scholarly journals Reply to Grigoriev et al., “Sequences of SARS-CoV-2 “Hybrids” with the Human Genome: Signs 1 of Non-coding RNA?”

2021 ◽  
Author(s):  
Bingyu Yan ◽  
Srishti Chakravorty ◽  
Carmen Mirabelli ◽  
Luopin Wang ◽  
Jorge L. Trujillo-Ochoa ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Host Cells ◽  
Viral Gene ◽  
Gene Expressions ◽  
Biologically Relevant ◽  
Sequencing Library ◽  
Infected Cells ◽  
Template Switch ◽  
Host Virus Interactions

High throughput sequencing reads from virally infected cells provide detailed information about both the infected host cells and invading viruses (1). For example, RNA-sequencing techniques from infected cells contains reads that unequivocally align to either the host or the viral transcriptomes, enabling quantification of host and viral gene expressions (2). Occasionally, there are reads with split characteristics, having one part (e.g., the 5’ end) unambiguously matching the host and another part (e.g., the 3’ end) clearly matching the viral genomes. The split characteristic with unambiguous matching on either part is the key here, typically requiring convincing stretches of sequence matches such as >30bp that we used in our analysis (3). Such reads are termed host-virus chimeric reads (HVCRs). Indeed, HVCRs that surpass statistical reproducibility and signal-to-noise standards might carry novel insights into the biology of host-virus interactions (4, 5). Thus, it is important to unambiguously detect statistically rigorous and biologically relevant HVCRs. We and others have shown that detection of relevant HVCRs is complicated by unfaithful reverse-transcriptase and polymerase enzymes that template-switch during typical high throughput sequencing library preparation protocols (6–9).

scholarly journals BK Polyomavirus MicroRNA Levels in Exosomes Are Modulated by Non-Coding Control Region Activity and Down-Regulate Viral Replication When Delivered to Non-Infected Cells Prior to Infection

Viruses ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 466 ◽  
Author(s):  
Francesco Martelli ◽  
Zongsong Wu ◽  
Serena Delbue ◽  
Fabian Weissbach ◽  
Maria Giulioli ◽  
...  
Keyword(s):  
Viral Replication ◽  
Point Mutations ◽  
T Antigen ◽  
Host Cells ◽  
Replication Capacity ◽  
Viral Gene ◽  
New Host ◽  
Infected Cells ◽  
Multiple Sclerosis Patients ◽  
Coding Control

In immunosuppressed patients, BKPyV-variants emerge carrying rearranged non-coding control-regions (rr-NCCRs) that increase early viral gene region (EVGR) expression and replication capacity. BKPyV also encodes microRNAs, which have been reported to downregulate EVGR-encoded large T-antigen transcripts, to decrease viral replication in infected cells and to be secreted in exosomes. To investigate the interplay of NCCR and microRNAs, we compared archetype- and rr-NCCR-BKPyV infection in cell culture. We found that laboratory and clinical rr-NCCR-BKPyV-strains show higher replication rates but significantly lower microRNA levels than archetype virus intracellularly and in exosomes. To investigate whether rr-NCCR or increased EVGR activity modulated microRNA levels, we examined the (sp1-4)NCCR-BKPyV, which has an archetype NCCR-architecture but shows increased EVGR expression due to point mutations inactivating one Sp1 binding site. We found that microRNA levels following (sp1-4)NCCR-BKPyV infection were as low as in rr-NCCR-variants. Thus, NCCR rearrangements are not required for lower miRNA levels. Accordingly, Sp1 siRNA knock-down decreased microRNA levels in archetype BKPyV infection but had no effect on (sp1-4)- or rr-NCCR-BKPyV. However, rr-NCCR-BKPyV replication was downregulated by exosome preparations carrying BKPyV-microRNA prior to infection. To explore the potential relevance in humans, urine samples from 12 natalizumab-treated multiple sclerosis patients were analysed. In 7 patients, rr-NCCR-BKPyV were detected showing high urine BKPyV loads but low microRNAs levels, whereas the opposite was seen in 5 patients with archetype BKPyV. We discuss the results in a dynamic model of BKPyV replication according to NCCR activity and exosome regulation, which integrates immune selection pressure, spread to new host cells and rr-NCCR emergence.

scholarly journals The promise of a DNA taxonomy

2004 ◽  
Vol 359 (1444) ◽  
pp. 669-679 ◽  
Author(s):  
Mark L. Blaxter
Keyword(s):  
High Throughput ◽  
Small Proportion ◽  
High Throughput Sequencing ◽  
Meta Analysis ◽  
Molecular Data ◽  
Similar Extent ◽  
Molecular Barcoding ◽  
Dna Taxonomy ◽  
Biologically Relevant ◽  
Operational Taxonomic Units

Not only is the number of described species a very small proportion of the estimated extant number of taxa, but it also appears that all concepts of the extent and boundaries of ‘species’ fail in many cases. Using conserved molecular sequences it is possible to define and diagnose molecular operational taxonomic units (MOTU) that have a similar extent to traditional ‘species’. Use of a MOTU system not only allows the rapid and effective identification of most taxa, including those not encountered before, but also allows investigation of the evolution of patterns of diversity. A MOTU approach is not without problems, particularly in the area of deciding what level of molecular difference defines a biologically relevant taxon, but has many benefits. Molecular data are extremely well suited to re–analysis and meta–analysis, and data from multiple independent studies can be readily collated and investigated by using new parameters and assumptions. Previous molecular taxonomic efforts have focused narrowly. Advances in high–throughput sequencing methodologies, however, place the idea of a universal, multi–locus molecular barcoding system in the realm of the possible.

scholarly journals Small RNA Expression Profiling by High-Throughput Sequencing: Implications of Enzymatic Manipulation

2012 ◽  
Vol 2012 ◽  
pp. 1-15 ◽  
Author(s):  
Fanglei Zhuang ◽  
Ryan T. Fuchs ◽  
G. Brett Robb
Keyword(s):  
High Throughput ◽  
Expression Profiling ◽  
Messenger Rna ◽  
High Throughput Sequencing ◽  
Biological Processes ◽  
Cellular Processes ◽  
Disease States ◽  
Powerful Approach ◽  
Sequencing Library ◽  
Rna Expression Profiling

Eukaryotic regulatory small RNAs (sRNAs) play significant roles in many fundamental cellular processes. As such, they have emerged as useful biomarkers for diseases and cell differentiation states. sRNA-based biomarkers outperform traditional messenger RNA-based biomarkers by testing fewer targets with greater accuracy and providing earlier detection for disease states. Therefore, expression profiling of sRNAs is fundamentally important to further advance the understanding of biological processes, as well as diagnosis and treatment of diseases. High-throughput sequencing (HTS) is a powerful approach for both sRNA discovery and expression profiling. Here, we discuss the general considerations for sRNA-based HTS profiling methods from RNA preparation to sequencing library construction, with a focus on the causes of systematic error. By examining the enzymatic manipulation steps of sRNA expression profiling, this paper aims to demystify current HTS-based sRNA profiling approaches and to aid researchers in the informed design and interpretation of profiling experiments.

scholarly journals Deubiquitinating Function of Adenovirus Proteinase

2002 ◽  
Vol 76 (12) ◽  
pp. 6323-6331 ◽  
Author(s):  
Maxim Y. Balakirev ◽  
Michel Jaquinod ◽  
Arthur L. Haas ◽  
Jadwiga Chroboczek
Keyword(s):  
Structural Model ◽  
Proteolytic Processing ◽  
Host Cells ◽  
Cellular Proteins ◽  
Viral Gene ◽  
Cellular Processes ◽  
Infected Cells ◽  
Substrate Binding Sites

ABSTRACT The invasion strategy of many viruses involves the synthesis of viral gene products that mimic the functions of the cellular proteins and thus interfere with the key cellular processes. Here we show that adenovirus infection is accompanied by an increased ubiquitin-cleaving (deubiquitinating) activity in the host cells. Affinity chromatography on ubiquitin aldehyde (Ubal), which was designed to identify the deubiquitinating proteases, revealed the presence of adenovirus L3 23K proteinase (Avp) in the eluate from adenovirus-infected cells. This proteinase is known to be necessary for the processing of viral precursor proteins during virion maturation. We show here that in vivo Avp deubiquitinates a number of cellular proteins. Analysis of the substrate specificity of Avp in vitro demonstrated that the protein deubiquitination by this enzyme could be as efficient as proteolytic processing of viral proteins. The structural model of the Ubal-Avp interaction revealed some similarity between S1-S4 substrate binding sites of Avp and ubiquitin hydrolases. These results may reflect the acquisition of an advantageous property by adenovirus and may indicate the importance of ubiquitin pathways in viral infection.

scholarly journals DNA extraction and sequencing of the Mawangdui ancient cadaver protected by formalin

2020 ◽  
Author(s):  
Hua-Lin Huang ◽  
Shikui Yin ◽  
Huifang Zhao ◽  
Chao Tian ◽  
Jufang Huang ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Pcr Amplification ◽  
Magnetic Bead ◽  
Sequencing Data ◽  
Sequencing Library ◽  
Bead Method ◽  
Formalin Fixed ◽  
Better Than

AbstractMawangdui ancient Cadaver is the first wet corpse found in the world, which is famous for being immortal for over two thousands of years. After being unearthed, the female corpse was immersed in the formalin protective solution for more than 40 years. We used magnetic bead method and formalin fixed paraffing (FFPE) method to extract the DNA of the female corpse, respectively. PCR amplification, sanger sequencing, library building, high throughput sequencing (testing) and data processing were carried out on the DNA samples, and about 0.5% of the whole genome coverage sequencing data was obtained. Comparing the results of DNA trough two extraction and sequencing methods. We found that the FFPE and high throughput sequencing methods is better than others for DNA extraction of the ancient samples which were preserved in formalin, providing a guidance for dealing with formalin preserved ancient samples in the future.

scholarly journals FLASH: ultra-fast protocol to identify RNA–protein interactions in cells

2019 ◽  
Vol 48 (3) ◽  
pp. e15-e15 ◽  
Author(s):  
Ibrahim Avsar Ilik ◽  
Tugce Aktas ◽  
Daniel Maticzka ◽  
Rolf Backofen ◽  
Asifa Akhtar
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
Binding Sites ◽  
Binding Proteins ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Affinity Purification ◽  
Sequencing Library

Abstract Determination of the in vivo binding sites of RNA-binding proteins (RBPs) is paramount to understanding their function and how they affect different aspects of gene regulation. With hundreds of RNA-binding proteins identified in human cells, a flexible, high-resolution, high-throughput, highly multiplexible and radioactivity-free method to determine their binding sites has not been described to date. Here we report FLASH (Fast Ligation of RNA after some sort of Affinity Purification for High-throughput Sequencing), which uses a special adapter design and an optimized protocol to determine protein–RNA interactions in living cells. The entire FLASH protocol, starting from cells on plates to a sequencing library, takes 1.5 days. We demonstrate the flexibility, speed and versatility of FLASH by using it to determine RNA targets of both tagged and endogenously expressed proteins under diverse conditions in vivo.

High throughput sequencing methods for microbiome profiling: application to food animal systems

2012 ◽  
Vol 13 (1) ◽  
pp. 40-53 ◽  
Author(s):  
Sarah K. Highlander
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Human Subjects ◽  
Human Microbiome ◽  
Host Cells ◽  
Rare Biosphere ◽  
Order Of Magnitude ◽  
Production Animals

AbstractAnalysis of microbial communities using high throughput sequencing methods began in the mid 2000s permitting the production of 1000s to 10,000s of sequence reads per sample and megabases of data per sequence run. This then unprecedented depth of sequencing allowed, for the first time, the discovery of the ‘rare biosphere’ in environmental samples. The technology was quickly applied to studies in several human subjects. Perhaps these early studies served as a reminder that though the microbes that inhabit mammals are known to outnumber host cells by an order of magnitude or more, most of these are unknown members of our second genome, or microbiome (as coined by Joshua Lederberg), because of our inability to culture them. High throughput methods for microbial 16S ribosomal RNA gene and whole genome shotgun (WGS) sequencing have now begun to reveal the composition and identity of archaeal, bacterial and viral communities at many sites, in and on the human body. Surveys of the microbiota of food production animals have been published in the past few years and future studies should benefit from protocols and tools developed from large-scale human microbiome studies. Nevertheless, production animal-related resources, such as improved host genome assemblies and increased numbers and diversity of host-specific microbial reference genome sequences, will be needed to permit meaningful and robust analysis of 16S rDNA and WGS sequence data.

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