scholarly journals A Multicenter Study To Evaluate the Performance of High-Throughput Sequencing for Virus Detection

mSphere ◽  
2017 ◽  
Vol 2 (5) ◽  
Author(s):  
Arifa S. Khan ◽  
Siemon H. S. Ng ◽  
Olivier Vandeputte ◽  
Aisha Aljanahi ◽  
Avisek Deyati ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Cell Line ◽  
High Throughput ◽  
Future Development ◽  
High Throughput Sequencing ◽  
Collaborative Study ◽  
Virus Detection ◽  
Biological Products ◽  
Reference Virus ◽  
Complex Technology

ABSTRACT Recent high-throughput sequencing (HTS) investigations have resulted in unexpected discoveries of known and novel viruses in a variety of sample types, including research materials, clinical materials, and biological products. Therefore, HTS can be a powerful tool for supplementing current methods for demonstrating the absence of adventitious or unwanted viruses in biological products, particularly when using a new cell line. However, HTS is a complex technology with different platforms, which needs standardization for evaluation of biologics. This collaborative study was undertaken to investigate detection of different virus types using two different HTS platforms. The results of the independently performed studies demonstrated a similar sensitivity of virus detection, regardless of the different sample preparation and processing procedures and bioinformatic analyses done in the three laboratories. Comparable HTS detection of different virus types supports future development of reference virus materials for standardization and validation of different HTS platforms. The capability of high-throughput sequencing (HTS) for detection of known and unknown viruses makes it a powerful tool for broad microbial investigations, such as evaluation of novel cell substrates that may be used for the development of new biological products. However, like any new assay, regulatory applications of HTS need method standardization. Therefore, our three laboratories initiated a study to evaluate performance of HTS for potential detection of viral adventitious agents by spiking model viruses in different cellular matrices to mimic putative materials for manufacturing of biologics. Four model viruses were selected based upon different physical and biochemical properties and commercial availability: human respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), feline leukemia virus (FeLV), and human reovirus (REO). Additionally, porcine circovirus (PCV) was tested by one laboratory. Independent samples were prepared for HTS by spiking intact viruses or extracted viral nucleic acids, singly or mixed, into different HeLa cell matrices (resuspended whole cells, cell lysate, or total cellular RNA). Data were obtained using different sequencing platforms (Roche 454, Illumina HiSeq1500 or HiSeq2500). Bioinformatic analyses were performed independently by each laboratory using available tools, pipelines, and databases. The results showed that comparable virus detection was obtained in the three laboratories regardless of sample processing, library preparation, sequencing platform, and bioinformatic analysis: between 0.1 and 3 viral genome copies per cell were detected for all of the model viruses used. This study highlights the potential for using HTS for sensitive detection of adventitious viruses in complex biological samples containing cellular background. IMPORTANCE Recent high-throughput sequencing (HTS) investigations have resulted in unexpected discoveries of known and novel viruses in a variety of sample types, including research materials, clinical materials, and biological products. Therefore, HTS can be a powerful tool for supplementing current methods for demonstrating the absence of adventitious or unwanted viruses in biological products, particularly when using a new cell line. However, HTS is a complex technology with different platforms, which needs standardization for evaluation of biologics. This collaborative study was undertaken to investigate detection of different virus types using two different HTS platforms. The results of the independently performed studies demonstrated a similar sensitivity of virus detection, regardless of the different sample preparation and processing procedures and bioinformatic analyses done in the three laboratories. Comparable HTS detection of different virus types supports future development of reference virus materials for standardization and validation of different HTS platforms.

scholarly journals Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparation

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 566 ◽  
Author(s):  
Siemon Ng ◽  
Cassandra Braxton ◽  
Marc Eloit ◽  
Szi Feng ◽  
Romain Fragnoud ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Nucleic Acids ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Virus Detection ◽  
Extraction Methods ◽  
Control Measures ◽  
Acid Extraction ◽  
Library Preparation ◽  
Sample Processing

A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG). The topics include: Use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation.

scholarly journals Adventitious Virus Detection in Cells by High-Throughput Sequencing of Newly Synthesized RNAs: Unambiguous Differentiation of Cell Infection from Carryover of Viral Nucleic Acids

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Justine Cheval ◽  
Erika Muth ◽  
Gaëlle Gonzalez ◽  
Muriel Coulpier ◽  
Pascale Beurdeley ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Rna Virus ◽  
Virus Detection ◽  
Analytical Sensitivity ◽  
Cell Infection ◽  
Biological Products ◽  
Viral Sequences ◽  
In Cells

ABSTRACTThe use of high-throughput sequencing (HTS) to identify viruses in biologicals differs from current molecular approaches, since its use enables an unbiased approach to detection without the need to design specific primers to preamplify target sequences. Its broad range of detection and analytical sensitivity make it an important tool to ensure that biologicals are free from adventitious viruses. Similar to other molecular methods, however, identification of viral sequences in cells by HTS does not prove viral infection, since this could reflect carryover of inert viral sequences from reagents or other sources or the presence of transcriptionally inactive cellular sequences. Due to the broad range of detection associated with HTS, the above can potentially be perceived as a drawback for the testing of pharmaceutical biological products using this method. In order to avoid the identification of inert viral sequences, we present a methodology based on metabolic RNA labeling and sequencing, which enables the specific identification of newly synthesized viral RNAs in infected cells, resulting in the ability to unambiguously distinguish active infection by DNA or RNA viruses from inert nucleic acids. In the present study, we report the ability to differentiate Vero cells acutely infected by a single-stranded positive-sense RNA virus (tick-borne encephalitis virus) from cells which have been in contact with nonreplicating virus particles. Additionally, we also found a laboratory contamination by the squirrel monkey retrovirus of our Vero cell line, which was derived from an Old World (African green) monkey, a type of contamination which until now has been identified only in cells derived from primates from the New World.IMPORTANCEThe use of high-throughput sequencing (HTS) to identify viral contamination of biological products is extremely sensitive and provides a broad range of detection. Nevertheless, viral sequences identified can also be inert. Examples include contamination resulting from reagents or the presence of inactivated viruses in animal-derived components of the cell culture medium. We therefore developed a method that relies on the sequencing of newly synthesized RNAs, an unequivocal sign of the presence of a transcriptionally active virus. This improvement in the specificity of viral testing increases the acceptability of HTS as a standard test for cells used in manufacturing biologicals and in biotherapies.

scholarly journals Interlaboratory Comparison Study on Ribodepleted Total RNA High-Throughput Sequencing for Plant Virus Diagnostics and Bioinformatic Competence

Pathogens ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1174
Author(s):  
Yahya Z. A. Gaafar ◽  
Marcel Westenberg ◽  
Marleen Botermans ◽  
Krizbai László ◽  
Kris De Jonghe ◽  
...  
Keyword(s):  
Sample Preparation ◽  
High Throughput ◽  
Ribosomal Rna ◽  
Diagnostic Tool ◽  
Plant Virus ◽  
High Throughput Sequencing ◽  
Interlaboratory Comparison ◽  
Virus Detection ◽  
Plant Viruses ◽  
Comparison Study

High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS workflows from sample preparation to data analysis and results interpretation for plant virus detection and identification must be evaluated (verified and validated) to approve this tool for diagnostics. Many different extraction methods, library preparation protocols, and sequence and bioinformatic pipelines are available for virus sequence detection. To assess the performance of plant virology diagnostic laboratories in using the HTS of ribosomal RNA depleted total RNA (ribodepleted totRNA) as a diagnostic tool, we carried out an interlaboratory comparison study in which eight participants were required to use the same samples, (RNA) extraction kit, ribosomal RNA depletion kit, and commercial sequencing provider, but also their own bioinformatics pipeline, for analysis. The accuracy of virus detection ranged from 65% to 100%. The false-positive detection rate was very low and was related to the misinterpretation of results as well as to possible cross-contaminations in the lab or sequencing provider. The bioinformatic pipeline used by each laboratory influenced the correct detection of the viruses of this study. The main difficulty was the detection of a novel virus as its sequence was not available in a publicly accessible database at the time. The raw data were reanalysed using Virtool to assess its ability for virus detection. All virus sequences were detected using Virtool in the different pools. This study revealed that the ribodepletion target enrichment for sample preparation is a reliable approach for the detection of plant viruses with different genomes. A significant level of virology expertise is needed to correctly interpret the results. It is also important to improve and complete the reference data

Comparison of high throughput sequencing to standard protocols for virus detection in berry crops

Plant Disease ◽  
2021 ◽  
Author(s):  
Dan Edward Veloso Villamor ◽  
Karen E Keller ◽  
Robert Martin ◽  
Ioannis Emmanouil Tzanetakis
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Wide Spectrum ◽  
Virus Detection ◽  
Rt Pcr ◽  
Host Interactions ◽  
Growing Seasons ◽  
Berry Crops ◽  
Sampling Times ◽  
Better Than

A comprehensive study comparing virus detection between high throughput sequencing (HTS) and standard protocols in 30 berry selections (12 Fragaria, 10 Vaccinium and 8 Rubus) with known virus profiles was completed. The study examined temporal detection of viruses at four sampling times encompassing two growing seasons. Within the standard protocols, RT-PCR proved better than biological indexing. Detection of known viruses by HTS and RT-PCR nearly mirrored each other. HTS provided superior detection compared to RT-PCR on a wide spectrum of virus variants and discovery of novel viruses. More importantly, in most cases where the two protocols showed parallel virus detection, 11 viruses in 16 berry selections were not consistently detected by both methods at all sampling points. Based on these data we propose a four sampling times/two-year testing requirement for berry and potentially other crops to ensure that no virus remains undetected independent of titer, distribution or other virus/virus or virus/host interactions.

scholarly journals CDK10 Regulates Gastric Cancer Metastasis By Inhibiting lncRNA-C5ORF42-5 Via Phosphorylation Level of AKT/ERK

2021 ◽  
Author(s):  
Zi-Jian Deng ◽  
Dong-Wen Chen ◽  
Xi-Jie Chen ◽  
Jia-Ming Fang ◽  
Liang Xv ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
High Throughput ◽  
Cancer Metastasis ◽  
High Throughput Sequencing ◽  
Gastric Cancer Cells ◽  
Related Proteins

Abstract Background: Gastric cancer is the fourth most common malignant disease. Both CDK10 and long noncoding RNAs (lncRNAs) have been found to exert biological functions in multiple cancers. However, it is still unclear whether CDK10 represses tumor progression in gastric cancer by reducing potential targeting lncRNAs.Methods: The functions of CDK10 and lncRNA-C5ORF42-5 in proliferation, invasion and migration were assessed by MTS assays, colony formation assays, cell cycle and apoptosis assays, Transwell assays, wound healing assays and animal experiments. We used high-throughput sequencing to confirm the existence of lncRNA-C5ORF42-5 and quantitative real-time PCR was used to evaluate lncRNA expression. Then, with RNA-seq sequencing as well as GO function and KEGG enrichment analysis, we identified the signaling pathways in which lncRNA-C5ORF42-5 was involved in gastric cancer. Finally, western blotting was used to identify the genes regulated by lncRNA-C5ORF42-5.Results: Our results showed that CDK10 is expressed at relatively low levels in gastric cancer cell lines and inhibits the progression of gastric cancer cells both in vitro and in vivo. Next, based on high-throughput sequencing, we identified a novel lncRNA, lncRNA-C5ORF42-5, in the stable CDK10-overexpressing cell line compared with the CDK-knockdown cell line and their controls. Additionally, we confirmed that lncRNA-C5ORF42-5 acts as an oncogene to promote metastasis in gastric cancer in vitro and in vivo. We then ascertained that lncRNA-C5ORF42-5 is a major contributor to the function of CDK10 in gastric cancer metastasis by upregulating lncRNA-C5ORF42-5 to reverse the effects of CDK10 overexpression. Finally, we explored the mechanism by which lncRNA-C5ORF42-5 overexpression affects gastric cancer cells to elucidate whether lncRNA-C5ORF42-5 may increase the activity of the SMAD pathway of BMP signaling and promote the expression of EMT-related proteins, such as E-cadherin. Additionally, overexpression of lncRNA-C5ORF42-5 affected the phosphorylation levels of AKT and ERK.Conclusion: Our findings suggest that CDK10 overexpression represses gastric cancer tumor progression by reducing lncRNA-C5ORF42-5 and hindering activation of the related proteins in metastatic signaling pathways, which provides new insight into developing effective therapeutic strategies in the treatment of metastatic gastric cancer.

scholarly journals High Throughput Sequencing For Plant Virus Detection and Discovery

2019 ◽  
Vol 109 (5) ◽  
pp. 716-725 ◽  
Author(s):  
D. E. V. Villamor ◽  
T. Ho ◽  
M. Al Rwahnih ◽  
R. R. Martin ◽  
I. E. Tzanetakis
Keyword(s):  
High Throughput ◽  
Plant Virus ◽  
High Throughput Sequencing ◽  
Virus Detection ◽  
Agricultural Crops ◽  
Virus Discovery ◽  
Plant Virus Detection ◽  
Virus Biology ◽  
Disease Causality

Over the last decade, virologists have discovered an unprecedented number of viruses using high throughput sequencing (HTS), which led to the advancement of our knowledge on the diversity of viruses in nature, particularly unraveling the virome of many agricultural crops. However, these new virus discoveries have often widened the gaps in our understanding of virus biology; the forefront of which is the actual role of a new virus in disease, if any. Yet, when used critically in etiological studies, HTS is a powerful tool to establish disease causality between the virus and its host. Conversely, with globalization, movement of plant material is increasingly more common and often a point of dispute between countries. HTS could potentially resolve these issues given its capacity to detect and discover. Although many pipelines are available for plant virus discovery, all share a common backbone. A description of the process of plant virus detection and discovery from HTS data are presented, providing a summary of the different pipelines available for scientists’ utility in their research.

Virus detection in high-throughput sequencing data without a reference genome of the host

2018 ◽  
Vol 66 ◽  
pp. 180-187 ◽  
Author(s):  
Jochen Kruppa ◽  
Wendy K. Jo ◽  
Erhard van der Vries ◽  
Martin Ludlow ◽  
Albert Osterhaus ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Reference Genome ◽  
Virus Detection ◽  
Sequencing Data ◽  
High Throughput Sequencing Data

scholarly journals High-throughput Sequencing for Species Authentication and Contamination Detection of 63 Cell Lines

2021 ◽  
Author(s):  
Oliver Lung ◽  
Rebecca Candlish ◽  
Michelle Nebroski ◽  
Peter Kruckiewicz ◽  
Cody Buchanan ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Classical Swine Fever ◽  
Virus Genome ◽  
Cox1 Gene ◽  
Species Identity ◽  
Cell Line Authentication ◽  
Dna And Rna

Abstract Cell lines are widely used in research and for diagnostic tests and are often shared between laboratories. Lack of cell line authentication can result in the use of contaminated or misidentified cell lines, potentially affecting the results from research and diagnostic activities. Cell line authentication and contamination detection based on metagenomic high-throughput sequencing (HTS) was tested on DNA and RNA from 63 cell lines available at the Canadian Food Inspection Agency’s National Centre for Foreign Animal Disease. Through sequence comparison of the cytochrome c oxidase subunit 1 (COX1) gene, the species identity of 53 cell lines was confirmed, and eight cell lines were found to show a greater pairwise nucleotide identity in the COX1 sequence of a different species within the same expected genus. Two cell lines, LFBK-αvβ6 and SCP-HS, were determined to be composed of cells from a different species and genus. Mycoplasma contamination was not detected in any cell lines. However, several expected and unexpected viral sequences were detected, including part of the classical swine fever virus genome in the IB-RS-2 Clone D10 cell line. Metagenomics-based HTS is a useful laboratory QA tool for cell line authentication and contamination detection that should be conducted regularly.

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