scholarly journals An improved primer set and amplification protocol with increased specificity and sensitivity targeting the Symbiodinium ITS2 region

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4816 ◽  
Author(s):  
Benjamin C.C. Hume ◽  
Maren Ziegler ◽  
Julie Poulain ◽  
Xavier Pochon ◽  
Sarah Romac ◽  
...  
Keyword(s):  
Large Scale ◽  
Marine Invertebrates ◽  
Temperature Cycle ◽  
Its2 Region ◽  
Rrna Gene ◽  
Surrounding Water ◽  
Cycle Number ◽  
Specificity And Sensitivity ◽  
Amplification Protocol ◽  
Generation Sequencing

The Internal Transcribed Spacer 2 (ITS2) rRNA gene is a commonly targeted genetic marker to assess diversity of Symbiodinium, a dinoflagellate genus of algal endosymbionts that is pervasively associated with marine invertebrates, and notably reef-building corals. Here we tested three commonly used ITS2 primer pairs (SYM_VAR_5.8S2/SYM_VAR_REV, ITSintfor2/ITSReverse, and ITS-DINO/ITS2Rev2) with regard to amplification specificity and sensitivity towards Symbiodinium, as well as sub-genera taxonomic bias. We tested these primers over a range of sample types including three coral species, coral surrounding water, reef surface water, and open ocean water to assess their suitability for use in large-scale next generation sequencing projects and to develop a standardised PCR protocol. We found the SYM_VAR_5.8S2/SYM_VAR_REV primers to perform superior to the other tested ITS2 primers. We therefore used this primer pair to develop a standardised PCR protocol. To do this, we tested the effect of PCR-to-PCR variation, annealing temperature, cycle number, and different polymerase systems on the PCR efficacy. The Symbiodinium ITS2 PCR protocol developed here delivers improved specificity and sensitivity towards Symbiodinium with apparent minimal sub-genera taxonomic bias across all sample types. In particular, the protocol’s ability to amplify Symbiodinium from a range of environmental sources will facilitate the study of Symbiodinium populations across biomes.

scholarly journals Tendentious effects of automated and manual metagenomic DNA purification protocols on broiler gut microbiome taxonomic profiling

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Gabor Fidler ◽  
Emese Tolnai ◽  
Aniko Stagel ◽  
Judit Remenyik ◽  
Laszlo Stundl ◽  
...  
Keyword(s):  
Large Scale ◽  
Magnetic Bead ◽  
Positive Influence ◽  
Optimal Choice ◽  
Rrna Gene ◽  
Taxonomic Profiling ◽  
High Consistency ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Significant Positive Influence

AbstractHere, we developed protocols to improve sensitivity, rigor and comparability of 16S rRNA gene amplification-based next-generation sequencing (NGS) results. A thorough study was performed by evaluating extraction efficiency with respect to the yield, purity, fragmentation of the purified DNA, and sequencing metrics considering the number of quality reads, amplicon sequence variants (ASVs), community structure and biodiversity. We identified batch-effects that significantly bias broiler gastrointestinal tract (GIT) community compositions and made recommendations to improve sensitivity, consistency, and cross-study comparability. We found that the purity of the extracted nucleic acid had a strong effect on the success rate of downstream library preparations. The preparation of stool bacterial suspensions from feces showed a significant positive influence on community biodiversity by enriching Gram-negative bacteria and cataloguing low abundant taxa with greater success than direct processing of fecal material. Applications relying on the automated Roche MagNa Pure 24 magnetic-bead based method provided results with high consistency therefore it seems to be the optimal choice in large-scale studies for investigating broiler GIT microbiota.

scholarly journals Exotic airborne bacteria identified in urban resuspended dust by next generation sequencing

2019 ◽  
Vol 99 ◽  
pp. 04009
Author(s):  
Nora Kováts ◽  
Eszter Horváth ◽  
Katalin Hubai ◽  
András Hoffer ◽  
Beatrix Jancsek-Turóczi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Large Scale ◽  
Rrna Gene ◽  
Pathogenic Microorganisms ◽  
Next Generation ◽  
Culture Independent ◽  
Next Generation Sequencing Ngs ◽  
Siberian Permafrost ◽  
Generation Sequencing ◽  
Airborne Transport

The airborne transport of bacteria is a well-known phenomenon, making it possible to exchange species between ecosystems, but it also provides a tool for spreading of pathogenic microorganisms. As part of a large-scale study, microbial community of inhalable and respirable fractions (PM1-10) of resuspended dust collected in Budapest (Hungary) has been characterised by culture-independent next generation sequencing (NGS) of variable 16S rRNA gene regions. Apart from common, mostly ubiqituos soil and organic material-dwelling bacteria, exotic airborne species have been identified, such as Variovorax ginsengisoli, previously isolated from Korean ginseng fields or Exiguobacterium sibiricum, isolated from the Siberian permafrost.

Methods for the Quantification of Salivary Cortisol and of a-amylase in Biosensors and Portable Devices

2018 ◽  
Vol 68 (12) ◽  
pp. 2857-2859
Author(s):  
Cristina Mihaela Ghiciuc ◽  
Andreea Silvana Szalontay ◽  
Luminita Radulescu ◽  
Sebastian Cozma ◽  
Catalina Elena Lupusoru ◽  
...  
Keyword(s):  
Real Time ◽  
Medical Practice ◽  
Salivary Cortisol ◽  
Clinical Studies ◽  
Large Scale ◽  
Psychological Research ◽  
Portable Devices ◽  
Salivary Amylase ◽  
Specificity And Sensitivity

There is an increasing interest in the analysis of salivary biomarkers for medical practice. The objective of this article was to identify the specificity and sensitivity of quantification methods used in biosensors or portable devices for the determination of salivary cortisol and salivary a-amylase. There are no biosensors and portable devices for salivary amylase and cortisol that are used on a large scale in clinical studies. These devices would be useful in assessing more real-time psychological research in the future.

Next-Generation Sequencing: An Emerging Tool for Drug Designing

2019 ◽  
Vol 25 (31) ◽  
pp. 3350-3357 ◽  
Author(s):  
Pooja Tripathi ◽  
Jyotsna Singh ◽  
Jonathan A. Lal ◽  
Vijay Tripathi
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Large Scale ◽  
Massively Parallel Sequencing ◽  
Genomic Research ◽  
Biological Research ◽  
Next Generation ◽  
Human Welfare ◽  
Drug Designing ◽  
Generation Sequencing

Background: With the outbreak of high throughput next-generation sequencing (NGS), the biological research of drug discovery has been directed towards the oncology and infectious disease therapeutic areas, with extensive use in biopharmaceutical development and vaccine production. Method: In this review, an effort was made to address the basic background of NGS technologies, potential applications of NGS in drug designing. Our purpose is also to provide a brief introduction of various Nextgeneration sequencing techniques. Discussions: The high-throughput methods execute Large-scale Unbiased Sequencing (LUS) which comprises of Massively Parallel Sequencing (MPS) or NGS technologies. The Next geneinvolved necessarily executes Largescale Unbiased Sequencing (LUS) which comprises of MPS or NGS technologies. These are related terms that describe a DNA sequencing technology which has revolutionized genomic research. Using NGS, an entire human genome can be sequenced within a single day. Conclusion: Analysis of NGS data unravels important clues in the quest for the treatment of various lifethreatening diseases and other related scientific problems related to human welfare.

scholarly journals Data analysis workflow for the detection of canine vector-borne pathogens using 16 S rRNA Next-Generation Sequencing

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Elton J. R. Vasconcelos ◽  
Chayan Roy ◽  
Joseph A. Geiger ◽  
Kristina M. Oney ◽  
Melody Koo ◽  
...  
Keyword(s):  
Veterinary Medicine ◽  
Next Generation Sequencing ◽  
Complete Analysis ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
Spotted Fever Group ◽  
Next Generation ◽  
Vector Borne ◽  
16 S Rrna ◽  
Generation Sequencing

Abstract Background Vector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP. Results We tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample. Conclusions Bloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.

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