scholarly journals Degenerate PCR Primers to Reveal the Diversity of Giant Viruses in Coastal Waters

Viruses ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 496 ◽  
Author(s):  
Yanze Li ◽  
Pascal Hingamp ◽  
Hiroyasu Watai ◽  
Hisashi Endo ◽  
Takashi Yoshida ◽  
...  
Keyword(s):  
Environmental Dna ◽  
Population Level ◽  
Pcr Primers ◽  
Natural Environments ◽  
Specific Primers ◽  
Degenerate Pcr ◽  
Operational Taxonomic Units ◽  
Structure And Dynamics ◽  
Order Of Magnitude ◽  
Giant Viruses

“Megaviridae” is a proposed family of giant viruses infecting unicellular eukaryotes. These viruses are ubiquitous in the sea and have impact on marine microbial community structure and dynamics through their lytic infection cycle. However, their diversity and biogeography have been poorly characterized due to the scarce detection of Megaviridae sequences in metagenomes, as well as the limitation of reference sequences used to design specific primers for this viral group. Here, we propose a set of 82 degenerated primers (referred to as MEGAPRIMER), targeting DNA polymerase genes (polBs) of Megaviridae. MEGAPRIMER was designed based on 921 Megaviridae polBs from sequenced genomes and metagenomes. By applying this primer set to environmental DNA meta-barcoding of a coastal seawater sample, we report 5595 non-singleton operational taxonomic units (OTUs) of Megaviridae at 97% nucleotide sequence identity. The majority of the OTUs were found to form diverse clades, which were phylogenetically distantly phylogenetically related to known viruses such as Mimivirus. The Megaviridae OTUs detected in this study outnumber the giant virus OTUs identified in previous individual studies by more than an order of magnitude. Hence, MEGAPRIMER represents a useful tool to study the diversity of Megaviridae at the population level in natural environments.

scholarly journals DNA from Uncultured Organisms as a Source of 2,5-Diketo-d-Gluconic Acid Reductases

2001 ◽  
Vol 67 (9) ◽  
pp. 4206-4214 ◽  
Author(s):  
William H. Eschenfeldt ◽  
Lucy Stols ◽  
Helga Rosenbaum ◽  
Zubin S. Khambatta ◽  
Elsie Quaite-Randall ◽  
...  
Keyword(s):  
Gluconic Acid ◽  
Environmental Dna ◽  
Pcr Primers ◽  
Sequence Information ◽  
Gene Products ◽  
Specific Primers ◽  
Degenerate Pcr ◽  
Genes Encoding ◽  
Internal Gene ◽  
Total Dna

ABSTRACT Total DNA of a population of uncultured organisms was extracted from soil samples, and by using PCR methods, the genes encoding two different 2,5-diketo-d-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primers based on published sequence information gave internal gene fragments homologous to known DKGRs. Nested primers specific for the internal fragments were combined with random primers to amplify flanking gene fragments from the environmental DNA, and two hypothetical full-length genes were predicted from the combined sequences. Based on these predictions, specific primers were used to amplify the two complete genes in single PCRs. These genes were cloned and expressed in Escherichia coli. The purified gene products catalyzed the reduction of 2,5-diketo-d-gluconic acid to 2-keto-l-gulonic acid. Compared to previously described DKGRs isolated fromCorynebacterium spp., these environmental reductases possessed some valuable properties. Both exhibited greater than 20-fold-higherk cat/K m values than those previously determined, primarily as a result of better binding of substrate. The K m values for the two new reductases were 57 and 67 μM, versus 2 and 13 mM for the Corynebacterium enzymes. Both environmental DKGRs accepted NADH as well as NADPH as a cosubstrate; other DKGRs and most related aldo-keto reductases use only NADPH. In addition, one of the new reductases was more thermostable than known DKGRs.

AFLP-derived, Codominant Markers for Locus-specific Applications

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 514e-514
Author(s):  
James M. Bradeen ◽  
Philipp W. Simon
Keyword(s):  
Linkage Mapping ◽  
Large Scale ◽  
Pcr Primers ◽  
Inverse Pcr ◽  
Sequence Information ◽  
Pcr Assay ◽  
Specific Primers ◽  
Simultaneous Evaluation ◽  
Feral Populations ◽  
Diversity Assessment

The amplified fragment length polymorphism (AFLP) is a powerful marker, allowing rapid and simultaneous evaluation of multiple potentially polymorphic sites. Although well-adapted to linkage mapping and diversity assessment, AFLPs are primarily dominant in nature. Dominance, relatively high cost, and technological difficulty limit use of AFLPs for marker-aided selection and other locus-specific applications. In carrot the Y2 locus conditions carotene accumulation in the root xylem. We identified AFLP fragments linked to the dominant Y2 allele and pursued conversion of those fragments to codominant, PCR-based forms useful for locus-specific applications. The short length of AFLPs (≈60 to 500 bp) precludes development of longer, more specific primers as in SCAR development. Instead, using sequence information from cloned AFLP fragments for primer design, regions outside of the original fragment were amplified by inverse PCR or ligation-mediated PCR, cloned, and sequenced. Differences in sequences associated with Y2 vs. y2 allowed development of simple PCR assays differentiating those alleles. PCR primers flanking an insertion associated with the recessive allele amplified differently sized products for the two Y2 alleles in one assay. This assay is rapid, technologically simple (requiring no radioactivity and little advanced training or equipment), reliable, inexpensive, and codominant. Our PCR assay has a variety of large scale, locus-specific applications including genotyping diverse carrot cultivars and wild and feral populations. Efforts are underway to improve upon conversion technology and to more extensively test the techniques we have developed.

scholarly journals Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
Ryohei Nakao ◽  
Hideyuki Doi ◽  
Toshifumi Minamoto
Keyword(s):  
Fish Species ◽  
High Throughput Sequencing ◽  
Correlation Coefficients ◽  
Environmental Dna ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Taqman Probe ◽  
Individual Species ◽  
Natural Environments ◽  
Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

scholarly journals Mutability of demographic noise in microbial range expansions

2021 ◽  
Author(s):  
QinQin Yu ◽  
Matti Gralka ◽  
Marie-Cécilia Duvernoy ◽  
Megan Sousa ◽  
Arbel Harpak ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Genetic Manipulation ◽  
Single Gene ◽  
Population Level ◽  
Growth Conditions ◽  
Establishment Probability ◽  
In The Wild ◽  
Order Of Magnitude ◽  
Demographic Noise ◽  
Single Gene Deletion

AbstractDemographic noise, the change in the composition of a population due to random birth and death events, is an important driving force in evolution because it reduces the efficacy of natural selection. Demographic noise is typically thought to be set by the population size and the environment, but recent experiments with microbial range expansions have revealed substantial strain-level differences in demographic noise under the same growth conditions. Many genetic and phenotypic differences exist between strains; to what extent do single mutations change the strength of demographic noise? To investigate this question, we developed a high-throughput method for measuring demographic noise in colonies without the need for genetic manipulation. By applying this method to 191 randomly-selected single gene deletion strains from the E. coli Keio collection, we find that a typical single gene deletion mutation decreases demographic noise by 8% (maximal decrease: 81%). We find that the strength of demographic noise is an emergent trait at the population level that can be predicted by colony-level traits but not cell-level traits. The observed differences in demographic noise from single gene deletions can increase the establishment probability of beneficial mutations by almost an order of magnitude (compared to in the wild type). Our results show that single mutations can substantially alter adaptation through their effects on demographic noise and suggest that demographic noise can be an evolvable trait of a population.

Exploring the plant environmental DNA diversity in soil from two sites on Deception Island (Antarctica, South Shetland Islands) using metabarcoding

2021 ◽  
pp. 1-10
Author(s):  
Micheline Carvalho-Silva ◽  
Luiz Henrique Rosa ◽  
Otávio H.B. Pinto ◽  
Thamar Holanda Da Silva ◽  
Diego Knop Henriques ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Crater Lake ◽  
Environmental Dna ◽  
Food Sources ◽  
South Shetland Islands ◽  
Deception Island ◽  
Shetland Islands ◽  
Operational Taxonomic Units ◽  
First Time ◽  
Impacted Site

Abstract The few Antarctic studies to date to have applied metabarcoding in Antarctica have primarily focused on microorganisms. In this study, for the first time, we apply high-throughput sequencing of environmental DNA to investigate the diversity of Embryophyta (Viridiplantae) DNA present in soil samples from two contrasting locations on Deception Island. The first was a relatively undisturbed site within an Antarctic Specially Protected Area at Crater Lake, and the second was a heavily human-impacted site in Whalers Bay. In samples obtained at Crater Lake, 84% of DNA reads represented fungi, 14% represented Chlorophyta and 2% represented Streptophyta, while at Whalers Bay, 79% of reads represented fungi, 20% represented Chlorophyta and < 1% represented Streptophyta, with ~1% of reads being unassigned. Among the Embryophyta we found 16 plant operational taxonomic units from three Divisions, including one Marchantiophyta, eight Bryophyta and seven Magnoliophyta. Sequences of six taxa were detected at both sampling sites, eight only at Whalers Bay and two only at Crater Lake. All of the Magnoliophyta sequences (flowering plants) represent species that are exotic to Antarctica, with most being plausibly linked to human food sources originating from local national research operator and tourism facilities.

scholarly journals High Diversity of Magnetotactic Deltaproteobacteria in a Freshwater Niche

2013 ◽  
Vol 79 (8) ◽  
pp. 2813-2817 ◽  
Author(s):  
Yinzhao Wang ◽  
Wei Lin ◽  
Jinhua Li ◽  
Yongxin Pan
Keyword(s):  
Magnetotactic Bacteria ◽  
Natural Environments ◽  
Content Type ◽  
Operational Taxonomic Units ◽  
Geological Processes ◽  
Freshwater Site ◽  
High Diversity

ABSTRACTKnowledge of the diversity of magnetotactic bacteria in natural environments is crucial for understanding their contribution to various biological and geological processes. Here we report a high diversity of magnetotactic bacteria in a freshwater site. Ten out of 18 operational taxonomic units (OTUs) were affiliated with theDeltaproteobacteria. Some rod-shaped bacteria simultaneously synthesized greigite and magnetite magnetosomes.

scholarly journals Estimation accuracy of species abundance based on environmental DNA with relation to its production source, state, and assay methodology suggested by meta-analyses

2021 ◽  
Author(s):  
Toshiaki Jo ◽  
Hiroki Yamanaka
Keyword(s):  
Meta Analysis ◽  
Empirical Studies ◽  
Species Abundance ◽  
Environmental Dna ◽  
Efficient Estimation ◽  
Estimation Accuracy ◽  
Accurate Estimation ◽  
Abundance Estimation ◽  
Natural Environments ◽  
Promising Tool

Environmental DNA (eDNA) analysis is a promising tool for non-disruptive and cost-efficient estimation of species abundance. However, its practical applicability in natural environments is limited because it is unclear whether eDNA concentrations actually represent species abundance in the field. Although the importance of accounting for eDNA dynamics, such as transport and degradation, has been discussed, the influences of eDNA characteristics, including production source and state, and methodology, including collection and quantification strategy and abundance metrics, on the accuracy of eDNA-based abundance estimation were entirely overlooked. We conducted a meta-analysis using 56 previous eDNA literature and investigated the relationships between the accuracy (R2) of eDNA-based abundance estimation and eDNA characteristics and methodology. Our meta-regression analysis found that R2 values were significantly lower for crustaceans than fish, suggesting that less frequent eDNA production owing to their external morphology and physiology may impede accurate estimation of their abundance via eDNA. Moreover, R2 values were positively associated with filter pore size, indicating that selective collection of larger-sized eDNA, which is typically fresher, could improve the estimation accuracy of species abundance. Furthermore, R2 values were significantly lower for natural than laboratory conditions, while there was no difference in the estimation accuracy among natural environments. Our findings shed a new light on the importance of what characteristics of eDNA should be targeted for more accurate estimation of species abundance. Further empirical studies are required to validate our findings and fully elucidate the relationship between eDNA characteristics and eDNA-based abundance estimation.

scholarly journals Evaluation of molecular characterization and phylogeny for quantification of Acanthamoeba and Naegleria fowleri in various water sources, Turkey

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0256659
Author(s):  
Mehmet Aykur ◽  
Hande Dagci
Keyword(s):  
Public Health ◽  
Public Awareness ◽  
Public Health Problem ◽  
Water Sources ◽  
Quantitative Detection ◽  
Human Populations ◽  
Rrna Gene ◽  
Natural Environments ◽  
Specific Primers ◽  
Important Public Health Problem

Free-living amoeba (FLA) is widely distributed in the natural environment. Since these amoebae are widely found in various waters, they pose an important public health problem. The aim of this study was to detect the presence of Acanthamoeba, B. mandrillaris, and N. fowleri in various water resources by qPCR in Izmir, Turkey. A total of (n = 27) 18.24% Acanthamoeba and (n = 4) 2.7% N. fowleri positives were detected in six different water sources using qPCR with ITS regions (ITS1) specific primers. The resulting concentrations varied in various water samples for Acanthamoeba in the range of 3.2x105-1.4x102 plasmid copies/l and for N. fowleri in the range of 8x103-11x102 plasmid copies/l. The highest concentration of Acanthamoeba and N. fowleri was found in seawater and damp samples respectively. All 27 Acanthamoeba isolates were identified in genotype level based on the 18S rRNA gene as T4 (51.85%), T5 (22.22%), T2 (14.81%) and T15 (11.11%). The four positive N. fowleri isolate was confirmed by sequencing the ITS1, ITS2 and 5.8S rRNA regions using specific primers. Four N. fowleri isolates were genotyped (three isolate as type 2 and one isolate as type 5) and detected for the first time from water sources in Turkey. Acanthamoeba and N. fowleri genotypes found in many natural environments are straightly related to human populations to have pathogenic potentials that may pose a risk to human health. Public health professionals should raise awareness on this issue, and public awareness education should be provided by the assistance of civil authorities. To the best of our knowledge, this is the first study on the quantitative detection and distribution of Acanthamoeba and N. fowleri genotypes in various water sources in Turkey.

scholarly journals Transcribed enhancers in the human brain identify novel disease risk mechanisms

2021 ◽  
Author(s):  
Pengfei Dong ◽  
Gabriel E. Hoffman ◽  
Pasha Apontes ◽  
Jaroslav Bendl ◽  
Samir Rahman ◽  
...  
Keyword(s):  
Human Brain ◽  
Disease Risk ◽  
Association Studies ◽  
Population Level ◽  
Cell Type ◽  
Level Variation ◽  
Functional Interpretation ◽  
Neuropsychiatric Diseases ◽  
Order Of Magnitude ◽  
Cell Type Specific

Enhancer RNAs (eRNAs) constitute an important tissue- and cell-type-specific layer of the regulome. Identification of risk variants for neuropsychiatric diseases within enhancers underscores the importance of understanding the population-level variation of eRNAs in the human brain. We jointly analyzed cell type-specific transcriptome and regulome data to identify 30,795 neuronal and 23,265 non-neuronal eRNAs, expanding the catalog of known human brain eRNAs by an order of magnitude. Examination of the population-level variation of the transcriptome and regulome in 1,382 brain samples identified reproducible changes affecting cis- and trans-co-regulation of eRNA-gene modules in schizophrenia. We show that 13% of schizophrenia heritability is jointly mediated in cis by brain gene and eRNA expression. Inclusion of eRNAs in transcriptome-wide association studies facilitated fine-mapping and functional interpretation of disease loci. Overall, our study characterizes the eRNA-gene regulome and genetic mechanisms in the human cortex in both healthy and disease states.

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