Koricheva, Julia; Stenlid, Jan

2017-01-01

Foliar fungi of silver birch (Betula pendula) in an experimental Finnish forest were investigated across a gradient of tree species richness using molecular high-throughput sequencing and visual macroscopic assessment. We hypothesized that the molecular approach detects more fungal taxa than visual assessment, and that there is a relationship among the most common fungal taxa detected by both techniques. Furthermore, we hypothesized that the fungal community composition, diversity, and distribution patterns are affected by changes in tree diversity. Sequencing revealed greater diversity of fungi on birch leaves than the visual assessment method. One species showed a linear relationship between the methods. Species-specific variation in fungal community composition could be partially explained by tree diversity, though overall fungal diversity was not affected by tree diversity. Analysis of specific fungal taxa indicated tree diversity effects at the local neighbourhood scale, where the proportion of birch among neighbouring trees varied, but not at the plot scale. In conclusion, both methods may be used to determine tree diversity effects on the foliar fungal community. However, high-throughput sequencing provided higher resolution of the fungal community, while the visual macroscopic assessment detected functionally active fungal species. PMID:28150710

Periodic Pattern of Genetic and Fitness Diversity during Evolution of an Artificial Cell-Like System.

PubMed

Ichihashi, Norikazu; Aita, Takuyo; Motooka, Daisuke; Nakamura, Shota; Yomo, Tetsuya

2015-12-01

Genetic and phenotypic diversity are the basis of evolution. Despite their importance, however, little is known about how they change over the course of evolution. In this study, we analyzed the dynamics of the adaptive evolution of a simple evolvable artificial cell-like system using single-molecule real-time sequencing technology that reads an entire single artificial genome. We found that the genomic RNA population increases in fitness intermittently, correlating with a periodic pattern of genetic and fitness diversity produced by repeated diversification and domination. In the diversification phase, a genomic RNA population spreads within a genetic space by accumulating mutations until mutants with higher fitness are generated, resulting in an increase in fitness diversity. In the domination phase, the mutants with higher fitness dominate, decreasing both the fitness and genetic diversity. This study reveals the dynamic nature of genetic and fitness diversity during adaptive evolution and demonstrates the utility of a simplified artificial cell-like system to study evolution at an unprecedented resolution. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Mucosal and Cutaneous Human Papillomaviruses Detected in Raw Sewages

PubMed Central

La Rosa, Giuseppina; Fratini, Marta; Accardi, Luisa; D'Oro, Graziana; Della Libera, Simonetta; Muscillo, Michele; Di Bonito, Paola

2013-01-01

Epitheliotropic viruses can find their way into sewage. The aim of the present study was to investigate the occurrence, distribution, and genetic diversity of Human Papillomaviruses (HPVs) in urban wastewaters. Sewage samples were collected from treatment plants distributed throughout Italy. The DNA extracted from these samples was analyzed by PCR using five PV-specific sets of primers targeting the L1 (GP5/GP6, MY09/MY11, FAP59/64, SKF/SKR) and E1 regions (PM-A/PM-B), according to the protocols previously validated for the detection of mucosal and cutaneous HPV genotypes. PCR products underwent sequencing analysis and the sequences were aligned to reference genomes from the Papillomavirus Episteme database. Phylogenetic analysis was then performed to assess the genetic relationships among the different sequences and between the sequences of the samples and those of the prototype strains. A broad spectrum of sequences related to mucosal and cutaneous HPV types was detected in 81% of the sewage samples analyzed. Surprisingly, sequences related to the anogenital HPV6 and 11 were detected in 19% of the samples, and sequences related to the “high risk” oncogenic HPV16 were identified in two samples. Sequences related to HPV9, HPV20, HPV25, HPV76, HPV80, HPV104, HPV110, HPV111, HPV120 and HPV145 beta Papillomaviruses were detected in 76% of the samples. In addition, similarity searches and phylogenetic analysis of some sequences suggest that they could belong to putative new genotypes of the beta genus. In this study, for the first time, the presence of HPV viruses strongly related to human cancer is reported in sewage samples. Our data increases the knowledge of HPV genomic diversity and suggests that virological analysis of urban sewage can provide key information useful in supporting epidemiological studies. PMID:23341898

Rising prevalence of non-B HIV-1 subtypes in North Carolina and evidence for local onward transmission.

PubMed

Dennis, Ann M; Hué, Stephane; Learner, Emily; Sebastian, Joseph; Miller, William C; Eron, Joseph J

2017-01-01

HIV-1 diversity is increasing in North American and European cohorts which may have public health implications. However, little is known about non-B subtype diversity in the southern United States, despite the region being the epicenter of the nation's epidemic. We characterized HIV-1 diversity and transmission clusters to identify the extent to which non-B strains are transmitted locally. We conducted cross-sectional analyses of HIV-1 partial pol sequences collected from 1997 to 2014 from adults accessing routine clinical care in North Carolina (NC). Subtypes were evaluated using COMET and phylogenetic analysis. Putative transmission clusters were identified using maximum-likelihood trees. Clusters involving non-B strains were confirmed and their dates of origin were estimated using Bayesian phylogenetics. Data were combined with demographic information collected at the time of sample collection and country of origin for a subset of patients. Among 24,972 sequences from 15,246 persons, the non-B subtype prevalence increased from 0% to 3.46% over the study period. Of 325 persons with non-B subtypes, diversity was high with over 15 pure subtypes and recombinants; subtype C (28.9%) and CRF02_AG (24.0%) were most common. While identification of transmission clusters was lower for persons with non-B versus B subtypes, several local transmission clusters (≥3 persons) involving non-B subtypes were identified and all were presumably due to heterosexual transmission. Prevalence of non-B subtype diversity remains low in NC but a statistically significant rise was identified over time which likely reflects multiple importation. However, the combined phylogenetic clustering analysis reveals evidence for local onward transmission. Detection of these non-B clusters suggests heterosexual transmission and may guide diagnostic and prevention interventions.

Sequencing Insights into Microbial Communities in the Water and Sediments of Fenghe River, China.

PubMed

Lu, Sidan; Sun, Yujiao; Zhao, Xuan; Wang, Lei; Ding, Aizhong; Zhao, Xiaohui

2016-07-01

The connection between microbial community structure and spatial variation and pollution in river waters has been widely investigated. However, water and sediments together have rarely been explored. In this study, Illumina high-throughput sequencing was performed to analyze microbes in 24 water and sediment samples from natural to anthropogenic sources and from headstream to downstream areas. These data were used to assess variability in microbial community structure and diversity along in the Fenghe River, China. The relationship between bacterial diversity and environmental parameters was statistically analyzed. An average of 1682 operational taxonomic units was obtained. Microbial diversity increased from the headstream to downstream and tended to be greater in sediment compared with water. The water samples near the headstream endured relatively low Shannon and Chao1 indices. These diversity indices and the number of observed species in the water and sediment samples increase downstream. The parameters also differ in the two river tributaries. Community structures shift based on the extent of nitrogen pollution variation in the sediment and water samples. The four most dominant genera in the water community were Escherichia, Acinetobacter, Comamonadaceae, and Pseudomonas. In the sediments, the most dominant genera were Stramenopiles, Flavobacterium, Pseudomonas, and Comamonadaceae. The number of ammonia-oxidizing archaea in the headstream water slightly differed from that in the sediment but varied considerably in the downstream sediments. Statistical analysis showed that community variation is correlated with changes in ammonia nitrogen, total nitrogen, and nitrate nitrogen. This study identified different microbial community structures in river water and sediments. Overall this study emphasized the need to elucidate spatial variations in bacterial diversity in water and sediments associated with physicochemical gradients and to show the effects of such variation on waterborne microbial community structures.

From algae to angiosperms–inferring the phylogeny of green plants (Viridiplantae) from 360 plastid genomes

PubMed Central

2014-01-01

Background Next-generation sequencing has provided a wealth of plastid genome sequence data from an increasingly diverse set of green plants (Viridiplantae). Although these data have helped resolve the phylogeny of numerous clades (e.g., green algae, angiosperms, and gymnosperms), their utility for inferring relationships across all green plants is uncertain. Viridiplantae originated 700-1500 million years ago and may comprise as many as 500,000 species. This clade represents a major source of photosynthetic carbon and contains an immense diversity of life forms, including some of the smallest and largest eukaryotes. Here we explore the limits and challenges of inferring a comprehensive green plant phylogeny from available complete or nearly complete plastid genome sequence data. Results We assembled protein-coding sequence data for 78 genes from 360 diverse green plant taxa with complete or nearly complete plastid genome sequences available from GenBank. Phylogenetic analyses of the plastid data recovered well-supported backbone relationships and strong support for relationships that were not observed in previous analyses of major subclades within Viridiplantae. However, there also is evidence of systematic error in some analyses. In several instances we obtained strongly supported but conflicting topologies from analyses of nucleotides versus amino acid characters, and the considerable variation in GC content among lineages and within single genomes affected the phylogenetic placement of several taxa. Conclusions Analyses of the plastid sequence data recovered a strongly supported framework of relationships for green plants. This framework includes: i) the placement of Zygnematophyceace as sister to land plants (Embryophyta), ii) a clade of extant gymnosperms (Acrogymnospermae) with cycads + Ginkgo sister to remaining extant gymnosperms and with gnetophytes (Gnetophyta) sister to non-Pinaceae conifers (Gnecup trees), and iii) within the monilophyte clade (Monilophyta), Equisetales + Psilotales are sister to Marattiales + leptosporangiate ferns. Our analyses also highlight the challenges of using plastid genome sequences in deep-level phylogenomic analyses, and we provide suggestions for future analyses that will likely incorporate plastid genome sequence data for thousands of species. We particularly emphasize the importance of exploring the effects of different partitioning and character coding strategies. PMID:24533922

HIV-1 envelope sequence-based diversity measures for identifying recent infections

PubMed Central

Kafando, Alexis; Fournier, Eric; Serhir, Bouchra; Martineau, Christine; Doualla-Bell, Florence; Sangaré, Mohamed Ndongo; Sylla, Mohamed; Chamberland, Annie; El-Far, Mohamed; Charest, Hugues

2017-01-01

Identifying recent HIV-1 infections is crucial for monitoring HIV-1 incidence and optimizing public health prevention efforts. To identify recent HIV-1 infections, we evaluated and compared the performance of 4 sequence-based diversity measures including percent diversity, percent complexity, Shannon entropy and number of haplotypes targeting 13 genetic segments within the env gene of HIV-1. A total of 597 diagnostic samples obtained in 2013 and 2015 from recently and chronically HIV-1 infected individuals were selected. From the selected samples, 249 (134 from recent versus 115 from chronic infections) env coding regions, including V1-C5 of gp120 and the gp41 ectodomain of HIV-1, were successfully amplified and sequenced by next generation sequencing (NGS) using the Illumina MiSeq platform. The ability of the four sequence-based diversity measures to correctly identify recent HIV infections was evaluated using the frequency distribution curves, median and interquartile range and area under the curve (AUC) of the receiver operating characteristic (ROC). Comparing the median and interquartile range and evaluating the frequency distribution curves associated with the 4 sequence-based diversity measures, we observed that the percent diversity, number of haplotypes and Shannon entropy demonstrated significant potential to discriminate recent from chronic infections (p<0.0001). Using the AUC of ROC analysis, only the Shannon entropy measure within three HIV-1 env segments could accurately identify recent infections at a satisfactory level. The env segments were gp120 C2_1 (AUC = 0.806), gp120 C2_3 (AUC = 0.805) and gp120 V3 (AUC = 0.812). Our results clearly indicate that the Shannon entropy measure represents a useful tool for predicting HIV-1 infection recency. PMID:29284009

HIV-1 envelope sequence-based diversity measures for identifying recent infections.

PubMed

Kafando, Alexis; Fournier, Eric; Serhir, Bouchra; Martineau, Christine; Doualla-Bell, Florence; Sangaré, Mohamed Ndongo; Sylla, Mohamed; Chamberland, Annie; El-Far, Mohamed; Charest, Hugues; Tremblay, Cécile L

2017-01-01

Identifying recent HIV-1 infections is crucial for monitoring HIV-1 incidence and optimizing public health prevention efforts. To identify recent HIV-1 infections, we evaluated and compared the performance of 4 sequence-based diversity measures including percent diversity, percent complexity, Shannon entropy and number of haplotypes targeting 13 genetic segments within the env gene of HIV-1. A total of 597 diagnostic samples obtained in 2013 and 2015 from recently and chronically HIV-1 infected individuals were selected. From the selected samples, 249 (134 from recent versus 115 from chronic infections) env coding regions, including V1-C5 of gp120 and the gp41 ectodomain of HIV-1, were successfully amplified and sequenced by next generation sequencing (NGS) using the Illumina MiSeq platform. The ability of the four sequence-based diversity measures to correctly identify recent HIV infections was evaluated using the frequency distribution curves, median and interquartile range and area under the curve (AUC) of the receiver operating characteristic (ROC). Comparing the median and interquartile range and evaluating the frequency distribution curves associated with the 4 sequence-based diversity measures, we observed that the percent diversity, number of haplotypes and Shannon entropy demonstrated significant potential to discriminate recent from chronic infections (p<0.0001). Using the AUC of ROC analysis, only the Shannon entropy measure within three HIV-1 env segments could accurately identify recent infections at a satisfactory level. The env segments were gp120 C2_1 (AUC = 0.806), gp120 C2_3 (AUC = 0.805) and gp120 V3 (AUC = 0.812). Our results clearly indicate that the Shannon entropy measure represents a useful tool for predicting HIV-1 infection recency.

[Ciliate diversity and spatiotemporal variation in surface sediments of Yangtze River estuary hypoxic zone].

PubMed

Feng, Zhao; Kui-Dong, Xu; Zhao-Cui, Meng

2012-12-01

By using denaturing gradient gel electrophoresis (DGGE) and sequencing as well as Ludox-QPS method, an investigation was made on the ciliate diversity and its spatiotemporal variation in the surface sediments at three sites of Yangtze River estuary hypoxic zone in April and August 2011. The ANOSIM analysis indicated that the ciliate diversity had significant difference among the sites (R = 0.896, P = 0.0001), but less difference among seasons (R = 0.043, P = 0.207). The sequencing of 18S rDNA DGGE bands revealed that the most predominant groups were planktonic Choreotrichia and Oligotrichia. The detection by Ludox-QPS method showed that the species number and abundance of active ciliates were maintained at a higher level, and increased by 2-5 times in summer, as compared with those in spring. Both the Ludox-QPS method and the DGGE technique detected that the ciliate diversity at the three sites had the similar variation trend, and the Ludox-QPS method detected that there was a significant variation in the ciliate species number and abundance between different seasons. The species number detected by Ludox-QPS method was higher than that detected by DGGE bands. Our study indicated that the ciliates in Yangtze River estuary hypoxic zone had higher diversity and abundance, with the potential to supply food for the polyps of jellyfish.

Links between sulphur oxidation and sulphur-oxidising bacteria abundance and diversity in soil microcosms based on soxB functional gene analysis.

PubMed

Tourna, Maria; Maclean, Paul; Condron, Leo; O'Callaghan, Maureen; Wakelin, Steven A

2014-06-01

Sulphur-oxidising bacteria (SOB) play a key role in the biogeochemical cycling of sulphur in soil ecosystems. However, the ecology of SOB is poorly understood, and there is little knowledge about the taxa capable of sulphur oxidation, their distribution, habitat preferences and ecophysiology. Furthermore, as yet there are no conclusive links between SOB community size or structure and rates of sulphur oxidation. We have developed a molecular approach based on primer design targeting the soxB functional gene of nonfilamentous chemolithotrophic SOB that allows assessment of both abundance and diversity. Cloning and sequencing revealed considerable diversity of known soxB genotypes from agricultural soils and also evidence for previously undescribed taxa. In a microcosm experiment, abundance of soxB genes increased with sulphur oxidation rate in soils amended with elemental sulphur. Addition of elemental sulphur to soil had a significant effect in the soxB gene diversity, with the chemolithotrophic Thiobacillus-like Betaproteobacteria sequences dominating clone libraries 6 days after sulphur application. Using culture-independent methodology, the study provides evidence for links between abundance and diversity of SOB and sulphur oxidation. The methodology provides a new tool for investigation of the ecology and role of SOB in soil sulphur biogeochemistry. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Stability of operational taxonomic units: an important but neglected property for analyzing microbial diversity.

PubMed

He, Yan; Caporaso, J Gregory; Jiang, Xiao-Tao; Sheng, Hua-Fang; Huse, Susan M; Rideout, Jai Ram; Edgar, Robert C; Kopylova, Evguenia; Walters, William A; Knight, Rob; Zhou, Hong-Wei

2015-01-01

The operational taxonomic unit (OTU) is widely used in microbial ecology. Reproducibility in microbial ecology research depends on the reliability of OTU-based 16S ribosomal subunit RNA (rRNA) analyses. Here, we report that many hierarchical and greedy clustering methods produce unstable OTUs, with membership that depends on the number of sequences clustered. If OTUs are regenerated with additional sequences or samples, sequences originally assigned to a given OTU can be split into different OTUs. Alternatively, sequences assigned to different OTUs can be merged into a single OTU. This OTU instability affects alpha-diversity analyses such as rarefaction curves, beta-diversity analyses such as distance-based ordination (for example, Principal Coordinate Analysis (PCoA)), and the identification of differentially represented OTUs. Our results show that the proportion of unstable OTUs varies for different clustering methods. We found that the closed-reference method is the only one that produces completely stable OTUs, with the caveat that sequences that do not match a pre-existing reference sequence collection are discarded. As a compromise to the factors listed above, we propose using an open-reference method to enhance OTU stability. This type of method clusters sequences against a database and includes unmatched sequences by clustering them via a relatively stable de novo clustering method. OTU stability is an important consideration when analyzing microbial diversity and is a feature that should be taken into account during the development of novel OTU clustering methods.

Population diversity of Diaphorina citri (Hemiptera: Liviidae) in China based on whole mitochondrial genome sequences.

PubMed

Wu, Fengnian; Jiang, Hongyan; Beattie, G Andrew C; Holford, Paul; Chen, Jianchi; Wallis, Christopher M; Zheng, Zheng; Deng, Xiaoling; Cen, Yijing

2018-04-24

Diaphorina citri (Asian citrus psyllid; ACP) transmits 'Candidatus Liberibacter asiaticus' associated with citrus Huanglongbing (HLB). ACP has been reported in 11 provinces/regions in China, yet its population diversity remains unclear. In this study, we evaluated ACP population diversity in China using representative whole mitochondrial genome (mitogenome) sequences. Additional mitogenome sequences outside China were also acquired and evaluated. The sizes of the 27 ACP mitogenome sequences ranged from 14 986 to 15 030 bp. Along with three previously published mitogenome sequences, the 30 sequences formed three major mitochondrial groups (MGs): MG1, present in southwestern China and occurring at elevations above 1000 m; MG2, present in southeastern China and Southeast Asia (Cambodia, Indonesia, Malaysia, and Vietnam) and occurring at elevations below 180 m; and MG3, present in the USA and Pakistan. Single nucleotide polymorphisms in five genes (cox2, atp8, nad3, nad1 and rrnL) contributed mostly in the ACP diversity. Among these genes, rrnL had the most variation. Mitogenome sequences analyses revealed two major phylogenetic groups of ACP present in China as well as a possible unique group present currently in Pakistan and the USA. The information could have significant implications for current ACP control and HLB management. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

Microbial and functional diversity of a subterrestrial high pH groundwater associated to serpentinization.

PubMed

Tiago, Igor; Veríssimo, António

2013-06-01

Microbial and functional diversity were assessed, from a serpentinization-driven subterrestrial alkaline aquifer - Cabeço de Vide Aquifer (CVA) in Portugal. DGGE analyses revealed the presence of a stable microbial community. By 16S rRNA gene libraries and pyrosequencing analyses, a diverse bacterial composition was determined, contrasting with low archaeal diversity. Within Bacteria the majority of the populations were related to organisms or sequences affiliated to class Clostridia, but members of classes Acidobacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Deinococci, Gammaproteobacteria and of the phyla Bacteroidetes, Chloroflexi and Nitrospira were also detected. Domain Archaea encompassed mainly sequences affiliated to Euryarchaeota. Only form I RuBisCO - cbbL was detected. Autotrophic carbon fixation via the rTCA, 3-HP and 3-HP/4H-B cycles could not be confirmed. The detected APS reductase alpha subunit - aprA sequences were phylogenetically related to sequences of sulfate-reducing bacteria belonging to Clostridia, and also to sequences of chemolithoautothrophic sulfur-oxidizing bacteria belonging to Betaproteobacteria. Sequences of methyl coenzyme M reductase - mcrA were phylogenetically affiliated to sequences belonging to Anaerobic Methanotroph group 1 (ANME-1). The populations found and the functional key markers detected in CVA suggest that metabolisms related to H2 , methane and/or sulfur may be the major driving forces in this environment. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Phylogenetic and ecological analyses of soil and sporocarp DNA sequences reveal high diversity and strong habitat partitioning in the boreal ectomycorrhizal genus Russula (Russulales; Basidiomycota)

Treesearch

József Geml; Gary A. Laursen; Ian C. Herriott; Jack M. McFarland; Michael G. Booth; Niall Lennon; H. Chad Nusbaum; D. Lee Taylor

2010-01-01

Although critical for the functioning of ecosystems, fungi are poorly known in high-latitude regions. Here, we provide the first genetic diversity assessment of one of the most diverse and abundant ectomycorrhizal genera in Alaska: Russula. We analyzed internal transcribed spacer rDNA sequences from sporocarps and soil samples using phylogenetic...

Temporal Changes in BEXSERO® Antigen Sequence Type Associated with Genetic Lineages of Neisseria meningitidis over a 15-Year Period in Western Australia

PubMed Central

Mowlaboccus, Shakeel; Perkins, Timothy T.; Smith, Helen; Sloots, Theo; Tozer, Sarah; Prempeh, Lydia-Jessica; Tay, Chin Yen; Peters, Fanny; Speers, David; Keil, Anthony D.; Kahler, Charlene M.

2016-01-01

Neisseria meningitidis is the causative agent of invasive meningococcal disease (IMD). The BEXSERO® vaccine which is used to prevent serogroup B disease is composed of four sub-capsular protein antigens supplemented with an outer membrane vesicle. Since the sub-capsular protein antigens are variably expressed and antigenically variable amongst meningococcal isolates, vaccine coverage can be estimated by the meningococcal antigen typing system (MATS) which measures the propensity of the strain to be killed by vaccinated sera. Whole genome sequencing (WGS) which identifies the alleles of the antigens that may be recognised by the antibody response could represent, in future, an alternative estimate of coverage. In this study, WGS of 278 meningococcal isolates responsible for 62% of IMD in Western Australia from 2000–2014 were analysed for association of genetic lineage (sequence type [ST], clonal complex [cc]) with BEXSERO® antigen sequence type (BAST) and MATS to predict the annual vaccine coverage. A hyper-endemic period of IMD between 2000–05 was caused by cc41/44 with the major sequence type of ST-146 which was not predicted by MATS or BAST to be covered by the vaccine. An increase in serogroup diversity was observed between 2010–14 with the emergence of cc11 serogroup W in the adolescent population and cc23 serogroup Y in the elderly. BASTs were statistically associated with clonal complex although individual antigens underwent antigenic drift from the major type. BAST and MATS predicted an annual range of 44–91% vaccine coverage. Periods of low vaccine coverage in years post-2005 were not a result of the resurgence of cc41/44:ST-146 but were characterised by increased diversity of clonal complexes expressing BASTs which were not predicted by MATS to be covered by the vaccine. The driving force behind the diversity of the clonal complex and BAST during these periods of low vaccine coverage is unknown, but could be due to immune selection and inter-strain competition with carriage of non-disease causing meningococci. PMID:27355628

A Diverse Range of Novel RNA Viruses in Geographically Distinct Honey Bee Populations

PubMed Central

Shi, Mang; Buchmann, Gabriele; Blacquière, Tjeerd; Beekman, Madeleine; Ashe, Alyson

2017-01-01

ABSTRACT Understanding the diversity and consequences of viruses present in honey bees is critical for maintaining pollinator health and managing the spread of disease. The viral landscape of honey bees (Apis mellifera) has changed dramatically since the emergence of the parasitic mite Varroa destructor, which increased the spread of virulent variants of viruses such as deformed wing virus. Previous genomic studies have focused on colonies suffering from infections by Varroa and virulent viruses, which could mask other viral species present in honey bees, resulting in a distorted view of viral diversity. To capture the viral diversity within colonies that are exposed to mites but do not suffer the ultimate consequences of the infestation, we examined populations of honey bees that have evolved naturally or have been selected for resistance to Varroa. This analysis revealed seven novel viruses isolated from honey bees sampled globally, including the first identification of negative-sense RNA viruses in honey bees. Notably, two rhabdoviruses were present in three geographically diverse locations and were also present in Varroa mites parasitizing the bees. To characterize the antiviral response, we performed deep sequencing of small RNA populations in honey bees and mites. This provided evidence of a Dicer-mediated immune response in honey bees, while the viral small RNA profile in Varroa mites was novel and distinct from the response observed in bees. Overall, we show that viral diversity in honey bee colonies is greater than previously thought, which encourages additional studies of the bee virome on a global scale and which may ultimately improve disease management. IMPORTANCE Honey bee populations have become increasingly susceptible to colony losses due to pathogenic viruses spread by parasitic Varroa mites. To date, 24 viruses have been described in honey bees, with most belonging to the order Picornavirales. Collapsing Varroa-infected colonies are often overwhelmed with high levels of picornaviruses. To examine the underlying viral diversity in honey bees, we employed viral metatranscriptomics analyses on three geographically diverse Varroa-resistant populations from Europe, Africa, and the Pacific. We describe seven novel viruses from a range of diverse viral families, including two viruses that are present in all three locations. In honey bees, small RNA sequences indicate that these viruses are processed by Dicer and the RNA interference pathway, whereas Varroa mites produce strikingly novel small RNA patterns. This work increases the number and diversity of known honey bee viruses and will ultimately contribute to improved disease management in our most important agricultural pollinator. PMID:28515299

A Diverse Range of Novel RNA Viruses in Geographically Distinct Honey Bee Populations.

PubMed

Remnant, Emily J; Shi, Mang; Buchmann, Gabriele; Blacquière, Tjeerd; Holmes, Edward C; Beekman, Madeleine; Ashe, Alyson

2017-08-15

Understanding the diversity and consequences of viruses present in honey bees is critical for maintaining pollinator health and managing the spread of disease. The viral landscape of honey bees ( Apis mellifera ) has changed dramatically since the emergence of the parasitic mite Varroa destructor , which increased the spread of virulent variants of viruses such as deformed wing virus. Previous genomic studies have focused on colonies suffering from infections by Varroa and virulent viruses, which could mask other viral species present in honey bees, resulting in a distorted view of viral diversity. To capture the viral diversity within colonies that are exposed to mites but do not suffer the ultimate consequences of the infestation, we examined populations of honey bees that have evolved naturally or have been selected for resistance to Varroa This analysis revealed seven novel viruses isolated from honey bees sampled globally, including the first identification of negative-sense RNA viruses in honey bees. Notably, two rhabdoviruses were present in three geographically diverse locations and were also present in Varroa mites parasitizing the bees. To characterize the antiviral response, we performed deep sequencing of small RNA populations in honey bees and mites. This provided evidence of a Dicer-mediated immune response in honey bees, while the viral small RNA profile in Varroa mites was novel and distinct from the response observed in bees. Overall, we show that viral diversity in honey bee colonies is greater than previously thought, which encourages additional studies of the bee virome on a global scale and which may ultimately improve disease management. IMPORTANCE Honey bee populations have become increasingly susceptible to colony losses due to pathogenic viruses spread by parasitic Varroa mites. To date, 24 viruses have been described in honey bees, with most belonging to the order Picornavirales Collapsing Varroa -infected colonies are often overwhelmed with high levels of picornaviruses. To examine the underlying viral diversity in honey bees, we employed viral metatranscriptomics analyses on three geographically diverse Varroa- resistant populations from Europe, Africa, and the Pacific. We describe seven novel viruses from a range of diverse viral families, including two viruses that are present in all three locations. In honey bees, small RNA sequences indicate that these viruses are processed by Dicer and the RNA interference pathway, whereas Varroa mites produce strikingly novel small RNA patterns. This work increases the number and diversity of known honey bee viruses and will ultimately contribute to improved disease management in our most important agricultural pollinator. Copyright © 2017 Remnant et al.

Ancient DNA from Giant Panda (Ailuropoda melanoleuca) of South-Western China Reveals Genetic Diversity Loss during the Holocene

PubMed Central

Barlow, Axel; Cooper, Alan; Hou, Xin-Dong; Ji, Xue-Ping; Zhong, Bo-Jian; Liu, Hong; Flynn, Lawrence J.; Yuan, Jun-Xia; Wang, Li-Rui; Basler, Nikolas; Westbury, Michael V.; Hofreiter, Michael; Lai, Xu-Long

2018-01-01

The giant panda was widely distributed in China and south-eastern Asia during the middle to late Pleistocene, prior to its habitat becoming rapidly reduced in the Holocene. While conservation reserves have been established and population numbers of the giant panda have recently increased, the interpretation of its genetic diversity remains controversial. Previous analyses, surprisingly, have indicated relatively high levels of genetic diversity raising issues concerning the efficiency and usefulness of reintroducing individuals from captive populations. However, due to a lack of DNA data from fossil specimens, it is unknown whether genetic diversity was even higher prior to the most recent population decline. We amplified complete cytb and 12s rRNA, partial 16s rRNA and ND1, and control region sequences from the mitochondrial genomes of two Holocene panda specimens. We estimated genetic diversity and population demography by analyzing the ancient mitochondrial DNA sequences alongside those from modern giant pandas, as well as from other members of the bear family (Ursidae). Phylogenetic analyses show that one of the ancient haplotypes is sister to all sampled modern pandas and the second ancient individual is nested among the modern haplotypes, suggesting that genetic diversity may indeed have been higher earlier during the Holocene. Bayesian skyline plot analysis supports this view and indicates a slight decline in female effective population size starting around 6000 years B.P., followed by a recovery around 2000 years ago. Therefore, while the genetic diversity of the giant panda has been affected by recent habitat contraction, it still harbors substantial genetic diversity. Moreover, while its still low population numbers require continued conservation efforts, there seem to be no immediate threats from the perspective of genetic evolutionary potential. PMID:29642393

Ancient DNA from Giant Panda (Ailuropoda melanoleuca) of South-Western China Reveals Genetic Diversity Loss during the Holocene.

PubMed

Sheng, Gui-Lian; Barlow, Axel; Cooper, Alan; Hou, Xin-Dong; Ji, Xue-Ping; Jablonski, Nina G; Zhong, Bo-Jian; Liu, Hong; Flynn, Lawrence J; Yuan, Jun-Xia; Wang, Li-Rui; Basler, Nikolas; Westbury, Michael V; Hofreiter, Michael; Lai, Xu-Long

2018-04-06

The giant panda was widely distributed in China and south-eastern Asia during the middle to late Pleistocene, prior to its habitat becoming rapidly reduced in the Holocene. While conservation reserves have been established and population numbers of the giant panda have recently increased, the interpretation of its genetic diversity remains controversial. Previous analyses, surprisingly, have indicated relatively high levels of genetic diversity raising issues concerning the efficiency and usefulness of reintroducing individuals from captive populations. However, due to a lack of DNA data from fossil specimens, it is unknown whether genetic diversity was even higher prior to the most recent population decline. We amplified complete cyt b and 12s rRNA, partial 16s rRNA and ND1 , and control region sequences from the mitochondrial genomes of two Holocene panda specimens. We estimated genetic diversity and population demography by analyzing the ancient mitochondrial DNA sequences alongside those from modern giant pandas, as well as from other members of the bear family (Ursidae). Phylogenetic analyses show that one of the ancient haplotypes is sister to all sampled modern pandas and the second ancient individual is nested among the modern haplotypes, suggesting that genetic diversity may indeed have been higher earlier during the Holocene. Bayesian skyline plot analysis supports this view and indicates a slight decline in female effective population size starting around 6000 years B.P., followed by a recovery around 2000 years ago. Therefore, while the genetic diversity of the giant panda has been affected by recent habitat contraction, it still harbors substantial genetic diversity. Moreover, while its still low population numbers require continued conservation efforts, there seem to be no immediate threats from the perspective of genetic evolutionary potential.

Genetic diversity of the merozoite surface protein-3 gene in Plasmodium falciparum populations in Thailand.

PubMed

Pattaradilokrat, Sittiporn; Sawaswong, Vorthon; Simpalipan, Phumin; Kaewthamasorn, Morakot; Siripoon, Napaporn; Harnyuttanakorn, Pongchai

2016-10-21

An effective malaria vaccine is an urgently needed tool to fight against human malaria, the most deadly parasitic disease of humans. One promising candidate is the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum. This antigenic protein, encoded by the merozoite surface protein (msp-3) gene, is polymorphic and classified according to size into the two allelic types of K1 and 3D7. A recent study revealed that both the K1 and 3D7 alleles co-circulated within P. falciparum populations in Thailand, but the extent of the sequence diversity and variation within each allelic type remains largely unknown. The msp-3 gene was sequenced from 59 P. falciparum samples collected from five endemic areas (Mae Hong Son, Kanchanaburi, Ranong, Trat and Ubon Ratchathani) in Thailand and analysed for nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity. The gene was also subject to population genetic analysis (F st ) and neutrality tests (Tajima's D, Fu and Li D* and Fu and Li' F* tests) to determine any signature of selection. The sequence analyses revealed eight unique DNA haplotypes and seven amino acid sequence variants, with a haplotype and nucleotide diversity of 0.828 and 0.049, respectively. Neutrality tests indicated that the polymorphism detected in the alanine heptad repeat region of MSP-3 was maintained by positive diversifying selection, suggesting its role as a potential target of protective immune responses and supporting its role as a vaccine candidate. Comparison of MSP-3 variants among parasite populations in Thailand, India and Nigeria also inferred a close genetic relationship between P. falciparum populations in Asia. This study revealed the extent of the msp-3 gene diversity in P. falciparum in Thailand, providing the fundamental basis for the better design of future blood stage malaria vaccines against P. falciparum.

Intermediary metabolism in protists: a sequence-based view of facultative anaerobic metabolism in evolutionarily diverse eukaryotes.

PubMed

Ginger, Michael L; Fritz-Laylin, Lillian K; Fulton, Chandler; Cande, W Zacheus; Dawson, Scott C

2010-12-01

Protists account for the bulk of eukaryotic diversity. Through studies of gene and especially genome sequences the molecular basis for this diversity can be determined. Evident from genome sequencing are examples of versatile metabolism that go far beyond the canonical pathways described for eukaryotes in textbooks. In the last 2-3 years, genome sequencing and transcript profiling has unveiled several examples of heterotrophic and phototrophic protists that are unexpectedly well-equipped for ATP production using a facultative anaerobic metabolism, including some protists that can (Chlamydomonas reinhardtii) or are predicted (Naegleria gruberi, Acanthamoeba castellanii, Amoebidium parasiticum) to produce H(2) in their metabolism. It is possible that some enzymes of anaerobic metabolism were acquired and distributed among eukaryotes by lateral transfer, but it is also likely that the common ancestor of eukaryotes already had far more metabolic versatility than was widely thought a few years ago. The discussion of core energy metabolism in unicellular eukaryotes is the subject of this review. Since genomic sequencing has so far only touched the surface of protist diversity, it is anticipated that sequences of additional protists may reveal an even wider range of metabolic capabilities, while simultaneously enriching our understanding of the early evolution of eukaryotes. Copyright © 2010 Elsevier GmbH. All rights reserved.

Intermediary Metabolism in Protists: a Sequence-based View of Facultative Anaerobic Metabolism in Evolutionarily Diverse Eukaryotes

PubMed Central

Ginger, Michael L.; Fritz-Laylin, Lillian K.; Fulton, Chandler; Cande, W. Zacheus; Dawson, Scott C.

2011-01-01

Protists account for the bulk of eukaryotic diversity. Through studies of gene and especially genome sequences the molecular basis for this diversity can be determined. Evident from genome sequencing are examples of versatile metabolism that go far beyond the canonical pathways described for eukaryotes in textbooks. In the last 2–3 years, genome sequencing and transcript profiling has unveiled several examples of heterotrophic and phototrophic protists that are unexpectedly well-equipped for ATP production using a facultative anaerobic metabolism, including some protists that can (Chlamydomonas reinhardtii) or are predicted (Naegleria gruberi, Acanthamoeba castellanii, Amoebidium parasiticum) to produce H2 in their metabolism. It is possible that some enzymes of anaerobic metabolism were acquired and distributed among eukaryotes by lateral transfer, but it is also likely that the common ancestor of eukaryotes already had far more metabolic versatility than was widely thought a few years ago. The discussion of core energy metabolism in unicellular eukaryotes is the subject of this review. Since genomic sequencing has so far only touched the surface of protist diversity, it is anticipated that sequences of additional protists may reveal an even wider range of metabolic capabilities, while simultaneously enriching our understanding of the early evolution of eukaryotes. PMID:21036663

Changes in community structure of active protistan assemblages from the lower Pearl River to coastal Waters of the South China Sea.

PubMed

Li, Ran; Jiao, Nianzhi; Warren, Alan; Xu, Dapeng

2018-04-01

Protists make up an important component of aquatic ecosystems, playing crucial roles in biogeochemical processes on local and global scales. To reveal the changes of diversity and community structure of protists along the salinity gradients, community compositions of active protistan assemblages were characterized along a transect from the lower Pearl River estuary to the open waters of the South China Sea (SCS), using high-throughput sequencing of the hyper-variable V9 regions of 18S rRNA. This study showed that the alpha diversity of protists, both in the freshwater and in the coastal SCS stations was higher than that in the estuary. The protist community structure also changed along the salinity gradient. The relative sequence abundance of Stramenopiles was highest at stations with lower salinity and decreased with the increasing of salinity. By contrast, the contributions of Alveolata, Hacrobia and Rhizaria to the protistan communities generally increased with the increasing of salinity. The composition of the active protistan community was strongly correlated with salinity, indicating that salinity was the dominant factor among measured environmental parameters affecting protistan community composition and structure. Copyright © 2018 Elsevier GmbH. All rights reserved.

Effect of magnesium oxide nanoparticles on microbial diversity and removal performance of sequencing batch reactor.

PubMed

Ma, Bingrui; Yu, Naling; Han, Yuetong; Gao, Mengchun; Wang, Sen; Li, Shanshan; Guo, Liang; She, Zonglian; Zhao, Yangguo; Jin, Chunji; Gao, Feng

2018-06-13

The performance, microbial enzymatic activity and microbial community of a sequencing batch reactor (SBR) have been explored under magnesium oxide nanoparticles (MgO NPs) stress. The NH 4 + -N removal efficiency kept relatively stable during the whole operational process. The MgO NPs at 30-60 mg/L slightly restrained the removal of chemical oxygen demand (COD), and the presence of MgO NPs also affected the denitrification and phosphorus removal. The specific oxygen uptake rate, nitrifying and denitrifying rates, phosphorus removal rate, and microbial enzymatic activities distinctly varied with the increase of MgO NPs concentration. The appearance of MgO NPs promoted more reactive oxygen species generation and lactate dehydrogenase leakage from activated sludge, suggesting that MgO NPs had obvious toxicity to activated sludge in the SBR. The protein and polysaccharide contents of extracellular polymeric substances from activated sludge increased with the increase of MgO NPs concentration. The microbial richness and diversity at different MgO NPs concentrations obviously varied at the phylum, class and genus levels due to the biological toxicity of MgO NPs. Copyright © 2018 Elsevier Ltd. All rights reserved.

A phylogenetic framework facilitates Y-STR variant discovery and classification via massively parallel sequencing.

PubMed

Huszar, Tunde I; Jobling, Mark A; Wetton, Jon H

2018-04-12

Short tandem repeats on the male-specific region of the Y chromosome (Y-STRs) are permanently linked as haplotypes, and therefore Y-STR sequence diversity can be considered within the robust framework of a phylogeny of haplogroups defined by single nucleotide polymorphisms (SNPs). Here we use massively parallel sequencing (MPS) to analyse the 23 Y-STRs in Promega's prototype PowerSeq™ Auto/Mito/Y System kit (containing the markers of the PowerPlex® Y23 [PPY23] System) in a set of 100 diverse Y chromosomes whose phylogenetic relationships are known from previous megabase-scale resequencing. Including allele duplications and alleles resulting from likely somatic mutation, we characterised 2311 alleles, demonstrating 99.83% concordance with capillary electrophoresis (CE) data on the same sample set. The set contains 267 distinct sequence-based alleles (an increase of 58% compared to the 169 detectable by CE), including 60 novel Y-STR variants phased with their flanking sequences which have not been reported previously to our knowledge. Variation includes 46 distinct alleles containing non-reference variants of SNPs/indels in both repeat and flanking regions, and 145 distinct alleles containing repeat pattern variants (RPV). For DYS385a,b, DYS481 and DYS390 we observed repeat count variation in short flanking segments previously considered invariable, and suggest new MPS-based structural designations based on these. We considered the observed variation in the context of the Y phylogeny: several specific haplogroup associations were observed for SNPs and indels, reflecting the low mutation rates of such variant types; however, RPVs showed less phylogenetic coherence and more recurrence, reflecting their relatively high mutation rates. In conclusion, our study reveals considerable additional diversity at the Y-STRs of the PPY23 set via MPS analysis, demonstrates high concordance with CE data, facilitates nomenclature standardisation, and places Y-STR sequence variants in their phylogenetic context. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Ancient diversity and geographical sub-structuring in African buffalo Theileria parva populations revealed through metagenetic analysis of antigen-encoding loci.

PubMed

Hemmink, Johanneke D; Sitt, Tatjana; Pelle, Roger; de Klerk-Lorist, Lin-Mari; Shiels, Brian; Toye, Philip G; Morrison, W Ivan; Weir, William

2018-03-01

An infection and treatment protocol involving infection with a mixture of three parasite isolates and simultaneous treatment with oxytetracycline is currently used to vaccinate cattle against Theileria parva. While vaccination results in high levels of protection in some regions, little or no protection is observed in areas where animals are challenged predominantly by parasites of buffalo origin. A previous study involving sequencing of two antigen-encoding genes from a series of parasite isolates indicated that this is associated with greater antigenic diversity in buffalo-derived T. parva. The current study set out to extend these analyses by applying high-throughput sequencing to ex vivo samples from naturally infected buffalo to determine the extent of diversity in a set of antigen-encoding genes. Samples from two populations of buffalo, one in Kenya and the other in South Africa, were examined to investigate the effect of geographical distance on the nature of sequence diversity. The results revealed a number of significant findings. First, there was a variable degree of nucleotide sequence diversity in all gene segments examined, with the percentage of polymorphic nucleotides ranging from 10% to 69%. Second, large numbers of allelic variants of each gene were found in individual animals, indicating multiple infection events. Third, despite the observed diversity in nucleotide sequences, several of the gene products had highly conserved amino acid sequences, and thus represent potential candidates for vaccine development. Fourth, although compelling evidence for population differentiation between the Kenyan and South African T. parva parasites was identified, analysis of molecular variance for each gene revealed that the majority of the underlying nucleotide sequence polymorphism was common to both areas, indicating that much of this aspect of genetic variation in the parasite population arose prior to geographic separation. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Infections by human gastrointestinal helminths are associated with changes in faecal microbiota diversity and composition.

PubMed

Jenkins, Timothy P; Rathnayaka, Yasara; Perera, Piyumali K; Peachey, Laura E; Nolan, Matthew J; Krause, Lutz; Rajakaruna, Rupika S; Cantacessi, Cinzia

2017-01-01

Investigations of the impact that patent infections by soil-transmitted gastrointestinal nematode parasites exert on the composition of the host gut commensal flora are attracting growing interest by the scientific community. However, information collected to date varies across experiments, and further studies are needed to identify consistent relationships between parasites and commensal microbial species. Here, we explore the qualitative and quantitative differences between the microbial community profiles of cohorts of human volunteers from Sri Lanka with patent infection by one or more parasitic nematode species (H+), as well as that of uninfected subjects (H-) and of volunteers who had been subjected to regular prophylactic anthelmintic treatment (Ht). High-throughput sequencing of the bacterial 16S rRNA gene, followed by bioinformatics and biostatistical analyses of sequence data revealed no significant differences in alpha diversity (Shannon) and richness between groups (P = 0.65, P = 0.13 respectively); however, beta diversity was significantly increased in H+ and Ht when individually compared to H-volunteers (P = 0.04). Among others, bacteria of the families Verrucomicrobiaceae and Enterobacteriaceae showed a trend towards increased abundance in H+, whereas the Leuconostocaceae and Bacteroidaceae showed a relative increase in H- and Ht respectively. Our findings add valuable knowledge to the vast, and yet little explored, research field of parasite-microbiota interactions and will provide a basis for the elucidation of the role such interactions play in pathogenic and immune-modulatory properties of parasitic nematodes in both human and animal hosts.

Infections by human gastrointestinal helminths are associated with changes in faecal microbiota diversity and composition

PubMed Central

Jenkins, Timothy P.; Rathnayaka, Yasara; Perera, Piyumali K.; Peachey, Laura E.; Nolan, Matthew J.; Krause, Lutz; Rajakaruna, Rupika S.

2017-01-01

Investigations of the impact that patent infections by soil-transmitted gastrointestinal nematode parasites exert on the composition of the host gut commensal flora are attracting growing interest by the scientific community. However, information collected to date varies across experiments, and further studies are needed to identify consistent relationships between parasites and commensal microbial species. Here, we explore the qualitative and quantitative differences between the microbial community profiles of cohorts of human volunteers from Sri Lanka with patent infection by one or more parasitic nematode species (H+), as well as that of uninfected subjects (H-) and of volunteers who had been subjected to regular prophylactic anthelmintic treatment (Ht). High-throughput sequencing of the bacterial 16S rRNA gene, followed by bioinformatics and biostatistical analyses of sequence data revealed no significant differences in alpha diversity (Shannon) and richness between groups (P = 0.65, P = 0.13 respectively); however, beta diversity was significantly increased in H+ and Ht when individually compared to H-volunteers (P = 0.04). Among others, bacteria of the families Verrucomicrobiaceae and Enterobacteriaceae showed a trend towards increased abundance in H+, whereas the Leuconostocaceae and Bacteroidaceae showed a relative increase in H- and Ht respectively. Our findings add valuable knowledge to the vast, and yet little explored, research field of parasite—microbiota interactions and will provide a basis for the elucidation of the role such interactions play in pathogenic and immune-modulatory properties of parasitic nematodes in both human and animal hosts. PMID:28892494

Applications of the rep-PCR DNA fingerprinting technique to study microbial diversity, ecology and evolution.

PubMed

Ishii, Satoshi; Sadowsky, Michael J

2009-04-01

A large number of repetitive DNA sequences are found in multiple sites in the genomes of numerous bacteria, archaea and eukarya. While the functions of many of these repetitive sequence elements are unknown, they have proven to be useful as the basis of several powerful tools for use in molecular diagnostics, medical microbiology, epidemiological analyses and environmental microbiology. The repetitive sequence-based PCR or rep-PCR DNA fingerprint technique uses primers targeting several of these repetitive elements and PCR to generate unique DNA profiles or 'fingerprints' of individual microbial strains. Although this technique has been extensively used to examine diversity among variety of prokaryotic microorganisms, rep-PCR DNA fingerprinting can also be applied to microbial ecology and microbial evolution studies since it has the power to distinguish microbes at the strain or isolate level. Recent advancement in rep-PCR methodology has resulted in increased accuracy, reproducibility and throughput. In this minireview, we summarize recent improvements in rep-PCR DNA fingerprinting methodology, and discuss its applications to address fundamentally important questions in microbial ecology and evolution.

Diversity of Babesia bovis merozoite surface antigen genes in the Philippines.

PubMed

Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Ybanez, Adrian Patalinghug; Ybanez, Rochelle Haidee Daclan; Perez, Zandro Obligado; Guswanto, Azirwan; Igarashi, Ikuo; Yokoyama, Naoaki

2014-02-01

Babesia bovis is the causative agent of fatal babesiosis in cattle. In the present study, we investigated the genetic diversity of B. bovis among Philippine cattle, based on the genes that encode merozoite surface antigens (MSAs). Forty-one B. bovis-positive blood DNA samples from cattle were used to amplify the msa-1, msa-2b, and msa-2c genes. In phylogenetic analyses, the msa-1, msa-2b, and msa-2c gene sequences generated from Philippine B. bovis-positive DNA samples were found in six, three, and four different clades, respectively. All of the msa-1 and most of the msa-2b sequences were found in clades that were formed only by Philippine msa sequences in the respective phylograms. While all the msa-1 sequences from the Philippines showed similarity to those formed by Australian msa-1 sequences, the msa-2b sequences showed similarity to either Australian or Mexican msa-2b sequences. In contrast, msa-2c sequences from the Philippines were distributed across all the clades of the phylogram, although one clade was formed exclusively by Philippine msa-2c sequences. Similarities among the deduced amino acid sequences of MSA-1, MSA-2b, and MSA-2c from the Philippines were 62.2-100, 73.1-100, and 67.3-100%, respectively. The present findings demonstrate that B. bovis populations are genetically diverse in the Philippines. This information will provide a good foundation for the future design and implementation of improved immunological preventive methodologies against bovine babesiosis in the Philippines. The study has also generated a set of data that will be useful for futher understanding of the global genetic diversity of this important parasite. © 2013.

[Community composition and diversity of endophytic fungi from roots of Sinopodophyllum hexandrum in forest of Upper-north mountain of Qinghai province].

PubMed

Ning, Yi; Li, Yan-Ling; Zhou, Guo-Ying; Yang, Lu-Cun; Xu, Wen-Hua

2016-04-01

High throughput sequencing technology is also called Next Generation Sequencing (NGS), which can sequence hundreds and thousands sequences in different samples at the same time. In the present study, the culture-independent high throughput sequencing technology was applied to sequence the fungi metagenomic DNA of the fungal internal transcribed spacer 1(ITS 1) in the root of Sinopodophyllum hexandrum. Sequencing data suggested that after the quality control, 22 565 reads were remained. Cluster similarity analysis was done based on 97% sequence similarity, which obtained 517 OTUs for the three samples (LD1, LD2 and LD3). All the fungi which identified from all the reads of OTUs based on 0.8 classification thresholds using the software of RDP classifier were classified as 13 classes, 35 orders, 44 family, 55 genera. Among these genera, the genus of Tetracladium was the dominant genera in all samples(35.49%, 68.55% and 12.96%).The Shannon's diversity indices and the Simpson indices of the endophytic fungi in the samples ranged from 1.75-2.92, 0.11-0.32, respectively.This is the first time for applying high through put sequencing technol-ogyto analyze the community composition and diversity of endophytic fungi in the medicinal plant, and the results showed that there were hyper diver sity and high community composition complexity of endophytic fungi in the root of S. hexandrum. It is also proved that the high through put sequencing technology has great advantage for analyzing ecommunity composition and diversity of endophtye in the plant. Copyright© by the Chinese Pharmaceutical Association.

Sequences of 95 human MHC haplotypes reveal extreme coding variation in genes other than highly polymorphic HLA class I and II

PubMed Central

Norman, Paul J.; Norberg, Steven J.; Guethlein, Lisbeth A.; Nemat-Gorgani, Neda; Royce, Thomas; Wroblewski, Emily E.; Dunn, Tamsen; Mann, Tobias; Alicata, Claudia; Hollenbach, Jill A.; Chang, Weihua; Shults Won, Melissa; Gunderson, Kevin L.; Abi-Rached, Laurent; Ronaghi, Mostafa; Parham, Peter

2017-01-01

The most polymorphic part of the human genome, the MHC, encodes over 160 proteins of diverse function. Half of them, including the HLA class I and II genes, are directly involved in immune responses. Consequently, the MHC region strongly associates with numerous diseases and clinical therapies. Notoriously, the MHC region has been intractable to high-throughput analysis at complete sequence resolution, and current reference haplotypes are inadequate for large-scale studies. To address these challenges, we developed a method that specifically captures and sequences the 4.8-Mbp MHC region from genomic DNA. For 95 MHC homozygous cell lines we assembled, de novo, a set of high-fidelity contigs and a sequence scaffold, representing a mean 98% of the target region. Included are six alternative MHC reference sequences of the human genome that we completed and refined. Characterization of the sequence and structural diversity of the MHC region shows the approach accurately determines the sequences of the highly polymorphic HLA class I and HLA class II genes and the complex structural diversity of complement factor C4A/C4B. It has also uncovered extensive and unexpected diversity in other MHC genes; an example is MUC22, which encodes a lung mucin and exhibits more coding sequence alleles than any HLA class I or II gene studied here. More than 60% of the coding sequence alleles analyzed were previously uncharacterized. We have created a substantial database of robust reference MHC haplotype sequences that will enable future population scale studies of this complicated and clinically important region of the human genome. PMID:28360230

Bacterial diversity in permanently cold and alkaline ikaite columns from Greenland.

PubMed

Schmidt, Mariane; Priemé, Anders; Stougaard, Peter

2006-12-01

Bacterial diversity in alkaline (pH 10.4) and permanently cold (4 degrees C) ikaite tufa columns from the Ikka Fjord, SW Greenland, was investigated using growth characterization of cultured bacterial isolates with Terminal-restriction fragment length polymorphism (T-RFLP) and sequence analysis of bacterial 16S rRNA gene fragments. More than 200 bacterial isolates were characterized with respect to pH and temperature tolerance, and it was shown that the majority were cold-active alkaliphiles. T-RFLP analysis revealed distinct bacterial communities in different fractions of three ikaite columns, and, along with sequence analysis, it showed the presence of rich and diverse bacterial communities. Rarefaction analysis showed that the 109 sequenced clones in the 16S rRNA gene library represented between 25 and 65% of the predicted species richness in the three ikaite columns investigated. Phylogenetic analysis of the 16S rRNA gene sequences revealed many sequences with similarity to alkaliphilic or psychrophilic bacteria, and showed that 33% of the cloned sequences and 33% of the cultured bacteria showed less than 97% sequence identity to known sequences in databases, and may therefore represent yet unknown species.

The Applied Development of a Tiered Multilocus Sequence Typing (MLST) Scheme for Dichelobacter nodosus.

PubMed

Blanchard, Adam M; Jolley, Keith A; Maiden, Martin C J; Coffey, Tracey J; Maboni, Grazieli; Staley, Ceri E; Bollard, Nicola J; Warry, Andrew; Emes, Richard D; Davies, Peers L; Tötemeyer, Sabine

2018-01-01

Dichelobacter nodosus ( D. nodosus ) is the causative pathogen of ovine footrot, a disease that has a significant welfare and financial impact on the global sheep industry. Previous studies into the phylogenetics of D. nodosus have focused on Australia and Scandinavia, meaning the current diversity in the United Kingdom (U.K.) population and its relationship globally, is poorly understood. Numerous epidemiological methods are available for bacterial typing; however, few account for whole genome diversity or provide the opportunity for future application of new computational techniques. Multilocus sequence typing (MLST) measures nucleotide variations within several loci with slow accumulation of variation to enable the designation of allele numbers to determine a sequence type. The usage of whole genome sequence data enables the application of MLST, but also core and whole genome MLST for higher levels of strain discrimination with a negligible increase in experimental cost. An MLST database was developed alongside a seven loci scheme using publically available whole genome data from the sequence read archive. Sequence type designation and strain discrimination was compared to previously published data to ensure reproducibility. Multiple D. nodosus isolates from U.K. farms were directly compared to populations from other countries. The U.K. isolates define new clades within the global population of D. nodosus and predominantly consist of serogroups A, B and H, however serogroups C, D, E, and I were also found. The scheme is publically available at https://pubmlst.org/dnodosus/.

High-throughput sequencing of complete human mtDNA genomes from the Caucasus and West Asia: high diversity and demographic inferences.

PubMed

Schönberg, Anna; Theunert, Christoph; Li, Mingkun; Stoneking, Mark; Nasidze, Ivan

2011-09-01

To investigate the demographic history of human populations from the Caucasus and surrounding regions, we used high-throughput sequencing to generate 147 complete mtDNA genome sequences from random samples of individuals from three groups from the Caucasus (Armenians, Azeri and Georgians), and one group each from Iran and Turkey. Overall diversity is very high, with 144 different sequences that fall into 97 different haplogroups found among the 147 individuals. Bayesian skyline plots (BSPs) of population size change through time show a population expansion around 40-50 kya, followed by a constant population size, and then another expansion around 15-18 kya for the groups from the Caucasus and Iran. The BSP for Turkey differs the most from the others, with an increase from 35 to 50 kya followed by a prolonged period of constant population size, and no indication of a second period of growth. An approximate Bayesian computation approach was used to estimate divergence times between each pair of populations; the oldest divergence times were between Turkey and the other four groups from the South Caucasus and Iran (~400-600 generations), while the divergence time of the three Caucasus groups from each other was comparable to their divergence time from Iran (average of ~360 generations). These results illustrate the value of random sampling of complete mtDNA genome sequences that can be obtained with high-throughput sequencing platforms.

Shifts in phylogenetic diversity of archaeal communities in mangrove sediments at different sites and depths in southeastern Brazil.

PubMed

Mendes, Lucas William; Taketani, Rodrigo Gouvêa; Navarrete, Acácio Aparecido; Tsai, Siu Mui

2012-06-01

This study focused on the structure and composition of archaeal communities in sediments of tropical mangroves in order to obtain sufficient insight into two Brazilian sites from different locations (one pristine and another located in an urban area) and at different depth levels from the surface. Terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified 16S rRNA gene fragments was used to scan the archaeal community structure, and 16S rRNA gene clone libraries were used to determine the community composition. Redundancy analysis of T-RFLP patterns revealed differences in archaeal community structure according to location, depth and soil attributes. Parameters such as pH, organic matter, potassium and magnesium presented significant correlation with general community structure. Furthermore, phylogenetic analysis revealed a community composition distributed differently according to depth where, in shallow samples, 74.3% of sequences were affiliated with Euryarchaeota and 25.7% were shared between Crenarchaeota and Thaumarchaeota, while for the deeper samples, 24.3% of the sequences were affiliated with Euryarchaeota and 75.7% with Crenarchaeota and Thaumarchaeota. Archaeal diversity measurements based on 16S rRNA gene clone libraries decreased with increasing depth and there was a greater difference between depths (<18% of sequences shared) than sites (>25% of sequences shared). Taken together, our findings indicate that mangrove ecosystems support a diverse archaeal community; it might possibly be involved in nutrient cycles and are affected by sediment properties, depth and distinct locations. Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Variation in the hindgut microbial communities of the Florida manatee, Trichechus manatus latirostris over winter in Crystal River, Florida.

PubMed

Merson, Samuel D; Ouwerkerk, Diane; Gulino, Lisa-Maree; Klieve, Athol; Bonde, Robert K; Burgess, Elizabeth A; Lanyon, Janet M

2014-03-01

The Florida manatee, Trichechus manatus latirostris, is a hindgut-fermenting herbivore. In winter, manatees migrate to warm water overwintering sites where they undergo dietary shifts and may suffer from cold-induced stress. Given these seasonally induced changes in diet, the present study aimed to examine variation in the hindgut bacterial communities of wild manatees overwintering at Crystal River, west Florida. Faeces were sampled from 36 manatees of known sex and body size in early winter when manatees were newly arrived and then in mid-winter and late winter when diet had probably changed and environmental stress may have increased. Concentrations of faecal cortisol metabolite, an indicator of a stress response, were measured by enzyme immunoassay. Using 454-pyrosequencing, 2027 bacterial operational taxonomic units were identified in manatee faeces following amplicon pyrosequencing of the 16S rRNA gene V3/V4 region. Classified sequences were assigned to eight previously described bacterial phyla; only 0.36% of sequences could not be classified to phylum level. Five core phyla were identified in all samples. The majority (96.8%) of sequences were classified as Firmicutes (77.3 ± 11.1% of total sequences) or Bacteroidetes (19.5 ± 10.6%). Alpha-diversity measures trended towards higher diversity of hindgut microbiota in manatees in mid-winter compared to early and late winter. Beta-diversity measures, analysed through PERMANOVA, also indicated significant differences in bacterial communities based on the season. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Severe chronic osteomyelitis caused by Morganella morganii with high population diversity.

PubMed

Zhu, Jialiang; Li, Haifeng; Feng, Li; Yang, Min; Yang, Ronggong; Yang, Lin; Li, Li; Li, Ruoyan; Liu, Minshan; Hou, Shuxun; Ke, Yuehua; Li, Wenfeng; Bai, Fan

2016-09-01

A case of chronic osteomyelitis probably caused by Morganella morganii, occurring over a period of 30 years, is reported. The organism was identified through a combination of sample culture, direct sequencing, and 16S RNA gene amplicon sequencing. Further whole-genome sequencing and population structure analysis of the isolates from the patient showed the bacterial population to be highly diverse. This case provides a valuable example of a long-term infection caused by an opportunistic pathogen, M. morganii, with high diversity, which might evolve during replication within the host. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Nitrogen deposition and management practices increase soil microbial biomass carbon but decrease diversity in Moso bamboo plantations

PubMed Central

Li, Quan; Song, Xinzhang; Gu, Honghao; Gao, Fei

2016-01-01

Because microbial communities play a key role in carbon (C) and nitrogen (N) cycling, changes in the soil microbial community may directly affect ecosystem functioning. However, the effects of N deposition and management practices on soil microbes are still poorly understood. We studied the effects of these two factors on soil microbial biomass carbon (MBC) and community composition in Moso bamboo plantations using high-throughput sequencing of the 16S rRNA gene. Plantations under conventional (CM) or intensive management (IM) were subjected to one of four N treatments for 30 months. IM and N addition, both separately and in combination, significantly increased soil MBC while decreasing bacterial diversity. However, increases in soil MBC were inhibited when N addition exceeded 60 kg N∙ha−1∙yr−1. IM increased the relative abundances of Actinobacteria and Crenarchaeota but decreased that of Acidobacteria. N addition increased the relative abundances of Acidobacteria, Crenarchaeota, and Actinobacteria but decreased that of Proteobacteria. Soil bacterial diversity was significantly related to soil pH, C/N ratio, and nitrogen and available phosphorus content. Management practices exerted a greater influence over regulation of the soil MBC and microbial diversity compared to that of N deposition in Moso bamboo plantations. PMID:27302857

Nitrogen deposition and management practices increase soil microbial biomass carbon but decrease diversity in Moso bamboo plantations

NASA Astrophysics Data System (ADS)

Li, Quan; Song, Xinzhang; Gu, Honghao; Gao, Fei

2016-06-01

Because microbial communities play a key role in carbon (C) and nitrogen (N) cycling, changes in the soil microbial community may directly affect ecosystem functioning. However, the effects of N deposition and management practices on soil microbes are still poorly understood. We studied the effects of these two factors on soil microbial biomass carbon (MBC) and community composition in Moso bamboo plantations using high-throughput sequencing of the 16S rRNA gene. Plantations under conventional (CM) or intensive management (IM) were subjected to one of four N treatments for 30 months. IM and N addition, both separately and in combination, significantly increased soil MBC while decreasing bacterial diversity. However, increases in soil MBC were inhibited when N addition exceeded 60 kg N•ha-1•yr-1. IM increased the relative abundances of Actinobacteria and Crenarchaeota but decreased that of Acidobacteria. N addition increased the relative abundances of Acidobacteria, Crenarchaeota, and Actinobacteria but decreased that of Proteobacteria. Soil bacterial diversity was significantly related to soil pH, C/N ratio, and nitrogen and available phosphorus content. Management practices exerted a greater influence over regulation of the soil MBC and microbial diversity compared to that of N deposition in Moso bamboo plantations.

Changes in bacterial and archaeal communities during the concentration of brine at the graduation towers in Ciechocinek spa (Poland).

PubMed

Kalwasińska, Agnieszka; Deja-Sikora, Edyta; Burkowska-But, Aleksandra; Szabó, Attila; Felföldi, Támas; Kosobucki, Przemysław; Krawiec, Arkadiusz; Walczak, Maciej

2018-03-01

This study evaluates the changes in bacterial and archaeal community structure during the gradual evaporation of water from the brine (extracted from subsurface Jurassic deposits) in the system of graduation towers located in Ciechocinek spa, Poland. The communities were assessed with 16S rRNA gene sequencing (MiSeq, Illumina) and microscopic methods. The microbial cell density determined by direct cell count was at the order of magnitude of 10 7 cells/mL. It was found that increasing salt concentration was positively correlated with both the cell counts, and species-level diversity of bacterial and archaeal communities. The archaeal community was mostly constituted by members of the phylum Euryarchaeota, class Halobacteria and was dominated by Halorubrum-related sequences. The bacterial community was more diverse, with representatives of the phyla Proteobacteria and Bacteroidetes as the most abundant. The proportion of Proteobacteria decreased with increasing salt concentration, while the proportion of Bacteroidetes increased significantly in the more concentrated samples. Representatives of the genera Idiomarina, Psychroflexus, Roseovarius, and Marinobacter appeared to be tolerant to changes of salinity. During the brine concentration, the relative abundances of Sphingobium and Sphingomonas were significantly decreased and the raised contributions of genera Fabibacter and Fodinibius were observed. The high proportion of novel (not identified at 97% similarity level) bacterial reads (up to 42%) in the 16S rRNA gene sequences indicated that potentially new bacterial taxa inhabit this unique environment.

Variation along ITS markers across strains of Fibrocapsa japonica (Raphidophyceae) suggests hybridisation events and recent range expansion

NASA Astrophysics Data System (ADS)

Kooistra, Wiebe H. C. F.; de Boer, M. Karin; Vrieling, Engel G.; Connell, Laurie B.; Gieskes, Winfried W. C.

2001-12-01

The flagellate micro-alga Fibrocapsa japonica can form harmful algal blooms along all temperate coastal regions of the world. The species was first observed in coastal waters of Japan and the western US in the 1970s; it has been reported regularly worldwide since. To unravel whether this apparent range expansion can be tracked, we assessed genetic variation among nuclear ribosomal DNA ITS sequences, obtained from sixteen global strains collected over the course of three decades. Ten sequence positions showed polymorphism across the strains. Nine out of these revealed ambiguities in several or most sequences sampled. The oldest strain collected (LB-2161) was the only one without such intra-individual polymorphism. In the others, the proportion of ambiguities at variable sites increased with more recent collection date. The pattern does not result from loss of variation due to sexual reproduction and random drift in culture because sister cultures CS-332 and NIES-136 showed virtually the same ITS-pattern after seven years of separation. Neither are the patterns explained by recent range expansion of a single genotype, because in that case one would expect lowest genetic diversity in the recently invaded North Sea; instead, polymorphism is highest there. Recent ballast-water-mediated mixing of formerly isolated populations and subsequent ongoing sexual reproduction among them can explain the increase in ambiguities. The species' capacity to form harmful blooms may well have been enhanced through increased genetic diversity of regional populations.

Metagenomic applications in environmental monitoring and bioremediation

DOE PAGES

Techtmann, Stephen M.; Hazen, Terry C.

2016-01-01

With the rapid advances in sequencing technology, the cost of sequencing has dramatically dropped and the scale of sequencing projects has increased accordingly. This has provided the opportunity for the routine use of sequencing techniques in the monitoring of environmental microbes. While metagenomic applications have been routinely applied to better understand the ecology and diversity of microbes, their use in environmental monitoring and bioremediation is increasingly common. In this review we seek to provide an overview of some of the metagenomic techniques used in environmental systems biology, addressing their application and limitation. We will also provide several recent examples ofmore » the application of metagenomics to bioremediation. We discuss examples where microbial communities have been used to predict the presence and extent of contamination, examples of how metagenomics can be used to characterize the process of natural attenuation by unculturable microbes, as well as examples detailing the use of metagenomics to understand the impact of biostimulation on microbial communities.« less

Assessing the Effect of Litter Species on the Dynamic of Bacterial and Fungal Communities during Leaf Decomposition in Microcosm by Molecular Techniques

PubMed Central

Xu, Wenjing; Shi, Lingling; Chan, Onchim; Li, Jiao; Casper, Peter; Zou, Xiaoming

2013-01-01

Although bacteria and fungi are well-known to be decomposers of leaf litter, few studies have examined their compositions and diversities during the decomposition process in tropical stream water. Xishuangbanna is a tropical region preserving one of the highest floristic diversity areas in China. In this study, leaf litter of four dominant plant species in Xishuangbanna was incubated in stream water for 42 days during which samples were taken regularly. Following DNA extraction, PCR-DGGE (denaturing gradient gel electrophoresis) and clone-sequencing analyses were performed using bacterial and fungal specific primers. Leaf species have slightly influences on bacterial community rather than fungal community. The richness and diversity of bacteria was higher than that of fungi, which increased towards the end of the 42-day-incubation. The bacterial community was initially more specific upon the type of leaves and gradually became similar at the later stage of decomposition with alpha-proteobacteria as major component. Sequences affiliated to methanotrophs were obtained that indicates potentially occurrence of methane oxidation and methanogenesis. For the fungal community, sequences affiliated to Aspergillus were predominant at the beginning and then shifted to Pleosporales. Our results suggest that the microorganisms colonizing leaf biofilm in tropical stream water were mostly generalists that could exploit the resources of leaves of various species equally well. PMID:24367682

[Research on soil bacteria under the impact of sealed CO2 leakage by high-throughput sequencing technology].

PubMed

Tian, Di; Ma, Xin; Li, Yu-E; Zha, Liang-Song; Wu, Yang; Zou, Xiao-Xia; Liu, Shuang

2013-10-01

Carbon dioxide Capture and Storage has provided a new option for mitigating global anthropogenic CO2 emission with its unique advantages. However, there is a risk of the sealed CO2 leakage, bringing a serious threat to the ecology system. It is widely known that soil microorganisms are closely related to soil health, while the study on the impact of sequestered CO2 leakage on soil microorganisms is quite deficient. In this study, the leakage scenarios of sealed CO2 were constructed and the 16S rRNA genes of soil bacteria were sequenced by Illumina high-throughput sequencing technology on Miseq platform, and related biological analysis was conducted to explore the changes of soil bacterial abundance, diversity and structure. There were 486,645 reads for 43,017 OTUs of 15 soil samples and the results of biological analysis showed that there were differences in the abundance, diversity and community structure of soil bacterial community under different CO, leakage scenarios while the abundance and diversity of the bacterial community declined with the amplification of CO2 leakage quantity and leakage time, and some bacteria species became the dominant bacteria species in the bacteria community, therefore the increase of Acidobacteria species would be a biological indicator for the impact of sealed CO2 leakage on soil ecology system.

MicRhoDE: a curated database for the analysis of microbial rhodopsin diversity and evolution

PubMed Central

Boeuf, Dominique; Audic, Stéphane; Brillet-Guéguen, Loraine; Caron, Christophe; Jeanthon, Christian

2015-01-01

Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life and in viruses. Today, microbial rhodopsin research is a flourishing research field in which new understandings of rhodopsin diversity, function and evolution are contributing to broader microbiological and molecular knowledge. Here, we describe MicRhoDE, a comprehensive, high-quality and freely accessible database that facilitates analysis of the diversity and evolution of microbial rhodopsins. Rhodopsin sequences isolated from a vast array of marine and terrestrial environments were manually collected and curated. To each rhodopsin sequence are associated related metadata, including predicted spectral tuning of the protein, putative activity and function, taxonomy for sequences that can be linked to a 16S rRNA gene, sampling date and location, and supporting literature. The database currently covers 7857 aligned sequences from more than 450 environmental samples or organisms. Based on a robust phylogenetic analysis, we introduce an operational classification system with multiple phylogenetic levels ranging from superclusters to species-level operational taxonomic units. An integrated pipeline for online sequence alignment and phylogenetic tree construction is also provided. With a user-friendly interface and integrated online bioinformatics tools, this unique resource should be highly valuable for upcoming studies of the biogeography, diversity, distribution and evolution of microbial rhodopsins. Database URL: http://micrhode.sb-roscoff.fr. PMID:26286928

Unveiling fungal zooflagellates as members of freshwater picoeukaryotes: evidence from a molecular diversity study in a deep meromictic lake.

PubMed

Lefèvre, Emilie; Bardot, Corinne; Noël, Christophe; Carrias, Jean-François; Viscogliosi, Eric; Amblard, Christian; Sime-Ngando, Télesphore

2007-01-01

This study presents an original 18S rRNA PCR survey of the freshwater picoeukaryote community, and was designed to detect unidentified heterotrophic picoflagellates (size range 0.6-5 microm) which are prevalent throughout the year within the heterotrophic flagellate assemblage in Lake Pavin. Four clone libraries were constructed from samples collected in two contrasting zones in the lake. Computerized statistic tools have suggested that sequence retrieval was representative of the in situ picoplankton diversity. The two sampling zones exhibited similar diversity patterns but shared only about 5% of the operational taxonomic units (OTUs). Phylogenetic analysis clustered our sequences into three taxonomic groups: Alveolates (30% of OTUs), Fungi (23%) and Cercozoa (19%). Fungi thus substantially contributed to the detected diversity, as was additionally supported by direct microscopic observations of fungal zoospores and sporangia. A large fraction of the sequences belonged to parasites, including Alveolate sequences affiliated to the genus Perkinsus known as zooparasites, and chytrids that include host-specific parasitic fungi of various freshwater phytoplankton species, primarily diatoms. Phylogenetic analysis revealed five novel clades that probably include typical freshwater environmental sequences. Overall, from the unsuspected fungal diversity unveiled, we think that fungal zooflagellates have been misidentified as phagotrophic nanoflagellates in previous studies. This is in agreement with a recent experimental demonstration that zoospore-producing fungi and parasitic activity may play an important role in aquatic food webs.

MicRhoDE: a curated database for the analysis of microbial rhodopsin diversity and evolution.

PubMed

Boeuf, Dominique; Audic, Stéphane; Brillet-Guéguen, Loraine; Caron, Christophe; Jeanthon, Christian

2015-01-01

Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life and in viruses. Today, microbial rhodopsin research is a flourishing research field in which new understandings of rhodopsin diversity, function and evolution are contributing to broader microbiological and molecular knowledge. Here, we describe MicRhoDE, a comprehensive, high-quality and freely accessible database that facilitates analysis of the diversity and evolution of microbial rhodopsins. Rhodopsin sequences isolated from a vast array of marine and terrestrial environments were manually collected and curated. To each rhodopsin sequence are associated related metadata, including predicted spectral tuning of the protein, putative activity and function, taxonomy for sequences that can be linked to a 16S rRNA gene, sampling date and location, and supporting literature. The database currently covers 7857 aligned sequences from more than 450 environmental samples or organisms. Based on a robust phylogenetic analysis, we introduce an operational classification system with multiple phylogenetic levels ranging from superclusters to species-level operational taxonomic units. An integrated pipeline for online sequence alignment and phylogenetic tree construction is also provided. With a user-friendly interface and integrated online bioinformatics tools, this unique resource should be highly valuable for upcoming studies of the biogeography, diversity, distribution and evolution of microbial rhodopsins. Database URL: http://micrhode.sb-roscoff.fr. © The Author(s) 2015. Published by Oxford University Press.

Problem Based Learning: Application to Technology Education in Three Countries

ERIC Educational Resources Information Center

Williams, P. John; Iglesias, Juan; Barak, Moshe

2008-01-01

An increasing variety of professional educational and training disciplines are now problem based (e.g., medicine, nursing, engineering, community health), and they may have a corresponding variety of educational objectives. However, they all have in common the use of problems in the instructional sequence. The problems may be as diverse as a…

DS-CDMA satellite diversity reception for personal satellite communication: Downlink performance analysis

NASA Technical Reports Server (NTRS)

DeGaudenzi, Riccardo; Giannetti, Filippo

1995-01-01

The downlink of a satellite-mobile personal communication system employing power-controlled Direct Sequence Code Division Multiple Access (DS-CDMA) and exploiting satellite-diversity is analyzed and its performance compared with a more traditional communication system utilizing single satellite reception. The analytical model developed has been thoroughly validated by means of extensive Monte Carlo computer simulations. It is shown how the capacity gain provided by diversity reception shrinks considerably in the presence of increasing traffic or in the case of light shadowing conditions. Moreover, the quantitative results tend to indicate that to combat system capacity reduction due to intra-system interference, no more than two satellites shall be active over the same region. To achieve higher system capacity, differently from terrestrial cellular systems, Multi-User Detection (MUD) techniques are likely to be required in the mobile user terminal, thus considerably increasing its complexity.

Large-Scale Sequencing: The Future of Genomic Sciences Colloquium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Margaret Riley; Merry Buckley

2009-01-01

Genetic sequencing and the various molecular techniques it has enabled have revolutionized the field of microbiology. Examining and comparing the genetic sequences borne by microbes - including bacteria, archaea, viruses, and microbial eukaryotes - provides researchers insights into the processes microbes carry out, their pathogenic traits, and new ways to use microorganisms in medicine and manufacturing. Until recently, sequencing entire microbial genomes has been laborious and expensive, and the decision to sequence the genome of an organism was made on a case-by-case basis by individual researchers and funding agencies. Now, thanks to new technologies, the cost and effort of sequencingmore » is within reach for even the smallest facilities, and the ability to sequence the genomes of a significant fraction of microbial life may be possible. The availability of numerous microbial genomes will enable unprecedented insights into microbial evolution, function, and physiology. However, the current ad hoc approach to gathering sequence data has resulted in an unbalanced and highly biased sampling of microbial diversity. A well-coordinated, large-scale effort to target the breadth and depth of microbial diversity would result in the greatest impact. The American Academy of Microbiology convened a colloquium to discuss the scientific benefits of engaging in a large-scale, taxonomically-based sequencing project. A group of individuals with expertise in microbiology, genomics, informatics, ecology, and evolution deliberated on the issues inherent in such an effort and generated a set of specific recommendations for how best to proceed. The vast majority of microbes are presently uncultured and, thus, pose significant challenges to such a taxonomically-based approach to sampling genome diversity. However, we have yet to even scratch the surface of the genomic diversity among cultured microbes. A coordinated sequencing effort of cultured organisms is an appropriate place to begin, since not only are their genomes available, but they are also accompanied by data on environment and physiology that can be used to understand the resulting data. As single cell isolation methods improve, there should be a shift toward incorporating uncultured organisms and communities into this effort. Efforts to sequence cultivated isolates should target characterized isolates from culture collections for which biochemical data are available, as well as other cultures of lasting value from personal collections. The genomes of type strains should be among the first targets for sequencing, but creative culture methods, novel cell isolation, and sorting methods would all be helpful in obtaining organisms we have not yet been able to cultivate for sequencing. The data that should be provided for strains targeted for sequencing will depend on the phylogenetic context of the organism and the amount of information available about its nearest relatives. Annotation is an important part of transforming genome sequences into useful resources, but it represents the most significant bottleneck to the field of comparative genomics right now and must be addressed. Furthermore, there is a need for more consistency in both annotation and achieving annotation data. As new annotation tools become available over time, re-annotation of genomes should be implemented, taking advantage of advancements in annotation techniques in order to capitalize on the genome sequences and increase both the societal and scientific benefit of genomics work. Given the proper resources, the knowledge and ability exist to be able to select model systems, some simple, some less so, and dissect them so that we may understand the processes and interactions at work in them. Colloquium participants suggest a five-pronged, coordinated initiative to exhaustively describe six different microbial ecosystems, designed to describe all the gene diversity, across genomes. In this effort, sequencing should be complemented by other experimental data, particularly transcriptomics and metabolomics data, all of which should be gathered and curated continuously. Systematic genomics efforts like the ones outlined in this document would significantly broaden our view of biological diversity and have major effects on science. This has to be backed up with examples. Considering these potential impacts and the need for acquiescence from both the public and scientists to get such projects funded and functioning, education and training will be crucial. New collaborations within the scientific community will also be necessary.« less

Towards a molecular taxonomy for protists: benefits, risks, and applications in plankton ecology.

PubMed

Caron, David A

2013-01-01

The increasing use of genetic information for the development of methods to study the diversity, distributions, and activities of protists in nature has spawned a new generation of powerful tools. For ecologists, one lure of these approaches lies in the potential for DNA sequences to provide the only immediately obvious means of normalizing the diverse criteria that presently exist for identifying and counting protists. A single, molecular taxonomy would allow studies of diversity across a broad range of species, as well as the detection and quantification of particular species of interest within complex, natural assemblages; goals that are not feasible using traditional methods. However, these advantages are not without their potential pitfalls and problems. Conflicts involving the species concept, disagreements over the true (physiological/ecological) meaning of genetic diversity, and a perceived threat by some that sequence information will displace knowledge regarding the morphologies, functions and physiologies of protistan taxa, have created debate and doubt regarding the efficacy and appropriateness of some genetic approaches. These concerns need continued discussion and eventual resolution as we move toward the irresistible attraction, and potentially enormous benefits, of the application of genetic approaches to protistan ecology. © 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists.

Effects of Predation by Protists on Prokaryotic Community Function, Structure, and Diversity in Anaerobic Granular Sludge.

PubMed

Hirakata, Yuga; Oshiki, Mamoru; Kuroda, Kyohei; Hatamoto, Masashi; Kubota, Kengo; Yamaguchi, Takashi; Harada, Hideki; Araki, Nobuo

2016-09-29

Predation by protists is top-down pressure that regulates prokaryotic abundance, community function, structure, and diversity in natural and artificial ecosystems. Although the effects of predation by protists have been studied in aerobic ecosystems, they are poorly understood in anoxic environments. We herein studied the influence of predation by Metopus and Caenomorpha ciliates-ciliates frequently found in anoxic ecosystems-on prokaryotic community function, structure, and diversity. Metopus and Caenomorpha ciliates were cocultivated with prokaryotic assemblages (i.e., anaerobic granular sludge) in an up-flow anaerobic sludge blanket (UASB) reactor for 171 d. Predation by these ciliates increased the methanogenic activities of granular sludge, which constituted 155% of those found in a UASB reactor without the ciliates (i.e., control reactor). Sequencing of 16S rRNA gene amplicons using Illumina MiSeq revealed that the prokaryotic community in the UASB reactor with the ciliates was more diverse than that in the control reactor; 2,885-3,190 and 2,387-2,426 operational taxonomic units (>97% sequence similarities), respectively. The effects of predation by protists in anaerobic engineered systems have mostly been overlooked, and our results show that the influence of predation by protists needs to be examined and considered in the future for a better understanding of prokaryotic community structure and function.

Fine-scale analysis of 16S rRNA sequences reveals a high level of taxonomic diversity among vaginal Atopobium spp.

PubMed Central

Mendes-Soares, Helena; Krishnan, Vandhana; Settles, Matthew L.; Ravel, Jacques; Brown, Celeste J.; Forney, Larry J.

2015-01-01

Although vaginal microbial communities of some healthy women have high proportions of Atopobium vaginae, the genus Atopobium is more commonly associated with bacterial vaginosis, a syndrome associated with an increased risk of adverse pregnancy outcomes and the transmission of sexually transmitted diseases. Genetic differences within Atopobium species may explain why single species can be associated with both health and disease. We used 16S rRNA gene sequences from previously published studies to explore the taxonomic diversity of the genus Atopobium in vaginal microbial communities of healthy women. Although A. vaginae was the species most commonly found, we also observed three other Atopobium species in the vaginal microbiota, one of which, A. parvulum, was not previously known to reside in the human vagina. Furthermore, we found several potential novel species of the genus Atopobium and multiple phylogenetic clades of A. vaginae. The diversity of Atopobium found in our study, which focused only on samples from healthy women, is greater than previously recognized, suggesting that analysis of samples from women with BV would yield even more diversity. Classification of microbes only to the genus level may thus obfuscate differences that might be important to better understand health or disease. PMID:25778779

Distinct composition signatures of archaeal and bacterial phylotypes in the Wanda Glacier forefield, Antarctic Peninsula.

PubMed

Pessi, Igor S; Osorio-Forero, César; Gálvez, Eric J C; Simões, Felipe L; Simões, Jefferson C; Junca, Howard; Macedo, Alexandre J

2015-01-01

Several studies have shown that microbial communities in Antarctic environments are highly diverse. However, considering that the Antarctic Peninsula is among the regions with the fastest warming rates, and that regional climate change has been linked to an increase in the mean rate of glacier retreat, the microbial diversity in Antarctic soil is still poorly understood. In this study, we analysed more than 40 000 sequences of the V5-V6 hypervariable region of the 16S rRNA gene obtained by 454 pyrosequencing from four soil samples from the Wanda Glacier forefield, King George Island, Antarctic Peninsula. Phylotype diversity and richness were surprisingly high, and taxonomic assignment of sequences revealed that communities are dominated by Proteobacteria, Bacteroidetes and Euryarchaeota, with a high frequency of archaeal and bacterial phylotypes unclassified at the genus level and without cultured representative strains, representing a distinct microbial community signature. Several phylotypes were related to marine microorganisms, indicating the importance of the marine environment as a source of colonizers for this recently deglaciated environment. Finally, dominant phylotypes were related to different microorganisms possessing a large array of metabolic strategies, indicating that early successional communities in Antarctic glacier forefield can be also functionally diverse. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Determination of a Screening Metric for High Diversity DNA Libraries.

PubMed

Guido, Nicholas J; Handerson, Steven; Joseph, Elaine M; Leake, Devin; Kung, Li A

2016-01-01

The fields of antibody engineering, enzyme optimization and pathway construction rely increasingly on screening complex variant DNA libraries. These highly diverse libraries allow researchers to sample a maximized sequence space; and therefore, more rapidly identify proteins with significantly improved activity. The current state of the art in synthetic biology allows for libraries with billions of variants, pushing the limits of researchers' ability to qualify libraries for screening by measuring the traditional quality metrics of fidelity and diversity of variants. Instead, when screening variant libraries, researchers typically use a generic, and often insufficient, oversampling rate based on a common rule-of-thumb. We have developed methods to calculate a library-specific oversampling metric, based on fidelity, diversity, and representation of variants, which informs researchers, prior to screening the library, of the amount of oversampling required to ensure that the desired fraction of variant molecules will be sampled. To derive this oversampling metric, we developed a novel alignment tool to efficiently measure frequency counts of individual nucleotide variant positions using next-generation sequencing data. Next, we apply a method based on the "coupon collector" probability theory to construct a curve of upper bound estimates of the sampling size required for any desired variant coverage. The calculated oversampling metric will guide researchers to maximize their efficiency in using highly variant libraries.

Diversity and evolution of centromere repeats in the maize genome.

PubMed

Bilinski, Paul; Distor, Kevin; Gutierrez-Lopez, Jose; Mendoza, Gabriela Mendoza; Shi, Jinghua; Dawe, R Kelly; Ross-Ibarra, Jeffrey

2015-03-01

Centromere repeats are found in most eukaryotes and play a critical role in kinetochore formation. Though centromere repeats exhibit considerable diversity both within and among species, little is understood about the mechanisms that drive centromere repeat evolution. Here, we use maize as a model to investigate how a complex history involving polyploidy, fractionation, and recent domestication has impacted the diversity of the maize centromeric repeat CentC. We first validate the existence of long tandem arrays of repeats in maize and other taxa in the genus Zea. Although we find considerable sequence diversity among CentC copies genome-wide, genetic similarity among repeats is highest within these arrays, suggesting that tandem duplications are the primary mechanism for the generation of new copies. Nonetheless, clustering analyses identify similar sequences among distant repeats, and simulations suggest that this pattern may be due to homoplasious mutation. Although the two ancestral subgenomes of maize have contributed nearly equal numbers of centromeres, our analysis shows that the majority of all CentC repeats derive from one of the parental genomes, with an even stronger bias when examining the largest assembled contiguous clusters. Finally, by comparing maize with its wild progenitor teosinte, we find that the abundance of CentC likely decreased after domestication, while the pericentromeric repeat Cent4 has drastically increased.

Genetic variation of Sargassum horneri populations detected by inter-simple sequence repeats.

PubMed

Ren, J R; Yang, R; He, Y Y; Sun, Q H

2015-01-30

The seaweed Sargassum horneri is an important brown alga in the marine environment, and it is an important raw material in the alginate industry. Unfortunately, the fixed resource that was originally reported is now reduced or disappeared, and increased floating populations have been reported in recent years. We sampled a floating population and 4 fixed cultivated populations of S. horneri along the coast of Zhejiang, China. Inter-simple sequence repeat (ISSR) markers were applied in this research to analyze the genetic variation between floating populations and fixed cultivated populations of S. horneri. In total, 220 loci were amplified with 23 ISSR primers. The percentage of polymorphic loci within each population ranged from 53.64 to 95.45%. The highest diversity was observed in population 3, which was the local species that was suspension cultured in the lab and then fixed cultivated in the Nanji Islands before sampling. The lowest diversity was obtained in the floating population 4. The genetic distances among the 5 S. horneri populations ranged from 0.0819 to 0.2889, and the distance tendency confirmed the genetic diversity. The results suggest that the floating population had the lowest genetic diversity and could not be joined into the cluster branch of the fixed cultivated populations.

Short communication: identification of a novel HIV type 1 subtype H/J recombinant in Canada with discordant HIV viral load (RNA) values in three different commercial assays.

PubMed

Kim, John E; Beckthold, Brenda; Chen, Zhaoxia; Mihowich, Jennifer; Malloch, Laurie; Gill, Michael John

2007-11-01

The presence of HIV-1 non-B subtypes is increasing worldwide. This poses challenges to commercial diagnostic and viral load (RNA) monitoring tests that are predominantly based on HIV-1 subtype B strains. Based on phylogenetic analysis of the gag, pol, and env gene regions, we describe the first HIV-1 H/J recombinant in Canada that presented divergent viral load values. DNA sequence analysis of the gag gene region further revealed that genetic diversity between this H/J recombinant and the primers and probes used in the bio-Merieux Nuclisens HIV-1 QT (Nuclisens) and Roche Amplicor Monitor HIV-1, v1.5 (Monitor) viral RNA assays can erroneously lead to undetectable viral load values. This observation appears to be more problematic in the Nuclisens assay. In light of increasing genetic diversity in HIV worldwide we recommend that DNA sequencing of HIV, especially in the gag gene region targeted by primers and probes used in molecular diagnostic and viral load tests, be incorporated into clinical monitoring practices.

Genetic diversity analysis of Leuconostoc mesenteroides from Korean vegetables and food products by multilocus sequence typing.

PubMed

Sharma, Anshul; Kaur, Jasmine; Lee, Sulhee; Park, Young-Seo

2018-06-01

In the present study, 35 Leuconostoc mesenteroides strains isolated from vegetables and food products from South Korea were studied by multilocus sequence typing (MLST) of seven housekeeping genes (atpA, groEL, gyrB, pheS, pyrG, rpoA, and uvrC). The fragment sizes of the seven amplified housekeeping genes ranged in length from 366 to 1414 bp. Sequence analysis indicated 27 different sequence types (STs) with 25 of them being represented by a single strain indicating high genetic diversity, whereas the remaining 2 were characterized by five strains each. In total, 220 polymorphic nucleotide sites were detected among seven housekeeping genes. The phylogenetic analysis based on the STs of the seven loci indicated that the 35 strains belonged to two major groups, A (28 strains) and B (7 strains). Split decomposition analysis showed that intraspecies recombination played a role in generating diversity among strains. The minimum spanning tree showed that the evolution of the STs was not correlated with food source. This study signifies that the multilocus sequence typing is a valuable tool to access the genetic diversity among L. mesenteroides strains from South Korea and can be used further to monitor the evolutionary changes.

Genetic diversity assessment of anoxygenic photosynthetic bacteria by distance-based grouping analysis of pufM sequences.

PubMed

Zeng, Y H; Chen, X H; Jiao, N Z

2007-12-01

To assess how completely the diversity of anoxygenic phototrophic bacteria (APB) was sampled in natural environments. All nucleotide sequences of the APB marker gene pufM from cultures and environmental clones were retrieved from the GenBank database. A set of cutoff values (sequence distances 0.06, 0.15 and 0.48 for species, genus, and (sub)phylum levels, respectively) was established using a distance-based grouping program. Analysis of the environmental clones revealed that current efforts on APB isolation and sampling in natural environments are largely inadequate. Analysis of the average distance between each identified genus and an uncultured environmental pufM sequence indicated that the majority of cultured APB genera lack environmental representatives. The distance-based grouping method is fast and efficient for bulk functional gene sequences analysis. The results clearly show that we are at a relatively early stage in sampling the global richness of APB species. Periodical assessment will undoubtedly facilitate in-depth analysis of potential biogeographical distribution pattern of APB. This is the first attempt to assess the present understanding of APB diversity in natural environments. The method used is also useful for assessing the diversity of other functional genes.

Deep Sequencing of the Trypanosoma cruzi GP63 Surface Proteases Reveals Diversity and Diversifying Selection among Chronic and Congenital Chagas Disease Patients

PubMed Central

Llewellyn, Martin S.; Messenger, Louisa A.; Luquetti, Alejandro O.; Garcia, Lineth; Torrico, Faustino; Tavares, Suelene B. N.; Cheaib, Bachar; Derome, Nicolas; Delepine, Marc; Baulard, Céline; Deleuze, Jean-Francois; Sauer, Sascha; Miles, Michael A.

2015-01-01

Background Chagas disease results from infection with the diploid protozoan parasite Trypanosoma cruzi. T. cruzi is highly genetically diverse, and multiclonal infections in individual hosts are common, but little studied. In this study, we explore T. cruzi infection multiclonality in the context of age, sex and clinical profile among a cohort of chronic patients, as well as paired congenital cases from Cochabamba, Bolivia and Goias, Brazil using amplicon deep sequencing technology. Methodology/ Principal Findings A 450bp fragment of the trypomastigote TcGP63I surface protease gene was amplified and sequenced across 70 chronic and 22 congenital cases on the Illumina MiSeq platform. In addition, a second, mitochondrial target—ND5—was sequenced across the same cohort of cases. Several million reads were generated, and sequencing read depths were normalized within patient cohorts (Goias chronic, n = 43, Goias congenital n = 2, Bolivia chronic, n = 27; Bolivia congenital, n = 20), Among chronic cases, analyses of variance indicated no clear correlation between intra-host sequence diversity and age, sex or symptoms, while principal coordinate analyses showed no clustering by symptoms between patients. Between congenital pairs, we found evidence for the transmission of multiple sequence types from mother to infant, as well as widespread instances of novel genotypes in infants. Finally, non-synonymous to synonymous (dn:ds) nucleotide substitution ratios among sequences of TcGP63Ia and TcGP63Ib subfamilies within each cohort provided powerful evidence of strong diversifying selection at this locus. Conclusions/Significance Our results shed light on the diversity of parasite DTUs within each patient, as well as the extent to which parasite strains pass between mother and foetus in congenital cases. Although we were unable to find any evidence that parasite diversity accumulates with age in our study cohorts, putative diversifying selection within members of the TcGP63I gene family suggests a link between genetic diversity within this gene family and survival in the mammalian host. PMID:25849488

Population-genomic variation within RNA viruses of the Western honey bee, Apis mellifera, inferred from deep sequencing

PubMed Central

2013-01-01

Background Deep sequencing of viruses isolated from infected hosts is an efficient way to measure population-genetic variation and can reveal patterns of dispersal and natural selection. In this study, we mined existing Illumina sequence reads to investigate single-nucleotide polymorphisms (SNPs) within two RNA viruses of the Western honey bee (Apis mellifera), deformed wing virus (DWV) and Israel acute paralysis virus (IAPV). All viral RNA was extracted from North American samples of honey bees or, in one case, the ectoparasitic mite Varroa destructor. Results Coverage depth was generally lower for IAPV than DWV, and marked gaps in coverage occurred in several narrow regions (< 50 bp) of IAPV. These coverage gaps occurred across sequencing runs and were virtually unchanged when reads were re-mapped with greater permissiveness (up to 8% divergence), suggesting a recurrent sequencing artifact rather than strain divergence. Consensus sequences of DWV for each sample showed little phylogenetic divergence, low nucleotide diversity, and strongly negative values of Fu and Li’s D statistic, suggesting a recent population bottleneck and/or purifying selection. The Kakugo strain of DWV fell outside of all other DWV sequences at 100% bootstrap support. IAPV consensus sequences supported the existence of multiple clades as had been previously reported, and Fu and Li’s D was closer to neutral expectation overall, although a sliding-window analysis identified a significantly positive D within the protease region, suggesting selection maintains diversity in that region. Within-sample mean diversity was comparable between the two viruses on average, although for both viruses there was substantial variation among samples in mean diversity at third codon positions and in the number of high-diversity sites. FST values were bimodal for DWV, likely reflecting neutral divergence in two low-diversity populations, whereas IAPV had several sites that were strong outliers with very low FST. Conclusions This initial survey of genetic variation within honey bee RNA viruses suggests future directions for studies examining the underlying causes of population-genetic structure in these economically important pathogens. PMID:23497218

Population-genomic variation within RNA viruses of the Western honey bee, Apis mellifera, inferred from deep sequencing.

PubMed

Cornman, Robert Scott; Boncristiani, Humberto; Dainat, Benjamin; Chen, Yanping; vanEngelsdorp, Dennis; Weaver, Daniel; Evans, Jay D

2013-03-07

Deep sequencing of viruses isolated from infected hosts is an efficient way to measure population-genetic variation and can reveal patterns of dispersal and natural selection. In this study, we mined existing Illumina sequence reads to investigate single-nucleotide polymorphisms (SNPs) within two RNA viruses of the Western honey bee (Apis mellifera), deformed wing virus (DWV) and Israel acute paralysis virus (IAPV). All viral RNA was extracted from North American samples of honey bees or, in one case, the ectoparasitic mite Varroa destructor. Coverage depth was generally lower for IAPV than DWV, and marked gaps in coverage occurred in several narrow regions (< 50 bp) of IAPV. These coverage gaps occurred across sequencing runs and were virtually unchanged when reads were re-mapped with greater permissiveness (up to 8% divergence), suggesting a recurrent sequencing artifact rather than strain divergence. Consensus sequences of DWV for each sample showed little phylogenetic divergence, low nucleotide diversity, and strongly negative values of Fu and Li's D statistic, suggesting a recent population bottleneck and/or purifying selection. The Kakugo strain of DWV fell outside of all other DWV sequences at 100% bootstrap support. IAPV consensus sequences supported the existence of multiple clades as had been previously reported, and Fu and Li's D was closer to neutral expectation overall, although a sliding-window analysis identified a significantly positive D within the protease region, suggesting selection maintains diversity in that region. Within-sample mean diversity was comparable between the two viruses on average, although for both viruses there was substantial variation among samples in mean diversity at third codon positions and in the number of high-diversity sites. FST values were bimodal for DWV, likely reflecting neutral divergence in two low-diversity populations, whereas IAPV had several sites that were strong outliers with very low FST. This initial survey of genetic variation within honey bee RNA viruses suggests future directions for studies examining the underlying causes of population-genetic structure in these economically important pathogens.

Biochar amendment decreases soil microbial biomass and increases bacterial diversity in Moso bamboo (Phyllostachys edulis) plantations under simulated nitrogen deposition

NASA Astrophysics Data System (ADS)

Li, Quan; Lei, Zhaofeng; Song, Xinzhang; Zhang, Zhiting; Ying, Yeqing; Peng, Changhui

2018-04-01

Biochar amendment has been proposed as a strategy to improve acidic soils after overuse of nitrogen fertilizers. However, little is known of the role of biochar in soil microbial biomass carbon (MBC) and bacterial community structure and diversity after soil acidification induced by nitrogen (N) deposition. Using high-throughput sequencing of the 16S rRNA gene, we determined the effects of biochar amendment (BC0, 0 t bamboo biochar ha‑1 BC20, 20 t bamboo biochar ha‑1 and BC40, 40 t bamboo biochar ha‑1) on the soil bacterial community structure and diversity in Moso bamboo plantations that had received simulated N deposition (N30, 30 kg N ha‑1 yr‑1 N60, 60 kg N ha‑1 yr‑1 N90, 90 kg N ha‑1 yr‑1 and N-free) for 21 months. After treatment of N-free plots, BC20 significantly increased soil MBC and bacterial diversity, while BC40 significantly decreased soil MBC but increased bacterial diversity. When used to amend N30 and N60 plots, biochar significantly decreased soil MBC and the reducing effect increased with biochar amendment amount. However, these significant effects were not observed in N90 plots. Under N deposition, biochar amendment largely increased soil bacterial diversity, and these effects depended on the rates of N deposition and biochar amendment. Soil bacterial diversity was significantly related to the soil C/N ratio, pH, and soil organic carbon content. These findings suggest an optimal approach for using biochar to offset the effects of N deposition in plantation soils and provide a new perspective for understanding the potential role of biochar amendments in plantation soil.

A Web-Based Monitoring System for Multidisciplinary Design Projects

NASA Technical Reports Server (NTRS)

Rogers, James L.; Salas, Andrea O.; Weston, Robert P.

1998-01-01

In today's competitive environment, both industry and government agencies are under pressure to reduce the time and cost of multidisciplinary design projects. New tools have been introduced to assist in this process by facilitating the integration of and communication among diverse disciplinary codes. One such tool, a framework for multidisciplinary computational environments, is defined as a hardware and software architecture that enables integration, execution, and communication among diverse disciplinary processes. An examination of current frameworks reveals weaknesses in various areas, such as sequencing, displaying, monitoring, and controlling the design process. The objective of this research is to explore how Web technology, integrated with an existing framework, can improve these areas of weakness. This paper describes a Web-based system that optimizes and controls the execution sequence of design processes; and monitors the project status and results. The three-stage evolution of the system with increasingly complex problems demonstrates the feasibility of this approach.

The Intestinal Eukaryotic and Bacterial Biome of Spotted Hyenas: The Impact of Social Status and Age on Diversity and Composition.

PubMed

Heitlinger, Emanuel; Ferreira, Susana C M; Thierer, Dagmar; Hofer, Heribert; East, Marion L

2017-01-01

In mammals, two factors likely to affect the diversity and composition of intestinal bacteria (bacterial microbiome) and eukaryotes (eukaryome) are social status and age. In species in which social status determines access to resources, socially dominant animals maintain better immune processes and health status than subordinates. As high species diversity is an index of ecosystem health, the intestinal biome of healthier, socially dominant animals should be more diverse than those of subordinates. Gradual colonization of the juvenile intestine after birth predicts lower intestinal biome diversity in juveniles than adults. We tested these predictions on the effect of: (1) age (juvenile/adult) and (2) social status (low/high) on bacterial microbiome and eukaryome diversity and composition in the spotted hyena ( Crocuta crocuta ), a highly social, female-dominated carnivore in which social status determines access to resources. We comprehensively screened feces from 35 individually known adult females and 7 juveniles in the Serengeti ecosystem for bacteria and eukaryotes, using a set of 48 different amplicons (4 for bacterial 16S, 44 for eukaryote 18S) in a multi-amplicon sequencing approach. We compared sequence abundances to classical coprological egg or oocyst counts. For all parasite taxa detected in more than six samples, the number of sequence reads significantly predicted the number of eggs or oocysts counted, underscoring the value of an amplicon sequencing approach for quantitative measurements of parasite load. In line with our predictions, our results revealed a significantly less diverse microbiome in juveniles than adults and a significantly higher diversity of eukaryotes in high-ranking than low-ranking animals. We propose that free-ranging wildlife can provide an intriguing model system to assess the adaptive value of intestinal biome diversity for both bacteria and eukaryotes.

The Intestinal Eukaryotic and Bacterial Biome of Spotted Hyenas: The Impact of Social Status and Age on Diversity and Composition

PubMed Central

Heitlinger, Emanuel; Ferreira, Susana C. M.; Thierer, Dagmar; Hofer, Heribert; East, Marion L.

2017-01-01

In mammals, two factors likely to affect the diversity and composition of intestinal bacteria (bacterial microbiome) and eukaryotes (eukaryome) are social status and age. In species in which social status determines access to resources, socially dominant animals maintain better immune processes and health status than subordinates. As high species diversity is an index of ecosystem health, the intestinal biome of healthier, socially dominant animals should be more diverse than those of subordinates. Gradual colonization of the juvenile intestine after birth predicts lower intestinal biome diversity in juveniles than adults. We tested these predictions on the effect of: (1) age (juvenile/adult) and (2) social status (low/high) on bacterial microbiome and eukaryome diversity and composition in the spotted hyena (Crocuta crocuta), a highly social, female-dominated carnivore in which social status determines access to resources. We comprehensively screened feces from 35 individually known adult females and 7 juveniles in the Serengeti ecosystem for bacteria and eukaryotes, using a set of 48 different amplicons (4 for bacterial 16S, 44 for eukaryote 18S) in a multi-amplicon sequencing approach. We compared sequence abundances to classical coprological egg or oocyst counts. For all parasite taxa detected in more than six samples, the number of sequence reads significantly predicted the number of eggs or oocysts counted, underscoring the value of an amplicon sequencing approach for quantitative measurements of parasite load. In line with our predictions, our results revealed a significantly less diverse microbiome in juveniles than adults and a significantly higher diversity of eukaryotes in high-ranking than low-ranking animals. We propose that free-ranging wildlife can provide an intriguing model system to assess the adaptive value of intestinal biome diversity for both bacteria and eukaryotes. PMID:28670573

Measuring the diversity of the human microbiota with targeted next-generation sequencing.

PubMed

Finotello, Francesca; Mastrorilli, Eleonora; Di Camillo, Barbara

2016-12-26

The human microbiota is a complex ecological community of commensal, symbiotic and pathogenic microorganisms harboured by the human body. Next-generation sequencing (NGS) technologies, in particular targeted amplicon sequencing of the 16S ribosomal RNA gene (16S-seq), are enabling the identification and quantification of human-resident microorganisms at unprecedented resolution, providing novel insights into the role of the microbiota in health and disease. Once microbial abundances are quantified through NGS data analysis, diversity indices provide valuable mathematical tools to describe the ecological complexity of a single sample or to detect species differences between samples. However, diversity is not a determined physical quantity for which a consensus definition and unit of measure have been established, and several diversity indices are currently available. Furthermore, they were originally developed for macroecology and their robustness to the possible bias introduced by sequencing has not been characterized so far. To assist the reader with the selection and interpretation of diversity measures, we review a panel of broadly used indices, describing their mathematical formulations, purposes and properties, and characterize their behaviour and criticalities in dependence of the data features using simulated data as ground truth. In addition, we make available an R package, DiversitySeq, which implements in a unified framework the full panel of diversity indices and a simulator of 16S-seq data, and thus represents a valuable resource for the analysis of diversity from NGS count data and for the benchmarking of computational methods for 16S-seq. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Microbial diversity of the hypersaline and lithium-rich Salar de Uyuni, Bolivia.

PubMed

Haferburg, Götz; Gröning, Janosch A D; Schmidt, Nadja; Kummer, Nicolai-Alexeji; Erquicia, Juan Carlos; Schlömann, Michael

2017-06-01

Salar de Uyuni, situated in the Southwest of the Bolivian Altiplano, is the largest salt flat on Earth. Brines of this athalassohaline hypersaline environment are rich in lithium and boron. Due to the ever- increasing commodity demand, the industrial exploitation of brines for metal recovery from the world's biggest lithium reservoir is likely to increase substantially in the near future. Studies on the composition of halophilic microbial communities in brines of the salar have not been published yet. Here we report for the first time on the prokaryotic diversity of four brine habitats across the salar. The brine is characterized by salinity values between 132 and 177 PSU, slightly acidic to near-neutral pH and lithium and boron concentrations of up to 2.0 and 1.4g/L, respectively. Community analysis was performed after sequencing the V3-V4 region of the 16S rRNA genes employing the Illumina MiSeq technology. The mothur software package was used for sequence processing and data analysis. Metagenomic analysis revealed the occurrence of an exclusively archaeal community comprising 26 halobacterial genera including only recently identified genera like Halapricum, Halorubellus and Salinarchaeum. Despite the high diversity of the halobacteria-dominated community in sample P3 (Shannon-Weaver index H'=3.12 at 3% OTU cutoff) almost 40% of the Halobacteriaceae-assigned sequences could not be classified on the genus level under stringent filtering conditions. Even if the limited taxonomic resolution of the V3-V4 region for halobacteria is considered, it seems likely to discover new, hitherto undescribed genera of the family halobacteriaceae in this particular habitat of Salar de Uyuni in future. Copyright © 2017 Elsevier GmbH. All rights reserved.

Assessing the impact of water treatment on bacterial biofilms in drinking water distribution systems using high-throughput DNA sequencing.

PubMed

Shaw, Jennifer L A; Monis, Paul; Fabris, Rolando; Ho, Lionel; Braun, Kalan; Drikas, Mary; Cooper, Alan

2014-12-01

Biofilm control in drinking water distribution systems (DWDSs) is crucial, as biofilms are known to reduce flow efficiency, impair taste and quality of drinking water and have been implicated in the transmission of harmful pathogens. Microorganisms within biofilm communities are more resistant to disinfection compared to planktonic microorganisms, making them difficult to manage in DWDSs. This study evaluates the impact of four unique drinking water treatments on biofilm community structure using metagenomic DNA sequencing. Four experimental DWDSs were subjected to the following treatments: (1) conventional coagulation, (2) magnetic ion exchange contact (MIEX) plus conventional coagulation, (3) MIEX plus conventional coagulation plus granular activated carbon, and (4) membrane filtration (MF). Bacterial biofilms located inside the pipes of each system were sampled under sterile conditions both (a) immediately after treatment application ('inlet') and (b) at a 1 km distance from the treatment application ('outlet'). Bacterial 16S rRNA gene sequencing revealed that the outlet biofilms were more diverse than those sampled at the inlet for all treatments. The lowest number of unique operational taxonomic units (OTUs) and lowest diversity was observed in the MF inlet. However, the MF system revealed the greatest increase in diversity and OTU count from inlet to outlet. Further, the biofilm communities at the outlet of each system were more similar to one another than to their respective inlet, suggesting that biofilm communities converge towards a common established equilibrium as distance from treatment application increases. Based on the results, MF treatment is most effective at inhibiting biofilm growth, but a highly efficient post-treatment disinfection regime is also critical in order to prevent the high rates of post-treatment regrowth. Copyright © 2014 Elsevier Ltd. All rights reserved.

Genetic diversity among pandemic 2009 influenza viruses isolated from a transmission chain

PubMed Central

2013-01-01

Background Influenza viruses such as swine-origin influenza A(H1N1) virus (A(H1N1)pdm09) generate genetic diversity due to the high error rate of their RNA polymerase, often resulting in mixed genotype populations (intra-host variants) within a single infection. This variation helps influenza to rapidly respond to selection pressures, such as those imposed by the immunological host response and antiviral therapy. We have applied deep sequencing to characterize influenza intra-host variation in a transmission chain consisting of three cases due to oseltamivir-sensitive viruses, and one derived oseltamivir-resistant case. Methods Following detection of the A(H1N1)pdm09 infections, we deep-sequenced the complete NA gene from two of the oseltamivir-sensitive virus-infected cases, and all eight gene segments of the viruses causing the remaining two cases. Results No evidence for the resistance-causing mutation (resulting in NA H275Y substitution) was observed in the oseltamivir-sensitive cases. Furthermore, deep sequencing revealed a subpopulation of oseltamivir-sensitive viruses in the case carrying resistant viruses. We detected higher levels of intra-host variation in the case carrying oseltamivir-resistant viruses than in those infected with oseltamivir-sensitive viruses. Conclusions Oseltamivir-resistance was only detected after prophylaxis with oseltamivir, suggesting that the mutation was selected for as a result of antiviral intervention. The persisting oseltamivir-sensitive virus population in the case carrying resistant viruses suggests either that a small proportion survive the treatment, or that the oseltamivir-sensitive virus rapidly re-establishes itself in the virus population after the bottleneck. Moreover, the increased intra-host variation in the oseltamivir-resistant case is consistent with the hypothesis that the population diversity of a RNA virus can increase rapidly following a population bottleneck. PMID:23587185

Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer

PubMed Central

Sanz, Yolanda

2017-01-01

Abstract The miniaturized and portable DNA sequencer MinION™ has demonstrated great potential in different analyses such as genome-wide sequencing, pathogen outbreak detection and surveillance, human genome variability, and microbial diversity. In this study, we tested the ability of the MinION™ platform to perform long amplicon sequencing in order to design new approaches to study microbial diversity using a multi-locus approach. After compiling a robust database by parsing and extracting the rrn bacterial region from more than 67000 complete or draft bacterial genomes, we demonstrated that the data obtained during sequencing of the long amplicon in the MinION™ device using R9 and R9.4 chemistries were sufficient to study 2 mock microbial communities in a multiplex manner and to almost completely reconstruct the microbial diversity contained in the HM782D and D6305 mock communities. Although nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, we presented a novel approach consisting of multi-locus and long amplicon sequencing using the MinION™ MkIb DNA sequencer and R9 and R9.4 chemistries that help to overcome the main disadvantage of this portable sequencing platform. Furthermore, the nanopore sequencing library, constructed with the last releases of pore chemistry (R9.4) and sequencing kit (SQK-LSK108), permitted the retrieval of the higher level of 1D read accuracy sufficient to characterize the microbial species present in each mock community analysed. Improvements in nanopore chemistry, such as minimizing base-calling errors and new library protocols able to produce rapid 1D libraries, will provide more reliable information in the near future. Such data will be useful for more comprehensive and faster specific detection of microbial species and strains in complex ecosystems. PMID:28605506

A national study of the molecular epidemiology of HIV-1 in Australia 2005–2012

PubMed Central

Castley, Alison; Sawleshwarkar, Shailendra; Varma, Rick; Herring, Belinda; Thapa, Kiran; Dwyer, Dominic; Chibo, Doris; Nguyen, Nam; Hawke, Karen; Ratcliff, Rodney; Garsia, Roger; Kelleher, Anthony; Nolan, David

2017-01-01

Introduction Rates of new HIV-1 diagnoses are increasing in Australia, with evidence of an increasing proportion of non-B HIV-1 subtypes reflecting a growing impact of migration and travel. The present study aims to define HIV-1 subtype diversity patterns and investigate possible HIV-1 transmission networks within Australia. Methods The Australian Molecular Epidemiology Network (AMEN) HIV collaborating sites in Western Australia, South Australia, Victoria, Queensland and western Sydney (New South Wales), provided baseline HIV-1 partial pol sequence, age and gender information for 4,873 patients who had genotypes performed during 2005–2012. HIV-1 phylogenetic analyses utilised MEGA V6, with a stringent classification of transmission pairs or clusters (bootstrap ≥98%, genetic distance ≤1.5% from at least one other sequence in the cluster). Results HIV-1 subtype B represented 74.5% of the 4,873 sequences (WA 59%, SA 68.4%, w-Syd 73.8%, Vic 75.6%, Qld 82.1%), with similar proportion of transmission pairs and clusters found in the B and non-B cohorts (23% vs 24.5% of sequences, p = 0.3). Significantly more subtype B clusters were comprised of ≥3 sequences compared with non-B clusters (45.0% vs 24.0%, p = 0.021) and significantly more subtype B pairs and clusters were male-only (88% compared to 53% CRF01_AE and 17% subtype C clusters). Factors associated with being in a cluster of any size included; being sequenced in a more recent time period (p<0.001), being younger (p<0.001), being male (p = 0.023) and having a B subtype (p = 0.02). Being in a larger cluster (>3) was associated with being sequenced in a more recent time period (p = 0.05) and being male (p = 0.008). Conclusion This nationwide HIV-1 study of 4,873 patient sequences highlights the increased diversity of HIV-1 subtypes within the Australian epidemic, as well as differences in transmission networks associated with these HIV-1 subtypes. These findings provide epidemiological insights not readily available using standard surveillance methods and can inform the development of effective public health strategies in the current paradigm of HIV prevention in Australia. PMID:28489920

A “Rosetta Stone” for metazoan zooplankton: DNA barcode analysis of species diversity of the Sargasso Sea (Northwest Atlantic Ocean)

NASA Astrophysics Data System (ADS)

Bucklin, Ann; Ortman, Brian D.; Jennings, Robert M.; Nigro, Lisa M.; Sweetman, Christopher J.; Copley, Nancy J.; Sutton, Tracey; Wiebe, Peter H.

2010-12-01

Species diversity of the metazoan holozooplankton assemblage of the Sargasso Sea, Northwest Atlantic Ocean, was examined through coordinated morphological taxonomic identification of species and DNA sequencing of a ˜650 base-pair region of mitochondrial cytochrome oxidase I (mtCOI) as a DNA barcode (i.e., short sequence for species recognition and discrimination). Zooplankton collections were made from the surface to 5,000 meters during April, 2006 on the R/V R.H. Brown. Samples were examined by a ship-board team of morphological taxonomists; DNA barcoding was carried out in both ship-board and land-based DNA sequencing laboratories. DNA barcodes were determined for a total of 297 individuals of 175 holozooplankton species in four phyla, including: Cnidaria (Hydromedusae, 4 species; Siphonophora, 47); Arthropoda (Amphipoda, 10; Copepoda, 34; Decapoda, 9; Euphausiacea, 10; Mysidacea, 1; Ostracoda, 27); and Mollusca (Cephalopoda, 8; Heteropoda, 6; Pteropoda, 15); and Chaetognatha (4). Thirty species of fish (Teleostei) were also barcoded. For all seven zooplankton groups for which sufficient data were available, Kimura-2-Parameter genetic distances were significantly lower between individuals of the same species (mean=0.0114; S.D. 0.0117) than between individuals of different species within the same group (mean=0.3166; S.D. 0.0378). This difference, known as the barcode gap, ensures that mtCOI sequences are reliable characters for species identification for the oceanic holozooplankton assemblage. In addition, DNA barcodes allow recognition of new or undescribed species, reveal cryptic species within known taxa, and inform phylogeographic and population genetic studies of geographic variation. The growing database of "gold standard" DNA barcodes serves as a Rosetta Stone for marine zooplankton, providing the key for decoding species diversity by linking species names, morphology, and DNA sequence variation. In light of the pivotal position of zooplankton in ocean food webs, their usefulness as rapid responders to environmental change, and the increasing scarcity of taxonomists, the use of DNA barcodes is an important and useful approach for rapid analysis of species diversity and distribution in the pelagic community.

PanFP: Pangenome-based functional profiles for microbial communities

DOE PAGES

Jun, Se -Ran; Hauser, Loren John; Schadt, Christopher Warren; ...

2015-09-26

For decades there has been increasing interest in understanding the relationships between microbial communities and ecosystem functions. Current DNA sequencing technologies allows for the exploration of microbial communities in two principle ways: targeted rRNA gene surveys and shotgun metagenomics. For large study designs, it is often still prohibitively expensive to sequence metagenomes at both the breadth and depth necessary to statistically capture the true functional diversity of a community. Although rRNA gene surveys provide no direct evidence of function, they do provide a reasonable estimation of microbial diversity, while being a very cost effective way to screen samples of interestmore » for later shotgun metagenomic analyses. However, there is a great deal of 16S rRNA gene survey data currently available from diverse environments, and thus a need for tools to infer functional composition of environmental samples based on 16S rRNA gene survey data. As a result, we present a computational method called pangenome based functional profiles (PanFP), which infers functional profiles of microbial communities from 16S rRNA gene survey data for Bacteria and Archaea. PanFP is based on pangenome reconstruction of a 16S rRNA gene operational taxonomic unit (OTU) from known genes and genomes pooled from the OTU s taxonomic lineage. From this lineage, we derive an OTU functional profile by weighting a pangenome s functional profile with the OTUs abundance observed in a given sample. We validated our method by comparing PanFP to the functional profiles obtained from the direct shotgun metagenomic measurement of 65 diverse communities via Spearman correlation coefficients. These correlations improved with increasing sequencing depth, within the range of 0.8 0.9 for the most deeply sequenced Human Microbiome Project mock community samples. PanFP is very similar in performance to another recently released tool, PICRUSt, for almost all of survey data analysed here. But, our method is unique in that any OTU building method can be used, as opposed to being limited to closed reference OTU picking strategies against specific reference sequence databases. In conclusion, we developed an automated computational method, which derives an inferred functional profile based on the 16S rRNA gene surveys of microbial communities. The inferred functional profile provides a cost effective way to study complex ecosystems through predicted comparative functional metagenomes and metadata analysis. All PanFP source code and additional documentation are freely available online at GitHub.« less

PanFP: pangenome-based functional profiles for microbial communities.

PubMed

Jun, Se-Ran; Robeson, Michael S; Hauser, Loren J; Schadt, Christopher W; Gorin, Andrey A

2015-09-26

For decades there has been increasing interest in understanding the relationships between microbial communities and ecosystem functions. Current DNA sequencing technologies allows for the exploration of microbial communities in two principle ways: targeted rRNA gene surveys and shotgun metagenomics. For large study designs, it is often still prohibitively expensive to sequence metagenomes at both the breadth and depth necessary to statistically capture the true functional diversity of a community. Although rRNA gene surveys provide no direct evidence of function, they do provide a reasonable estimation of microbial diversity, while being a very cost-effective way to screen samples of interest for later shotgun metagenomic analyses. However, there is a great deal of 16S rRNA gene survey data currently available from diverse environments, and thus a need for tools to infer functional composition of environmental samples based on 16S rRNA gene survey data. We present a computational method called pangenome-based functional profiles (PanFP), which infers functional profiles of microbial communities from 16S rRNA gene survey data for Bacteria and Archaea. PanFP is based on pangenome reconstruction of a 16S rRNA gene operational taxonomic unit (OTU) from known genes and genomes pooled from the OTU's taxonomic lineage. From this lineage, we derive an OTU functional profile by weighting a pangenome's functional profile with the OTUs abundance observed in a given sample. We validated our method by comparing PanFP to the functional profiles obtained from the direct shotgun metagenomic measurement of 65 diverse communities via Spearman correlation coefficients. These correlations improved with increasing sequencing depth, within the range of 0.8-0.9 for the most deeply sequenced Human Microbiome Project mock community samples. PanFP is very similar in performance to another recently released tool, PICRUSt, for almost all of survey data analysed here. But, our method is unique in that any OTU building method can be used, as opposed to being limited to closed-reference OTU picking strategies against specific reference sequence databases. We developed an automated computational method, which derives an inferred functional profile based on the 16S rRNA gene surveys of microbial communities. The inferred functional profile provides a cost effective way to study complex ecosystems through predicted comparative functional metagenomes and metadata analysis. All PanFP source code and additional documentation are freely available online at GitHub ( https://github.com/srjun/PanFP ).

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jun, Se -Ran; Hauser, Loren John; Schadt, Christopher Warren

For decades there has been increasing interest in understanding the relationships between microbial communities and ecosystem functions. Current DNA sequencing technologies allows for the exploration of microbial communities in two principle ways: targeted rRNA gene surveys and shotgun metagenomics. For large study designs, it is often still prohibitively expensive to sequence metagenomes at both the breadth and depth necessary to statistically capture the true functional diversity of a community. Although rRNA gene surveys provide no direct evidence of function, they do provide a reasonable estimation of microbial diversity, while being a very cost effective way to screen samples of interestmore » for later shotgun metagenomic analyses. However, there is a great deal of 16S rRNA gene survey data currently available from diverse environments, and thus a need for tools to infer functional composition of environmental samples based on 16S rRNA gene survey data. As a result, we present a computational method called pangenome based functional profiles (PanFP), which infers functional profiles of microbial communities from 16S rRNA gene survey data for Bacteria and Archaea. PanFP is based on pangenome reconstruction of a 16S rRNA gene operational taxonomic unit (OTU) from known genes and genomes pooled from the OTU s taxonomic lineage. From this lineage, we derive an OTU functional profile by weighting a pangenome s functional profile with the OTUs abundance observed in a given sample. We validated our method by comparing PanFP to the functional profiles obtained from the direct shotgun metagenomic measurement of 65 diverse communities via Spearman correlation coefficients. These correlations improved with increasing sequencing depth, within the range of 0.8 0.9 for the most deeply sequenced Human Microbiome Project mock community samples. PanFP is very similar in performance to another recently released tool, PICRUSt, for almost all of survey data analysed here. But, our method is unique in that any OTU building method can be used, as opposed to being limited to closed reference OTU picking strategies against specific reference sequence databases. In conclusion, we developed an automated computational method, which derives an inferred functional profile based on the 16S rRNA gene surveys of microbial communities. The inferred functional profile provides a cost effective way to study complex ecosystems through predicted comparative functional metagenomes and metadata analysis. All PanFP source code and additional documentation are freely available online at GitHub.« less

Complete sequence and diversity of a maize-associated Polerovirus in East Africa

USDA-ARS?s Scientific Manuscript database

Since 2011-2012, Maize lethal necrosis (MLN) has emerged in East Africa, causing massive yield loss and propelling research to identify viruses and virus populations present in maize. As expected, next generation sequencing (NGS) has revealed diverse and abundant viruses from the family Potyviridae,...

The complete genome sequences of 65 Campylobacter jejuni and C. coli strains

USDA-ARS?s Scientific Manuscript database

Campylobacter jejuni (Cj) and C. coli (Cc) are genetically highly diverse based on various molecular methods including MLST, microarray-based comparisons and the whole genome sequences of a few strains. Cj and Cc diversity is also exhibited by variable capsular polysaccharides (CPS) that are the maj...

Maize HapMap2 identifies extant variation from a genome in flux

USDA-ARS?s Scientific Manuscript database

The maize genome is the largest, most diverse and complex plant genome sequenced to date. Using high-throughput sequencing to access genetic variation and a population genetics model to score the polymorphisms, we characterize and unite the diversity of the world’s key breeding germplasm, wild rela...

High throughput SNP discovery and genotyping in grapevine (Vitis vinifera L.) by combining a re-sequencing approach and SNPlex technology

PubMed Central

Lijavetzky, Diego; Cabezas, José Antonio; Ibáñez, Ana; Rodríguez, Virginia; Martínez-Zapater, José M

2007-01-01

Background Single-nucleotide polymorphisms (SNPs) are the most abundant type of DNA sequence polymorphisms. Their higher availability and stability when compared to simple sequence repeats (SSRs) provide enhanced possibilities for genetic and breeding applications such as cultivar identification, construction of genetic maps, the assessment of genetic diversity, the detection of genotype/phenotype associations, or marker-assisted breeding. In addition, the efficiency of these activities can be improved thanks to the ease with which SNP genotyping can be automated. Expressed sequence tags (EST) sequencing projects in grapevine are allowing for the in silico detection of multiple putative sequence polymorphisms within and among a reduced number of cultivars. In parallel, the sequence of the grapevine cultivar Pinot Noir is also providing thousands of polymorphisms present in this highly heterozygous genome. Still the general application of those SNPs requires further validation since their use could be restricted to those specific genotypes. Results In order to develop a large SNP set of wide application in grapevine we followed a systematic re-sequencing approach in a group of 11 grape genotypes corresponding to ancient unrelated cultivars as well as wild plants. Using this approach, we have sequenced 230 gene fragments, what represents the analysis of over 1 Mb of grape DNA sequence. This analysis has allowed the discovery of 1573 SNPs with an average of one SNP every 64 bp (one SNP every 47 bp in non-coding regions and every 69 bp in coding regions). Nucleotide diversity in grape (π = 0.0051) was found to be similar to values observed in highly polymorphic plant species such as maize. The average number of haplotypes per gene sequence was estimated as six, with three haplotypes representing over 83% of the analyzed sequences. Short-range linkage disequilibrium (LD) studies within the analyzed sequences indicate the existence of a rapid decay of LD within the selected grapevine genotypes. To validate the use of the detected polymorphisms in genetic mapping, cultivar identification and genetic diversity studies we have used the SNPlex™ genotyping technology in a sample of grapevine genotypes and segregating progenies. Conclusion These results provide accurate values for nucleotide diversity in coding sequences and a first estimate of short-range LD in grapevine. Using SNPlex™ genotyping we have shown the application of a set of discovered SNPs as molecular markers for cultivar identification, linkage mapping and genetic diversity studies. Thus, the combination a highly efficient re-sequencing approach and the SNPlex™ high throughput genotyping technology provide a powerful tool for grapevine genetic analysis. PMID:18021442

MHC class I diversity in chimpanzees and bonobos.

PubMed

Maibach, Vincent; Hans, Jörg B; Hvilsom, Christina; Marques-Bonet, Tomas; Vigilant, Linda

2017-10-01

Major histocompatibility complex (MHC) class I genes are critically involved in the defense against intracellular pathogens. MHC diversity comparisons among samples of closely related taxa may reveal traces of past or ongoing selective processes. The bonobo and chimpanzee are the closest living evolutionary relatives of humans and last shared a common ancestor some 1 mya. However, little is known concerning MHC class I diversity in bonobos or in central chimpanzees, the most numerous and genetically diverse chimpanzee subspecies. Here, we used a long-read sequencing technology (PacBio) to sequence the classical MHC class I genes A, B, C, and A-like in 20 and 30 wild-born bonobos and chimpanzees, respectively, with a main focus on central chimpanzees to assess and compare diversity in those two species. We describe in total 21 and 42 novel coding region sequences for the two species, respectively. In addition, we found evidence for a reduced MHC class I diversity in bonobos as compared to central chimpanzees as well as to western chimpanzees and humans. The reduced bonobo MHC class I diversity may be the result of a selective process in their evolutionary past since their split from chimpanzees.

Effect of the natural arsenic gradient on the diversity and arsenic resistance of bacterial communities of the sediments of Camarones River (Atacama Desert, Chile)

PubMed Central

Leon, Carla G.; Moraga, Ruben; Valenzuela, Cristian; Gugliandolo, Concetta; Lo Giudice, Angelina; Papale, Maria; Vilo, Claudia; Dong, Qunfeng; Smith, Carlos T.; Rossello-Mora, Ramon; Yañez, Jorge

2018-01-01

Arsenic (As), a highly toxic metalloid, naturally present in Camarones River (Atacama Desert, Chile) is a great health concern for the local population and authorities. In this study, the taxonomic and functional characterization of bacterial communities associated to metal-rich sediments from three sites of the river (sites M1, M2 and M3), showing different arsenic concentrations, were evaluated using a combination of approaches. Diversity of bacterial communities was evaluated by Illumina sequencing. Strains resistant to arsenic concentrations varying from 0.5 to 100 mM arsenite or arsenate were isolated and the presence of genes coding for enzymes involved in arsenic oxidation (aio) or reduction (arsC) investigated. Bacterial communities showed a moderate diversity which increased as arsenic concentrations decreased along the river. Sequences of the dominant taxonomic groups (abundances ≥1%) present in all three sites were affiliated to Proteobacteria (range 40.3–47.2%), Firmicutes (8.4–24.8%), Acidobacteria (10.4–17.1%), Actinobacteria (5.4–8.1%), Chloroflexi (3.9–7.5%), Planctomycetes (1.2–5.3%), Gemmatimonadetes (1.2–1.5%), and Nitrospirae (1.1–1.2%). Bacterial communities from sites M2 and M3 showed no significant differences in diversity between each other (p = 0.9753) but they were significantly more diverse than M1 (p<0.001 and p<0.001, respectively). Sequences affiliated with Proteobacteria, Firmicutes, Acidobacteria, Chloroflexi and Actinobacteria at M1 accounted for more than 89% of the total classified bacterial sequences present but these phyla were present in lesser proportions in M2 and M3 sites. Strains isolated from the sediment of sample M1, having the greatest arsenic concentration (498 mg kg-1), showed the largest percentages of arsenic oxidation and reduction. Genes aio were more frequently detected in isolates from M1 (54%), whereas arsC genes were present in almost all isolates from all three sediments, suggesting that bacterial communities play an important role in the arsenic biogeochemical cycle and detoxification of arsenical compounds. Overall, results provide further knowledge on the microbial diversity of arsenic contaminated fresh-water sediments. PMID:29715297

Novel molecular markers of Chlamydia pecorum genetic diversity in the koala (Phascolarctos cinereus)

PubMed Central

2011-01-01

Background Chlamydia pecorum is an obligate intracellular bacterium and the causative agent of reproductive and ocular disease in several animal hosts including koalas, sheep, cattle and goats. C. pecorum strains detected in koalas are genetically diverse, raising interesting questions about the origin and transmission of this species within koala hosts. While the ompA gene remains the most widely-used target in C. pecorum typing studies, it is generally recognised that surface protein encoding genes are not suited for phylogenetic analysis and it is becoming increasingly apparent that the ompA gene locus is not congruent with the phylogeny of the C. pecorum genome. Using the recently sequenced C. pecorum genome sequence (E58), we analysed 10 genes, including ompA, to evaluate the use of ompA as a molecular marker in the study of koala C. pecorum genetic diversity. Results Three genes (incA, ORF663, tarP) were found to contain sufficient nucleotide diversity and discriminatory power for detailed analysis and were used, with ompA, to genotype 24 C. pecorum PCR-positive koala samples from four populations. The most robust representation of the phylogeny of these samples was achieved through concatenation of all four gene sequences, enabling the recreation of a "true" phylogenetic signal. OmpA and incA were of limited value as fine-detailed genetic markers as they were unable to confer accurate phylogenetic distinctions between samples. On the other hand, the tarP and ORF663 genes were identified as useful "neutral" and "contingency" markers respectively, to represent the broad evolutionary history and intra-species genetic diversity of koala C. pecorum. Furthermore, the concatenation of ompA, incA and ORF663 sequences highlighted the monophyletic nature of koala C. pecorum infections by demonstrating a single evolutionary trajectory for koala hosts that is distinct from that seen in non-koala hosts. Conclusions While the continued use of ompA as a fine-detailed molecular marker for epidemiological analysis appears justified, the tarP and ORF663 genes also appear to be valuable markers of phylogenetic or biogeographic divisions at the C. pecorum intra-species level. This research has significant implications for future typing studies to understand the phylogeny, genetic diversity, and epidemiology of C. pecorum infections in the koala and other animal species. PMID:21496349

Novel molecular markers of Chlamydia pecorum genetic diversity in the koala (Phascolarctos cinereus).

PubMed

Marsh, James; Kollipara, Avinash; Timms, Peter; Polkinghorne, Adam

2011-04-18

Chlamydia pecorum is an obligate intracellular bacterium and the causative agent of reproductive and ocular disease in several animal hosts including koalas, sheep, cattle and goats. C. pecorum strains detected in koalas are genetically diverse, raising interesting questions about the origin and transmission of this species within koala hosts. While the ompA gene remains the most widely-used target in C. pecorum typing studies, it is generally recognised that surface protein encoding genes are not suited for phylogenetic analysis and it is becoming increasingly apparent that the ompA gene locus is not congruent with the phylogeny of the C. pecorum genome. Using the recently sequenced C. pecorum genome sequence (E58), we analysed 10 genes, including ompA, to evaluate the use of ompA as a molecular marker in the study of koala C. pecorum genetic diversity. Three genes (incA, ORF663, tarP) were found to contain sufficient nucleotide diversity and discriminatory power for detailed analysis and were used, with ompA, to genotype 24 C. pecorum PCR-positive koala samples from four populations. The most robust representation of the phylogeny of these samples was achieved through concatenation of all four gene sequences, enabling the recreation of a "true" phylogenetic signal. OmpA and incA were of limited value as fine-detailed genetic markers as they were unable to confer accurate phylogenetic distinctions between samples. On the other hand, the tarP and ORF663 genes were identified as useful "neutral" and "contingency" markers respectively, to represent the broad evolutionary history and intra-species genetic diversity of koala C. pecorum. Furthermore, the concatenation of ompA, incA and ORF663 sequences highlighted the monophyletic nature of koala C. pecorum infections by demonstrating a single evolutionary trajectory for koala hosts that is distinct from that seen in non-koala hosts. While the continued use of ompA as a fine-detailed molecular marker for epidemiological analysis appears justified, the tarP and ORF663 genes also appear to be valuable markers of phylogenetic or biogeographic divisions at the C. pecorum intra-species level. This research has significant implications for future typing studies to understand the phylogeny, genetic diversity, and epidemiology of C. pecorum infections in the koala and other animal species.

High-accuracy identification of incident HIV-1 infections using a sequence clustering based diversity measure.

PubMed

Xia, Xia-Yu; Ge, Meng; Hsi, Jenny H; He, Xiang; Ruan, Yu-Hua; Wang, Zhi-Xin; Shao, Yi-Ming; Pan, Xian-Ming

2014-01-01

Accurate estimates of HIV-1 incidence are essential for monitoring epidemic trends and evaluating intervention efforts. However, the long asymptomatic stage of HIV-1 infection makes it difficult to effectively distinguish incident infections from chronic ones. Current incidence assays based on serology or viral sequence diversity are both still lacking in accuracy. In the present work, a sequence clustering based diversity (SCBD) assay was devised by utilizing the fact that viral sequences derived from each transmitted/founder (T/F) strain tend to cluster together at early stage, and that only the intra-cluster diversity is correlated with the time since HIV-1 infection. The dot-matrix pairwise alignment was used to eliminate the disproportional impact of insertion/deletions (indels) and recombination events, and so was the proportion of clusterable sequences (Pc) as an index to identify late chronic infections with declined viral genetic diversity. Tested on a dataset containing 398 incident and 163 chronic infection cases collected from the Los Alamos HIV database (last modified 2/8/2012), our SCBD method achieved 99.5% sensitivity and 98.8% specificity, with an overall accuracy of 99.3%. Further analysis and evaluation also suggested its performance was not affected by host factors such as the viral subtypes and transmission routes. The SCBD method demonstrated the potential of sequencing based techniques to become useful for identifying incident infections. Its use may be most advantageous for settings with low to moderate incidence relative to available resources. The online service is available at http://www.bioinfo.tsinghua.edu.cn:8080/SCBD/index.jsp.

454 Pyrosequencing to Describe Microbial Eukaryotic Community Composition, Diversity and Relative Abundance: A Test for Marine Haptophytes

PubMed Central

Egge, Elianne; Bittner, Lucie; Andersen, Tom; Audic, Stéphane; de Vargas, Colomban; Edvardsen, Bente

2013-01-01

Next generation sequencing of ribosomal DNA is increasingly used to assess the diversity and structure of microbial communities. Here we test the ability of 454 pyrosequencing to detect the number of species present, and assess the relative abundance in terms of cell numbers and biomass of protists in the phylum Haptophyta. We used a mock community consisting of equal number of cells of 11 haptophyte species and compared targeting DNA and RNA/cDNA, and two different V4 SSU rDNA haptophyte-biased primer pairs. Further, we tested four different bioinformatic filtering methods to reduce errors in the resulting sequence dataset. With sequencing depth of 11000–20000 reads and targeting cDNA with Haptophyta specific primers Hap454 we detected all 11 species. A rarefaction analysis of expected number of species recovered as a function of sampling depth suggested that minimum 1400 reads were required here to recover all species in the mock community. Relative read abundance did not correlate to relative cell numbers. Although the species represented with the largest biomass was also proportionally most abundant among the reads, there was generally a weak correlation between proportional read abundance and proportional biomass of the different species, both with DNA and cDNA as template. The 454 sequencing generated considerable spurious diversity, and more with cDNA than DNA as template. With initial filtering based only on match with barcode and primer we observed 100-fold more operational taxonomic units (OTUs) at 99% similarity than the number of species present in the mock community. Filtering based on quality scores, or denoising with PyroNoise resulted in ten times more OTU99% than the number of species. Denoising with AmpliconNoise reduced the number of OTU99% to match the number of species present in the mock community. Based on our analyses, we propose a strategy to more accurately depict haptophyte diversity using 454 pyrosequencing. PMID:24069303

[Current applications of high-throughput DNA sequencing technology in antibody drug research].

PubMed

Yu, Xin; Liu, Qi-Gang; Wang, Ming-Rong

2012-03-01

Since the publication of a high-throughput DNA sequencing technology based on PCR reaction was carried out in oil emulsions in 2005, high-throughput DNA sequencing platforms have been evolved to a robust technology in sequencing genomes and diverse DNA libraries. Antibody libraries with vast numbers of members currently serve as a foundation of discovering novel antibody drugs, and high-throughput DNA sequencing technology makes it possible to rapidly identify functional antibody variants with desired properties. Herein we present a review of current applications of high-throughput DNA sequencing technology in the analysis of antibody library diversity, sequencing of CDR3 regions, identification of potent antibodies based on sequence frequency, discovery of functional genes, and combination with various display technologies, so as to provide an alternative approach of discovery and development of antibody drugs.

Elevated Genetic Diversity in the Emerging Blueberry Pathogen Exobasidium maculosum.

PubMed

Stewart, Jane E; Brooks, Kyle; Brannen, Phillip M; Cline, William O; Brewer, Marin T

2015-01-01

Emerging diseases caused by fungi are increasing at an alarming rate. Exobasidium leaf and fruit spot of blueberry, caused by the fungus Exobasidium maculosum, is an emerging disease that has rapidly increased in prevalence throughout the southeastern USA, severely reducing fruit quality in some plantings. The objectives of this study were to determine the genetic diversity of E. maculosum in the southeastern USA to elucidate the basis of disease emergence and to investigate if populations of E. maculosum are structured by geography, host species, or tissue type. We sequenced three conserved loci from 82 isolates collected from leaves and fruit of rabbiteye blueberry (Vaccinium virgatum), highbush blueberry (V. corymbosum), and southern highbush blueberry (V. corymbosum hybrids) from commercial fields in Georgia and North Carolina, USA, and 6 isolates from lowbush blueberry (V. angustifolium) from Maine, USA, and Nova Scotia, Canada. Populations of E. maculosum from the southeastern USA and from lowbush blueberry in Maine and Nova Scotia are distinct, but do not represent unique species. No difference in genetic structure was detected between different host tissues or among different host species within the southeastern USA; however, differentiation was detected between populations in Georgia and North Carolina. Overall, E. maculosum showed extreme genetic diversity within the conserved loci with 286 segregating sites among the 1,775 sequenced nucleotides and each isolate representing a unique multilocus haplotype. However, 94% of the nucleotide substitutions were silent, so despite the high number of mutations, selective constraints have limited changes to the amino acid sequences of the housekeeping genes. Overall, these results suggest that the emergence of Exobasidium leaf and fruit spot is not due to a recent introduction or host shift, or the recent evolution of aggressive genotypes of E. maculosum, but more likely as a result of an increasing host population or an environmental change.

Elevated Genetic Diversity in the Emerging Blueberry Pathogen Exobasidium maculosum

PubMed Central

Stewart, Jane E.; Brooks, Kyle; Brannen, Phillip M.; Cline, William O.; Brewer, Marin T.

2015-01-01

Emerging diseases caused by fungi are increasing at an alarming rate. Exobasidium leaf and fruit spot of blueberry, caused by the fungus Exobasidium maculosum, is an emerging disease that has rapidly increased in prevalence throughout the southeastern USA, severely reducing fruit quality in some plantings. The objectives of this study were to determine the genetic diversity of E. maculosum in the southeastern USA to elucidate the basis of disease emergence and to investigate if populations of E. maculosum are structured by geography, host species, or tissue type. We sequenced three conserved loci from 82 isolates collected from leaves and fruit of rabbiteye blueberry (Vaccinium virgatum), highbush blueberry (V. corymbosum), and southern highbush blueberry (V. corymbosum hybrids) from commercial fields in Georgia and North Carolina, USA, and 6 isolates from lowbush blueberry (V. angustifolium) from Maine, USA, and Nova Scotia, Canada. Populations of E. maculosum from the southeastern USA and from lowbush blueberry in Maine and Nova Scotia are distinct, but do not represent unique species. No difference in genetic structure was detected between different host tissues or among different host species within the southeastern USA; however, differentiation was detected between populations in Georgia and North Carolina. Overall, E. maculosum showed extreme genetic diversity within the conserved loci with 286 segregating sites among the 1,775 sequenced nucleotides and each isolate representing a unique multilocus haplotype. However, 94% of the nucleotide substitutions were silent, so despite the high number of mutations, selective constraints have limited changes to the amino acid sequences of the housekeeping genes. Overall, these results suggest that the emergence of Exobasidium leaf and fruit spot is not due to a recent introduction or host shift, or the recent evolution of aggressive genotypes of E. maculosum, but more likely as a result of an increasing host population or an environmental change. PMID:26207812

MicroRNA repertoire for functional genome research in tilapia identified by deep sequencing.

PubMed

Yan, Biao; Wang, Zhen-Hua; Zhu, Chang-Dong; Guo, Jin-Tao; Zhao, Jin-Liang

2014-08-01

The Nile tilapia (Oreochromis niloticus; Cichlidae) is an economically important species in aquaculture and occupies a prominent position in the aquaculture industry. MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression involved in diverse biological and metabolic processes. To increase the repertoire of miRNAs characterized in tilapia, we used the Illumina/Solexa sequencing technology to sequence a small RNA library using pooled RNA sample isolated from the different developmental stages of tilapia. Bioinformatic analyses suggest that 197 conserved and 27 novel miRNAs are expressed in tilapia. Sequence alignments indicate that all tested miRNAs and miRNAs* are highly conserved across many species. In addition, we characterized the tissue expression patterns of five miRNAs using real-time quantitative PCR. We found that miR-1/206, miR-7/9, and miR-122 is abundantly expressed in muscle, brain, and liver, respectively, implying a potential role in the regulation of tissue differentiation or the maintenance of tissue identity. Overall, our results expand the number of tilapia miRNAs, and the discovery of miRNAs in tilapia genome contributes to a better understanding the role of miRNAs in regulating diverse biological processes.

High-throughput nucleotide sequence analysis of diverse bacterial communities in leachates of decomposing pig carcasses

PubMed Central

Yang, Seung Hak; Lim, Joung Soo; Khan, Modabber Ahmed; Kim, Bong Soo; Choi, Dong Yoon; Lee, Eun Young; Ahn, Hee Kwon

2015-01-01

The leachate generated by the decomposition of animal carcass has been implicated as an environmental contaminant surrounding the burial site. High-throughput nucleotide sequencing was conducted to investigate the bacterial communities in leachates from the decomposition of pig carcasses. We acquired 51,230 reads from six different samples (1, 2, 3, 4, 6 and 14 week-old carcasses) and found that sequences representing the phylum Firmicutes predominated. The diversity of bacterial 16S rRNA gene sequences in the leachate was the highest at 6 weeks, in contrast to those at 2 and 14 weeks. The relative abundance of Firmicutes was reduced, while the proportion of Bacteroidetes and Proteobacteria increased from 3–6 weeks. The representation of phyla was restored after 14 weeks. However, the community structures between the samples taken at 1–2 and 14 weeks differed at the bacterial classification level. The trend in pH was similar to the changes seen in bacterial communities, indicating that the pH of the leachate could be related to the shift in the microbial community. The results indicate that the composition of bacterial communities in leachates of decomposing pig carcasses shifted continuously during the study period and might be influenced by the burial site. PMID:26500442

Construction of a scFv Library with Synthetic, Non-combinatorial CDR Diversity.

PubMed

Bai, Xuelian; Shim, Hyunbo

2017-01-01

Many large synthetic antibody libraries have been designed, constructed, and successfully generated high-quality antibodies suitable for various demanding applications. While synthetic antibody libraries have many advantages such as optimized framework sequences and a broader sequence landscape than natural antibodies, their sequence diversities typically are generated by random combinatorial synthetic processes which cause the incorporation of many undesired CDR sequences. Here, we describe the construction of a synthetic scFv library using oligonucleotide mixtures that contain predefined, non-combinatorially synthesized CDR sequences. Each CDR is first inserted to a master scFv framework sequence and the resulting single-CDR libraries are subjected to a round of proofread panning. The proofread CDR sequences are assembled to produce the final scFv library with six diversified CDRs.

Conservation and variability of West Nile virus proteins.

PubMed

Koo, Qi Ying; Khan, Asif M; Jung, Keun-Ok; Ramdas, Shweta; Miotto, Olivo; Tan, Tin Wee; Brusic, Vladimir; Salmon, Jerome; August, J Thomas

2009-01-01

West Nile virus (WNV) has emerged globally as an increasingly important pathogen for humans and domestic animals. Studies of the evolutionary diversity of the virus over its known history will help to elucidate conserved sites, and characterize their correspondence to other pathogens and their relevance to the immune system. We describe a large-scale analysis of the entire WNV proteome, aimed at identifying and characterizing evolutionarily conserved amino acid sequences. This study, which used 2,746 WNV protein sequences collected from the NCBI GenPept database, focused on analysis of peptides of length 9 amino acids or more, which are immunologically relevant as potential T-cell epitopes. Entropy-based analysis of the diversity of WNV sequences, revealed the presence of numerous evolutionarily stable nonamer positions across the proteome (entropy value of < or = 1). The representation (frequency) of nonamers variant to the predominant peptide at these stable positions was, generally, low (< or = 10% of the WNV sequences analyzed). Eighty-eight fragments of length 9-29 amino acids, representing approximately 34% of the WNV polyprotein length, were identified to be identical and evolutionarily stable in all analyzed WNV sequences. Of the 88 completely conserved sequences, 67 are also present in other flaviviruses, and several have been associated with the functional and structural properties of viral proteins. Immunoinformatic analysis revealed that the majority (78/88) of conserved sequences are potentially immunogenic, while 44 contained experimentally confirmed human T-cell epitopes. This study identified a comprehensive catalogue of completely conserved WNV sequences, many of which are shared by other flaviviruses, and majority are potential epitopes. The complete conservation of these immunologically relevant sequences through the entire recorded WNV history suggests they will be valuable as components of peptide-specific vaccines or other therapeutic applications, for sequence-specific diagnosis of a wide-range of Flavivirus infections, and for studies of homologous sequences among other flaviviruses.

Sequence diversity within the reovirus S2 gene: reovirus genes reassort in nature, and their termini are predicted to form a panhandle motif.

PubMed Central

Chapell, J D; Goral, M I; Rodgers, S E; dePamphilis, C W; Dermody, T S

1994-01-01

To better understand genetic diversity within mammalian reoviruses, we determined S2 nucleotide and deduced sigma 2 amino acid sequences of nine reovirus strains and compared these sequences with those of prototype strains of the three reovirus serotypes. The S2 gene and sigma 2 protein are highly conserved among the four type 1, one type 2, and seven type 3 strains studied. Phylogenetic analyses based on S2 nucleotide sequences of the 12 reovirus strains indicate that diversity within the S2 gene is independent of viral serotype. Additionally, we found marked topological differences between phylogenetic trees generated from S1 and S2 gene nucleotide sequences of the seven type 3 strains. These results demonstrate that reovirus S1 and S2 genes have distinct evolutionary histories, thus providing phylogenetic evidence for lateral transfer of reovirus genes in nature. When variability among the 12 sigma 2-encoding S2 nucleotide sequences was analyzed at synonymous positions, we found that approximately 60 nucleotides at the 5' terminus and 30 nucleotides at the 3' terminus were markedly conserved in comparison with other sigma 2-encoding regions of S2. Predictions of RNA secondary structures indicate that the more conserved S2 sequences participate in the formation of an extended region of duplex RNA interrupted by a pair of stem-loops. Among the 12 deduced sigma 2 amino acid sequences examined, substitutions were observed at only 11% of amino acid positions. This finding suggests that constraints on the structure or function of sigma 2, perhaps in part because of its location in the virion core, have limited sequence diversity within this protein. PMID:8289378

Salmonella enterica Prophage Sequence Profiles Reflect Genome Diversity and Can Be Used for High Discrimination Subtyping.

PubMed

Mottawea, Walid; Duceppe, Marc-Olivier; Dupras, Andrée A; Usongo, Valentine; Jeukens, Julie; Freschi, Luca; Emond-Rheault, Jean-Guillaume; Hamel, Jeremie; Kukavica-Ibrulj, Irena; Boyle, Brian; Gill, Alexander; Burnett, Elton; Franz, Eelco; Arya, Gitanjali; Weadge, Joel T; Gruenheid, Samantha; Wiedmann, Martin; Huang, Hongsheng; Daigle, France; Moineau, Sylvain; Bekal, Sadjia; Levesque, Roger C; Goodridge, Lawrence D; Ogunremi, Dele

2018-01-01

Non-typhoidal Salmonella is a leading cause of foodborne illness worldwide. Prompt and accurate identification of the sources of Salmonella responsible for disease outbreaks is crucial to minimize infections and eliminate ongoing sources of contamination. Current subtyping tools including single nucleotide polymorphism (SNP) typing may be inadequate, in some instances, to provide the required discrimination among epidemiologically unrelated Salmonella strains. Prophage genes represent the majority of the accessory genes in bacteria genomes and have potential to be used as high discrimination markers in Salmonella . In this study, the prophage sequence diversity in different Salmonella serovars and genetically related strains was investigated. Using whole genome sequences of 1,760 isolates of S. enterica representing 151 Salmonella serovars and 66 closely related bacteria, prophage sequences were identified from assembled contigs using PHASTER. We detected 154 different prophages in S. enterica genomes. Prophage sequences were highly variable among S. enterica serovars with a median ± interquartile range (IQR) of 5 ± 3 prophage regions per genome. While some prophage sequences were highly conserved among the strains of specific serovars, few regions were lineage specific. Therefore, strains belonging to each serovar could be clustered separately based on their prophage content. Analysis of S . Enteritidis isolates from seven outbreaks generated distinct prophage profiles for each outbreak. Taken altogether, the diversity of the prophage sequences correlates with genome diversity. Prophage repertoires provide an additional marker for differentiating S. enterica subtypes during foodborne outbreaks.

Diversity and function in microbial mats from the Lucky Strike hydrothermal vent field.

PubMed

Crépeau, Valentin; Cambon Bonavita, Marie-Anne; Lesongeur, Françoise; Randrianalivelo, Henintsoa; Sarradin, Pierre-Marie; Sarrazin, Jozée; Godfroy, Anne

2011-06-01

Diversity and function in microbial mats from the Lucky Strike hydrothermal vent field (Mid-Atlantic Ridge) were investigated using molecular approaches. DNA and RNA were extracted from mat samples overlaying hydrothermal deposits and Bathymodiolus azoricus mussel assemblages. We constructed and analyzed libraries of 16S rRNA gene sequences and sequences of functional genes involved in autotrophic carbon fixation [forms I and II RuBisCO (cbbL/M), ATP-citrate lyase B (aclB)]; methane oxidation [particulate methane monooxygenase (pmoA)] and sulfur oxidation [adenosine-5'-phosphosulfate reductase (aprA) and soxB]. To gain new insights into the relationships between mats and mussels, we also used new domain-specific 16S rRNA gene primers targeting Bathymodiolus sp. symbionts. All identified archaeal sequences were affiliated with a single group: the marine group 1 Thaumarchaeota. In contrast, analyses of bacterial sequences revealed much higher diversity, although two phyla Proteobacteria and Bacteroidetes were largely dominant. The 16S rRNA gene sequence library revealed that species affiliated to Beggiatoa Gammaproteobacteria were the dominant active population. Analyses of DNA and RNA functional gene libraries revealed a diverse and active chemolithoautotrophic population. Most of these sequences were affiliated with Gammaproteobacteria, including hydrothermal fauna symbionts, Thiotrichales and Methylococcales. PCR and reverse transcription-PCR using 16S rRNA gene primers targeted to Bathymodiolus sp. symbionts revealed sequences affiliated with both methanotrophic and thiotrophic endosymbionts. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

PCR Primers to Study the Diversity of Expressed Fungal Genes Encoding Lignocellulolytic Enzymes in Soils Using High-Throughput Sequencing

PubMed Central

Barbi, Florian; Bragalini, Claudia; Vallon, Laurent; Prudent, Elsa; Dubost, Audrey; Fraissinet-Tachet, Laurence; Marmeisse, Roland; Luis, Patricia

2014-01-01

Plant biomass degradation in soil is one of the key steps of carbon cycling in terrestrial ecosystems. Fungal saprotrophic communities play an essential role in this process by producing hydrolytic enzymes active on the main components of plant organic matter. Open questions in this field regard the diversity of the species involved, the major biochemical pathways implicated and how these are affected by external factors such as litter quality or climate changes. This can be tackled by environmental genomic approaches involving the systematic sequencing of key enzyme-coding gene families using soil-extracted RNA as material. Such an approach necessitates the design and evaluation of gene family-specific PCR primers producing sequence fragments compatible with high-throughput sequencing approaches. In the present study, we developed and evaluated PCR primers for the specific amplification of fungal CAZy Glycoside Hydrolase gene families GH5 (subfamily 5) and GH11 encoding endo-β-1,4-glucanases and endo-β-1,4-xylanases respectively as well as Basidiomycota class II peroxidases, corresponding to the CAZy Auxiliary Activity family 2 (AA2), active on lignin. These primers were experimentally validated using DNA extracted from a wide range of Ascomycota and Basidiomycota species including 27 with sequenced genomes. Along with the published primers for Glycoside Hydrolase GH7 encoding enzymes active on cellulose, the newly design primers were shown to be compatible with the Illumina MiSeq sequencing technology. Sequences obtained from RNA extracted from beech or spruce forest soils showed a high diversity and were uniformly distributed in gene trees featuring the global diversity of these gene families. This high-throughput sequencing approach using several degenerate primers constitutes a robust method, which allows the simultaneous characterization of the diversity of different fungal transcripts involved in plant organic matter degradation and may lead to the discovery of complex patterns in gene expression of soil fungal communities. PMID:25545363

Investigation of Microbial Diversity in Geothermal Hot Springs in Unkeshwar, India, Based on 16S rRNA Amplicon Metagenome Sequencing

PubMed Central

Mehetre, Gajanan T.; Paranjpe, Aditi; Dastager, Syed G.

2016-01-01

Microbial diversity in geothermal waters of the Unkeshwar hot springs in Maharashtra, India, was studied using 16S rRNA amplicon metagenomic sequencing. Taxonomic analysis revealed the presence of Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, Archeae, and OD1 phyla. Metabolic function prediction analysis indicated a battery of biological information systems indicating rich and novel microbial diversity, with potential biotechnological applications in this niche. PMID:26950332

Compound haplotypes at Xp11.23 and human population growth in Eurasia.

PubMed

Alonso, S; Armour, J A L

2004-09-01

To investigate patterns of diversity and the evolutionary history of Eurasians, we have sequenced a 2.8 kb region at Xp11.23 in a sample of African and Eurasian chromosomes. This region is in a long intron of CLCN5 and is immediately flanked by a highly variable minisatellite, DXS255, and a human-specific Ta0 LINE. Compared to Africans, Eurasians showed a marked reduction in sequence diversity. The main Euro-Asiatic haplotype seems to be the ancestral haplotype for the whole sample. Coalescent simulations, including recombination and exponential growth, indicate a median length of strong linkage disequilibrium, up to approximately 9 kb for this area. The Ka/Ks ratio between the coding sequence of human CLCN5 and its mouse orthologue is much less than 1. This implies that the region sequenced is unlikely to be under the strong influence of positive selective processes on CLCN5, mutations in which have been associated with disorders such as Dent's disease. In contrast, a scenario based on a population bottleneck and exponential growth seems a more likely explanation for the reduced diversity observed in Eurasians. Coalescent analysis and linked minisatellite diversity (which reaches a gene diversity value greater than 98% in Eurasians) suggest an estimated age of origin of the Euro-Asiatic diversity compatible with a recent out-of-Africa model for colonization of Eurasia by modern Homo sapiens.

Genetic diversity of Babesia bovis in virulent and attenuated strains.

PubMed

Mazuz, M L; Molad, T; Fish, L; Leibovitz, B; Wolkomirsky, R; Fleiderovitz, L; Shkap, V

2012-03-01

The aim of this study was to compare the genetic diversity of the single copy Bv80 gene sequences of Babesia bovis in populations of attenuated and virulent parasites. PCR/ RT-PCR followed by cloning and sequence analyses of 4 attenuated and 4 virulent strains were performed. Multiple fragments in the range of 420 to 744 bp were amplified by PCR or RT-PCR. Cloning of the PCR fragments and sequence analyses revealed the presence of mixed subpopulations in either virulent or attenuated parasites with a total of 19 variants with 12 different sequences that differed in number and type of tandem repeats. High levels of intra- and inter-strain diversity of the Bv80 gene, with the presence of mixed populations of parasites were found in both the virulent field isolates and the attenuated vaccine strains. In addition, during the attenuation process, sequence analyses showed changes in the pattern of the parasite subpopulations. Despite high polymorphism found by sequence analyses, the patterns observed and the number of repeats, order, or motifs found could not discriminate between virulent field isolates and attenuated vaccine strains of the parasite.

Diversity of phytases in the rumen.

PubMed

Nakashima, Brenda A; McAllister, Tim A; Sharma, Ranjana; Selinger, L Brent

2007-01-01

Examples of a new class of phytase related to protein tyrosine phosphatases (PTP) were recently isolated from several anaerobic bacteria from the rumen of cattle. In this study, the diversity of PTP-like phytase gene sequences in the rumen was surveyed by using the polymerase chain reaction (PCR). Two sets of degenerate primers were used to amplify sequences from rumen fluid total community DNA and genomic DNA from nine bacterial isolates. Four novel PTP-like phytase sequences were retrieved from rumen fluid, whereas all nine of the anaerobic bacterial isolates investigated in this work contained PTP-like phytase sequences. One isolate, Selenomonas lacticifex, contained two distinct PTP-like phytase sequences, suggesting that multiple phytate hydrolyzing enzymes are present in this bacterium. The degenerate primer and PCR conditions described here, as well as novel sequences obtained in this study, will provide a valuable resource for future studies on this new class of phytase. The observed diversity of microbial phytases in the rumen may account for the ability of ruminants to derive a significant proportion of their phosphorus requirements from phytate.

Novel chytrid lineages dominate fungal sequences in diverse marine and freshwater habitats

NASA Astrophysics Data System (ADS)

Comeau, André M.; Vincent, Warwick F.; Bernier, Louis; Lovejoy, Connie

2016-07-01

In aquatic environments, fungal communities remain little studied despite their taxonomic and functional diversity. To extend the ecological coverage of this group, we conducted an in-depth analysis of fungal sequences within our collection of 3.6 million V4 18S rRNA pyrosequences originating from 319 individual marine (including sea-ice) and freshwater samples from libraries generated within diverse projects studying Arctic and temperate biomes in the past decade. Among the ~1.7 million post-filtered reads of highest taxonomic and phylogenetic quality, 23,263 fungal sequences were identified. The overall mean proportion was 1.35%, but with large variability; for example, from 0.01 to 59% of total sequences for Arctic seawater samples. Almost all sample types were dominated by Chytridiomycota-like sequences, followed by moderate-to-minor contributions of Ascomycota, Cryptomycota and Basidiomycota. Species and/or strain richness was high, with many novel sequences and high niche separation. The affinity of the most common reads to phytoplankton parasites suggests that aquatic fungi deserve renewed attention for their role in algal succession and carbon cycling.

Universal Influenza B Virus Genomic Amplification Facilitates Sequencing, Diagnostics, and Reverse Genetics

PubMed Central

Zhou, Bin; Lin, Xudong; Wang, Wei; Halpin, Rebecca A.; Bera, Jayati; Stockwell, Timothy B.; Barr, Ian G.

2014-01-01

Although human influenza B virus (IBV) is a significant human pathogen, its great genetic diversity has limited our ability to universally amplify the entire genome for subsequent sequencing or vaccine production. The generation of sequence data via next-generation approaches and the rapid cloning of viral genes are critical for basic research, diagnostics, antiviral drugs, and vaccines to combat IBV. To overcome the difficulty of amplifying the diverse and ever-changing IBV genome, we developed and optimized techniques that amplify the complete segmented negative-sense RNA genome from any IBV strain in a single tube/well (IBV genomic amplification [IBV-GA]). Amplicons for >1,000 diverse IBV genomes from different sample types (e.g., clinical specimens) were generated and sequenced using this robust technology. These approaches are sensitive, robust, and sequence independent (i.e., universally amplify past, present, and future IBVs), which facilitates next-generation sequencing and advanced genomic diagnostics. Importantly, special terminal sequences engineered into the optimized IBV-GA2 products also enable ligation-free cloning to rapidly generate reverse-genetics plasmids, which can be used for the rescue of recombinant viruses and/or the creation of vaccine seed stock. PMID:24501036

Nucleotide diversity maps reveal variation in diversity among wheat genomes and chromosomes

PubMed Central

2010-01-01

Background A genome-wide assessment of nucleotide diversity in a polyploid species must minimize the inclusion of homoeologous sequences into diversity estimates and reliably allocate individual haplotypes into their respective genomes. The same requirements complicate the development and deployment of single nucleotide polymorphism (SNP) markers in polyploid species. We report here a strategy that satisfies these requirements and deploy it in the sequencing of genes in cultivated hexaploid wheat (Triticum aestivum, genomes AABBDD) and wild tetraploid wheat (Triticum turgidum ssp. dicoccoides, genomes AABB) from the putative site of wheat domestication in Turkey. Data are used to assess the distribution of diversity among and within wheat genomes and to develop a panel of SNP markers for polyploid wheat. Results Nucleotide diversity was estimated in 2114 wheat genes and was similar between the A and B genomes and reduced in the D genome. Within a genome, diversity was diminished on some chromosomes. Low diversity was always accompanied by an excess of rare alleles. A total of 5,471 SNPs was discovered in 1791 wheat genes. Totals of 1,271, 1,218, and 2,203 SNPs were discovered in 488, 463, and 641 genes of wheat putative diploid ancestors, T. urartu, Aegilops speltoides, and Ae. tauschii, respectively. A public database containing genome-specific primers, SNPs, and other information was constructed. A total of 987 genes with nucleotide diversity estimated in one or more of the wheat genomes was placed on an Ae. tauschii genetic map, and the map was superimposed on wheat deletion-bin maps. The agreement between the maps was assessed. Conclusions In a young polyploid, exemplified by T. aestivum, ancestral species are the primary source of genetic diversity. Low effective recombination due to self-pollination and a genetic mechanism precluding homoeologous chromosome pairing during polyploid meiosis can lead to the loss of diversity from large chromosomal regions. The net effect of these factors in T. aestivum is large variation in diversity among genomes and chromosomes, which impacts the development of SNP markers and their practical utility. Accumulation of new mutations in older polyploid species, such as wild emmer, results in increased diversity and its more uniform distribution across the genome. PMID:21156062

Genetic diversity based on 28S rDNA sequences among populations of Culex quinquefasciatus collected at different locations in Tamil Nadu, India.

PubMed

Sakthivelkumar, S; Ramaraj, P; Veeramani, V; Janarthanan, S

2015-09-01

The basis of the present study was to distinguish the existence of any genetic variability among populations of Culex quinquefasciatus which would be a valuable tool in the management of mosquito control programmes. In the present study, population of Cx. quinquefasciatus collected at different locations in Tamil Nadu were analyzed for their genetic variation based on 28S rDNA D2 region nucleotide sequences. A high degree of genetic polymorphism was detected in the sequences of D2 region of 28S rDNA on the predicted secondary structures in spite of high nucleotide sequence similarity. The findings based on secondary structure using rDNA sequences suggested the existence of a complex genotypic diversity of Cx. quinquefasciatus population collected at different locations of Tamil Nadu, India. This complexity in genetic diversity in a single mosquito population collected at different locations is considered an important issue towards their influence and nature of vector potential of these mosquitoes.

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era

PubMed Central

d’Avila-Levy, Claudia Masini; Boucinha, Carolina; Kostygov, Alexei; Santos, Helena Lúcia Carneiro; Morelli, Karina Alessandra; Grybchuk-Ieremenko, Anastasiia; Duval, Linda; Votýpka, Jan; Yurchenko, Vyacheslav; Grellier, Philippe; Lukeš, Julius

2015-01-01

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists. PMID:26602872

Assessing the genetic diversity of Cu resistance in mine tailings through high-throughput recovery of full-length copA genes

PubMed Central

Li, Xiaofang; Zhu, Yong-Guan; Shaban, Babak; Bruxner, Timothy J. C.; Bond, Philip L.; Huang, Longbin

2015-01-01

Characterizing the genetic diversity of microbial copper (Cu) resistance at the community level remains challenging, mainly due to the polymorphism of the core functional gene copA. In this study, a local BLASTN method using a copA database built in this study was developed to recover full-length putative copA sequences from an assembled tailings metagenome; these sequences were then screened for potentially functioning CopA using conserved metal-binding motifs, inferred by evolutionary trace analysis of CopA sequences from known Cu resistant microorganisms. In total, 99 putative copA sequences were recovered from the tailings metagenome, out of which 70 were found with high potential to be functioning in Cu resistance. Phylogenetic analysis of selected copA sequences detected in the tailings metagenome showed that topology of the copA phylogeny is largely congruent with that of the 16S-based phylogeny of the tailings microbial community obtained in our previous study, indicating that the development of copA diversity in the tailings might be mainly through vertical descent with few lateral gene transfer events. The method established here can be used to explore copA (and potentially other metal resistance genes) diversity in any metagenome and has the potential to exhaust the full-length gene sequences for downstream analyses. PMID:26286020

Fungal communities from the calcareous deep-sea sediments in the Southwest India Ridge revealed by Illumina sequencing technology.

PubMed

Zhang, Likui; Kang, Manyu; Huang, Yangchao; Yang, Lixiang

2016-05-01

The diversity and ecological significance of bacteria and archaea in deep-sea environments have been thoroughly investigated, but eukaryotic microorganisms in these areas, such as fungi, are poorly understood. To elucidate fungal diversity in calcareous deep-sea sediments in the Southwest India Ridge (SWIR), the internal transcribed spacer (ITS) regions of rRNA genes from two sediment metagenomic DNA samples were amplified and sequenced using the Illumina sequencing platform. The results revealed that 58-63 % and 36-42 % of the ITS sequences (97 % similarity) belonged to Basidiomycota and Ascomycota, respectively. These findings suggest that Basidiomycota and Ascomycota are the predominant fungal phyla in the two samples. We also found that Agaricomycetes, Leotiomycetes, and Pezizomycetes were the major fungal classes in the two samples. At the species level, Thelephoraceae sp. and Phialocephala fortinii were major fungal species in the two samples. Despite the low relative abundance, unidentified fungal sequences were also observed in the two samples. Furthermore, we found that there were slight differences in fungal diversity between the two sediment samples, although both were collected from the SWIR. Thus, our results demonstrate that calcareous deep-sea sediments in the SWIR harbor diverse fungi, which augment the fungal groups in deep-sea sediments. This is the first report of fungal communities in calcareous deep-sea sediments in the SWIR revealed by Illumina sequencing.

Functional diversity of microbial communities in pristine aquifers inferred by PLFA- and sequencing-based approaches

NASA Astrophysics Data System (ADS)

Schwab, Valérie F.; Herrmann, Martina; Roth, Vanessa-Nina; Gleixner, Gerd; Lehmann, Robert; Pohnert, Georg; Trumbore, Susan; Küsel, Kirsten; Totsche, Kai U.

2017-05-01

Microorganisms in groundwater play an important role in aquifer biogeochemical cycles and water quality. However, the mechanisms linking the functional diversity of microbial populations and the groundwater physico-chemistry are still not well understood due to the complexity of interactions between surface and subsurface. Within the framework of Hainich (north-western Thuringia, central Germany) Critical Zone Exploratory of the Collaborative Research Centre AquaDiva, we used the relative abundances of phospholipid-derived fatty acids (PLFAs) to link specific biochemical markers within the microbial communities to the spatio-temporal changes of the groundwater physico-chemistry. The functional diversities of the microbial communities were mainly correlated with groundwater chemistry, including dissolved O2, Fet and NH4+ concentrations. Abundances of PLFAs derived from eukaryotes and potential nitrite-oxidizing bacteria (11Me16:0 as biomarker for Nitrospira moscoviensis) were high at sites with elevated O2 concentration where groundwater recharge supplies bioavailable substrates. In anoxic groundwaters more rich in Fet, PLFAs abundant in sulfate-reducing bacteria (SRB), iron-reducing bacteria and fungi increased with Fet and HCO3- concentrations, suggesting the occurrence of active iron reduction and the possible role of fungi in meditating iron solubilization and transport in those aquifer domains. In more NH4+-rich anoxic groundwaters, anammox bacteria and SRB-derived PLFAs increased with NH4+ concentration, further evidencing the dependence of the anammox process on ammonium concentration and potential links between SRB and anammox bacteria. Additional support of the PLFA-based bacterial communities was found in DNA- and RNA-based Illumina MiSeq amplicon sequencing of bacterial 16S rRNA genes, which showed high predominance of nitrite-oxidizing bacteria Nitrospira, e.g. Nitrospira moscoviensis, in oxic aquifer zones and of anammox bacteria in more NH4+-rich anoxic groundwater. Higher relative abundances of sequence reads in the RNA-based datasets affiliated with iron-reducing bacteria in more Fet-rich groundwater supported the occurrence of active dissimilatory iron reduction. The functional diversity of the microbial communities in the biogeochemically distinct groundwater assemblages can be largely attributed to the redox conditions linked to changes in bioavailable substrates and input of substrates with the seepage. Our results demonstrate the power of complementary information derived from PLFA-based and sequencing-based approaches.

Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data.

PubMed

Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne-Vibeke; Kruse, Torben A; Larsen, Martin Jakob

2016-01-01

Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths.

Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data

PubMed Central

Krøigård, Anne Bruun; Thomassen, Mads; Lænkholm, Anne-Vibeke; Kruse, Torben A.; Larsen, Martin Jakob

2016-01-01

Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths. PMID:27002637

Biofacies expression of Upper Cretaceous sequences in the Rock Springs uplift

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Y.Y.; Pflum, C.E.; Wright, R.C.

1991-03-01

The sequence-stratigraphic framework and vertical succession of depositional environments in the Upper Cretaceous section of the Rock Springs uplift is expressed in the biofacies patterns as well as in the stratal stacking patterns. Vertical trends in six biofacies parameters track affinities to marine and nonmarine environments as well as proximity to the paleoshoreline. These six parameters and their environmental significance include the relative proportion of herbaceous kerogen (land-derived), amorphous kerogen (marine), dinoflagellates (marine), bisaccate pollen (land-derived but buoyant and easily transported offshore), and the abundance and diversity of benthic foraminifera (both increase offshore). Shoaling marine environments are characterized by anmore » increasing proportion of herbaceous kerogen and decreasing proportions of amorphous kerogen, dinoflagellated, bisaccates, and the abundance and diversity of benthic foraminifera. Conversely, a deepening-upward marine sedimentary succession is characterized by an opposite trend in these parameters. A synthesis of the six biofacies parameters emphasizes the third-order cyclicity of the stratal succession as reflected in the well-developed third-order downlap surfaces and condensed sections. The biofacies trends indicate the transgressive nature of the lower Rock Springs and lower Lewis formations, and the progradational nature of the upper arts of the Baxter, Blair, and Rock Springs formations. An overall progradational (i.e., shoaling) character is exhibited in the three lower sequences (Baxter through Rock Springs) by the progressively decreasing abundance of amorphous kerogen, dinoflagellates, and foraminifera.« less

Comparative Analysis of the Gut Microbial Communities in Forest and Alpine Musk Deer Using High-Throughput Sequencing

PubMed Central

Hu, Xiaolong; Liu, Gang; Shafer, Aaron B. A.; Wei, Yuting; Zhou, Juntong; Lin, Shaobi; Wu, Haibin; Zhou, Mi; Hu, Defu; Liu, Shuqiang

2017-01-01

The gut ecosystem is characterized by dynamic and reciprocal interactions between the host and bacteria. Although characterizing microbiota for herbivores has become recognized as important tool for gauging species health, no study to date has investigated the bacterial communities and evaluated the age-related bacterial dynamics of musk deer. Moreover, gastrointestinal diseases have been hypothesized to be a limiting factor of population growth in captive musk deer. Here, high-throughput sequencing of the bacterial 16S rRNA gene was used to profile the fecal bacterial communities in juvenile and adult alpine and forest musk deer. The two musk deer species harbored similar bacterial communities at the phylum level, whereas the key genera for the two species were distinct. The bacterial communities were dominated by Firmicutes and Bacteroidetes, with the bacterial diversity being higher in forest musk deer. The Firmicutes to Bacteroidetes ratio also increased from juvenile to adult, while the bacterial diversity, within-group and between-group similarity, all increased with age. This work serves as the first sequence-based analysis of variation in bacterial communities within and between musk deer species, and demonstrates how the gut microbial community dynamics vary among closely related species and shift with age. As gastrointestinal diseases have been observed in captive populations, this study provides valuable data that might benefit captive management and future reintroduction programs. PMID:28421061

Effects of 16S rDNA sampling on estimates of the number of endosymbiont lineages in sucking lice

PubMed Central

Burleigh, J. Gordon; Light, Jessica E.; Reed, David L.

2016-01-01

Phylogenetic trees can reveal the origins of endosymbiotic lineages of bacteria and detect patterns of co-evolution with their hosts. Although taxon sampling can greatly affect phylogenetic and co-evolutionary inference, most hypotheses of endosymbiont relationships are based on few available bacterial sequences. Here we examined how different sampling strategies of Gammaproteobacteria sequences affect estimates of the number of endosymbiont lineages in parasitic sucking lice (Insecta: Phthirapatera: Anoplura). We estimated the number of louse endosymbiont lineages using both newly obtained and previously sequenced 16S rDNA bacterial sequences and more than 42,000 16S rDNA sequences from other Gammaproteobacteria. We also performed parametric and nonparametric bootstrapping experiments to examine the effects of phylogenetic error and uncertainty on these estimates. Sampling of 16S rDNA sequences affects the estimates of endosymbiont diversity in sucking lice until we reach a threshold of genetic diversity, the size of which depends on the sampling strategy. Sampling by maximizing the diversity of 16S rDNA sequences is more efficient than randomly sampling available 16S rDNA sequences. Although simulation results validate estimates of multiple endosymbiont lineages in sucking lice, the bootstrap results suggest that the precise number of endosymbiont origins is still uncertain. PMID:27547523

Molecular diversity and distribution pattern of ciliates in sediments from deep-sea hydrothermal vents in the Okinawa Trough and adjacent sea areas

NASA Astrophysics Data System (ADS)

Zhao, Feng; Xu, Kuidong

2016-10-01

In comparison with the macrobenthos and prokaryotes, patterns of diversity and distribution of microbial eukaryotes in deep-sea hydrothermal vents are poorly known. The widely used high-throughput sequencing of 18S rDNA has revealed a high diversity of microeukaryotes yielded from both living organisms and buried DNA in marine sediments. More recently, cDNA surveys have been utilized to uncover the diversity of active organisms. However, both methods have never been used to evaluate the diversity of ciliates in hydrothermal vents. By using high-throughput DNA and cDNA sequencing of 18S rDNA, we evaluated the molecular diversity of ciliates, a representative group of microbial eukaryotes, from the sediments of deep-sea hydrothermal vents in the Okinawa Trough and compared it with that of an adjacent deep-sea area about 15 km away and that of an offshore area of the Yellow Sea about 500 km away. The results of DNA sequencing showed that Spirotrichea and Oligohymenophorea were the most diverse and abundant groups in all the three habitats. The proportion of sequences of Oligohymenophorea was the highest in the hydrothermal vents whereas Spirotrichea was the most diverse group at all three habitats. Plagiopyleans were found only in the hydrothermal vents but with low diversity and abundance. By contrast, the cDNA sequencing showed that Plagiopylea was the most diverse and most abundant group in the hydrothermal vents, followed by Spirotrichea in terms of diversity and Oligohymenophorea in terms of relative abundance. A novel group of ciliates, distinctly separate from the 12 known classes, was detected in the hydrothermal vents, indicating undescribed, possibly highly divergent ciliates may inhabit this environment. Statistical analyses showed that: (i) the three habitats differed significantly from one another in terms of diversity of both the rare and the total ciliate taxa, and; (ii) the adjacent deep sea was more similar to the offshore area than to the hydrothermal vents. In terms of the diversity of abundant taxa, however, there was no significant difference between the hydrothermal vents and the adjacent deep sea, both of which differed significantly from the offshore area. As abundant ciliate taxa can be found in several sampling sites, they are likely adapted to large environmental variations, while rare taxa are found in specific habitat and thus are potentially more sensitive to varying environmental conditions.

Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection.

PubMed

Weiss, Eric R; Lamers, Susanna L; Henderson, Jennifer L; Melnikov, Alexandre; Somasundaran, Mohan; Garber, Manuel; Selin, Liisa; Nusbaum, Chad; Luzuriaga, Katherine

2018-01-15

Over 90% of the world's population is persistently infected with Epstein-Barr virus. While EBV does not cause disease in most individuals, it is the common cause of acute infectious mononucleosis (AIM) and has been associated with several cancers and autoimmune diseases, highlighting a need for a preventive vaccine. At present, very few primary, circulating EBV genomes have been sequenced directly from infected individuals. While low levels of diversity and low viral evolution rates have been predicted for double-stranded DNA (dsDNA) viruses, recent studies have demonstrated appreciable diversity in common dsDNA pathogens (e.g., cytomegalovirus). Here, we report 40 full-length EBV genome sequences obtained from matched oral wash and B cell fractions from a cohort of 10 AIM patients. Both intra- and interpatient diversity were observed across the length of the entire viral genome. Diversity was most pronounced in viral genes required for establishing latent infection and persistence, with appreciable levels of diversity also detected in structural genes, including envelope glycoproteins. Interestingly, intrapatient diversity declined significantly over time ( P < 0.01), and this was particularly evident on comparison of viral genomes sequenced from B cell fractions in early primary infection and convalescence ( P < 0.001). B cell-associated viral genomes were observed to converge, becoming nearly identical to the B95.8 reference genome over time (Spearman rank-order correlation test; r = -0.5589, P = 0.0264). The reduction in diversity was most marked in the EBV latency genes. In summary, our data suggest independent convergence of diverse viral genome sequences toward a reference-like strain within a relatively short period following primary EBV infection. IMPORTANCE Identification of viral proteins with low variability and high immunogenicity is important for the development of a protective vaccine. Knowledge of genome diversity within circulating viral populations is a key step in this process, as is the expansion of intrahost genomic variation during infection. We report full-length EBV genomes sequenced from the blood and oral wash of 10 individuals early in primary infection and during convalescence. Our data demonstrate considerable diversity within the pool of circulating EBV strains, as well as within individual patients. Overall viral diversity decreased from early to persistent infection, particularly in latently infected B cells, which serve as the viral reservoir. Reduction in B cell-associated viral genome diversity coincided with a convergence toward a reference-like EBV genotype. Greater convergence positively correlated with time after infection, suggesting that the reference-like genome is the result of selection. Copyright © 2018 American Society for Microbiology.

Genome Sequence of Micromonospora lupini Lupac 08, Isolated from Root Nodules of Lupinus angustifolius

PubMed Central

Alonso-Vega, Pablo; Normand, Philippe; Bacigalupe, Rodrigo; Pujic, Petar; Lajus, Aurelie; Vallenet, David; Carro, Lorena; Coll, Pedro

2012-01-01

Micromonospora strains have been isolated from diverse niches, including soil, water, and marine sediments and root nodules of diverse symbiotic plants. In this work, we report the genome sequence of Micromonospora lupini Lupac 08 isolated from root nodules of the wild legume Lupinus angustifolious. PMID:22815450

Genome re-sequencing reveals the history of apple and supports a two-stage model for fruit enlargement

USDA-ARS?s Scientific Manuscript database

Human selection has reshaped crop genomes. Here we report an apple genome variation map generated through genome sequencing of 117 diverse accessions. A comprehensive model of apple speciation and domestication along the Silk Road was proposed based on evidence from diverse genomic analyses. Cultiva...

Natural Variation in Brachypodium disctachyon: Deep Sequencing of Highly Diverse Natural Accessions (2013 DOE JGI Genomics of Energy and Environment 8th Annual User Meeting)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gordon, Sean

2013-03-01

Sean Gordon of the USDA on Natural variation in Brachypodium disctachyon: Deep Sequencing of Highly Diverse Natural Accessions at the 8th Annual Genomics of Energy Environment Meeting on March 27, 2013 in Walnut Creek, CA.

Diversity of Frankia populations in root nodules of geographically isolated Arizona alder trees in central Arizona (United States)

Treesearch

Allana K. Welsh; Jeffrey O. Dawson; Gerald J. Gottfried; Dittmar Hahn

2009-01-01

The diversity of uncultured Frankia populations in root nodules of Alnus oblongifolia trees geographically isolated on mountaintops of central Arizona was analyzed by comparative sequence analyses of nifH gene fragments. Sequences were retrieved from Frankia populations in nodules of four trees from each of...

PHYLOGENETIC ANALYSIS OF 16S RRNA GENE SEQUENCES REVEALS THE PREVALENCE OF MYCOBACTERIA SP., ALPHA-PROTEOBACTERIA, AND UNCULTURED BACTERIA IN DRINKING WATER MICROBIAL COMMUNITIES

EPA Science Inventory

Previous studies have shown that culture-based methods tend to underestimate the densities and diversity of bacterial populations inhabiting water distribution systems (WDS). In this study, the phylogenetic diversity of drinking water bacteria was assessed using sequence analysis...

Ibrutinib Therapy Increases T Cell Repertoire Diversity in Patients with Chronic Lymphocytic Leukemia.

PubMed

Yin, Qingsong; Sivina, Mariela; Robins, Harlan; Yusko, Erik; Vignali, Marissa; O'Brien, Susan; Keating, Michael J; Ferrajoli, Alessandra; Estrov, Zeev; Jain, Nitin; Wierda, William G; Burger, Jan A

2017-02-15

The Bruton's tyrosine kinase inhibitor ibrutinib is a highly effective, new targeted therapy for chronic lymphocytic leukemia (CLL) that thwarts leukemia cell survival, growth, and tissue homing. The effects of ibrutinib treatment on the T cell compartment, which is clonally expanded and thought to support the growth of malignant B cells in CLL, are not fully characterized. Using next-generation sequencing technology, we characterized the diversity of TCRβ-chains in peripheral blood T cells from 15 CLL patients before and after 1 y of ibrutinib therapy. We noted elevated CD4 + and CD8 + T cell numbers and a restricted TCRβ repertoire in all pretreatment samples. After 1 y of ibrutinib therapy, elevated peripheral blood T cell numbers and T cell-related cytokine levels had normalized, and T cell repertoire diversity increased significantly. Dominant TCRβ clones in pretreatment samples declined or became undetectable, and the number of productive unique clones increased significantly during ibrutinib therapy, with the emergence of large numbers of low-frequency TCRβ clones. Importantly, broader TCR repertoire diversity was associated with clinical efficacy and lower rates of infections during ibrutinib therapy. These data demonstrate that ibrutinib therapy increases diversification of the T cell compartment in CLL patients, which contributes to cellular immune reconstitution. Copyright © 2017 by The American Association of Immunologists, Inc.

Ibrutinib therapy increases T cell repertoire diversity in patients with chronic lymphocytic leukemia

PubMed Central

Yin, Qingsong; Sivina, Mariela; Robins, Harlan; Yusko, Erik; Vignali, Marissa; O’Brien, Susan; Keating, Michael J.; Ferrajoli, Alessandra; Estrov, Zeev; Jain, Nitin; Wierda, William G.; Burger, Jan A.

2017-01-01

The BTK inhibitor ibrutinib is a highly effective, new targeted therapy for chronic lymphocytic leukemia (CLL) that thwarts leukemia cell survival, growth, and tissue homing. The effects of ibrutinib treatment on the T cell compartment, which is clonally expanded and thought to support the growth of the malignant B cells in CLL, are not fully characterized. Using next-generation sequencing technology we characterized the diversity of TCRβ chains in peripheral blood T cells from 15 CLL patients before and after one year of ibrutinib therapy. We noted elevated CD4+ and CD8+ T cell numbers and a restricted TCRβ repertoire in all pretreatment samples. After one year of ibrutinib therapy, elevated PB T cell numbers and T-cell related cytokine levels had normalized and T cell repertoire diversity significantly increased. Dominant TCRβ clones in pretreatment samples declined or became undetectable, and the number of productive unique clones significantly increased during ibrutinib therapy, with the emergence of large numbers of low-frequency TCRβ clones. Importantly, broader TCR repertoire diversity was associated with clinical efficacy and lower rates of infections during ibrutinib therapy. These data demonstrate that ibrutinib therapy increases diversification of the T cell compartment in CLL patients, which contributes to cellular immune reconstitution. PMID:28077600

[Study on Microbial Diversity of Peri-implantitis Subgingival by High-throughput Sequencing].

PubMed

Li, Zhi-jie; Wang, Shao-guo; Li, Yue-hong; Tu, Dong-xiang; Liu, Shi-yun; Nie, Hong-bing; Li, Zhi-qiang; Zhang, Ju-mei

2015-07-01

To study microbial diversity of peri-implantitis subgingival with high-throughput sequencing, and investigate microbiological etiology of peri-implantitis. Subgingival plaques were sampled from the patients with peri-implantitis (D group) and non-peri-implantitis subjects (N group). The microbiological diversity of the subgingival plaques was detected by sequencing V4 region of 16S rRNA with Illumina Miseq platform. The diversity of the community structure was analyzed using Mothur software. A total of 156 507 gene sequences were detected in nine samples and 4 402 operational taxonomic units (OTUs) were found. Selenomonas, Pseudomonas, and Fusobacterium were dominant bacteria in D group, while Fusobacterium, Veillonella and Streptococcus were dominant bacteria in N group. Differences between peri-implantitis and non-peri-implantitis bacterial communities were observed at all phylogenetic levels by LEfSe, which was also found in PcoA test. The occurrence of peri-implantitis is not only related to periodontitis pathogenic microbe, but also related with the changes of oral microbial community structure. Treponema, Herbaspirillum, Butyricimonas and Phaeobacte may be closely related to the occurrence and development of peri-implantitis.

Coastal bacterioplankton community diversity along a latitudinal gradient in Latin America by means of V6 tag pyrosequencing.

PubMed

Thompson, Fabiano L; Bruce, Thiago; Gonzalez, Alessandra; Cardoso, Alexander; Clementino, Maysa; Costagliola, Marcela; Hozbor, Constanza; Otero, Ernesto; Piccini, Claudia; Peressutti, Silvia; Schmieder, Robert; Edwards, Robert; Smith, Mathew; Takiyama, Luis Roberto; Vieira, Ricardo; Paranhos, Rodolfo; Artigas, Luis Felipe

2011-02-01

The bacterioplankton diversity of coastal waters along a latitudinal gradient between Puerto Rico and Argentina was analyzed using a total of 134,197 high-quality sequences from the V6 hypervariable region of the small-subunit ribosomal RNA gene (16S rRNA) (mean length of 60 nt). Most of the OTUs were identified into Proteobacteria, Bacteriodetes, Cyanobacteria, and Actinobacteria, corresponding to approx. 80% of the total number of sequences. The number of OTUs corresponding to species varied between 937 and 1946 in the seven locations. Proteobacteria appeared at high frequency in the seven locations. An enrichment of Cyanobacteria was observed in Puerto Rico, whereas an enrichment of Bacteroidetes was detected in the Argentinian shelf and Uruguayan coastal lagoons. The highest number of sequences of Actinobacteria and Acidobacteria were obtained in the Amazon estuary mouth. The rarefaction curves and Good coverage estimator for species diversity suggested a significant coverage, with values ranging between 92 and 97% for Good coverage. Conserved taxa corresponded to aprox. 52% of all sequences. This study suggests that human-contaminated environments may influence bacterioplankton diversity.

Diversity of the Cronobacter Genus as Revealed by Multilocus Sequence Typing

PubMed Central

Joseph, S.; Sonbol, H.; Hariri, S.; Desai, P.; McClelland, M.

2012-01-01

Cronobacter (previously known as Enterobacter sakazakii) is a diverse bacterial genus consisting of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. universalis, C. muytjensii, C. dublinensis, and C. condimenti. In this study, we have used a multilocus sequence typing (MLST) approach employing the alleles of 7 genes (atpD, fusA, glnS, gltB, gyrB, infB, and ppsA; total length, 3,036 bp) to investigate the phylogenetic relationship of 325 Cronobacter species isolates. Strains were chosen on the basis of their species, geographic and temporal distribution, source, and clinical outcome. The earliest strain was isolated from milk powder in 1950, and the earliest clinical strain was isolated in 1953. The existence of seven species was supported by MLST. Intraspecific variation ranged from low diversity in C. sakazakii to extensive diversity within some species, such as C. muytjensii and C. dublinensis, including evidence of gene conversion between species. The predominant species from clinical sources was found to be C. sakazakii. C. sakazakii sequence type 4 (ST4) was the predominant sequence type of cerebral spinal fluid isolates from cases of meningitis. PMID:22785185

HIV populations are large and accumulate high genetic diversity in a nonlinear fashion.

PubMed

Maldarelli, Frank; Kearney, Mary; Palmer, Sarah; Stephens, Robert; Mican, JoAnn; Polis, Michael A; Davey, Richard T; Kovacs, Joseph; Shao, Wei; Rock-Kress, Diane; Metcalf, Julia A; Rehm, Catherine; Greer, Sarah E; Lucey, Daniel L; Danley, Kristen; Alter, Harvey; Mellors, John W; Coffin, John M

2013-09-01

HIV infection is characterized by rapid and error-prone viral replication resulting in genetically diverse virus populations. The rate of accumulation of diversity and the mechanisms involved are under intense study to provide useful information to understand immune evasion and the development of drug resistance. To characterize the development of viral diversity after infection, we carried out an in-depth analysis of single genome sequences of HIV pro-pol to assess diversity and divergence and to estimate replicating population sizes in a group of treatment-naive HIV-infected individuals sampled at single (n = 22) or multiple, longitudinal (n = 11) time points. Analysis of single genome sequences revealed nonlinear accumulation of sequence diversity during the course of infection. Diversity accumulated in recently infected individuals at rates 30-fold higher than in patients with chronic infection. Accumulation of synonymous changes accounted for most of the diversity during chronic infection. Accumulation of diversity resulted in population shifts, but the rates of change were low relative to estimated replication cycle times, consistent with relatively large population sizes. Analysis of changes in allele frequencies revealed effective population sizes that are substantially higher than previous estimates of approximately 1,000 infectious particles/infected individual. Taken together, these observations indicate that HIV populations are large, diverse, and slow to change in chronic infection and that the emergence of new mutations, including drug resistance mutations, is governed by both selection forces and drift.

The complicated substrates enhance the microbial diversity and zinc leaching efficiency in sphalerite bioleaching system.

PubMed

Xiao, Yunhua; Xu, YongDong; Dong, Weiling; Liang, Yili; Fan, Fenliang; Zhang, Xiaoxia; Zhang, Xian; Niu, Jiaojiao; Ma, Liyuan; She, Siyuan; He, Zhili; Liu, Xueduan; Yin, Huaqun

2015-12-01

This study used an artificial enrichment microbial consortium to examine the effects of different substrate conditions on microbial diversity, composition, and function (e.g., zinc leaching efficiency) through adding pyrite (SP group), chalcopyrite (SC group), or both (SPC group) in sphalerite bioleaching systems. 16S rRNA gene sequencing analysis showed that microbial community structures and compositions dramatically changed with additions of pyrite or chalcopyrite during the sphalerite bioleaching process. Shannon diversity index showed a significantly increase in the SP (1.460), SC (1.476), and SPC (1.341) groups compared with control (sphalerite group, 0.624) on day 30, meanwhile, zinc leaching efficiencies were enhanced by about 13.4, 2.9, and 13.2%, respectively. Also, additions of pyrite or chalcopyrite could increase electric potential (ORP) and the concentrations of Fe3+ and H+, which were the main factors shaping microbial community structures by Mantel test analysis. Linear regression analysis showed that ORP, Fe3+ concentration, and pH were significantly correlated to zinc leaching efficiency and microbial diversity. In addition, we found that leaching efficiency showed a positive and significant relationship with microbial diversity. In conclusion, our results showed that the complicated substrates could significantly enhance microbial diversity and activity of function.

Whole-Genome Sequencing and Variant Analysis of Human Papillomavirus 16 Infections.

PubMed

van der Weele, Pascal; Meijer, Chris J L M; King, Audrey J

2017-10-01

Human papillomavirus (HPV) is a strongly conserved DNA virus, high-risk types of which can cause cervical cancer in persistent infections. The most common type found in HPV-attributable cancer is HPV16, which can be subdivided into four lineages (A to D) with different carcinogenic properties. Studies have shown HPV16 sequence diversity in different geographical areas, but only limited information is available regarding HPV16 diversity within a population, especially at the whole-genome level. We analyzed HPV16 major variant diversity and conservation in persistent infections and performed a single nucleotide polymorphism (SNP) comparison between persistent and clearing infections. Materials were obtained in the Netherlands from a cohort study with longitudinal follow-up for up to 3 years. Our analysis shows a remarkably large variant diversity in the population. Whole-genome sequences were obtained for 57 persistent and 59 clearing HPV16 infections, resulting in 109 unique variants. Interestingly, persistent infections were completely conserved through time. One reinfection event was identified where the initial and follow-up samples clustered differently. Non-A1/A2 variants seemed to clear preferentially ( P = 0.02). Our analysis shows that population-wide HPV16 sequence diversity is very large. In persistent infections, the HPV16 sequence was fully conserved. Sequencing can identify HPV16 reinfections, although occurrence is rare. SNP comparison identified no strongly acting effect of the viral genome affecting HPV16 infection clearance or persistence in up to 3 years of follow-up. These findings suggest the progression of an early HPV16 infection could be host related. IMPORTANCE Human papillomavirus 16 (HPV16) is the predominant type found in cervical cancer. Progression of initial infection to cervical cancer has been linked to sequence properties; however, knowledge of variants circulating in European populations, especially with longitudinal follow-up, is limited. By sequencing a number of infections with known follow-up for up to 3 years, we gained initial insights into the genetic diversity of HPV16 and the effects of the viral genome on the persistence of infections. A SNP comparison between sequences obtained from clearing and persistent infections did not identify strongly acting DNA variations responsible for these infection outcomes. In addition, we identified an HPV16 reinfection event where sequencing of initial and follow-up samples showed different HPV16 variants. Based on conventional genotyping, this infection would incorrectly be considered a persistent HPV16 infection. In the context of vaccine efficacy and monitoring studies, such infections could potentially cause reduced reported efficacy or efficiency. Copyright © 2017 van der Weele et al.

Diversity, expression and mRNA targeting abilities of Argonaute-targeting miRNAs among selected vascular plants.

PubMed

Jagtap, Soham; Shivaprasad, Padubidri V

2014-12-02

Micro (mi)RNAs are important regulators of plant development. Across plant lineages, Dicer-like 1 (DCL1) proteins process long ds-like structures to produce micro (mi) RNA duplexes in a stepwise manner. These miRNAs are incorporated into Argonaute (AGO) proteins and influence expression of RNAs that have sequence complementarity with miRNAs. Expression levels of AGOs are greatly regulated by plants in order to minimize unwarranted perturbations using miRNAs to target mRNAs coding for AGOs. AGOs may also have high promoter specificity-sometimes expression of AGO can be limited to just a few cells in a plant. Viral pathogens utilize various means to counter antiviral roles of AGOs including hijacking the host encoded miRNAs to target AGOs. Two host encoded miRNAs namely miR168 and miR403 that target AGOs have been described in the model plant Arabidopsis and such a mechanism is thought to be well conserved across plants because AGO sequences are well conserved. We show that the interaction between AGO mRNAs and miRNAs is species-specific due to the diversity in sequences of two miRNAs that target AGOs, sequence diversity among corresponding target regions in AGO mRNAs and variable expression levels of these miRNAs among vascular plants. We used miRNA sequences from 68 plant species representing 31 plant families for this analysis. Sequences of miR168 and miR403 are not conserved among plant lineages, but surprisingly they differ drastically in their sequence diversity and expression levels even among closely related plants. Variation in miR168 expression among plants correlates well with secondary structures/length of loop sequences of their precursors. Our data indicates a complex AGO targeting interaction among plant lineages due to miRNA sequence diversity and sequences of miRNA targeting regions among AGO mRNAs, thus leading to the assumption that the perturbations by viruses that use host miRNAs to target antiviral AGOs can only be species-specific. We also show that rapid evolution and likely loss of expression of miR168 isoforms in tobacco is related to the insertion of MITE-like transposons between miRNA and miRNA* sequences, a possible mechanism showing how miRNAs are lost in few plant lineages even though other close relatives have abundantly expressing miRNAs.

Investigation of Microbial Diversity in Geothermal Hot Springs in Unkeshwar, India, Based on 16S rRNA Amplicon Metagenome Sequencing.

PubMed

Mehetre, Gajanan T; Paranjpe, Aditi; Dastager, Syed G; Dharne, Mahesh S

2016-02-25

Microbial diversity in geothermal waters of the Unkeshwar hot springs in Maharashtra, India, was studied using 16S rRNA amplicon metagenomic sequencing. Taxonomic analysis revealed the presence of Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, Archeae, and OD1 phyla. Metabolic function prediction analysis indicated a battery of biological information systems indicating rich and novel microbial diversity, with potential biotechnological applications in this niche. Copyright © 2016 Mehetre et al.

Clustering, haplotype diversity and locations of MIC-3: a unique root-specific defense-related gene family in upland cotton (Gossypium hirsutum L.)

USDA-ARS?s Scientific Manuscript database

MIC-3-related genes of cotton (Gossypium spp.) were identified and shown to have root-specific expression, associated with pathogen defense-related function and specifically increased expression in root-knot nematode (RKN) resistant plants after nematode infection. Here we cloned and sequenced MIC-...

Are we there yet? Tracking the development of new model systems

Treesearch

A. Abzhanov; C. Extavour; A. Groover; S. Hodges; H. Hoekstra; E. Kramer; A. Monteiro

2008-01-01

It is increasingly clear that additional ‘model’ systems are needed to elucidate the genetic and developmental basis of organismal diversity. Whereas model system development previously required enormous investment, recent advances including the decreasing cost of DNA sequencing and the power of reverse genetics to study gene function are greatly facilitating...

Exploring the Inner and Outer Cultural Landscapes of Counseling Candidates towards Diverse Students and Families through Self-Reflection

ERIC Educational Resources Information Center

Montes, Adonay A.; Rodriguez-Valls, Fernando; Schroeder, Laurie

2014-01-01

This article presents an interpersonal methodology designed to increase the cultural awareness of counselor candidates. This methodology was implemented through a sequence of activities, which was part of a multicultural course in the counseling credential program in a university located in Southern California. The goal was to enrich future…

Double-stranded telomeric DNA binding proteins: Diversity matters.

PubMed

Červenák, Filip; Juríková, Katarína; Sepšiová, Regina; Neboháčová, Martina; Nosek, Jozef; Tomáška, L'ubomír

2017-01-01

Telomeric sequences constitute only a small fraction of the whole genome yet they are crucial for ensuring genomic stability. This function is in large part mediated by protein complexes recruited to telomeric sequences by specific telomere-binding proteins (TBPs). Although the principal tasks of nuclear telomeres are the same in all eukaryotes, TBPs in various taxa exhibit a surprising diversity indicating their distinct evolutionary origin. This diversity is especially pronounced in ascomycetous yeasts where they must have co-evolved with rapidly diversifying sequences of telomeric repeats. In this article we (i) provide a historical overview of the discoveries leading to the current list of TBPs binding to double-stranded (ds) regions of telomeres, (ii) describe examples of dsTBPs highlighting their diversity in even closely related species, and (iii) speculate about possible evolutionary trajectories leading to a long list of various dsTBPs fulfilling the same general role(s) in their own unique ways.

[Genetic diversity of psbA of cyanophage from paddy floodwater in northeast China].

PubMed

Jing, Ruiyong; Cao, Kun; Liu, Junjie; Liu, Judong; Jin, Jian; Liu, Xiaobing; Wang, Guanghua

2017-01-04

To provide scientific data for studying the ecology of cyanophage, we studied the genetic diversity of psbA of cyanophage from paddy floodwater in northeast China and its phylogenetic positions. Membrane separation and concentration of cyanophage, PCR-cloning-sequencing were applied to study the diversity of psbA of cyanophage from paddy floodwater in northeast China. In total 17 psbA sequences of cyanophage were obtained. Novel cyanophages were found by phylogenetic analysis. Compared to those of Japanese paddy floodwater, marine and lakes, psbA gene assemblage of paddy floodwater in northeast China was significantly different. This is the first report to study genetic diversity of cyanophage from paddy floodwater in northeast China with a molecular marker of psbA by PCR-cloning-sequencing. The novel psbA assembly of cyanophage was found in paddy floodwater in northeast China.

Understanding the enormous diversity of bacteriophages: the tailed phages that infect the bacterial family Enterobacteriaceae.

PubMed

Grose, Julianne H; Casjens, Sherwood R

2014-11-01

Bacteriophages are the predominant biological entity on the planet. The recent explosion of sequence information has made estimates of their diversity possible. We describe the genomic comparison of 337 fully sequenced tailed phages isolated on 18 genera and 31 species of bacteria in the Enterobacteriaceae. These phages were largely unambiguously grouped into 56 diverse clusters (32 lytic and 24 temperate) that have syntenic similarity over >50% of the genomes within each cluster, but substantially less sequence similarity between clusters. Most clusters naturally break into sets of more closely related subclusters, 78% of which are correlated with their host genera. The largest groups of related phages are superclusters united by genome synteny to lambda (81 phages) and T7 (51 phages). This study forms a robust framework for understanding diversity and evolutionary relationships of existing tailed phages, for relating newly discovered phages and for determining host/phage relationships.

Understanding the enormous diversity of bacteriophages: the tailed phages that infect the bacterial family Enterobacteriaceae

PubMed Central

Grose, Julianne H.; Casjens, Sherwood R.

2014-01-01

Bacteriophages are the predominant biological entity on the planet. The recent explosion of sequence information has made estimates of their diversity possible. We describe the genomic comparison of 337 fully sequenced tailed phages isolated on 18 genera and 31 species of bacteria in the Enterobacteriaceae. These phages were largely unambiguously grouped into 56 diverse clusters (32 lytic and 24 temperate) that have syntenic similarity over >50% of the genomes within each cluster, but substantially less sequence similarity between clusters. Most clusters naturally break into sets of more closely related subclusters, 78% of which are correlated with their host genera. The largest groups of related phages are superclusters united by genome synteny to lambda (81 phages) and T7 (51 phages). This study forms a robust framework for understanding diversity and evolutionary relationships of existing tailed phages, for relating newly discovered phages and for determining host/phage relationships. PMID:25240328

Increasing Clinical Severity during a Dengue Virus Type 3 Cuban Epidemic: Deep Sequencing of Evolving Viral Populations

PubMed Central

Blanc, Hervé; Bordería, Antonio V.; Díaz, Gisell; Henningsson, Rasmus; Gonzalez, Daniel; Santana, Emidalys; Alvarez, Mayling; Castro, Osvaldo; Fontes, Magnus; Vignuzzi, Marco; Guzman, Maria G.

2016-01-01

ABSTRACT During the dengue virus type 3 (DENV-3) epidemic that occurred in Havana in 2001 to 2002, severe disease was associated with the infection sequence DENV-1 followed by DENV-3 (DENV-1/DENV-3), while the sequence DENV-2/DENV-3 was associated with mild/asymptomatic infections. To determine the role of the virus in the increasing severity demonstrated during the epidemic, serum samples collected at different time points were studied. A total of 22 full-length sequences were obtained using a deep-sequencing approach. Bayesian phylogenetic analysis of consensus sequences revealed that two DENV-3 lineages were circulating in Havana at that time, both grouped within genotype III. The predominant lineage is closely related to Peruvian and Ecuadorian strains, while the minor lineage is related to Venezuelan strains. According to consensus sequences, relatively few nonsynonymous mutations were observed; only one was fixed during the epidemic at position 4380 in the NS2B gene. Intrahost genetic analysis indicated that a significant minor population was selected and became predominant toward the end of the epidemic. In conclusion, greater variability was detected during the epidemic's progression in terms of significant minority variants, particularly in the nonstructural genes. An increasing trend of genetic diversity toward the end of the epidemic was observed only for synonymous variant allele rates, with higher variability in secondary cases. Remarkably, significant intrahost genetic variation was demonstrated within the same patient during the course of secondary infection with DENV-1/DENV-3, including changes in the structural proteins premembrane (PrM) and envelope (E). Therefore, the dynamic of evolving viral populations in the context of heterotypic antibodies could be related to the increasing clinical severity observed during the epidemic. IMPORTANCE Based on the evidence that DENV fitness is context dependent, our research has focused on the study of viral factors associated with intraepidemic increasing severity in a unique epidemiological setting. Here, we investigated the intrahost genetic diversity in acute human samples collected at different time points during the DENV-3 epidemic that occurred in Cuba in 2001 to 2002 using a deep-sequencing approach. We concluded that greater variability in significant minor populations occurred as the epidemic progressed, particularly in the nonstructural genes, with higher variability observed in secondary infection cases. Remarkably, for the first time significant intrahost genetic variation was demonstrated within the same patient during the course of secondary infection with DENV-1/DENV-3, including changes in structural proteins. These findings indicate that high-resolution approaches are needed to unravel molecular mechanisms involved in dengue pathogenesis. PMID:26889031

Metagenomic study of bacterial microbiota in persistent endodontic infections using Next-generation sequencing.

PubMed

Sánchez-Sanhueza, G; Bello-Toledo, H; González-Rocha, G; Gonçalves, A T; Valenzuela, V; Gallardo-Escárate, C

2018-05-22

To determine the bacterial microbiota in root canals associated with persistent apical periodontitis and their relationship with the clinical characteristics of patients using next-generation sequencing (NGS). Bacterial samples from root canals associated with teeth having persistent apical periodontitis were taken from 24 patients undergoing root canal retreatment. Bacterial DNA was extracted, and V3-V4 variable regions of the 16S rRNA gene were amplified. The amplification was deep sequenced by Illumina technology to establish the metagenetic relationships among the bacterial species identified. The composition and diversity of microbial communities in the root canal and their relationships with clinical features were analysed. Parametric and nonparametric tests were used to analyse differences between patient characteristics and microbial data. A total of 86 different operational taxonomic units (OTUs) were identified and Good's nonparametric coverage estimator method indicated that 99.9 ± 0.00001% diversity was recovered per sample. The largest number of bacteria belonged to the phylum Proteobacteria. According to the medical history from the American Society of Anesthesiologists (ASA) Classification System, ASA II-III had higher richness estimates and distinct phylogenetic relationships compared to ASA I individuals (P < 0.05). Periapical index (PAI) score 5 was associated with increased microbiota diversity in comparison to PAI score 4, and this index was reduced in symptomatic patients. Based on the findings of this study, it is possible to suggest a close relationship between several clinical features and greater microbiota diversity with persistent endodontic infections. This work provides a better understanding on how microbial communities interact with their host and vice versa. © 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Genomic diversity in switchgrass (Panicum virgatum): from the continental scale to a dune landscape

PubMed Central

Morris, Geoffrey P.; Grabowski, Paul; Borevitz, Justin O.

2011-01-01

Connecting broad-scale patterns of genetic variation and population structure to genetic diversity on a landscape is a key step towards understanding historical processes of migration and adaptation. New genomic approaches can be used to increase the resolution of phylogeographic studies while reducing locus sampling effects and circumventing ascertainment bias. Here, we use a novel approach based on high-throughput sequencing to characterize genetic diversity in complete chloroplast genomes and >10,000 nuclear loci in switchgrass, across a continental and landscape scale. Switchgrass is a North American tallgrass species, which is widely used in conservation and perennial biomass production, and shows strong ecotypic adaptation and population structure across the continental range. We sequenced 40.9 billion base pairs from 24 individuals from across the species’ range and 20 individuals from the Indiana Dunes. Analysis of plastome sequence revealed 203 variable SNP sites that define eight haplogroups, which are differentiated by 4 to 127 SNPs and confirmed by patterns of indel variation. These include three deeply divergent haplogroups, which correspond to the previously described lowland-upland ecotypic split and a novel upland haplogroup split that dates to the mid-Pleistoscene. Most of the plastome haplogroup diversity present in the northern switchgrass range, including in the Indiana Dunes, originated in the mid- or upper-Pleistocene prior to the most recent postglacial recolonization. Furthermore, a recently colonized landscape feature (~150 ya) in the Indiana Dunes contains several deeply divergent upland haplogroups. Nuclear markers also support a deep lowland-upland split, followed by limited gene flow, and show extensive gene flow in the local population of the Indiana Dunes. PMID:22060816

Genetic diversity and differentiation in reef-building Millepora species, as revealed by cross-species amplification of fifteen novel microsatellite loci.

PubMed

Dubé, Caroline E; Planes, Serge; Zhou, Yuxiang; Berteaux-Lecellier, Véronique; Boissin, Emilie

2017-01-01

Quantifying the genetic diversity in natural populations is crucial to address ecological and evolutionary questions. Despite recent advances in whole-genome sequencing, microsatellite markers have remained one of the most powerful tools for a myriad of population genetic approaches. Here, we used the 454 sequencing technique to develop microsatellite loci in the fire coral Millepora platyphylla , an important reef-builder of Indo-Pacific reefs . We tested the cross-species amplification of these loci in five other species of the genus Millepora and analysed its success in correlation with the genetic distances between species using mitochondrial 16S sequences. We succeeded in discovering fifteen microsatellite loci in our target species M. platyphylla, among which twelve were polymorphic with 2-13 alleles and a mean observed heterozygosity of 0.411. Cross-species amplification in the five other Millepora species revealed a high probability of amplification success (71%) and polymorphism (59%) of the loci. Our results show no evidence of decreased heterozygosity with increasing genetic distance. However, only one locus enabled measures of genetic diversity in the Caribbean species M. complanata due to high proportions of null alleles for most of the microsatellites. This result indicates that our novel markers may only be useful for the Indo-Pacific species of Millepora. Measures of genetic diversity revealed significant linkage disequilibrium, moderate levels of observed heterozygosity (0.323-0.496) and heterozygote deficiencies for the Indo-Pacific species. The accessibility to new polymorphic microsatellite markers for hydrozoan Millepora species creates new opportunities for future research on processes driving the complexity of their colonisation success on many Indo-Pacific reefs.

Pivoting the Plant Immune System from Dissection to Deployment

PubMed Central

Dangl, Jeffery L.; Horvath, Diana M.; Staskawicz, Brian J.

2013-01-01

Diverse and rapidly evolving pathogens cause plant diseases and epidemics that threaten crop yield and food security around the world. Research over the last 25 years has led to an increasingly clear conceptual understanding of the molecular components of the plant immune system. Combined with ever-cheaper DNA-sequencing technology and the rich diversity of germ plasm manipulated for over a century by plant breeders, we now have the means to begin development of durable (long-lasting) disease resistance beyond the limits imposed by conventional breeding and in a manner that will replace costly and unsustainable chemical controls. PMID:23950531

Genetic diversity of mtDNA D-loop sequences in four native Chinese chicken breeds.

PubMed

Guo, H W; Li, C; Wang, X N; Li, Z J; Sun, G R; Li, G X; Liu, X J; Kang, X T; Han, R L

2017-10-01

1. To explore the genetic diversity of Chinese indigenous chicken breeds, a 585 bp fragment of the mitochondrial DNA (mtDNA) region was sequenced in 102 birds from the Xichuan black-bone chicken, Yunyang black-bone chicken and Lushi chicken. In addition, 30 mtDNA D-loop sequences of Silkie fowls were downloaded from NCBI. The mtDNA D-loop sequence polymorphism and maternal origin of 4 chicken breeds were analysed in this study. 2. The results showed that a total of 33 mutation sites and 28 haplotypes were detected in the 4 chicken breeds. The haplotype diversity and nucleotide diversity of these 4 native breeds were 0.916 ± 0.014 and 0.012 ± 0.002, respectively. Three clusters were formed in 4 Chinese native chickens and 12 reference breeds. Both the Xichuan black-bone chicken and Yunyang black-bone chicken were grouped into one cluster. Four haplogroups (A, B, C and E) emerged in the median-joining network in these breeds. 3. It was concluded that these 4 Chinese chicken breeds had high genetic diversity. The phylogenetic tree and median network profiles showed that Chinese native chickens and its neighbouring countries had at least two maternal origins, one from Yunnan, China and another from Southeast Asia or its surrounding area.

Increasing Sequence Diversity with Flexible Backbone Protein Design: The Complete Redesign of a Protein Hydrophobic Core

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, Grant S.; Mills, Jeffrey L.; Miley, Michael J.

2015-10-15

Protein design tests our understanding of protein stability and structure. Successful design methods should allow the exploration of sequence space not found in nature. However, when redesigning naturally occurring protein structures, most fixed backbone design algorithms return amino acid sequences that share strong sequence identity with wild-type sequences, especially in the protein core. This behavior places a restriction on functional space that can be explored and is not consistent with observations from nature, where sequences of low identity have similar structures. Here, we allow backbone flexibility during design to mutate every position in the core (38 residues) of a four-helixmore » bundle protein. Only small perturbations to the backbone, 12 {angstrom}, were needed to entirely mutate the core. The redesigned protein, DRNN, is exceptionally stable (melting point >140C). An NMR and X-ray crystal structure show that the side chains and backbone were accurately modeled (all-atom RMSD = 1.3 {angstrom}).« less

Nucleotide Sequence Diversity and Linkage Disequilibrium of Four Nuclear Loci in Foxtail Millet (Setaria italica).

PubMed

He, Shui-Lian; Yang, Yang; Morrell, Peter L; Yi, Ting-Shuang

2015-01-01

Foxtail millet (Setaria italica (L.) Beauv) is one of the earliest domesticated grains, which has been cultivated in northern China by 8,700 years before present (YBP) and across Eurasia by 4,000 YBP. Owing to a small genome and diploid nature, foxtail millet is a tractable model crop for studying functional genomics of millets and bioenergy grasses. In this study, we examined nucleotide sequence diversity, geographic structure, and levels of linkage disequilibrium at four nuclear loci (ADH1, G3PDH, IGS1 and TPI1) in representative samples of 311 landrace accessions across its cultivated range. Higher levels of nucleotide sequence and haplotype diversity were observed in samples from China relative to other sampled regions. Genetic assignment analysis classified the accessions into seven clusters based on nucleotide sequence polymorphisms. Intralocus LD decayed rapidly to half the initial value within ~1.2 kb or less.

Integrating metagenomic and amplicon databases to resolve the phylogenetic and ecological diversity of the Chlamydiae

PubMed Central

Lagkouvardos, Ilias; Weinmaier, Thomas; Lauro, Federico M; Cavicchioli, Ricardo; Rattei, Thomas; Horn, Matthias

2014-01-01

In the era of metagenomics and amplicon sequencing, comprehensive analyses of available sequence data remain a challenge. Here we describe an approach exploiting metagenomic and amplicon data sets from public databases to elucidate phylogenetic diversity of defined microbial taxa. We investigated the phylum Chlamydiae whose known members are obligate intracellular bacteria that represent important pathogens of humans and animals, as well as symbionts of protists. Despite their medical relevance, our knowledge about chlamydial diversity is still scarce. Most of the nine known families are represented by only a few isolates, while previous clone library-based surveys suggested the existence of yet uncharacterized members of this phylum. Here we identified more than 22 000 high quality, non-redundant chlamydial 16S rRNA gene sequences in diverse databases, as well as 1900 putative chlamydial protein-encoding genes. Even when applying the most conservative approach, clustering of chlamydial 16S rRNA gene sequences into operational taxonomic units revealed an unexpectedly high species, genus and family-level diversity within the Chlamydiae, including 181 putative families. These in silico findings were verified experimentally in one Antarctic sample, which contained a high diversity of novel Chlamydiae. In our analysis, the Rhabdochlamydiaceae, whose known members infect arthropods, represents the most diverse and species-rich chlamydial family, followed by the protist-associated Parachlamydiaceae, and a putative new family (PCF8) with unknown host specificity. Available information on the origin of metagenomic samples indicated that marine environments contain the majority of the newly discovered chlamydial lineages, highlighting this environment as an important chlamydial reservoir. PMID:23949660

Fungal diversity in deep-sea sediments of a hydrothermal vent system in the Southwest Indian Ridge

NASA Astrophysics Data System (ADS)

Xu, Wei; Gong, Lin-feng; Pang, Ka-Lai; Luo, Zhu-Hua

2018-01-01

Deep-sea hydrothermal sediment is known to support remarkably diverse microbial consortia. In deep sea environments, fungal communities remain less studied despite their known taxonomic and functional diversity. High-throughput sequencing methods have augmented our capacity to assess eukaryotic diversity and their functions in microbial ecology. Here we provide the first description of the fungal community diversity found in deep sea sediments collected at the Southwest Indian Ridge (SWIR) using culture-dependent and high-throughput sequencing approaches. A total of 138 fungal isolates were cultured from seven different sediment samples using various nutrient media, and these isolates were identified to 14 fungal taxa, including 11 Ascomycota taxa (7 genera) and 3 Basidiomycota taxa (2 genera) based on internal transcribed spacers (ITS1, ITS2 and 5.8S) of rDNA. Using illumina HiSeq sequencing, a total of 757,467 fungal ITS2 tags were recovered from the samples and clustered into 723 operational taxonomic units (OTUs) belonging to 79 taxa (Ascomycota and Basidiomycota contributed to 99% of all samples) based on 97% sequence similarity. Results from both approaches suggest that there is a high fungal diversity in the deep-sea sediments collected in the SWIR and fungal communities were shown to be slightly different by location, although all were collected from adjacent sites at the SWIR. This study provides baseline data of the fungal diversity and biogeography, and a glimpse to the microbial ecology associated with the deep-sea sediments of the hydrothermal vent system of the Southwest Indian Ridge.

Low level of sequence diversity at merozoite surface protein-1 locus of Plasmodium ovale curtisi and P. ovale wallikeri from Thai isolates.

PubMed

Putaporntip, Chaturong; Hughes, Austin L; Jongwutiwes, Somchai

2013-01-01

The merozoite surface protein-1 (MSP-1) is a candidate target for the development of blood stage vaccines against malaria. Polymorphism in MSP-1 can be useful as a genetic marker for strain differentiation in malarial parasites. Although sequence diversity in the MSP-1 locus has been extensively analyzed in field isolates of Plasmodium falciparum and P. vivax, the extent of variation in its homologues in P. ovale curtisi and P. ovale wallikeri, remains unknown. Analysis of the mitochondrial cytochrome b sequences of 10 P. ovale isolates from symptomatic malaria patients from diverse endemic areas of Thailand revealed co-existence of P. ovale curtisi (n = 5) and P. ovale wallikeri (n = 5). Direct sequencing of the PCR-amplified products encompassing the entire coding region of MSP-1 of P. ovale curtisi (PocMSP-1) and P. ovale wallikeri (PowMSP-1) has identified 3 imperfect repeated segments in the former and one in the latter. Most amino acid differences between these proteins were located in the interspecies variable domains of malarial MSP-1. Synonymous nucleotide diversity (πS) exceeded nonsynonymous nucleotide diversity (πN) for both PocMSP-1 and PowMSP-1, albeit at a non-significant level. However, when MSP-1 of both these species was considered together, πS was significantly greater than πN (p<0.0001), suggesting that purifying selection has shaped diversity at this locus prior to speciation. Phylogenetic analysis based on conserved domains has placed PocMSP-1 and PowMSP-1 in a distinct bifurcating branch that probably diverged from each other around 4.5 million years ago. The MSP-1 sequences support that P. ovale curtisi and P. ovale wallikeri are distinct species. Both species are sympatric in Thailand. The low level of sequence diversity in PocMSP-1 and PowMSP-1 among Thai isolates could stem from persistent low prevalence of these species, limiting the chance of outcrossing at this locus.

Low Level of Sequence Diversity at Merozoite Surface Protein-1 Locus of Plasmodium ovale curtisi and P. ovale wallikeri from Thai Isolates

PubMed Central

Putaporntip, Chaturong; Hughes, Austin L.; Jongwutiwes, Somchai

2013-01-01

Background The merozoite surface protein-1 (MSP-1) is a candidate target for the development of blood stage vaccines against malaria. Polymorphism in MSP-1 can be useful as a genetic marker for strain differentiation in malarial parasites. Although sequence diversity in the MSP-1 locus has been extensively analyzed in field isolates of Plasmodium falciparum and P. vivax, the extent of variation in its homologues in P. ovale curtisi and P. ovale wallikeri, remains unknown. Methodology/Principal Findings Analysis of the mitochondrial cytochrome b sequences of 10 P. ovale isolates from symptomatic malaria patients from diverse endemic areas of Thailand revealed co-existence of P. ovale curtisi (n = 5) and P. ovale wallikeri (n = 5). Direct sequencing of the PCR-amplified products encompassing the entire coding region of MSP-1 of P. ovale curtisi (PocMSP-1) and P. ovale wallikeri (PowMSP-1) has identified 3 imperfect repeated segments in the former and one in the latter. Most amino acid differences between these proteins were located in the interspecies variable domains of malarial MSP-1. Synonymous nucleotide diversity (πS) exceeded nonsynonymous nucleotide diversity (πN) for both PocMSP-1 and PowMSP-1, albeit at a non-significant level. However, when MSP-1 of both these species was considered together, πS was significantly greater than πN (p<0.0001), suggesting that purifying selection has shaped diversity at this locus prior to speciation. Phylogenetic analysis based on conserved domains has placed PocMSP-1 and PowMSP-1 in a distinct bifurcating branch that probably diverged from each other around 4.5 million years ago. Conclusion/Significance The MSP-1 sequences support that P. ovale curtisi and P. ovale wallikeri are distinct species. Both species are sympatric in Thailand. The low level of sequence diversity in PocMSP-1 and PowMSP-1 among Thai isolates could stem from persistent low prevalence of these species, limiting the chance of outcrossing at this locus. PMID:23536840

Evolution and Diversity in Human Herpes Simplex Virus Genomes

PubMed Central

Gatherer, Derek; Ochoa, Alejandro; Greenbaum, Benjamin; Dolan, Aidan; Bowden, Rory J.; Enquist, Lynn W.; Legendre, Matthieu; Davison, Andrew J.

2014-01-01

Herpes simplex virus 1 (HSV-1) causes a chronic, lifelong infection in >60% of adults. Multiple recent vaccine trials have failed, with viral diversity likely contributing to these failures. To understand HSV-1 diversity better, we comprehensively compared 20 newly sequenced viral genomes from China, Japan, Kenya, and South Korea with six previously sequenced genomes from the United States, Europe, and Japan. In this diverse collection of passaged strains, we found that one-fifth of the newly sequenced members share a gene deletion and one-third exhibit homopolymeric frameshift mutations (HFMs). Individual strains exhibit genotypic and potential phenotypic variation via HFMs, deletions, short sequence repeats, and single-nucleotide polymorphisms, although the protein sequence identity between strains exceeds 90% on average. In the first genome-scale analysis of positive selection in HSV-1, we found signs of selection in specific proteins and residues, including the fusion protein glycoprotein H. We also confirmed previous results suggesting that recombination has occurred with high frequency throughout the HSV-1 genome. Despite this, the HSV-1 strains analyzed clustered by geographic origin during whole-genome distance analysis. These data shed light on likely routes of HSV-1 adaptation to changing environments and will aid in the selection of vaccine antigens that are invariant worldwide. PMID:24227835

Neotropical Bats from Costa Rica harbour Diverse Coronaviruses.

PubMed

Moreira-Soto, A; Taylor-Castillo, L; Vargas-Vargas, N; Rodríguez-Herrera, B; Jiménez, C; Corrales-Aguilar, E

2015-11-01

Bats are hosts of diverse coronaviruses (CoVs) known to potentially cross the host-species barrier. For analysing coronavirus diversity in a bat species-rich country, a total of 421 anal swabs/faecal samples from Costa Rican bats were screened for CoV RNA-dependent RNA polymerase (RdRp) gene sequences by a pancoronavirus PCR. Six families, 24 genera and 41 species of bats were analysed. The detection rate for CoV was 1%. Individuals (n = 4) from four different species of frugivorous (Artibeus jamaicensis, Carollia perspicillata and Carollia castanea) and nectivorous (Glossophaga soricina) bats were positive for coronavirus-derived nucleic acids. Analysis of 440 nt. RdRp sequences allocated all Costa Rican bat CoVs to the α-CoV group. Several CoVs sequences clustered near previously described CoVs from the same species of bat, but were phylogenetically distant from the human CoV sequences identified to date, suggesting no recent spillover events. The Glossophaga soricina CoV sequence is sufficiently dissimilar (26% homology to the closest known bat CoVs) to represent a unique coronavirus not clustering near other CoVs found in the same bat species so far, implying an even higher CoV diversity than previously suspected. © 2015 Blackwell Verlag GmbH.

Genetic variation in potential Giardia vaccine candidates cyst wall protein 2 and α1-giardin.

PubMed

Radunovic, Matej; Klotz, Christian; Saghaug, Christina Skår; Brattbakk, Hans-Richard; Aebischer, Toni; Langeland, Nina; Hanevik, Kurt

2017-08-01

Giardia is a prevalent intestinal parasitic infection. The trophozoite structural protein a1-giardin (a1-g) and the cyst protein cyst wall protein 2 (CWP2) have shown promise as Giardia vaccine antigen candidates in murine models. The present study assesses the genetic diversity of a1-g and CWP2 between and within assemblages A and B in human clinical isolates. a1-g and CWP2 sequences were acquired from 15 Norwegian isolates by PCR amplification and 20 sequences from German cultured isolates by whole genome sequencing. Sequences were aligned to reference genomes from assemblage A2 and B to identify genetic variance. Genetic diversity was found between assemblage A and B reference sequences for both a1-g (90.8% nucleotide identity) and CWP2 (82.5% nucleotide identity). However, for a1-g, this translated into only 3 amino acid (aa) substitutions, while for CWP2 there were 41 aa substitutions, and also one aa deletion. Genetic diversity within assemblage B was larger; nucleotide identity 92.0% for a1-g and 94.3% for CWP2, than within assemblage A (nucleotide identity 99.0% for a1-g and 99.7% for CWP2). For CWP2, the diversity on both nucleotide and protein level was higher in the C-terminal end. Predicted antigenic epitopes were not affected for a1-g, but partially for CWP2. Despite genetic diversity in a1-g, we found aa sequence, characteristics, and antigenicity to be well preserved. CWP2 showed more aa variance and potential antigenic differences. Several CWP2 antigens might be necessary in a future Giardia vaccine to provide cross protection against both Giardia assemblages infecting humans.

Variation in Symbiodinium ITS2 sequence assemblages among coral colonies.

PubMed

Stat, Michael; Bird, Christopher E; Pochon, Xavier; Chasqui, Luis; Chauka, Leonard J; Concepcion, Gregory T; Logan, Dan; Takabayashi, Misaki; Toonen, Robert J; Gates, Ruth D

2011-01-05

Endosymbiotic dinoflagellates in the genus Symbiodinium are fundamentally important to the biology of scleractinian corals, as well as to a variety of other marine organisms. The genus Symbiodinium is genetically and functionally diverse and the taxonomic nature of the union between Symbiodinium and corals is implicated as a key trait determining the environmental tolerance of the symbiosis. Surprisingly, the question of how Symbiodinium diversity partitions within a species across spatial scales of meters to kilometers has received little attention, but is important to understanding the intrinsic biological scope of a given coral population and adaptations to the local environment. Here we address this gap by describing the Symbiodinium ITS2 sequence assemblages recovered from colonies of the reef building coral Montipora capitata sampled across Kāne'ohe Bay, Hawai'i. A total of 52 corals were sampled in a nested design of Coral Colony(Site(Region)) reflecting spatial scales of meters to kilometers. A diversity of Symbiodinium ITS2 sequences was recovered with the majority of variance partitioning at the level of the Coral Colony. To confirm this result, the Symbiodinium ITS2 sequence diversity in six M. capitata colonies were analyzed in much greater depth with 35 to 55 clones per colony. The ITS2 sequences and quantitative composition recovered from these colonies varied significantly, indicating that each coral hosted a different assemblage of Symbiodinium. The diversity of Symbiodinium ITS2 sequence assemblages retrieved from individual colonies of M. capitata here highlights the problems inherent in interpreting multi-copy and intra-genomically variable molecular markers, and serves as a context for discussing the utility and biological relevance of assigning species names based on Symbiodinium ITS2 genotyping.

Variation in Symbiodinium ITS2 Sequence Assemblages among Coral Colonies

PubMed Central

Stat, Michael; Bird, Christopher E.; Pochon, Xavier; Chasqui, Luis; Chauka, Leonard J.; Concepcion, Gregory T.; Logan, Dan; Takabayashi, Misaki; Toonen, Robert J.; Gates, Ruth D.

2011-01-01

Endosymbiotic dinoflagellates in the genus Symbiodinium are fundamentally important to the biology of scleractinian corals, as well as to a variety of other marine organisms. The genus Symbiodinium is genetically and functionally diverse and the taxonomic nature of the union between Symbiodinium and corals is implicated as a key trait determining the environmental tolerance of the symbiosis. Surprisingly, the question of how Symbiodinium diversity partitions within a species across spatial scales of meters to kilometers has received little attention, but is important to understanding the intrinsic biological scope of a given coral population and adaptations to the local environment. Here we address this gap by describing the Symbiodinium ITS2 sequence assemblages recovered from colonies of the reef building coral Montipora capitata sampled across Kāne'ohe Bay, Hawai'i. A total of 52 corals were sampled in a nested design of Coral Colony(Site(Region)) reflecting spatial scales of meters to kilometers. A diversity of Symbiodinium ITS2 sequences was recovered with the majority of variance partitioning at the level of the Coral Colony. To confirm this result, the Symbiodinium ITS2 sequence diversity in six M. capitata colonies were analyzed in much greater depth with 35 to 55 clones per colony. The ITS2 sequences and quantitative composition recovered from these colonies varied significantly, indicating that each coral hosted a different assemblage of Symbiodinium. The diversity of Symbiodinium ITS2 sequence assemblages retrieved from individual colonies of M. capitata here highlights the problems inherent in interpreting multi-copy and intra-genomically variable molecular markers, and serves as a context for discussing the utility and biological relevance of assigning species names based on Symbiodinium ITS2 genotyping. PMID:21246044

Characterization of Vibrio parahaemolyticus clinical strains from Maryland (2012-2013) and comparisons to a locally and globally diverse V. parahaemolyticus strains by whole-genome sequence analysis.

PubMed

Haendiges, Julie; Timme, Ruth; Allard, Marc W; Myers, Robert A; Brown, Eric W; Gonzalez-Escalona, Narjol

2015-01-01

Vibrio parahaemolyticus is the leading cause of foodborne illnesses in the US associated with the consumption of raw shellfish. Previous population studies of V. parahaemolyticus have used Multi-Locus Sequence Typing (MLST) or Pulsed Field Gel Electrophoresis (PFGE). Whole genome sequencing (WGS) provides a much higher level of resolution, but has been used to characterize only a few United States (US) clinical isolates. Here we report the WGS characterization of 34 genomes of V. parahaemolyticus strains that were isolated from clinical cases in the state of Maryland (MD) during 2 years (2012-2013). These 2 years saw an increase of V. parahaemolyticus cases compared to previous years. Among these MD isolates, 28% were negative for tdh and trh, 8% were tdh positive only, 11% were trh positive only, and 53% contained both genes. We compared this set of V. parahaemolyticus genomes to those of a collection of 17 archival strains from the US (10 previously sequenced strains and 7 from NCBI, collected between 1988 and 2004) and 15 international strains, isolated from geographically-diverse environmental and clinical sources (collected between 1980 and 2010). A WGS phylogenetic analysis of these strains revealed the regional outbreak strains from MD are highly diverse and yet genetically distinct from the international strains. Some MD strains caused outbreaks 2 years in a row, indicating a local source of contamination (e.g., ST631). Advances in WGS will enable this type of analysis to become routine, providing an excellent tool for improved surveillance. Databases built with phylogenetic data will help pinpoint sources of contamination in future outbreaks and contribute to faster outbreak control.

Characterization of Vibrio parahaemolyticus clinical strains from Maryland (2012–2013) and comparisons to a locally and globally diverse V. parahaemolyticus strains by whole-genome sequence analysis

PubMed Central

Haendiges, Julie; Timme, Ruth; Allard, Marc W.; Myers, Robert A.; Brown, Eric W.; Gonzalez-Escalona, Narjol

2015-01-01

Vibrio parahaemolyticus is the leading cause of foodborne illnesses in the US associated with the consumption of raw shellfish. Previous population studies of V. parahaemolyticus have used Multi-Locus Sequence Typing (MLST) or Pulsed Field Gel Electrophoresis (PFGE). Whole genome sequencing (WGS) provides a much higher level of resolution, but has been used to characterize only a few United States (US) clinical isolates. Here we report the WGS characterization of 34 genomes of V. parahaemolyticus strains that were isolated from clinical cases in the state of Maryland (MD) during 2 years (2012–2013). These 2 years saw an increase of V. parahaemolyticus cases compared to previous years. Among these MD isolates, 28% were negative for tdh and trh, 8% were tdh positive only, 11% were trh positive only, and 53% contained both genes. We compared this set of V. parahaemolyticus genomes to those of a collection of 17 archival strains from the US (10 previously sequenced strains and 7 from NCBI, collected between 1988 and 2004) and 15 international strains, isolated from geographically-diverse environmental and clinical sources (collected between 1980 and 2010). A WGS phylogenetic analysis of these strains revealed the regional outbreak strains from MD are highly diverse and yet genetically distinct from the international strains. Some MD strains caused outbreaks 2 years in a row, indicating a local source of contamination (e.g., ST631). Advances in WGS will enable this type of analysis to become routine, providing an excellent tool for improved surveillance. Databases built with phylogenetic data will help pinpoint sources of contamination in future outbreaks and contribute to faster outbreak control. PMID:25745421

Quantifying selection and diversity in viruses by entropy methods, with application to the haemagglutinin of H3N2 influenza

PubMed Central

Pan, Keyao; Deem, Michael W.

2011-01-01

Many viruses evolve rapidly. For example, haemagglutinin (HA) of the H3N2 influenza A virus evolves to escape antibody binding. This evolution of the H3N2 virus means that people who have previously been exposed to an influenza strain may be infected by a newly emerged virus. In this paper, we use Shannon entropy and relative entropy to measure the diversity and selection pressure by an antibody in each amino acid site of H3 HA between the 1992–1993 season and the 2009–2010 season. Shannon entropy and relative entropy are two independent state variables that we use to characterize H3N2 evolution. The entropy method estimates future H3N2 evolution and migration using currently available H3 HA sequences. First, we show that the rate of evolution increases with the virus diversity in the current season. The Shannon entropy of the sequence in the current season predicts relative entropy between sequences in the current season and those in the next season. Second, a global migration pattern of H3N2 is assembled by comparing the relative entropy flows of sequences sampled in China, Japan, the USA and Europe. We verify this entropy method by describing two aspects of historical H3N2 evolution. First, we identify 54 amino acid sites in HA that have evolved in the past to evade the immune system. Second, the entropy method shows that epitopes A and B on the top of HA evolve most vigorously to escape antibody binding. Our work provides a novel entropy-based method to predict and quantify future H3N2 evolution and to describe the evolutionary history of H3N2. PMID:21543352

Maize Endophytic Bacterial Diversity as Affected by Soil Cultivation History.

PubMed

Correa-Galeote, David; Bedmar, Eulogio J; Arone, Gregorio J

2018-01-01

The bacterial endophytic communities residing within roots of maize ( Zea mays L.) plants cultivated by a sustainable management in soils from the Quechua maize belt (Peruvian Andes) were examined using tags pyrosequencing spanning the V4 and V5 hypervariable regions of the 16S rRNA. Across four replicate libraries, two corresponding to sequences of endophytic bacteria from long time maize-cultivated soils and the other two obtained from fallow soils, 793 bacterial sequences were found that grouped into 188 bacterial operational taxonomic units (OTUs, 97% genetic similarity). The numbers of OTUs in the libraries from the maize-cultivated soils were significantly higher than those found in the libraries from fallow soils. A mean of 30 genera were found in the fallow soil libraries and 47 were in those from the maize-cultivated soils. Both alpha and beta diversity indexes showed clear differences between bacterial endophytic populations from plants with different soil cultivation history and that the soils cultivated for long time requires a higher diversity of endophytes. The number of sequences corresponding to main genera Sphingomonas, Herbaspirillum, Bradyrhizobium and Methylophilus in the maize-cultivated libraries were statistically more abundant than those from the fallow soils. Sequences of genera Dyella and Sreptococcus were significantly more abundant in the libraries from the fallow soils. Relative abundance of genera Burkholderia, candidatus Glomeribacter, Staphylococcus, Variovorax, Bacillus and Chitinophaga were similar among libraries. A canonical correspondence analysis of the relative abundance of the main genera showed that the four libraries distributed in two clearly separated groups. Our results suggest that cultivation history is an important driver of endophytic colonization of maize and that after a long time of cultivation of the soil the maize plants need to increase the richness of the bacterial endophytes communities.

Maize Endophytic Bacterial Diversity as Affected by Soil Cultivation History

PubMed Central

Correa-Galeote, David; Bedmar, Eulogio J.; Arone, Gregorio J.

2018-01-01

The bacterial endophytic communities residing within roots of maize (Zea mays L.) plants cultivated by a sustainable management in soils from the Quechua maize belt (Peruvian Andes) were examined using tags pyrosequencing spanning the V4 and V5 hypervariable regions of the 16S rRNA. Across four replicate libraries, two corresponding to sequences of endophytic bacteria from long time maize-cultivated soils and the other two obtained from fallow soils, 793 bacterial sequences were found that grouped into 188 bacterial operational taxonomic units (OTUs, 97% genetic similarity). The numbers of OTUs in the libraries from the maize-cultivated soils were significantly higher than those found in the libraries from fallow soils. A mean of 30 genera were found in the fallow soil libraries and 47 were in those from the maize-cultivated soils. Both alpha and beta diversity indexes showed clear differences between bacterial endophytic populations from plants with different soil cultivation history and that the soils cultivated for long time requires a higher diversity of endophytes. The number of sequences corresponding to main genera Sphingomonas, Herbaspirillum, Bradyrhizobium and Methylophilus in the maize-cultivated libraries were statistically more abundant than those from the fallow soils. Sequences of genera Dyella and Sreptococcus were significantly more abundant in the libraries from the fallow soils. Relative abundance of genera Burkholderia, candidatus Glomeribacter, Staphylococcus, Variovorax, Bacillus and Chitinophaga were similar among libraries. A canonical correspondence analysis of the relative abundance of the main genera showed that the four libraries distributed in two clearly separated groups. Our results suggest that cultivation history is an important driver of endophytic colonization of maize and that after a long time of cultivation of the soil the maize plants need to increase the richness of the bacterial endophytes communities. PMID:29662471

Prevalence, distribution, and sequence diversity of hmwA among commensal and otitis media non-typeable Haemophilus influenzae.

PubMed

Davis, Gregg S; Patel, May; Hammond, James; Zhang, Lixin; Dawid, Suzanne; Marrs, Carl F; Gilsdorf, Janet R

2014-12-01

Nontypeable Haemophilus influenzae (NTHi) are Gram-negative coccobacilli that colonize the human pharynx, their only known natural reservoir. Adherence to the host epithelium facilitates NTHi colonization and marks one of the first steps in NTHi pathogenesis. Epithelial cell attachment is mediated, in part, by a pair of high molecular weight (HMW) adhesins that are highly immunogenic, antigenically diverse, and display a wide range of amino acid diversity both within and between isolates. In this study, the prevalence of hmwA, which encodes the HMW adhesin, was determined for a collection of 170 NTHi isolates recovered from the middle ears of children with otitis media (OM isolates) or throats or nasopharynges of healthy children (commensal isolates) from Finland, Israel, and the U.S. Overall, hmwA was detected in 61% of NTHi isolates and was significantly more prevalent (P=0.004) among OM isolates than among commensal isolates; the prevalence ratio comparing hmwA prevalence among ear isolates with that of commensal isolates was 1.47 (95% CI (1.12, 1.92)). Ninety-five percent (98/103) of the hmwA-positive NTHi isolates possessed two hmw loci. To advance our understanding of hmwA binding sequence diversity, we determined the DNA sequence of the hmwA binding region of 33 isolates from this collection. The average amino acid identity across all hmwA sequences was 62%. Phylogenetic analyses of the hmwA binding revealed four distinct sequence clusters, and the majority of hmwA sequences (83%) belonged to one of two dominant sequence clusters. hmwA sequences did not cluster by chromosomal location, geographic region, or disease status. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of microRNAs in color space.

PubMed

Marco, Antonio; Griffiths-Jones, Sam

2012-02-01

Deep sequencing provides inexpensive opportunities to characterize the transcriptional diversity of known genomes. The AB SOLiD technology generates millions of short sequencing reads in color-space; that is, the raw data is a sequence of colors, where each color represents 2 nt and each nucleotide is represented by two consecutive colors. This strategy is purported to have several advantages, including increased ability to distinguish sequencing errors from polymorphisms. Several programs have been developed to map short reads to genomes in color space. However, a number of previously unexplored technical issues arise when using SOLiD technology to characterize microRNAs. Here we explore these technical difficulties. First, since the sequenced reads are longer than the biological sequences, every read is expected to contain linker fragments. The color-calling error rate increases toward the 3(') end of the read such that recognizing the linker sequence for removal becomes problematic. Second, mapping in color space may lead to the loss of the first nucleotide of each read. We propose a sequential trimming and mapping approach to map small RNAs. Using our strategy, we reanalyze three published insect small RNA deep sequencing datasets and characterize 22 new microRNAs. A bash shell script to perform the sequential trimming and mapping procedure, called SeqTrimMap, is available at: http://www.mirbase.org/tools/seqtrimmap/ antonio.marco@manchester.ac.uk Supplementary data are available at Bioinformatics online.

Evaluating the use of diversity indices to distinguish between microbial communities with different traits.

PubMed

Feranchuk, Sergey; Belkova, Natalia; Potapova, Ulyana; Kuzmin, Dmitry; Belikov, Sergei

2018-05-23

Several measures of biodiversity are commonly used to describe microbial communities, analyzed using 16S gene sequencing. A wide range of available experiments on 16S gene sequencing allows us to present a framework for a comparison of various diversity indices. The criterion for the comparison is the statistical significance of the difference in index values for microbial communities with different traits, within the same experiment. The results of the evaluation indicate that Shannon diversity is the most effective measure among the commonly used diversity indices. The results also indicate that, within the present framework, the Gini coefficient as a diversity index is comparable to Shannon diversity, despite the fact that the Gini coefficient, as a diversity estimator, is far less popular in microbiology than several other measures. Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

How Much Do rRNA Gene Surveys Underestimate Extant Bacterial Diversity?

PubMed

Rodriguez-R, Luis M; Castro, Juan C; Kyrpides, Nikos C; Cole, James R; Tiedje, James M; Konstantinidis, Konstantinos T

2018-03-15

The most common practice in studying and cataloguing prokaryotic diversity involves the grouping of sequences into operational taxonomic units (OTUs) at the 97% 16S rRNA gene sequence identity level, often using partial gene sequences, such as PCR-generated amplicons. Due to the high sequence conservation of rRNA genes, organisms belonging to closely related yet distinct species may be grouped under the same OTU. However, it remains unclear how much diversity has been underestimated by this practice. To address this question, we compared the OTUs of genomes defined at the 97% or 98.5% 16S rRNA gene identity level against OTUs of the same genomes defined at the 95% whole-genome average nucleotide identity (ANI), which is a much more accurate proxy for species. Our results show that OTUs resulting from a 98.5% 16S rRNA gene identity cutoff are more accurate than 97% compared to 95% ANI (90.5% versus 89.9% accuracy) but indistinguishable from any other threshold in the 98.29 to 98.78% range. Even with the more stringent thresholds, however, the 16S rRNA gene-based approach commonly underestimates the number of OTUs by ∼12%, on average, compared to the ANI-based approach (∼14% underestimation when using the 97% identity threshold). More importantly, the degree of underestimation can become 50% or more for certain taxa, such as the genera Pseudomonas , Burkholderia , Escherichia , Campylobacter , and Citrobacter These results provide a quantitative view of the degree of underestimation of extant prokaryotic diversity by 16S rRNA gene-defined OTUs and suggest that genomic resolution is often necessary. IMPORTANCE Species diversity is one of the most fundamental pieces of information for community ecology and conservational biology. Therefore, employing accurate proxies for what a species or the unit of diversity is are cornerstones for a large set of microbial ecology and diversity studies. The most common proxies currently used rely on the clustering of 16S rRNA gene sequences at some threshold of nucleotide identity, typically 97% or 98.5%. Here, we explore how well this strategy reflects the more accurate whole-genome-based proxies and determine the frequency with which the high conservation of 16S rRNA sequences masks substantial species-level diversity. Copyright © 2018 American Society for Microbiology.

Sequence Variation of the tRNALeu Intron as a Marker for Genetic Diversity and Specificity of Symbiotic Cyanobacteria in Some Lichens

PubMed Central

Paulsrud, Per; Lindblad, Peter

1998-01-01

We examined the genetic diversity of Nostoc symbionts in some lichens by using the tRNALeu (UAA) intron as a genetic marker. The nucleotide sequence was analyzed in the context of the secondary structure of the transcribed intron. Cyanobacterial tRNALeu (UAA) introns were specifically amplified from freshly collected lichen samples without previous DNA extraction. The lichen species used in the present study were Nephroma arcticum, Peltigera aphthosa, P. membranacea, and P. canina. Introns with different sizes around 300 bp were consistently obtained. Multiple clones from single PCRs were screened by using their single-stranded conformational polymorphism pattern, and the nucleotide sequence was determined. No evidence for sample heterogenity was found. This implies that the symbiont in situ is not a diverse community of cyanobionts but, rather, one Nostoc strain. Furthermore, each lichen thallus contained only one intron type, indicating that each thallus is colonized only once or that there is a high degree of specificity. The same cyanobacterial intron sequence was also found in samples of one lichen species from different localities. In a phylogenetic analysis, the cyanobacterial lichen sequences grouped together with the sequences from two free-living Nostoc strains. The size differences in the intron were due to insertions and deletions in highly variable regions. The sequence data were used in discussions concerning specificity and biology of the lichen symbiosis. It is concluded that the tRNALeu (UAA) intron can be of great value when examining cyanobacterial diversity. PMID:9435083

Taxonomy, Epidemiology, and Clinical Relevance of the Genus Arcobacter

PubMed Central

Collado, Luis; Figueras, Maria José

2011-01-01

Summary: The genus Arcobacter, defined almost 20 years ago from members of the genus Campylobacter, has become increasingly important because its members are being considered emergent enteropathogens and/or potential zoonotic agents. Over recent years information that is relevant for microbiologists, especially those working in the medical and veterinary fields and in the food safety sector, has accumulated. Recently, the genus has been enlarged with several new species. The complete genomes of Arcobacter butzleri and Arcobacter nitrofigilis are available, with the former revealing diverse pathways characteristic of free-living microbes and virulence genes homologous to those of Campylobacter. The first multilocus sequence typing analysis showed a great diversity of sequence types, with no association with specific hosts or geographical regions. Advances in detection and identification techniques, mostly based on molecular methods, have been made. These microbes have been associated with water outbreaks and with indicators of fecal pollution, with food products and water as the suspected routes of transmission. This review updates this knowledge and provides the most recent data on the taxonomy, species diversity, methods of detection, and identification of these microbes as well as on their virulence potential and implication in human and animal diseases. PMID:21233511

Population Structure, Genetic Diversity, and Evolutionary History of Kleinia neriifolia (Asteraceae) on the Canary Islands.

PubMed

Sun, Ye; Vargas-Mendoza, Carlos F

2017-01-01

Kleinia neriifolia Haw. is an endemic species on the Canarian archipelago, this species is widespread in the coastal thicket of all the Canarian islands. In the present study, genetic diversity and population structure of K. neriifolia were investigated using chloroplast gene sequences and nuclear SSR (simple sequence repeat). The differentiation among island populations, the historical demography, and the underlying evolutionary scenarios of this species are further tested based on the genetic data. Chloroplast diversity reveals a strong genetic divergence between eastern islands (Gran Canaria, Fuerteventura, and Lanzarote) and western islands (EI Hierro, La Palma, La Gomera, Tenerife), this west-east genetic divergence may reflect a very beginning of speciation. The evolutionary scenario with highest posterior probabilities suggests Gran Canaria as oldest population with a westward colonization path to Tenerife, La Gomera, La Palma, and EI Hierro, and eastward dispersal path to Lanzarote through Fuerteventura. In the western islands, there is a slight decrease in the effective population size toward areas of recent colonization. However, in the eastern islands, the effective population size increase in Lanzarote relative to Gran Canaria and Fuerteventura. These results further our understanding of the evolution of widespread endemic plants within Canarian archipelago.

Population Structure, Genetic Diversity, and Evolutionary History of Kleinia neriifolia (Asteraceae) on the Canary Islands

PubMed Central

Sun, Ye; Vargas-Mendoza, Carlos F.

2017-01-01

Kleinia neriifolia Haw. is an endemic species on the Canarian archipelago, this species is widespread in the coastal thicket of all the Canarian islands. In the present study, genetic diversity and population structure of K. neriifolia were investigated using chloroplast gene sequences and nuclear SSR (simple sequence repeat). The differentiation among island populations, the historical demography, and the underlying evolutionary scenarios of this species are further tested based on the genetic data. Chloroplast diversity reveals a strong genetic divergence between eastern islands (Gran Canaria, Fuerteventura, and Lanzarote) and western islands (EI Hierro, La Palma, La Gomera, Tenerife), this west–east genetic divergence may reflect a very beginning of speciation. The evolutionary scenario with highest posterior probabilities suggests Gran Canaria as oldest population with a westward colonization path to Tenerife, La Gomera, La Palma, and EI Hierro, and eastward dispersal path to Lanzarote through Fuerteventura. In the western islands, there is a slight decrease in the effective population size toward areas of recent colonization. However, in the eastern islands, the effective population size increase in Lanzarote relative to Gran Canaria and Fuerteventura. These results further our understanding of the evolution of widespread endemic plants within Canarian archipelago. PMID:28713419

Cyanobacterial Diversity in Biological Soil Crusts along a Precipitation Gradient, Northwest Negev Desert, Israel.

PubMed

Hagemann, Martin; Henneberg, Manja; Felde, Vincent J M N L; Drahorad, Sylvie L; Berkowicz, Simon M; Felix-Henningsen, Peter; Kaplan, Aaron

2015-07-01

Cyanobacteria occur worldwide but play an important role in the formation and primary activity of biological soil crusts (BSCs) in arid and semi-arid ecosystems. The cyanobacterial diversity in BSCs of the northwest Negev desert of Israel was surveyed at three fixed sampling stations situated along a precipitation gradient in the years 2010 to 2012. The three stations also are characterized by marked differences in soil features such as soil carbon, nitrogen, or electrical conductivity. The cyanobacterial biodiversity was analyzed by sequencing inserts of clone libraries harboring partial 16S rRNA gene sequences obtained with cyanobacteria-specific primers. Filamentous, non-diazotrophic strains (subsection III), particularly Microcoleus-like, dominated the cyanobacterial community (30% proportion) in all years. Specific cyanobacterial groups showed increased (e.g., Chroococcidiopsis, Leptolyngbya, and Nostoc strains) or decreased (e.g., unicellular strains belonging to the subsection I and Scytonema strains) abundances with declining water availability at the most arid, southern station, whereas many cyanobacterial strains were frequently found in the soils of all three stations. The cyanobacterial diversity at the three sampling stations appears dependent on the available precipitation, whereas the differences in soil chemistry were of lower importance.

Helminth Colonization Is Associated with Increased Diversity of the Gut Microbiota

PubMed Central

Lee, Soo Ching; Tang, Mei San; Lim, Yvonne A. L.; Choy, Seow Huey; Kurtz, Zachary D.; Cox, Laura M.; Gundra, Uma Mahesh; Cho, Ilseung; Bonneau, Richard; Blaser, Martin J.; Chua, Kek Heng; Loke, P'ng

2014-01-01

Soil-transmitted helminths colonize more than 1.5 billion people worldwide, yet little is known about how they interact with bacterial communities in the gut microbiota. Differences in the gut microbiota between individuals living in developed and developing countries may be partly due to the presence of helminths, since they predominantly infect individuals from developing countries, such as the indigenous communities in Malaysia we examine in this work. We compared the composition and diversity of bacterial communities from the fecal microbiota of 51 people from two villages in Malaysia, of which 36 (70.6%) were infected by helminths. The 16S rRNA V4 region was sequenced at an average of nineteen thousand sequences per samples. Helminth-colonized individuals had greater species richness and number of observed OTUs with enrichment of Paraprevotellaceae, especially with Trichuris infection. We developed a new approach of combining centered log-ratio (clr) transformation for OTU relative abundances with sparse Partial Least Squares Discriminant Analysis (sPLS-DA) to enable more robust predictions of OTU interrelationships. These results suggest that helminths may have an impact on the diversity, bacterial community structure and function of the gut microbiota. PMID:24851867

T7 lytic phage-displayed peptide libraries exhibit less sequence bias than M13 filamentous phage-displayed peptide libraries.

PubMed

Krumpe, Lauren R H; Atkinson, Andrew J; Smythers, Gary W; Kandel, Andrea; Schumacher, Kathryn M; McMahon, James B; Makowski, Lee; Mori, Toshiyuki

2006-08-01

We investigated whether the T7 system of phage display could produce peptide libraries of greater diversity than the M13 system of phage display due to the differing processes of lytic and filamentous phage morphogenesis. Using a bioinformatics-assisted computational approach, collections of random peptide sequences obtained from a T7 12-mer library (X(12)) and a T7 7-mer disulfide-constrained library (CX(7)C) were analyzed and compared with peptide populations obtained from New England BioLabs' M13 Ph.D.-12 and Ph.D.-C7C libraries. Based on this analysis, peptide libraries constructed with the T7 system have fewer amino acid biases, increased peptide diversity, and more normal distributions of peptide net charge and hydropathy than the M13 libraries. The greater diversity of T7-displayed libraries provides a potential resource of novel binding peptides for new as well as previously studied molecular targets. To demonstrate their utility, several of the T7-displayed peptide libraries were screened for streptavidin- and neutravidin-binding phage. Novel binding motifs were identified for each protein.

Screening and Characterization of RAPD Markers in Viscerotropic Leishmania Parasites

PubMed Central

Mkada–Driss, Imen; Talbi, Chiraz; Guerbouj, Souheila; Driss, Mehdi; Elamine, Elwaleed M.; Cupolillo, Elisa; Mukhtar, Moawia M.; Guizani, Ikram

2014-01-01

Visceral leishmaniasis (VL) is mainly due to the Leishmania donovani complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL Leishmania parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 L. donovani, L. archibaldi and L. infantum strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5′ end transversions, and presence of inter– and intra– taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between L. infantum and L. donovani. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents. PMID:25313833

Genes encoding two Theileria parva antigens recognized by CD8+ T-cells exhibit sequence diversity in South Sudanese cattle populations but the majority of alleles are similar to the Muguga component of the live vaccine cocktail

PubMed Central

Pelle, Roger; Mwacharo, Joram M.; Njahira, Moses N.; Marcellino, Wani L.; Kiara, Henry; Malak, Agol K.; EL Hussein, Abdel Rahim M.; Bishop, Richard; Skilton, Robert A.

2017-01-01

East Coast fever (ECF), caused by Theileria parva infection, is a frequently fatal disease of cattle in eastern, central and southern Africa, and an emerging disease in South Sudan. Immunization using the infection and treatment method (ITM) is increasingly being used for control in countries affected by ECF, but not yet in South Sudan. It has been reported that CD8+ T-cell lymphocytes specific for parasitized cells play a central role in the immunity induced by ITM and a number of T. parva antigens recognized by parasite-specific CD8+ T-cells have been identified. In this study we determined the sequence diversity among two of these antigens, Tp1 and Tp2, which are under evaluation as candidates for inclusion in a sub-unit vaccine. T. parva samples (n = 81) obtained from cattle in four geographical regions of South Sudan were studied for sequence polymorphism in partial sequences of the Tp1 and Tp2 genes. Eight positions (1.97%) in Tp1 and 78 positions (15.48%) in Tp2 were shown to be polymorphic, giving rise to four and 14 antigen variants in Tp1 and Tp2, respectively. The overall nucleotide diversity in the Tp1 and Tp2 genes was π = 1.65% and π = 4.76%, respectively. The parasites were sampled from regions approximately 300 km apart, but there was limited evidence for genetic differentiation between populations. Analyses of the sequences revealed limited numbers of amino acid polymorphisms both overall and in residues within the mapped CD8+ T-cell epitopes. Although novel epitopes were identified in the samples from South Sudan, a large number of the samples harboured several epitopes in both antigens that were similar to those in the T. parva Muguga reference stock, which is a key component in the widely used live vaccine cocktail. PMID:28231338

Characterization of bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, as determined by 16S rDNA analysis.

PubMed

Escalante, Adelfo; Rodríguez, María Elena; Martínez, Alfredo; López-Munguía, Agustín; Bolívar, Francisco; Gosset, Guillermo

2004-06-15

The bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, was studied in 16S rDNA clone libraries from three pulque samples. Sequenced clones identified as Lactobacillus acidophilus, Lactobacillus strain ASF360, L. kefir, L. acetotolerans, L. hilgardii, L. plantarum, Leuconostoc pseudomesenteroides, Microbacterium arborescens, Flavobacterium johnsoniae, Acetobacter pomorium, Gluconobacter oxydans, and Hafnia alvei, were detected for the first time in pulque. Identity of 16S rDNA sequenced clones showed that bacterial diversity present among pulque samples is dominated by Lactobacillus species (80.97%). Seventy-eight clones exhibited less than 95% of relatedness to NCBI database sequences, which may indicate the presence of new species in pulque samples.

Sequence diversity and evolution of antimicrobial peptides in invertebrates.

PubMed

Tassanakajon, Anchalee; Somboonwiwat, Kunlaya; Amparyup, Piti

2015-02-01

Antimicrobial peptides (AMPs) are evolutionarily ancient molecules that act as the key components in the invertebrate innate immunity against invading pathogens. Several AMPs have been identified and characterized in invertebrates, and found to display considerable diversity in their amino acid sequence, structure and biological activity. AMP genes appear to have rapidly evolved, which might have arisen from the co-evolutionary arms race between host and pathogens, and enabled organisms to survive in different microbial environments. Here, the sequence diversity of invertebrate AMPs (defensins, cecropins, crustins and anti-lipopolysaccharide factors) are presented to provide a better understanding of the evolution pattern of these peptides that play a major role in host defense mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Impact of selected non-steroidal anti-inflammatory pharmaceuticals on microbial community assembly and activity in sequencing batch reactors

PubMed Central

Jiang, Cong; Hu, Haidong; Ma, Haijun; Gao, Xingsheng; Ren, Hongqiang

2017-01-01

This study covers three widely detected non-steroidal anti-inflammatory pharmaceuticals (NSAIDs), diclofenac (DCF), ibuprofen (IBP) and naproxen (NPX), as NSAIDs pollutants. The objective is to evaluate the impact of NSAIDs at their environmental concentrations on microbial community assembly and activity. The exposure experiments were conducted under three conditions (5 μg L-1 DCF, 5 μg L-1 DCF+5 μg L-1 IBP and 5 μg L-1 DCF+5 μg L-1 IBP+ 5 μg L-1 NPX) in sequencing batch reactors (SBRs) for 130 days. Removals of COD and NH4+-N were not affected but total nitrogen (TN) removal decreased. IBP and NPX had the high removal efficiencies (79.96% to 85.64%), whereas DCF was more persistent (57.24% to 64.12%). In addition, the decreased removals of TN remained the same under the three conditions (p > 0.05). The results of oxidizing enzyme activities, live cell percentages and extracellular polymeric substances (EPS) indicated that NSAIDs damaged the cell walls or microorganisms and the mixtures of the three NSAIDs increased the toxicity. The increased Shannon-Wiener diversity index suggested that bacterial diversity was increased with the addition of selected NSAIDs. Bacterial ribosomal RNA small subunit (16S) gene sequencing results indicated that Actinobacteria and Bacteroidetes were enriched, while Micropruina and Nakamurella decreased with the addition of NSAIDs. The enrichment of Actinobacteria and Bacteroidetes indicated that both of them might have the ability to degrade NSAIDs and thereby could adapt well with the presence of NSAIDs. PMID:28640897

Impact of selected non-steroidal anti-inflammatory pharmaceuticals on microbial community assembly and activity in sequencing batch reactors.

PubMed

Jiang, Cong; Geng, Jinju; Hu, Haidong; Ma, Haijun; Gao, Xingsheng; Ren, Hongqiang

2017-01-01

This study covers three widely detected non-steroidal anti-inflammatory pharmaceuticals (NSAIDs), diclofenac (DCF), ibuprofen (IBP) and naproxen (NPX), as NSAIDs pollutants. The objective is to evaluate the impact of NSAIDs at their environmental concentrations on microbial community assembly and activity. The exposure experiments were conducted under three conditions (5 μg L-1 DCF, 5 μg L-1 DCF+5 μg L-1 IBP and 5 μg L-1 DCF+5 μg L-1 IBP+ 5 μg L-1 NPX) in sequencing batch reactors (SBRs) for 130 days. Removals of COD and NH4+-N were not affected but total nitrogen (TN) removal decreased. IBP and NPX had the high removal efficiencies (79.96% to 85.64%), whereas DCF was more persistent (57.24% to 64.12%). In addition, the decreased removals of TN remained the same under the three conditions (p > 0.05). The results of oxidizing enzyme activities, live cell percentages and extracellular polymeric substances (EPS) indicated that NSAIDs damaged the cell walls or microorganisms and the mixtures of the three NSAIDs increased the toxicity. The increased Shannon-Wiener diversity index suggested that bacterial diversity was increased with the addition of selected NSAIDs. Bacterial ribosomal RNA small subunit (16S) gene sequencing results indicated that Actinobacteria and Bacteroidetes were enriched, while Micropruina and Nakamurella decreased with the addition of NSAIDs. The enrichment of Actinobacteria and Bacteroidetes indicated that both of them might have the ability to degrade NSAIDs and thereby could adapt well with the presence of NSAIDs.

K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries.

PubMed

Jangid, Kamlesh; Kao, Ming-Hung; Lahamge, Aishwarya; Williams, Mark A; Rathbun, Stephen L; Whitman, William B

2016-01-01

K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. Inspired by Ripley's K-function for spatial point pattern analysis, the Intra K-function or IKF measures the structural diversity, including both the richness and overall similarity of the sequences, within a library. The Cross K-function or CKF measures the compositional diversity between gene libraries, reflecting both the number of OTUs shared as well as the overall similarity in OTUs. A Monte Carlo testing procedure then enables statistical evaluation of both the structural and compositional diversity between gene libraries. For 16S rRNA gene libraries from complex bacterial communities such as those found in seawater, salt marsh sediments, and soils, K-shuff yields reproducible estimates of structural and compositional diversity with libraries greater than 50 sequences. Similarly, for pyrosequencing libraries generated from a glacial retreat chronosequence and Illumina® libraries generated from US homes, K-shuff required >300 and 100 sequences per sample, respectively. Power analyses demonstrated that K-shuff is sensitive to small differences in Sanger or Illumina® libraries. This extra sensitivity of K-shuff enabled examination of compositional differences at much deeper taxonomic levels, such as within abundant OTUs. This is especially useful when comparing communities that are compositionally very similar but functionally different. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.

Soil Bacterial Community Shifts after Chitin Enrichment: An Integrative Metagenomic Approach

PubMed Central

Jacquiod, Samuel; Franqueville, Laure; Cécillon, Sébastien; M. Vogel, Timothy; Simonet, Pascal

2013-01-01

Chitin is the second most produced biopolymer on Earth after cellulose. Chitin degrading enzymes are promising but untapped sources for developing novel industrial biocatalysts. Hidden amongst uncultivated micro-organisms, new bacterial enzymes can be discovered and exploited by metagenomic approaches through extensive cloning and screening. Enrichment is also a well-known strategy, as it allows selection of organisms adapted to feed on a specific compound. In this study, we investigated how the soil bacterial community responded to chitin enrichment in a microcosm experiment. An integrative metagenomic approach coupling phylochips and high throughput shotgun pyrosequencing was established in order to assess the taxonomical and functional changes in the soil bacterial community. Results indicate that chitin enrichment leads to an increase of Actinobacteria, γ-proteobacteria and β-proteobacteria suggesting specific selection of chitin degrading bacteria belonging to these classes. Part of enriched bacterial genera were not yet reported to be involved in chitin degradation, like the members from the Micrococcineae sub-order (Actinobacteria). An increase of the observed bacterial diversity was noticed, with detection of specific genera only in chitin treated conditions. The relative proportion of metagenomic sequences related to chitin degradation was significantly increased, even if it represents only a tiny fraction of the sequence diversity found in a soil metagenome. PMID:24278158

Functional Anthology of Intrinsic Disorder. II. Cellular Components, Domains, Technical Terms, Developmental Processes and Coding Sequence Diversities Correlated with Long Disordered Regions

PubMed Central

Vucetic, Slobodan; Xie, Hongbo; Iakoucheva, Lilia M.; Oldfield, Christopher J.; Dunker, A. Keith; Obradovic, Zoran; Uversky, Vladimir N.

2008-01-01

Biologically active proteins without stable ordered structure (i.e., intrinsically disordered proteins) are attracting increased attention. Functional repertoires of ordered and disordered proteins are very different, and the ability to differentiate whether a given function is associated with intrinsic disorder or with a well-folded protein is crucial for modern protein science. However, there is a large gap between the number of proteins experimentally confirmed to be disordered and their actual number in nature. As a result, studies of functional properties of confirmed disordered proteins, while helpful in revealing the functional diversity of protein disorder, provide only a limited view. To overcome this problem, a bioinformatics approach for comprehensive study of functional roles of protein disorder was proposed in the first paper of this series (Xie H., Vucetic S., Iakoucheva L.M., Oldfield C.J., Dunker A.K., Obradovic Z., Uversky V.N. (2006) Functional anthology of intrinsic disorder. I. Biological processes and functions of proteins with long disordered regions. J. Proteome Res.). Applying this novel approach to Swiss-Prot sequences and functional keywords, we found over 238 and 302 keywords to be strongly positively or negatively correlated, respectively, with long intrinsically disordered regions. This paper describes ~90 Swiss-Prot keywords attributed to the cellular components, domains, technical terms, developmental processes and coding sequence diversities possessing strong positive and negative correlation with long disordered regions. PMID:17391015

Functional anthology of intrinsic disorder. 2. Cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions.

PubMed

Vucetic, Slobodan; Xie, Hongbo; Iakoucheva, Lilia M; Oldfield, Christopher J; Dunker, A Keith; Obradovic, Zoran; Uversky, Vladimir N

2007-05-01

Biologically active proteins without stable ordered structure (i.e., intrinsically disordered proteins) are attracting increased attention. Functional repertoires of ordered and disordered proteins are very different, and the ability to differentiate whether a given function is associated with intrinsic disorder or with a well-folded protein is crucial for modern protein science. However, there is a large gap between the number of proteins experimentally confirmed to be disordered and their actual number in nature. As a result, studies of functional properties of confirmed disordered proteins, while helpful in revealing the functional diversity of protein disorder, provide only a limited view. To overcome this problem, a bioinformatics approach for comprehensive study of functional roles of protein disorder was proposed in the first paper of this series (Xie, H.; Vucetic, S.; Iakoucheva, L. M.; Oldfield, C. J.; Dunker, A. K.; Obradovic, Z.; Uversky, V. N. Functional anthology of intrinsic disorder. 1. Biological processes and functions of proteins with long disordered regions. J. Proteome Res. 2007, 5, 1882-1898). Applying this novel approach to Swiss-Prot sequences and functional keywords, we found over 238 and 302 keywords to be strongly positively or negatively correlated, respectively, with long intrinsically disordered regions. This paper describes approximately 90 Swiss-Prot keywords attributed to the cellular components, domains, technical terms, developmental processes, and coding sequence diversities possessing strong positive and negative correlation with long disordered regions.

Genetic diversity and antigenicity variation of Babesia bovis merozoite surface antigen-1 (MSA-1) in Thailand.

PubMed

Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Takemae, Hitoshi; Simking, Pacharathon; Jittapalapong, Sathaporn; Igarashi, Ikuo; Yokoyama, Naoaki

2016-07-01

Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand. Copyright © 2016 Elsevier B.V. All rights reserved.

Phylogenetic analysis of feline immunodeficiency virus strains from naturally infected cats in Belgium and The Netherlands.

PubMed

Roukaerts, Inge D M; Theuns, Sebastiaan; Taffin, Elien R L; Daminet, Sylvie; Nauwynck, Hans J

2015-01-22

Feline immunodeficiency virus (FIV) is a major pathogen in feline populations worldwide, with seroprevalences up to 26%. Virus strains circulating in domestic cats are subdivided into different phylogenetic clades (A-E), based on the genetic diversity of the V3-V4 region of the env gene. In this report, a phylogenetic analysis of the V3-V4 env region, and a variable region in the gag gene was made for 36 FIV strains isolated in Belgium and The Netherlands. All newly generated gag sequences clustered together with previously known clade A FIV viruses, confirming the dominance of clade A viruses in Northern Europe. The same was true for the obtained env sequences, with only one sample of an unknown env subtype. Overall, the genetic diversity of FIV strains sequenced in this report was low. This indicates a relatively recent introduction of FIV in Belgium and The Netherlands. However, the sample with an unknown env subtype indicates that new introductions of FIV from unknown origin do occur and this will likely increase genetic variability in time. Copyright © 2014 Elsevier B.V. All rights reserved.

Selection on hemagglutinin imposes a bottleneck during mammalian transmission of reassortant H5N1 influenza viruses

PubMed Central

Wilker, Peter R.; Dinis, Jorge M.; Starrett, Gabriel; Imai, Masaki; Hatta, Masato; Nelson, Chase W.; O’Connor, David H.; Hughes, Austin L.; Neumann, Gabriele; Kawaoka, Yoshihiro; Friedrich, Thomas C.

2013-01-01

The emergence of human-transmissible H5N1 avian influenza viruses poses a major pandemic threat. H5N1 viruses are thought to be highly genetically diverse both among and within hosts, but the effects of this diversity on viral replication and transmission are poorly understood. Here we use deep sequencing to investigate the impact of within-host viral variation on adaptation and transmission of H5N1 viruses in ferrets. We show that although within-host genetic diversity in hemagglutinin (HA) increases during replication in inoculated ferrets, HA diversity is dramatically reduced upon respiratory droplet transmission, where infection is established by only 1–2 distinct HA segments from a diverse source virus population in transmitting animals. Moreover, minor HA variants present in as little as 5.9% of viruses within the source animal become dominant in ferrets infected via respiratory droplets. These findings demonstrate that selective pressures acting during influenza virus transmission among mammals impose a significant bottleneck. PMID:24149915

Transcriptomics and molecular evolutionary rate analysis of the bladderwort (Utricularia), a carnivorous plant with a minimal genome

PubMed Central

2011-01-01

Background The carnivorous plant Utricularia gibba (bladderwort) is remarkable in having a minute genome, which at ca. 80 megabases is approximately half that of Arabidopsis. Bladderworts show an incredible diversity of forms surrounding a defined theme: tiny, bladder-like suction traps on terrestrial, epiphytic, or aquatic plants with a diversity of unusual vegetative forms. Utricularia plants, which are rootless, are also anomalous in physiological features (respiration and carbon distribution), and highly enhanced molecular evolutionary rates in chloroplast, mitochondrial and nuclear ribosomal sequences. Despite great interest in the genus, no genomic resources exist for Utricularia, and the substitution rate increase has received limited study. Results Here we describe the sequencing and analysis of the Utricularia gibba transcriptome. Three different organs were surveyed, the traps, the vegetative shoot bodies, and the inflorescence stems. We also examined the bladderwort transcriptome under diverse stress conditions. We detail aspects of functional classification, tissue similarity, nitrogen and phosphorus metabolism, respiration, DNA repair, and detoxification of reactive oxygen species (ROS). Long contigs of plastid and mitochondrial genomes, as well as sequences for 100 individual nuclear genes, were compared with those of other plants to better establish information on molecular evolutionary rates. Conclusion The Utricularia transcriptome provides a detailed genomic window into processes occurring in a carnivorous plant. It contains a deep representation of the complex metabolic pathways that characterize a putative minimal plant genome, permitting its use as a source of genomic information to explore the structural, functional, and evolutionary diversity of the genus. Vegetative shoots and traps are the most similar organs by functional classification of their transcriptome, the traps expressing hydrolytic enzymes for prey digestion that were previously thought to be encoded by bacteria. Supporting physiological data, global gene expression analysis shows that traps significantly over-express genes involved in respiration and that phosphate uptake might occur mainly in traps, whereas nitrogen uptake could in part take place in vegetative parts. Expression of DNA repair and ROS detoxification enzymes may be indicative of a response to increased respiration. Finally, evidence from the bladderwort transcriptome, direct measurement of ROS in situ, and cross-species comparisons of organellar genomes and multiple nuclear genes supports the hypothesis that increased nucleotide substitution rates throughout the plant may be due to the mutagenic action of amplified ROS production. PMID:21639913

A not-so-big crisis: re-reading Silurian conodont diversity in a sequence-stratigraphic framework

NASA Astrophysics Data System (ADS)

Jarochowska, Emilia; Munnecke, Axel

2016-04-01

Conodonts are extensively used in Ordovician through Triassic biostratigraphy and fossil-based geochemistry. However, their distribution in rock successions is commonly taken at face value, without taking into account their diverse and poorly understood ecology. Multielement taxonomy, ontogenetic and environmental variability, difficulties in extraction, and relative rarity all contribute to the general lack of quantitative studies on conodont stratigraphic distribution and temporal turnover. With respect to Silurian conodonts, the concept of recurrent conodont extinction events - the so called Ireviken, Mulde and Lau events - has become a standard in the stratigraphic literature. The concept has been proposed based on qualitative observations of local extirpations of open-marine pelagic or nekto-benthic taxa and temporary dominance of shallow-water species in the Silurian succession of the Swedish island of Gotland. These changes coincided with positive carbon isotope excursions, abrupt facies shifts, "blooms" of benthic fauna, and changes in reef communities, which have all been combined into a general view of Silurian bio-geochemical events. This view posits a deterministic, reproducible pattern in Silurian conodont diversity, attributed to recurrent ecological or geochemical conditions. The growing body of sequence-stratigraphic interpretations across these events in Gotland and other sections worldwide indicate that in all cases the Silurian "events" are associated with rapid global regressions. This suggests that faunal changes such as the dominance of shallow-water, low-diversity conodont fauna and the increase of benthic invertebrate diversity and abundance represent predictable consequences of the variation in the completeness of the rock record and preservation potential of different environments. Our studies in Poland and Ukraine indicate that the magnitude of change in the taxonomic composition of conodont assemblages across the middle Silurian global regression and the hypothesized Mulde Event is proportional to the associated facies shift. Quantitative data on facies distribution of individual conodont species combined with sequence stratigraphic architecture provides a testable model for the impact of sea-level changes on perceived conodont diversity in a section or basin. This approach highlights the need for quantitative data on conodont distribution in their environmental context, their integration into conodont-based stratigraphy and geochemistry, and for the regular use of Occam's razor to interpretations of paleobiodiversity.

Diverse archaeal community of a bat guano pile in Domica Cave (Slovak Karst, Slovakia).

PubMed

Chronáková, A; Horák, A; Elhottová, D; Kristůfek, V

2009-09-01

The molecular diversity of Archaea in a bat guano pile in Cave Domica (Slovakia), temperate cave ecosystem with significant bat colony (about 1600 individuals), was examined. The guano pile was created mainly by an activity of the Mediterranean horseshoe bat (Rhinolophus euryale) and provides a source of organic carbon and other nutrients in the oligotrophic subsurface ecosystem. The upper and the basal parts of guano surface were sampled where the latter one had higher pH and higher admixture of limestone bedrock and increased colonization of invertebrates. The relative proportion of Archaea determined using CARD-FISH in both parts was 3.5-3.9 % (the basal and upper part, respectively). The archaeal community was dominated by non-thermophilic Crenarchaeota (99 % of clones). Phylogenetic analysis of 115 16S rDNA sequences revealed the presence of Crenarchaeota previously isolated from temperate surface soils (group 1.1b, 62 clones), deep subsurface acid waters (group 1.1a, 52 clones) and Euryarchaeota (1 clone). Four of the analyzed sequences were found to have little similarity to those in public databases. The composition of both archaeal communities differed, with respect to higher diversity of Archaea in the upper part of the bat guano pile. High diversity archaeal population is present in the bat guano deposit and consists of both soil- and subsurface-born Crenarchaeota.

Diverse deep-sea fungi from the South China Sea and their antimicrobial activity.

PubMed

Zhang, Xiao-Yong; Zhang, Yun; Xu, Xin-Ya; Qi, Shu-Hua

2013-11-01

We investigated the diversity of fungal communities in nine different deep-sea sediment samples of the South China Sea by culture-dependent methods followed by analysis of fungal internal transcribed spacer (ITS) sequences. Although 14 out of 27 identified species were reported in a previous study, 13 species were isolated from sediments of deep-sea environments for the first report. Moreover, these ITS sequences of six isolates shared 84-92 % similarity with their closest matches in GenBank, which suggested that they might be novel phylotypes of genera Ajellomyces, Podosordaria, Torula, and Xylaria. The antimicrobial activities of these fungal isolates were explored using a double-layer technique. A relatively high proportion (56 %) of fungal isolates exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among four marine pathogenic microbes (Micrococcus luteus, Pseudoaltermonas piscida, Aspergerillus versicolor, and A. sydowii). Out of these antimicrobial fungi, the genera Arthrinium, Aspergillus, and Penicillium exhibited antibacterial and antifungal activities, while genus Aureobasidium displayed only antibacterial activity, and genera Acremonium, Cladosporium, Geomyces, and Phaeosphaeriopsis displayed only antifungal activity. To our knowledge, this is the first report to investigate the diversity and antimicrobial activity of culturable deep-sea-derived fungi in the South China Sea. These results suggest that diverse deep-sea fungi from the South China Sea are a potential source for antibiotics' discovery and further increase the pool of fungi available for natural bioactive product screening.

Photoautotrophic symbiont and geography are major factors affecting highly structured and diverse bacterial communities in the lichen microbiome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hodkinson, Brendan P; Gottel, Neil R; Schadt, Christopher Warren

2011-01-01

Although common knowledge dictates that the lichen thallus is formed solely by a fungus (mycobiont) that develops a symbiotic relationship with an alga and/or cyanobacterium (photobiont), the non-photoautotrophic bacteria found in lichen microbiomes are increasingly regarded as integral components of lichen thalli. For this study, comparative analyses were conducted on lichen-associated bacterial communities to test for effects of photobiont-types (i.e. green algal vs. cyanobacterial), mycobiont-types and large-scale spatial distances (from tropical to arctic latitudes). Amplicons of the 16S (SSU) rRNA gene were examined using both Sanger sequencing of cloned fragments and barcoded pyrosequencing. Rhizobiales is typically the most abundant andmore » taxonomically diverse order in lichen microbiomes; however, overall bacterial diversity in lichens is shown to be much higher than previously reported. Members of Acidobacteriaceae, Acetobacteraceae, Brucellaceae and sequence group LAR1 are the most commonly found groups across the phylogenetically and geographically broad array of lichens examined here. Major bacterial community trends are significantly correlated with differences in large-scale geography, photobiont-type and mycobiont-type. The lichen as a microcosm represents a structured, unique microbial habitat with greater ecological complexity and bacterial diversity than previously appreciated and can serve as a model system for studying larger ecological and evolutionary principles.« less

A Pan-HIV Strategy for Complete Genome Sequencing

PubMed Central

Yamaguchi, Julie; Alessandri-Gradt, Elodie; Tell, Robert W.; Brennan, Catherine A.

2015-01-01

Molecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e., switching mechanism at 5′ end of RNA transcript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median ∼2,000× depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of ≤4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance. PMID:26699702

Evolution of Enzyme Superfamilies: Comprehensive Exploration of Sequence-Function Relationships.

PubMed

Baier, F; Copp, J N; Tokuriki, N

2016-11-22

The sequence and functional diversity of enzyme superfamilies have expanded through billions of years of evolution from a common ancestor. Understanding how protein sequence and functional "space" have expanded, at both the evolutionary and molecular level, is central to biochemistry, molecular biology, and evolutionary biology. Integrative approaches that examine protein sequence, structure, and function have begun to provide comprehensive views of the functional diversity and evolutionary relationships within enzyme superfamilies. In this review, we outline the recent advances in our understanding of enzyme evolution and superfamily functional diversity. We describe the tools that have been used to comprehensively analyze sequence relationships and to characterize sequence and function relationships. We also highlight recent large-scale experimental approaches that systematically determine the activity profiles across enzyme superfamilies. We identify several intriguing insights from this recent body of work. First, promiscuous activities are prevalent among extant enzymes. Second, many divergent proteins retain "function connectivity" via enzyme promiscuity, which can be used to probe the evolutionary potential and history of enzyme superfamilies. Finally, we discuss open questions regarding the intricacies of enzyme divergence, as well as potential research directions that will deepen our understanding of enzyme superfamily evolution.

Exploitation of data from breeding programs supports rapid implementation of genomic selection for key agronomic traits in perennial ryegrass.

PubMed

Pembleton, Luke W; Inch, Courtney; Baillie, Rebecca C; Drayton, Michelle C; Thakur, Preeti; Ogaji, Yvonne O; Spangenberg, German C; Forster, John W; Daetwyler, Hans D; Cogan, Noel O I

2018-06-02

Exploitation of data from a ryegrass breeding program has enabled rapid development and implementation of genomic selection for sward-based biomass yield with a twofold-to-threefold increase in genetic gain. Genomic selection, which uses genome-wide sequence polymorphism data and quantitative genetics techniques to predict plant performance, has large potential for the improvement in pasture plants. Major factors influencing the accuracy of genomic selection include the size of reference populations, trait heritability values and the genetic diversity of breeding populations. Global diversity of the important forage species perennial ryegrass is high and so would require a large reference population in order to achieve moderate accuracies of genomic selection. However, diversity of germplasm within a breeding program is likely to be lower. In addition, de novo construction and characterisation of reference populations are a logistically complex process. Consequently, historical phenotypic records for seasonal biomass yield and heading date over a 18-year period within a commercial perennial ryegrass breeding program have been accessed, and target populations have been characterised with a high-density transcriptome-based genotyping-by-sequencing assay. Ability to predict observed phenotypic performance in each successive year was assessed by using all synthetic populations from previous years as a reference population. Moderate and high accuracies were achieved for the two traits, respectively, consistent with broad-sense heritability values. The present study represents the first demonstration and validation of genomic selection for seasonal biomass yield within a diverse commercial breeding program across multiple years. These results, supported by previous simulation studies, demonstrate the ability to predict sward-based phenotypic performance early in the process of individual plant selection, so shortening the breeding cycle, increasing the rate of genetic gain and allowing rapid adoption in ryegrass improvement programs.

Direct sequencing of hepatitis A virus and norovirus RT-PCR products from environmentally contaminated oyster using M13-tailed primers.

PubMed

Williams-Woods, Jacquelina; González-Escalona, Narjol; Burkhardt, William

2011-12-01

Human norovirus (HuNoV) and hepatitis A (HAV) are recognized as leading causes of non-bacterial foodborne associated illnesses in the United States. DNA sequencing is generally considered the standard for accurate viral genotyping in support of epidemiological investigations. Due to the genetic diversity of noroviruses (NoV), degenerate primer sets are often used in conventional reverse transcription (RT) PCR and real-time RT-quantitative PCR (RT-qPCR) for the detection of these viruses and cDNA fragments are generally cloned prior to sequencing. HAV detection methods that are sensitive and specific for real-time RT-qPCR yields small fragments sizes of 89-150bp, which can be difficult to sequence. In order to overcome these obstacles, norovirus and HAV primers were tailed with M13 forward and reverse primers. This modification increases the sequenced product size and allows for direct sequencing of the amplicons utilizing complementary M13 primers. HuNoV and HAV cDNA products from environmentally contaminated oysters were analyzed using this method. Alignments of the sequenced samples revealed ≥95% nucleotide identities. Tailing NoV and HAV primers with M13 sequence increases the cDNA product size, offers an alternative to cloning, and allows for rapid, accurate and direct sequencing of cDNA products produced by conventional or real time RT-qPCR assays. Published by Elsevier B.V.

Linking secondary metabolites to gene clusters through genome sequencing of six diverse Aspergillus species

DOE PAGES

Kjerbolling, Inge; Vesth, Tammi C.; Frisvad, Jens C.; ...

2018-01-09

The fungal genus of Aspergillus is highly interesting, containing everything from industrial cell factories over model organisms to human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverse Aspergillus species (A. campestris, A. novofumigatus, A. ochraceoroseus and A. steynii) has been whole genome PacBio sequenced to provide genetic references in three Aspergillus sections. Additionally, A. taichungensis and A. candidus were sequenced for SM elucidation. Thirteen Aspergillus genomes were analysed with comparative genomics to determine phylogeny and genetic diversity, showing that each new genome contains 15–27% genes not found in othermore » sequenced Aspergilli. In particular, the new species A. novofumigatus was compared to the pathogenic species A. fumigatus. This suggests that A. novofumigatus can produce most of the same allergens, virulence and pathogenicity factors as A. fumigatus suggesting that A. novofumigatus could be as pathogenic as A. fumigatus. Furthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences and predictive algorithms.« less

Linking secondary metabolites to gene clusters through genome sequencing of six diverse Aspergillus species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kjerbolling, Inge; Vesth, Tammi C.; Frisvad, Jens C.

The fungal genus of Aspergillus is highly interesting, containing everything from industrial cell factories over model organisms to human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverse Aspergillus species (A. campestris, A. novofumigatus, A. ochraceoroseus and A. steynii) has been whole genome PacBio sequenced to provide genetic references in three Aspergillus sections. Additionally, A. taichungensis and A. candidus were sequenced for SM elucidation. Thirteen Aspergillus genomes were analysed with comparative genomics to determine phylogeny and genetic diversity, showing that each new genome contains 15–27% genes not found in othermore » sequenced Aspergilli. In particular, the new species A. novofumigatus was compared to the pathogenic species A. fumigatus. This suggests that A. novofumigatus can produce most of the same allergens, virulence and pathogenicity factors as A. fumigatus suggesting that A. novofumigatus could be as pathogenic as A. fumigatus. Furthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences and predictive algorithms.« less

[Phylogenetic and diversity analysis of Acidithiobacillus spp. based on 16S rRNA and RubisCO genes homologues].

PubMed

Liu, Minrui; Lin, Pengwu; Qi, Xing'e; Ni, Yongqing

2016-04-14

The purpose of the study was to reveal geographic region-related Acidithiobacillus spp. distribution and allopatric speciation. Phylogenetic and diversity analysis was done to expand our knowledge on microbial phylogeography, diversity-maintaining mechanisms and molecular biogeography. We amplified 16S rRNA gene and RubisCO genes to construct corresponding phylogenetic trees based on the sequence homology and analyzed genetic diversity of Acidithiobacillus spp.. Thirty-five strains were isolated from three different regions in China (Yunnan, Hubei, Xinjiang). The whole isolates were classified into five groups. Four strains were identified as A. ferrivorans, six as A. ferridurans, YNTR4-15 Leptspirillum ferrooxidans and HBDY3-31 as Leptospirillum ferrodiazotrophum. The remaining strains were identified as A. ferrooxidans. Analysis of cbbL and cbbM genes sequences of representative 26 strains indicated that cbbL gene of 19 were two copies (cbbL1 and cbbL2) and 7 possessed only cbbL1. cbbM gene was single copy. In nucleotide-based trees, cbbL1 gene sequences of strains were separated into three sequence types, and the cbbL2 was similar to cbbL1 with three types. Codon bias of RubisCO genes was not obvious in Acidithiobacillus spp.. Strains isolated from three different regions in China indicated a great genetic diversity in Acidithiobacillus spp. and their 16S rRNA/RubisCO genes sequence was of significant difference. Phylogenetic tree based on 16S rRNA genes and RubisCO genes was different in Acidithiobacillus spp..

Diversity of Bacteria at Healthy Human Conjunctiva

PubMed Central

Dong, Qunfeng; Brulc, Jennifer M.; Iovieno, Alfonso; Bates, Brandon; Garoutte, Aaron; Miller, Darlene; Revanna, Kashi V.; Gao, Xiang; Antonopoulos, Dionysios A.; Slepak, Vladlen Z.

2011-01-01

Purpose. Ocular surface (OS) microbiota contributes to infectious and autoimmune diseases of the eye. Comprehensive analysis of microbial diversity at the OS has been impossible because of the limitations of conventional cultivation techniques. This pilot study aimed to explore true diversity of human OS microbiota using DNA sequencing-based detection and identification of bacteria. Methods. Composition of the bacterial community was characterized using deep sequencing of the 16S rRNA gene amplicon libraries generated from total conjunctival swab DNA. The DNA sequences were classified and the diversity parameters measured using bioinformatics software ESPRIT and MOTHUR and tools available through the Ribosomal Database Project-II (RDP-II). Results. Deep sequencing of conjunctival rDNA from four subjects yielded a total of 115,003 quality DNA reads, corresponding to 221 species-level phylotypes per subject. The combined bacterial community classified into 5 phyla and 59 distinct genera. However, 31% of all DNA reads belonged to unclassified or novel bacteria. The intersubject variability of individual OS microbiomes was very significant. Regardless, 12 genera—Pseudomonas, Propionibacterium, Bradyrhizobium, Corynebacterium, Acinetobacter, Brevundimonas, Staphylococci, Aquabacterium, Sphingomonas, Streptococcus, Streptophyta, and Methylobacterium—were ubiquitous among the analyzed cohort and represented the putative “core” of conjunctival microbiota. The other 47 genera accounted for <4% of the classified portion of this microbiome. Unexpectedly, healthy conjunctiva contained many genera that are commonly identified as ocular surface pathogens. Conclusions. The first DNA sequencing-based survey of bacterial population at the conjunctiva have revealed an unexpectedly diverse microbial community. All analyzed samples contained ubiquitous (core) genera that included commensal, environmental, and opportunistic pathogenic bacteria. PMID:21571682

Unraveling Haplotype Diversity of the Apical Membrane Antigen-1 Gene in Plasmodium falciparum Populations in Thailand

PubMed Central

Lumkul, Lalita; Sawaswong, Vorthon; Simpalipan, Phumin; Kaewthamasorn, Morakot; Harnyuttanakorn, Pongchai; Pattaradilokrat, Sittiporn

2018-01-01

Development of an effective vaccine is critically needed for the prevention of malaria. One of the key antigens for malaria vaccines is the apical membrane antigen 1 (AMA-1) of the human malaria parasite Plasmodium falciparum, the surface protein for erythrocyte invasion of the parasite. The gene encoding AMA-1 has been sequenced from populations of P. falciparum worldwide, but the haplotype diversity of the gene in P. falciparum populations in the Greater Mekong Subregion (GMS), including Thailand, remains to be characterized. In the present study, the AMA-1 gene was PCR amplified and sequenced from the genomic DNA of 65 P. falciparum isolates from 5 endemic areas in Thailand. The nearly full-length 1,848 nucleotide sequence of AMA-1 was subjected to molecular analyses, including nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity and neutrality tests. Phylogenetic analysis and pairwise population differentiation (Fst indices) were performed to infer the population structure. The analyses identified 60 single nucleotide polymorphic loci, predominately located in domain I of AMA-1. A total of 31 unique AMA-1 haplotypes were identified, which included 11 novel ones. The phylogenetic tree of the AMA-1 haplotypes revealed multiple clades of AMA-1, each of which contained parasites of multiple geographical origins, consistent with the Fst indices indicating genetic homogeneity or gene flow among geographically distinct populations of P. falciparum in Thailand’s borders with Myanmar, Laos and Cambodia. In summary, the study revealed novel haplotypes and population structure needed for the further advancement of AMA-1-based malaria vaccines in the GMS. PMID:29742870

Leveraging genome-wide datasets to quantify the functional role of the anti-Shine-Dalgarno sequence in regulating translation efficiency.

PubMed

Hockenberry, Adam J; Pah, Adam R; Jewett, Michael C; Amaral, Luís A N

2017-01-01

Studies dating back to the 1970s established that sequence complementarity between the anti-Shine-Dalgarno (aSD) sequence on prokaryotic ribosomes and the 5' untranslated region of mRNAs helps to facilitate translation initiation. The optimal location of aSD sequence binding relative to the start codon, the full extents of the aSD sequence and the functional form of the relationship between aSD sequence complementarity and translation efficiency have not been fully resolved. Here, we investigate these relationships by leveraging the sequence diversity of endogenous genes and recently available genome-wide estimates of translation efficiency. We show that-after accounting for predicted mRNA structure-aSD sequence complementarity increases the translation of endogenous mRNAs by roughly 50%. Further, we observe that this relationship is nonlinear, with translation efficiency maximized for mRNAs with intermediate levels of aSD sequence complementarity. The mechanistic insights that we observe are highly robust: we find nearly identical results in multiple datasets spanning three distantly related bacteria. Further, we verify our main conclusions by re-analysing a controlled experimental dataset. © 2017 The Authors.

An empirical evaluation of two-stage species tree inference strategies using a multilocus dataset from North American pines

Treesearch

Michael DeGiorgio; John Syring; Andrew J. Eckert; Aaron Liston; Richard Cronn; David B. Neale; Noah A. Rosenberg

2014-01-01

Background: As it becomes increasingly possible to obtain DNA sequences of orthologous genes from diverse sets of taxa, species trees are frequently being inferred from multilocus data. However, the behavior of many methods for performing this inference has remained largely unexplored. Some methods have been proven to be consistent given certain evolutionary models,...

High Diversity of the Saliva Microbiome in Batwa Pygmies

PubMed Central

Schroeder, Roland; Creasey, Jean L.; Li, Mingkun; Stoneking, Mark

2011-01-01

We describe the saliva microbiome diversity in Batwa Pygmies, a former hunter-gatherer group from Uganda, using next-generation sequencing of partial 16S rRNA sequences. Microbial community diversity in the Batwa is significantly higher than in agricultural groups from Sierra Leone and the Democratic Republic of Congo. We found 40 microbial genera in the Batwa, which have previously not been described in the human oral cavity. The distinctive composition of the salvia microbiome of the Batwa may have been influenced by their recent different lifestyle and diet. PMID:21858083

Diverse Lifestyles and Strategies of Plant Pathogenesis Encoded in the Genomes of Eighteen Dothideomycetes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohm, Robin A.; Feau, Nicolas; Henrissat, Bernard

2013-03-05

The class of Dothideomycetes is one of the largest and most diverse groups of fungi. Many are plant pathogens and pose a serious threat to agricultural crops that are grown for biofuel, food or feed. Most Dothideomycetes have only a single host plant, and related species can have very diverse hosts. Eighteen genomes of Dothideomycetes have currently been sequenced by the Joint Genome Institute and other sequencing centers. Here we describe the results of comparative analyses of the fungi in this group.

Microbial genomics, transcriptomics and proteomics: new discoveries in decomposition research using complementary methods.

PubMed

Baldrian, Petr; López-Mondéjar, Rubén

2014-02-01

Molecular methods for the analysis of biomolecules have undergone rapid technological development in the last decade. The advent of next-generation sequencing methods and improvements in instrumental resolution enabled the analysis of complex transcriptome, proteome and metabolome data, as well as a detailed annotation of microbial genomes. The mechanisms of decomposition by model fungi have been described in unprecedented detail by the combination of genome sequencing, transcriptomics and proteomics. The increasing number of available genomes for fungi and bacteria shows that the genetic potential for decomposition of organic matter is widespread among taxonomically diverse microbial taxa, while expression studies document the importance of the regulation of expression in decomposition efficiency. Importantly, high-throughput methods of nucleic acid analysis used for the analysis of metagenomes and metatranscriptomes indicate the high diversity of decomposer communities in natural habitats and their taxonomic composition. Today, the metaproteomics of natural habitats is of interest. In combination with advanced analytical techniques to explore the products of decomposition and the accumulation of information on the genomes of environmentally relevant microorganisms, advanced methods in microbial ecophysiology should increase our understanding of the complex processes of organic matter transformation.

High-throughput sequencing of TCR repertoires in multiple sclerosis reveals intrathecal enrichment of EBV-reactive CD8+ T cells.

PubMed

Lossius, Andreas; Johansen, Jorunn N; Vartdal, Frode; Robins, Harlan; Jūratė Šaltytė, Benth; Holmøy, Trygve; Olweus, Johanna

2014-11-01

Epstein-Barr virus (EBV) has long been suggested as a pathogen in multiple sclerosis (MS). Here, we used high-throughput sequencing to determine the diversity, compartmentalization, persistence, and EBV-reactivity of the T-cell receptor (TCR) repertoires in MS. TCR-β genes were sequenced in paired samples of cerebrospinal fluid (CSF) and blood from patients with MS and controls with other inflammatory neurological diseases. The TCR repertoires were highly diverse in both compartments and patient groups. Expanded T-cell clones, represented by TCR-β sequences >0.1%, were of different identity in CSF and blood of MS patients, and persisted for more than a year. Reference TCR-β libraries generated from peripheral blood T cells reactive against autologous EBV-transformed B cells were highly enriched for public EBV-specific sequences and were used to quantify EBV-reactive TCR-β sequences in CSF. TCR-β sequences of EBV-reactive CD8+ T cells, including several public EBV-specific sequences, were intrathecally enriched in MS patients only, whereas those of EBV-reactive CD4+ T cells were also enriched in CSF of controls. These data provide evidence for a clonally diverse, yet compartmentalized and persistent, intrathecal T-cell response in MS. The presented strategy links TCR sequence to intrathecal T-cell specificity, demonstrating enrichment of EBV-reactive CD8+ T cells in MS. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The microbiome associated with equine periodontitis and oral health.

PubMed

Kennedy, Rebekah; Lappin, David Francis; Dixon, Padraic Martin; Buijs, Mark Johannes; Zaura, Egija; Crielaard, Wim; O'Donnell, Lindsay; Bennett, David; Brandt, Bernd Willem; Riggio, Marcello Pasquale

2016-04-14

Equine periodontal disease is a common and painful condition and its severe form, periodontitis, can lead to tooth loss. Its aetiopathogenesis remains poorly understood despite recent increased awareness of this disorder amongst the veterinary profession. Bacteria have been found to be causative agents of the disease in other species, but current understanding of their role in equine periodontitis is extremely limited. The aim of this study was to use high-throughput sequencing to identify the microbiome associated with equine periodontitis and oral health. Subgingival plaque samples from 24 horses with periodontitis and gingival swabs from 24 orally healthy horses were collected. DNA was extracted from samples, the V3-V4 region of the bacterial 16S rRNA gene amplified by PCR and amplicons sequenced using Illumina MiSeq. Data processing was conducted using USEARCH and QIIME. Diversity analyses were performed with PAST v3.02. Linear discriminant analysis effect size (LEfSe) was used to determine differences between the groups. In total, 1308 OTUs were identified and classified into 356 genera or higher taxa. Microbial profiles at health differed significantly from periodontitis, both in their composition (p < 0.0001, F = 12.24; PERMANOVA) and in microbial diversity (p < 0.001; Mann-Whitney test). Samples from healthy horses were less diverse (1.78, SD 0.74; Shannon diversity index) and were dominated by the genera Gemella and Actinobacillus, while the periodontitis group samples showed higher diversity (3.16, SD 0.98) and were dominated by the genera Prevotella and Veillonella. It is concluded that the microbiomes associated with equine oral health and periodontitis are distinct, with the latter displaying greater microbial diversity.

Status of the Microbial Census

PubMed Central

Schloss, Patrick D.; Handelsman, Jo

2004-01-01

Over the past 20 years, more than 78,000 16S rRNA gene sequences have been deposited in GenBank and the Ribosomal Database Project, making the 16S rRNA gene the most widely studied gene for reconstructing bacterial phylogeny. While there is a general appreciation that these sequences are largely unique and derived from diverse species of bacteria, there has not been a quantitative attempt to describe the extent of sequencing efforts to date. We constructed rarefaction curves for each bacterial phylum and for the entire bacterial domain to assess the current state of sampling and the relative taxonomic richness of each phylum. This analysis quantifies the general sense among microbiologists that we are a long way from a complete census of the bacteria on Earth. Moreover, the analysis indicates that current sampling strategies might not be the most effective ones to describe novel diversity because there remain numerous phyla that are globally distributed yet poorly sampled. Based on the current level of sampling, it is not possible to estimate the total number of bacterial species on Earth, but the minimum species richness is 35,498. Considering previous global species richness estimates of 107 to 109, we are certain that this estimate will increase with additional sequencing efforts. The data support previous calls for extensive surveys of multiple chemically disparate environments and of specific phylogenetic groups to advance the census most rapidly. PMID:15590780

The History of Bordetella pertussis Genome Evolution Includes Structural Rearrangement

PubMed Central

Peng, Yanhui; Loparev, Vladimir; Batra, Dhwani; Bowden, Katherine E.; Burroughs, Mark; Cassiday, Pamela K.; Davis, Jamie K.; Johnson, Taccara; Juieng, Phalasy; Knipe, Kristen; Mathis, Marsenia H.; Pruitt, Andrea M.; Rowe, Lori; Sheth, Mili; Tondella, M. Lucia; Williams, Margaret M.

2017-01-01

ABSTRACT Despite high pertussis vaccine coverage, reported cases of whooping cough (pertussis) have increased over the last decade in the United States and other developed countries. Although Bordetella pertussis is well known for its limited gene sequence variation, recent advances in long-read sequencing technology have begun to reveal genomic structural heterogeneity among otherwise indistinguishable isolates, even within geographically or temporally defined epidemics. We have compared rearrangements among complete genome assemblies from 257 B. pertussis isolates to examine the potential evolution of the chromosomal structure in a pathogen with minimal gene nucleotide sequence diversity. Discrete changes in gene order were identified that differentiated genomes from vaccine reference strains and clinical isolates of various genotypes, frequently along phylogenetic boundaries defined by single nucleotide polymorphisms. The observed rearrangements were primarily large inversions centered on the replication origin or terminus and flanked by IS481, a mobile genetic element with >240 copies per genome and previously suspected to mediate rearrangements and deletions by homologous recombination. These data illustrate that structural genome evolution in B. pertussis is not limited to reduction but also includes rearrangement. Therefore, although genomes of clinical isolates are structurally diverse, specific changes in gene order are conserved, perhaps due to positive selection, providing novel information for investigating disease resurgence and molecular epidemiology. IMPORTANCE Whooping cough, primarily caused by Bordetella pertussis, has resurged in the United States even though the coverage with pertussis-containing vaccines remains high. The rise in reported cases has included increased disease rates among all vaccinated age groups, provoking questions about the pathogen's evolution. The chromosome of B. pertussis includes a large number of repetitive mobile genetic elements that obstruct genome analysis. However, these mobile elements facilitate large rearrangements that alter the order and orientation of essential protein-encoding genes, which otherwise exhibit little nucleotide sequence diversity. By comparing the complete genome assemblies from 257 isolates, we show that specific rearrangements have been conserved throughout recent evolutionary history, perhaps by eliciting changes in gene expression, which may also provide useful information for molecular epidemiology. PMID:28167525

The importance of standardization for biodiversity comparisons: A case study using autonomous reef monitoring structures (ARMS) and metabarcoding to measure cryptic diversity on Mo’orea coral reefs, French Polynesia

PubMed Central

Geller, Jonathan B.; Timmers, Molly; Leray, Matthieu; Mahardini, Angka; Sembiring, Andrianus; Collins, Allen G.; Meyer, Christopher P.

2017-01-01

The advancement of metabarcoding techniques, declining costs of high-throughput sequencing and development of systematic sampling devices, such as autonomous reef monitoring structures (ARMS), have provided the means to gather a vast amount of diversity data from cryptic marine communities. However, such increased capability could also lead to analytical challenges if the methods used to examine these communities across local and global scales are not standardized. Here we compare and assess the underlying biases of four ARMS field processing methods, preservation media, and current bioinformatic pipelines in evaluating diversity from cytochrome c oxidase I metabarcoding data. Illustrating the ability of ARMS-based metabarcoding to capture a wide spectrum of biodiversity, 3,372 OTUs and twenty-eight phyla, including 17 of 33 marine metazoan phyla, were detected from 3 ARMS (2.607 m2 area) collected on coral reefs in Mo’orea, French Polynesia. Significant differences were found between processing and preservation methods, demonstrating the need to standardize methods for biodiversity comparisons. We recommend the use of a standardized protocol (NOAA method) combined with DMSO preservation of tissues for sessile macroorganisms because it gave a more accurate representation of the underlying communities, is cost effective and removes chemical restrictions associated with sample transportation. We found that sequences identified at ≥ 97% similarity increased more than 7-fold (5.1% to 38.6%) using a geographically local barcode inventory, highlighting the importance of local species inventories. Phylogenetic approaches that assign higher taxonomic ranks accrued phylum identification errors (9.7%) due to sparse taxonomic coverage of the understudied cryptic coral reef community in public databases. However, a ≥ 85% sequence identity cut-off provided more accurate results (0.7% errors) and enabled phylum level identifications of 86.3% of the sequence reads. With over 1600 ARMS deployed, standardizing methods and improving databases are imperative to provide unprecedented global baseline assessments of understudied cryptic marine species in a rapidly changing world. PMID:28430780

Design and Synthesis of a Library of Tetracyclic Hydroazulenoisoindoles

PubMed Central

Brummond, Kay M.; Mao, Shuli; Shinde, Sunita N.; Johnston, Paul J.; Day, Billy W.

2009-01-01

Forty-four tetracyclic hydroazulenoisoindoles were synthesized via a tandem cyclopropanation/Cope rearrangement followed by a Diels-Alder sequence from easily available five-membered cyclic cross-conjugated trienones. These trienones were obtained from two different routes depending upon whether R1 and R2 are alkyl or amino acid derived functional groups, via a rhodium(I)-catalyzed cycloisomerization reaction. In order to increase diversity, four maleimides and two 1,2,4-triazoline-3,5-diones were used as dienophiles in the Diels-Alder step. Several Diels-Alder adducts were further reacted under palladium-catalyzed hydrogenation conditions, leading to a diastereoselective reduction of the trisubstituted double bond. This library has demonstrated rapid access to a variety of structurally complex natural product-like compounds via stereochemical diversity and building block diversity approaches. PMID:19366169

Bacterial diversity along a 2600 km river continuum

PubMed Central

Savio, Domenico; Sinclair, Lucas; Ijaz, Umer Z.; Parajka, Juraj; Reischer, Georg H.; Stadler, Philipp; Blaschke, Alfred P.; Blöschl, Günter; Mach, Robert L.; Kirschner, Alexander K. T.; Farnleitner, Andreas H.

2015-01-01

Summary The bacterioplankton diversity in large rivers has thus far been under‐sampled despite the importance of streams and rivers as components of continental landscapes. Here, we present a comprehensive dataset detailing the bacterioplankton diversity along the midstream of the Danube River and its tributaries. Using 16S rRNA‐gene amplicon sequencing, our analysis revealed that bacterial richness and evenness gradually declined downriver in both the free‐living and particle‐associated bacterial communities. These shifts were also supported by beta diversity analysis, where the effects of tributaries were negligible in regards to the overall variation. In addition, the river was largely dominated by bacteria that are commonly observed in freshwaters. Dominated by the acI lineage, the freshwater SAR11 (LD12) and the P olynucleobacter group, typical freshwater taxa increased in proportion downriver and were accompanied by a decrease in soil and groundwater‐affiliated bacteria. Based on views of the meta‐community and River Continuum Concept, we interpret the observed taxonomic patterns and accompanying changes in alpha and beta diversity with the intention of laying the foundation for a unified concept for river bacterioplankton diversity. PMID:25922985

Metagenomic Evaluation of Bacterial and Archaeal Diversity in the Geothermal Hot Springs of Manikaran, India

PubMed Central

Pathak, Ashish; Green, Stefan J.; Joshi, Amit; Chauhan, Ashvini

2015-01-01

Bacterial and archaeal diversity in geothermal spring water were investigated using 16S rRNA gene amplicon metagenomic sequencing. This revealed the dominance of Firmicutes, Aquificae, and the Deinococcus-Thermus group in this thermophilic environment. A number of sequences remained taxonomically unresolved, indicating the presence of potentially novel microbes in this unique habitat. PMID:25700403

Nucleotide diversity maps reveal variation in diversity among wheat genomes and chromosomes

USDA-ARS?s Scientific Manuscript database

Technical Abstract: 20-75 CHARACTER LINES A strategy for a genome-wide assessment of nucleotide diversity in a polyploid species must minimize the inclusion of homoeologous sequences into diversity estimates and reliably allocate individual haplotypes into respective genomes. In this study, nucle...