scholarly journals Nucleotide-Resolution Profiling of RNA Recombination in the Encapsidated Genome of a Eukaryotic RNA Virus by Next-Generation Sequencing

2012 ◽  
Vol 424 (5) ◽  
pp. 257-269 ◽  
Author(s):  
Andrew Routh ◽  
Phillip Ordoukhanian ◽  
John E. Johnson
Keyword(s):  
Next Generation Sequencing ◽  
Rna Virus ◽  
Next Generation ◽  
Rna Recombination ◽  
Nucleotide Resolution ◽  
Generation Sequencing

scholarly journals Mapping DNA Topoisomerase Binding and Cleavage Genome Wide Using Next-Generation Sequencing Techniques

Genes ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 92 ◽  
Author(s):  
Shannon J. McKie ◽  
Anthony Maxwell ◽  
Keir C. Neuman
Keyword(s):  
Next Generation Sequencing ◽  
Dna Cleavage ◽  
Next Generation ◽  
Dna Breaks ◽  
Genome Wide ◽  
Extension Activity ◽  
Nucleotide Resolution ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Next-generation sequencing (NGS) platforms have been adapted to generate genome-wide maps and sequence context of binding and cleavage of DNA topoisomerases (topos). Continuous refinements of these techniques have resulted in the acquisition of data with unprecedented depth and resolution, which has shed new light on in vivo topo behavior. Topos regulate DNA topology through the formation of reversible single- or double-stranded DNA breaks. Topo activity is critical for DNA metabolism in general, and in particular to support transcription and replication. However, the binding and activity of topos over the genome in vivo was difficult to study until the advent of NGS. Over and above traditional chromatin immunoprecipitation (ChIP)-seq approaches that probe protein binding, the unique formation of covalent protein–DNA linkages associated with DNA cleavage by topos affords the ability to probe cleavage and, by extension, activity over the genome. NGS platforms have facilitated genome-wide studies mapping the behavior of topos in vivo, how the behavior varies among species and how inhibitors affect cleavage. Many NGS approaches achieve nucleotide resolution of topo binding and cleavage sites, imparting an extent of information not previously attainable. We review the development of NGS approaches to probe topo interactions over the genome in vivo and highlight general conclusions and quandaries that have arisen from this rapidly advancing field of topoisomerase research.

scholarly journals A Retrospective Paired Comparison Between Untargeted Next Generation Sequencing and Conventional Microbiology Tests With Wisely Chosen Metagenomic Sequencing Positive Criteria

2021 ◽  
Vol 8 ◽  
Author(s):  
Hanyu Qin ◽  
Jinmin Peng ◽  
Ling Liu ◽  
Jing Wu ◽  
Lingai Pan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Fungal Infections ◽  
Rna Virus ◽  
Paired Comparison ◽  
Final Analysis ◽  
Metagenomic Sequencing ◽  
Next Generation ◽  
Dna Virus ◽  
Virus Identification ◽  
Generation Sequencing

Objectives: To evaluate the performance of metagenomic next generation sequencing (mNGS) using adequate criteria for the detection of pathogens in lower respiratory tract (LRT) samples with a paired comparison to conventional microbiology tests (CMT).Methods: One hundred sixty-seven patients were reviewed from four different intensive care units (ICUs) in mainland China during 2018 with both mNGS and CMT results of LRT samples available. The reads per million ratio (RPMsample/RPMnon−template−control ratio) and standardized strictly mapped reads number (SDSMRN) were the two criteria chosen for identifying positive pathogens reported from mNGS. A McNemar test was used for a paired comparison analysis between mNGS and CMT.Results: One hundred forty-nine cases were counted into the final analysis. The RPMsample/RPMNTC ratio criterion performed better with a higher accuracy for bacteria, fungi, and virus than SDSMRN criterion [bacteria (RPMsample/RPMNTC ratio vs. SDSMRN), 65.1 vs. 55.7%; fungi, 75.8 vs. 71.1%; DNA virus, 86.3 vs. 74.5%; RNA virus, 90.9 vs. 81.8%]. The mNGS was also superior in bacteria detection only if an SDSMRN ≥3 was used as a positive criterion with a paired comparison to culture [SDSMRN positive, 92/149 (61.7%); culture positive, 54/149 (36.2%); p < 0.001]; however, it was outperformed with significantly more fungi and DNA virus identification when choosing both criteria for positive outliers [fungi (RPMsample/RPMNTC ratio vs. SDSMRN vs. culture), 23.5 vs. 29.5 vs. 8.7%, p < 0.001; DNA virus (RPMsample/RPMNTC ratio vs. SDSMRN vs. PCR), 14.1 vs. 20.8 vs. 11.8%, p < 0.05].Conclusions: Metagenomic next generation sequencing may contribute to revealing the LRT infection etiology in hospitalized groups of potential fungal infections and in situations with less access to the multiplex PCR of LRT samples from the laboratory by choosing a wise criterion like the RPMsample/RPMNTC ratio.

scholarly journals A beginner’s guide for FMDV quasispecies analysis: sub-consensus variant detection and haplotype reconstruction using next-generation sequencing

2019 ◽  
Vol 21 (5) ◽  
pp. 1766-1775 ◽  
Author(s):  
Marco Cacciabue ◽  
Anabella Currá ◽  
Elisa Carrillo ◽  
Guido König ◽  
María Inés Gismondi
Keyword(s):  
Next Generation Sequencing ◽  
Rna Virus ◽  
Foot And Mouth Disease ◽  
Next Generation Sequencing Data ◽  
Complete Analysis ◽  
Viral Quasispecies ◽  
Next Generation ◽  
Sequencing Data ◽  
Viral Genomes ◽  
Generation Sequencing

Abstract Deep sequencing of viral genomes is a powerful tool to study RNA virus complexity. However, the analysis of next-generation sequencing data might be challenging for researchers who have never approached the study of viral quasispecies by this methodology. In this work we present a suitable and affordable guide to explore the sub-consensus variability and to reconstruct viral quasispecies from Illumina sequencing data. The guide includes a complete analysis pipeline along with user-friendly descriptions of software and file formats. In addition, we assessed the feasibility of the workflow proposed by analyzing a set of foot-and-mouth disease viruses (FMDV) with different degrees of variability. This guide introduces the analysis of quasispecies of FMDV and other viruses through this kind of approach.

scholarly journals Using Next Generation Sequencing to Study the Genetic Diversity of Candidate Live Attenuated Zika Vaccines

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 161
Author(s):  
Natalie D. Collins ◽  
Chao Shan ◽  
Bruno T.D. Nunes ◽  
Steven G. Widen ◽  
Pei-Yong Shi ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Next Generation Sequencing ◽  
Rna Virus ◽  
Nonstructural Protein ◽  
Sequencing Analysis ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Live Attenuated Vaccine ◽  
Generation Sequencing

Zika virus (ZIKV) is a mosquito-transmitted positive-sense RNA virus in the family Flaviviridae. Candidate live-attenuated vaccine (LAV) viruses with engineered deletions in the 3’ untranslated region (UTR) provide immunity and protection in animal models of ZIKV infection, and phenotypic studies show that LAVs retain protective abilities following in vitro passage. The present study investigated the genetic diversity of wild-type (WT) parent ZIKV and its candidate LAVs using next generation sequencing analysis of five sequential in vitro passages. The results show that genomic entropy of WT ZIKV steadily increases during in vitro passage, whereas that of LAVs also increased by passage number five but was variable throughout passaging. Additionally, clusters of single nucleotide variants (SNVs) were found to be present in the pre-membrane/membrane (prM), envelope (E), nonstructural protein NS1 (NS1), and other nonstructural protein genes, depending on the specific deletion, whereas in the parent WT ZIKV, they are more abundant in prM and NS1. Ultimately, both the parental WT and LAV derivatives increase in genetic diversity, with evidence of adaptation following passage.

scholarly journals A Pan-HIV Strategy for Complete Genome Sequencing

2015 ◽  
Vol 54 (4) ◽  
pp. 868-882 ◽  
Author(s):  
Michael G. Berg ◽  
Julie Yamaguchi ◽  
Elodie Alessandri-Gradt ◽  
Robert W. Tell ◽  
Jean-Christophe Plantier ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rna Virus ◽  
Consensus Sequence ◽  
Sample Type ◽  
Full Genome ◽  
Next Generation ◽  
Viral Loads ◽  
Subtype B ◽  
Generation Sequencing ◽  
Hiv 1

Molecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e.,switchingmechanismat 5′ end ofRNAtranscript]) 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.

scholarly journals Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing

2013 ◽  
Vol 10 (4) ◽  
pp. 361-365 ◽  
Author(s):  
Nicola Crosetto ◽  
Abhishek Mitra ◽  
Maria Joao Silva ◽  
Magda Bienko ◽  
Norbert Dojer ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Strand Break ◽  
Double Strand Break ◽  
Next Generation ◽  
Dna Double Strand Break ◽  
Nucleotide Resolution ◽  
Generation Sequencing
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