scholarly journals Metagenomic Analysis of the Virome of Mosquito Excreta

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Ana L. Ramírez ◽  
Agathe M. G. Colmant ◽  
David Warrilow ◽  
Bixing Huang ◽  
Alyssa T. Pyke ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rna Viruses ◽  
Full Length ◽  
Metagenomic Analysis ◽  
Virus Discovery ◽  
Next Generation ◽  
West Nile ◽  
Arbovirus Surveillance ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

ABSTRACT Traditional screening for arboviruses in mosquitoes requires a priori knowledge and the utilization of appropriate assays for their detection. Mosquitoes can also provide other valuable information, including unexpected or novel arboviruses, nonarboviral pathogens ingested from hosts they feed on, and their own genetic material. Metagenomic analysis using next-generation sequencing (NGS) is a rapidly advancing technology that allows us to potentially obtain all this information from a mosquito sample without any prior knowledge of virus, host, or vector. Moreover, it has been recently demonstrated that pathogens, including arboviruses and parasites, can be detected in mosquito excreta by molecular methods. In this study, we investigated whether RNA viruses could be detected in mosquito excreta by NGS. Excreta samples were collected from Aedes vigilax and Culex annulirostris experimentally exposed to either Ross River or West Nile viruses and from field mosquitoes collected across Queensland, Australia. Total RNA was extracted from the excreta samples, reverse transcribed to cDNA, and sequenced using the Illumina NextSeq 500 platform. Bioinformatic analyses from the generated reads demonstrate that mosquito excreta provide sufficient RNA for NGS, allowing the assembly of near-full-length viral genomes. We detected Australian Anopheles totivirus, Wuhan insect virus 33, and Hubei odonate virus 5 and identified seven potentially novel viruses closely related to members of the order Picornavirales (2/7) and to previously described, but unclassified, RNA viruses (5/7). Our results suggest that metagenomic analysis of mosquito excreta has great potential for virus discovery and for unbiased arbovirus surveillance in the near future. IMPORTANCE When a mosquito feeds on a host, it ingests not only its blood meal but also an assortment of microorganisms that are present in the blood, thus acting as an environmental sampler. By using specific tests, it is possible to detect arthropod-borne viruses (arboviruses) like dengue and West Nile viruses in mosquito excreta. Here, we explored the use of next-generation sequencing (NGS) for unbiased detection of RNA viruses present in excreta from experimentally infected and field-collected mosquitoes. We have demonstrated that mosquito excreta provide a suitable template for NGS and that it is possible to recover and assemble near-full-length genomes of both arboviruses and insect-borne viruses, including potentially novel ones. These results importantly show the direct practicality of the use of mosquito excreta for NGS, which in the future could be used for virus discovery, environmental virome sampling, and arbovirus surveillance.

scholarly journals A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

mSystems ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Lindsey A. Moser ◽  
Lisbeth Ramirez-Carvajal ◽  
Vinita Puri ◽  
Steven J. Pauszek ◽  
Krystal Matthews ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Operating Procedure ◽  
Rna Viruses ◽  
Standard Operating Procedure ◽  
Next Generation ◽  
Standard Operating ◽  
Level 3 ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Biosafety Level

ABSTRACT This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories. Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories.

Initial Next-Generation Sequencing (NGS) Results of Alport Syndrome

2019 ◽  
Author(s):  
Altuğ Koç ◽  
Elçin Bora ◽  
Tayfun Cinleti ◽  
Gizem Yıldız ◽  
Meral Torun Bayram ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Alport Syndrome ◽  
Next Generation ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

The applications of next-generation sequencing (NGS) in drug development for cancer therapy

2020 ◽  
Vol 16 ◽  
Author(s):  
Pelin Telkoparan-Akillilar ◽  
Dilek Cevik
Keyword(s):  
Next Generation Sequencing ◽  
Drug Discovery ◽  
Cancer Therapy ◽  
Target Identification ◽  
Rapid Development ◽  
Next Generation ◽  
Biomedical Sciences ◽  
Dna And Rna ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Background: Numerous sequencing techniques have been progressed since the 1960s with the rapid development of molecular biology studies focusing on DNA and RNA. Methods: a great number of articles, book chapters, websites are reviewed, and the studies covering NGS history, technology and applications to cancer therapy are included in the present article. Results: High throughput next-generation sequencing (NGS) technologies offer many advantages over classical Sanger sequencing with decreasing cost per base and increasing sequencing efficiency. NGS technologies are combined with bioinformatics software to sequence genomes to be used in diagnostics, transcriptomics, epidemiologic and clinical trials in biomedical sciences. The NGS technology has also been successfully used in drug discovery for the treatment of different cancer types. Conclusion: This review focuses on current and potential applications of NGS in various stages of drug discovery process, from target identification through to personalized medicine.

scholarly journals Next-Generation Sequencing in Tumor Diagnosis and Treatment

Diagnostics ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 962
Author(s):  
Dario de Biase ◽  
Matteo Fassan ◽  
Umberto Malapelle
Keyword(s):  
Next Generation Sequencing ◽  
Tumor Diagnosis ◽  
Diagnosis And Treatment ◽  
Next Generation ◽  
Multiple Genes ◽  
Next Generation Sequencing Ngs ◽  
Very High ◽  
Generation Sequencing ◽  
High Depth

Next-Generation Sequencing (NGS) allows for the sequencing of multiple genes at a very high depth of coverage [...]

scholarly journals Evaluation of Ion Torrent next-generation sequencing for thalassemia diagnosis

2020 ◽  
Vol 48 (12) ◽  
pp. 030006052096777
Author(s):  
Peisong Chen ◽  
Xuegao Yu ◽  
Hao Huang ◽  
Wentao Zeng ◽  
Xiaohong He ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Ion Torrent ◽  
Next Generation ◽  
Large Deletions ◽  
Different Types ◽  
Variant Detection ◽  
Polymerase Chain ◽  
Next Generation Sequencing Ngs ◽  
Accuracy And Repeatability ◽  
Generation Sequencing

Introduction To evaluate a next-generation sequencing (NGS) workflow in the screening and diagnosis of thalassemia. Methods In this prospective study, blood samples were obtained from people undergoing genetic screening for thalassemia at our centre in Guangzhou, China. Genomic DNA was polymerase chain reaction (PCR)-amplified and sequenced using the Ion Torrent system and results compared with traditional genetic analyses. Results Of the 359 subjects, 148 (41%) were confirmed to have thalassemia. Variant detection identified 35 different types including the most common. Identification of the mutational sites by NGS were consistent with those identified by Sanger sequencing and Gap-PCR. The sensitivity and specificities of the Ion Torrent NGS were 100%. In a separate test of 16 samples, results were consistent when repeated ten times. Conclusion Our NGS workflow based on the Ion Torrent sequencer was successful in the detection of large deletions and non-deletional defects in thalassemia with high accuracy and repeatability.

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