scholarly journals High-throughput identification of viral termini and packaging mechanisms in virome datasets using PhageTermVirome

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Julian R. Garneau ◽  
Véronique Legrand ◽  
Martial Marbouty ◽  
Maximilian O. Press ◽  
Dean R. Vik ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Computing Time ◽  
Rapid Identification ◽  
Viral Metagenomics ◽  
Genome Packaging ◽  
Viral Origin ◽  
Virus Identification ◽  
Viral Particles

AbstractViruses that infect bacteria (phages) are increasingly recognized for their importance in diverse ecosystems but identifying and annotating them in large-scale sequence datasets is still challenging. Although efficient scalable virus identification tools are emerging, defining the exact ends (termini) of phage genomes is still particularly difficult. The proper identification of termini is crucial, as it helps in characterizing the packaging mechanism of bacteriophages and provides information on various aspects of phage biology. Here, we introduce PhageTermVirome (PTV) as a tool for the easy and rapid high-throughput determination of phage termini and packaging mechanisms using modern large-scale metagenomics datasets. We successfully tested the PTV algorithm on a mock virome dataset and then used it on two real virome datasets to achieve the rapid identification of more than 100 phage termini and packaging mechanisms, with just a few hours of computing time. Because PTV allows the identification of free fully formed viral particles (by recognition of termini present only in encapsidated DNA), it can also complement other virus identification softwares to predict the true viral origin of contigs in viral metagenomics datasets. PTV is a novel and unique tool for high-throughput characterization of phage genomes, including phage termini identification and characterization of genome packaging mechanisms. This software should help researchers better visualize, map and study the virosphere. PTV is freely available for downloading and installation at https://gitlab.pasteur.fr/vlegrand/ptv.

scholarly journals Large-Scale, High-Throughput Validation of Short Hairpin RNA Sequences for RNA Interference

2006 ◽  
Vol 11 (3) ◽  
pp. 236-246 ◽  
Author(s):  
Laurence H. Lamarcq ◽  
Bradley J. Scherer ◽  
Michael L. Phelan ◽  
Nikolai N. Kalnine ◽  
Yen H. Nguyen ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Strong Support ◽  
Gc Content ◽  
Rapid Identification ◽  
Hairpin Rna ◽  
Rna Sequences ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Interfering Rna

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for positionspecific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNAinterference (RNAi).

scholarly journals Technological advancements and their importance for nematode identification

SOIL ◽  
2016 ◽  
Vol 2 (2) ◽  
pp. 257-270 ◽  
Author(s):  
Mohammed Ahmed ◽  
Melanie Sapp ◽  
Thomas Prior ◽  
Gerrit Karssen ◽  
Matthew Alan Back
Keyword(s):  
High Throughput ◽  
Crop Production ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Biological Indicators ◽  
Rapid Identification ◽  
Community Studies ◽  
Terrestrial Environments ◽  
Traditional Taxonomy ◽  
Sequencing Platforms

Abstract. Nematodes represent a species-rich and morphologically diverse group of metazoans known to inhabit both aquatic and terrestrial environments. Their role as biological indicators and as key players in nutrient cycling has been well documented. Some plant-parasitic species are also known to cause significant losses to crop production. In spite of this, there still exists a huge gap in our knowledge of their diversity due to the enormity of time and expertise often involved in characterising species using phenotypic features. Molecular methodology provides useful means of complementing the limited number of reliable diagnostic characters available for morphology-based identification. We discuss herein some of the limitations of traditional taxonomy and how molecular methodologies, especially the use of high-throughput sequencing, have assisted in carrying out large-scale nematode community studies and characterisation of phytonematodes through rapid identification of multiple taxa. We also provide brief descriptions of some the current and almost-outdated high-throughput sequencing platforms and their applications in both plant nematology and soil ecology.

scholarly journals Crowdsourced RNA design discovers diverse, reversible, efficient, self-contained molecular sensors

2019 ◽  
Author(s):  
Johan O. L. Andreasson ◽  
Michael R. Gotrik ◽  
Michelle J. Wu ◽  
Hannah K. Wayment-Steele ◽  
Wipapat Kladwang ◽  
...  
Keyword(s):  
High Throughput ◽  
Online Community ◽  
Large Scale ◽  
Functional Characterization ◽  
New Paradigm ◽  
Molecular Sensors ◽  
Rna Switches ◽  
Rna Sensors ◽  
Rna Design

AbstractInternet-based scientific communities promise a means to apply distributed, diverse human intelligence towards previously intractable scientific problems. However, current implementations have not allowed communities to propose experiments to test all emerging hypotheses at scale or to modify hypotheses in response to experiments. We report high-throughput methods for molecular characterization of nucleic acids that enable the large-scale videogame-based crowdsourcing of functional RNA sensor design, followed by high-throughput functional characterization. Iterative design testing of thousands of crowdsourced RNA sensor designs produced near-thermodynamically optimal and reversible RNA switches that act as self-contained molecular sensors and couple five distinct small molecule inputs to three distinct protein binding and fluorogenic outputs—results that surpass computational and expert-based design. This work represents a new paradigm for widely distributed experimental bioscience.One Sentence SummaryOnline community discovers standalone RNA sensors.

scholarly journals High-throughput synthetic rescue for exhaustive characterization of suppressor mutations in human genes

2020 ◽  
Vol 77 (21) ◽  
pp. 4209-4222
Author(s):  
Farah Kobaisi ◽  
Nour Fayyad ◽  
Eric Sulpice ◽  
Bassam Badran ◽  
Hussein Fayyad-Kazan ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Random Mutagenesis ◽  
Screening Methods ◽  
Knock Out ◽  
Suppressor Mutations ◽  
Human Genes ◽  
Cultured Human Cells

Abstract Inherited or acquired mutations can lead to pathological outcomes. However, in a process defined as synthetic rescue, phenotypic outcome created by primary mutation is alleviated by suppressor mutations. An exhaustive characterization of these mutations in humans is extremely valuable to better comprehend why patients carrying the same detrimental mutation exhibit different pathological outcomes or different responses to treatment. Here, we first review all known suppressor mutations’ mechanisms characterized by genetic screens on model species like yeast or flies. However, human suppressor mutations are scarce, despite some being discovered based on orthologue genes. Because of recent advances in high-throughput screening, developing an inventory of human suppressor mutations for pathological processes seems achievable. In addition, we review several screening methods for suppressor mutations in cultured human cells through knock-out, knock-down or random mutagenesis screens on large scale. We provide examples of studies published over the past years that opened new therapeutic avenues, particularly in oncology.

scholarly journals Dub-seq: dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits

2018 ◽  
Author(s):  
Vivek K. Mutalik ◽  
Pavel S. Novichkov ◽  
Morgan N. Price ◽  
Trenton K. Owens ◽  
Mark Callaghan ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Dna Barcode ◽  
Gene Overexpression ◽  
Expression Library ◽  
Gain Of Function ◽  
Experimental Conditions ◽  
Genome Wide ◽  
Barcode Sequencing

AbstractA major challenge in genomics is the knowledge gap between sequence and its encoded function. Gain-of-function methods based on gene overexpression are attractive avenues for phenotype-based functional screens, but are not easily applied in high-throughput across many experimental conditions. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a method that greatly increases the throughput of genome-wide overexpression assays. In Dub-seq, a shotgun expression library is cloned between dual random DNA barcodes and the precise breakpoints of DNA fragments are associated to the barcode sequences prior to performing assays. To assess the fitness of individual strains carrying these plasmids, we use DNA barcode sequencing (BarSeq), which is amenable to large-scale sample multiplexing. As a demonstration of this approach, we constructed a Dub-seq library with total Escherichia coli genomic DNA, performed 155 genome-wide fitness assays in 52 experimental conditions, and identified 813 genes with high-confidence overexpression phenotypes across 4,151 genes assayed. We show that Dub-seq data is reproducible, accurately recapitulates known biology, and identifies hundreds of novel gain-of-function phenotypes for E. coli genes, a subset of which we verified with assays of individual strains. Dub-seq provides complementary information to loss-of-function approaches such as transposon site sequencing or CRISPRi and will facilitate rapid and systematic functional characterization of microbial genomes.ImportanceMeasuring the phenotypic consequences of overexpressing genes is a classic genetic approach for understanding protein function; for identifying drug targets, antibiotic and metal resistance mechanisms; and for optimizing strains for metabolic engineering. In microorganisms, these gain-of-function assays are typically done using laborious protocols with individually archived strains or in low-throughput following qualitative selection for a phenotype of interest, such as antibiotic resistance. However, many microbial genes are poorly characterized and the importance of a given gene may only be apparent under certain conditions. Therefore, more scalable approaches for gain-of-function assays are needed. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a strategy that couples systematic gene overexpression with DNA barcode sequencing for large-scale interrogation of gene fitness under many experimental conditions at low cost. Dub-seq can be applied to many microorganisms and is a valuable new tool for large-scale gene function characterization.

scholarly journals Edwin

2016 ◽  
Vol 22 (1) ◽  
pp. 50-62 ◽  
Author(s):  
Aitor de las Heras ◽  
Weike Xiao ◽  
Vlastimil Sren ◽  
Alistair Elfick
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Posttranscriptional Regulation ◽  
Large Scale ◽  
Rna Extraction ◽  
Automated Analysis ◽  
Regulatory Elements ◽  
Fluorescence Signal ◽  
Microbial Cells

Characterization of gene expression is a central tenet of the synthetic biology design cycle. Sometimes it requires high-throughput approaches that allow quantification of the gene expression of different elements in diverse conditions. Recently, several large-scale studies have highlighted the importance of posttranscriptional regulation mechanisms and their impact on correlations between mRNA and protein abundance. Here, we introduce Edwin, a robotic workstation that enables the automated propagation of microbial cells and the dynamic characterization of gene expression. We developed an automated procedure that integrates customized RNA extraction and analysis into the typical high-throughput characterization of reporter gene expression. To test the system, we engineered Escherichia coli strains carrying different promoter/ gfp fusions. We validated Edwin’s abilities: (1) preparation of custom cultures of microbial cells and (2) dynamic quantification of fluorescence signal and bacterial growth and simultaneous RNA extraction and analysis at different time points. We confirmed that RNA obtained during this automated process was suitable for use in qPCR analysis. Our results established that Edwin is a powerful platform for the automated analysis of microbial gene expression at the protein and RNA level. This platform could be used in a high-throughput manner to characterize not only natural regulatory elements but also synthetic ones.

scholarly journals Large-Scale Structural Characterization of Drug and Drug-Like Compounds by High-Throughput Ion Mobility-Mass Spectrometry

2017 ◽  
Vol 89 (17) ◽  
pp. 9023-9030 ◽  
Author(s):  
Kelly M. Hines ◽  
Dylan H. Ross ◽  
Kimberly L. Davidson ◽  
Matthew F. Bush ◽  
Libin Xu
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Structural Characterization ◽  
Ion Mobility ◽  
Large Scale ◽  
Ion Mobility Mass Spectrometry

High-throughput genotyping assay for the large-scale genetic characterization ofCryptosporidiumparasites from human and bovine samples

Parasitology ◽  
2013 ◽  
Vol 141 (4) ◽  
pp. 491-500 ◽  
Author(s):  
J. L. ABAL-FABEIRO ◽  
X. MASIDE ◽  
J. LLOVO ◽  
X. BELLO ◽  
M. TORRES ◽  
...  
Keyword(s):  
High Throughput ◽  
Sanger Sequencing ◽  
Large Scale ◽  
Genetic Characterization ◽  
Low Cost ◽  
Cost Effective ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Identification Of Species

SUMMARYThe epidemiological study of human cryptosporidiosis requires the characterization of species and subtypes involved in human disease in large sample collections. Molecular genotyping is costly and time-consuming, making the implementation of low-cost, highly efficient technologies increasingly necessary. Here, we designed a protocol based on MALDI-TOF mass spectrometry for the high-throughput genotyping of a panel of 55 single nucleotide variants (SNVs) selected as markers for the identification of commongp60subtypes of fourCryptosporidiumspecies that infect humans. The method was applied to a panel of 608 human and 63 bovine isolates and the results were compared with control samples typed by Sanger sequencing. The method allowed the identification of species in 610 specimens (90·9%) andgp60subtype in 605 (90·2%). It displayed excellent performance, with sensitivity and specificity values of 87·3 and 98·0%, respectively. Up to nine genotypes from four differentCryptosporidiumspecies (C. hominis, C. parvum, C. meleagridisandC. felis) were detected in humans; the most common ones wereC. hominissubtype Ib, andC. parvumIIa (61·3 and 28·3%, respectively). 96·5% of the bovine samples were typed as IIa. The method performs as well as the widely used Sanger sequencing and is more cost-effective and less time consuming.

scholarly journals Platon: identification and characterization of bacterial plasmid contigs in short-read draft assemblies exploiting protein sequence-based replicon distribution scores

2020 ◽  
Vol 6 (10) ◽  
Author(s):  
Oliver Schwengers ◽  
Patrick Barth ◽  
Linda Falgenhauer ◽  
Torsten Hain ◽  
Trinad Chakraborty ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Software Tool ◽  
High Sensitivity ◽  
Independent Manner ◽  
Short Read ◽  
Content Type ◽  
Link Type ◽  
Antimicrobial Resistance Genes

Plasmids are extrachromosomal genetic elements that replicate independently of the chromosome and play a vital role in the environmental adaptation of bacteria. Due to potential mobilization or conjugation capabilities, plasmids are important genetic vehicles for antimicrobial resistance genes and virulence factors with huge and increasing clinical implications. They are therefore subject to large genomic studies within the scientific community worldwide. As a result of rapidly improving next-generation sequencing methods, the quantity of sequenced bacterial genomes is constantly increasing, in turn raising the need for specialized tools to (i) extract plasmid sequences from draft assemblies, (ii) derive their origin and distribution, and (iii) further investigate their genetic repertoire. Recently, several bioinformatic methods and tools have emerged to tackle this issue; however, a combination of high sensitivity and specificity in plasmid sequence identification is rarely achieved in a taxon-independent manner. In addition, many software tools are not appropriate for large high-throughput analyses or cannot be included in existing software pipelines due to their technical design or software implementation. In this study, we investigated differences in the replicon distributions of protein-coding genes on a large scale as a new approach to distinguish plasmid-borne from chromosome-borne contigs. We defined and computed statistical discrimination thresholds for a new metric: the replicon distribution score (RDS), which achieved an accuracy of 96.6 %. The final performance was further improved by the combination of the RDS metric with heuristics exploiting several plasmid-specific higher-level contig characterizations. We implemented this workflow in a new high-throughput taxon-independent bioinformatics software tool called Platon for the recruitment and characterization of plasmid-borne contigs from short-read draft assemblies. Compared to PlasFlow, Platon achieved a higher accuracy (97.5 %) and more balanced predictions (F1=82.6 %) tested on a broad range of bacterial taxa and better or equal performance against the targeted tools PlasmidFinder and PlaScope on sequenced Escherichia coli isolates. Platon is available at: http://platon.computational.bio/.

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