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Related Concept Videos

RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
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Next-generation Sequencing03:00

Next-generation Sequencing

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The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
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RACE - Rapid Amplification of cDNA Ends02:35

RACE - Rapid Amplification of cDNA Ends

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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific...
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Real Time RT-PCR02:57

Real Time RT-PCR

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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Low-Input, High-Resolution 5' Terminal Filovirus RNA Sequencing with ViBE-Seq.

Stephen J Ross1,2,3, Adam J Hume1,2, Judith Olejnik1,2

  • 1Department of Virology, Immunology & Microbiology, Chobanian and Avedisian School of Medicine, Boston University, Boston, MA 02215, USA.

Viruses
|July 27, 2024
PubMed
Summary
This summary is machine-generated.

ViBE-Seq is a novel method for sequencing the ends of viral RNA genomes, crucial for understanding viral replication. This technique provides high-resolution, accurate terminal sequence data for emerging viruses like Ebola.

Keywords:
5′ end RNA sequencingEbola virusMarburg virusViBE-Seqemerging virusesfilovirusterminal deoxynucleotidyl transferaseviral bona fide end sequencing

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Area of Science:

  • Virology
  • Molecular Biology
  • Genomics

Background:

  • Next-generation sequencing (NGS) often provides insufficient coverage of viral genome 5' and 3' ends.
  • Terminal sequences are critical for RNA virus transcription and replication.
  • Lack of terminal sequence data impedes the study of emerging and re-emerging viruses.

Purpose of the Study:

  • To develop a novel, high-resolution method for sequencing the 5' and 3' ends of viral RNA genomes.
  • To overcome limitations in current sequencing techniques for viral genome termini.
  • To facilitate comprehensive genomic analysis of emerging RNA viruses.

Main Methods:

  • Development of ViBE-Seq (Viral Bona Fide End Sequencing) protocol.
  • Application of ViBE-Seq to sequence 5' ends of viral RNA genomes and mRNAs.
  • Utilized Ebola virus and Marburg virus as model systems.
  • Tested ViBE-Seq with various RNA sources: virions, infected cells, and animal tissues.

Main Results:

  • ViBE-Seq achieves high-resolution sequencing of filoviral genome ends with high fidelity.
  • The method requires as little as 50 ng of total RNA for 5' end sequencing.
  • Demonstrated reliability and accuracy for sequencing RNA from diverse sources.
  • ViBE-Seq can identify terminal deoxynucleotidyl transferase activity in reverse transcriptases.

Conclusions:

  • ViBE-Seq is a robust and reliable technique for accurate 5' end sequencing of filovirus RNA.
  • This method enables access to complete viral genome sequences, essential for studying replication and transcription.
  • ViBE-Seq significantly advances the study of emerging and re-emerging RNA viruses.