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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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High-throughput Detection Method for Influenza Virus
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Improved influenza A whole-genome sequencing protocol.

Iryna V Goraichuk1, Jacquline Risalvato1, Mary Pantin-Jackwood1

  • 1Southeast Poultry Research Laboratory, U.S. National Poultry Research Center, Agriculture Research Service, U.S Department of Agriculture, Athens, GA, United States.

Frontiers in Cellular and Infection Microbiology
|December 13, 2024
PubMed
Summary

Optimizing whole-genome sequencing (WGS) for Influenza A virus enhances pathogen surveillance. This refined protocol improves read depth and efficiency for rapid outbreak response and broader pathogen monitoring.

Keywords:
IlluminaMinIONNGSRT-PCRWGSinfluenzananoporenext-generation sequencing

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Area of Science:

  • Virology
  • Genomics
  • Bioinformatics

Background:

  • Influenza A virus presents significant public health challenges due to rapid mutation and zoonotic potential.
  • Whole-genome sequencing (WGS) is essential for tracking and understanding influenza virus evolution.
  • Next-generation sequencing (NGS) platforms like Oxford Nanopore Technologies (ONT) and Illumina are vital tools for WGS.

Purpose of the Study:

  • To optimize the ONT Ligation Sequencing Influenza A Whole Genome protocol for improved sequencing quality and efficiency.
  • To evaluate the impact of alternative RT-PCR kits, primers, and purification methods on influenza A WGS.
  • To assess the potential for automation in high-throughput processing of influenza A WGS.

Main Methods:

  • Refinement of reverse transcription PCR (RT-PCR) kits and primer sets for targeted influenza A WGS.
  • Optimization of purification methods to enhance sequencing library preparation.
  • Evaluation of automated workflows for high-throughput sequencing applications.

Main Results:

  • Alternative RT-PCR kits and primers significantly improved read depth coverage across the influenza A genome compared to the original ONT protocol.
  • Reduced incidence of short, untargeted reads was observed with the optimized protocol.
  • Enhanced uniform coverage, particularly for challenging polymerase segments, was achieved, with improved 5' and 3' termini coverage.

Conclusions:

  • The optimized protocol enhances the quality and efficiency of targeted influenza A WGS.
  • This protocol is adaptable to various NGS platforms, aiding influenza adaptation studies and surveillance.
  • The protocol's adaptability extends to the sequencing of other pathogens, broadening its utility in infectious disease monitoring.