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Related Concept Videos

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
Although all next-generation methods use different technologies, they all share a set of standard features....
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Retroviruses02:33

Retroviruses

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Retroviruses and retrotransposons both insert copies of their genetic elements into the genome of the host cell. Thus, the viral genes are passed on when the host genome is replicated or translated. A typical retroviral DNA sequence contains 3-4 genes that encode the different proteins required for its structural assembly and function as a molecular parasite. This DNA is transcribed into a single mRNA, which is very similar in structure to conventional mRNAs, i.e., it is capped at the 5’...
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Retrovirus Life Cycles01:10

Retrovirus Life Cycles

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Retroviruses have a single-stranded RNA genome that undergoes a special form of replication. Once the retrovirus has entered the host cell, an enzyme called reverse transcriptase synthesizes double-stranded DNA from the retroviral RNA genome. This DNA copy of the genome is then integrated into the host’s genome inside the nucleus via an enzyme called integrase. Consequently, the retroviral genome is transcribed into RNA whenever the host’s genome is transcribed, allowing the...
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Related Experiment Video

Updated: May 25, 2025

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells
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Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

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Next-Generation Sequencing Methods to Determine the Accuracy of Retroviral Reverse Transcriptases: Advantages and

Javier Martínez Del Río1, Luis Menéndez-Arias1

  • 1Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid, c/Nicolás Cabrera 1, 28049 Madrid, Spain.

Viruses
|February 26, 2025
PubMed
Summary

This review details how next-generation sequencing methods accurately measure reverse transcriptase (RT) error rates. These advanced techniques overcome limitations of older assays, improving our understanding of viral mutation and biotechnological applications.

Keywords:
cDNA synthesisfidelity of DNA synthesisnext-generation sequencingretrovirusreverse transcriptase

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Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
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Area of Science:

  • Molecular Biology
  • Virology
  • Biotechnology

Background:

  • Retroviruses exhibit high mutation rates due to error-prone viral reverse transcriptase (RT).
  • RTs are crucial tools in biotechnology for RNA detection and gene cloning.
  • Traditional methods for assessing RT fidelity are laborious and limited.

Purpose of the Study:

  • To review advancements in determining RNA-dependent DNA synthesis error rates using next-generation sequencing (NGS).
  • To compare various NGS-based methods for RT error rate determination.
  • To present and compare cDNA synthesis error rates from diverse studies.

Main Methods:

  • Summary of NGS-based methodologies including PRIMER IDs, REP-SEQ, ARC-SEQ, CIR-SEQ, SMRT-SEQ, and ROLL-SEQ.
  • Discussion of the advantages and limitations of each NGS method.
  • Compilation and comparison of reported cDNA synthesis error rates.

Main Results:

  • NGS technologies enable high-throughput determination of RT error rates.
  • Various NGS protocols offer different advantages for assessing reverse transcription fidelity.
  • Data from multiple studies provide a comparative overview of RT error rates.

Conclusions:

  • NGS methods have revolutionized the assessment of RT fidelity.
  • Accurate measurement of RT error rates is vital for understanding viral evolution and optimizing biotechnological applications.
  • Future methodological improvements are needed for precise mutation identification, including modified RNA bases.