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Related Experiment Video

Updated: May 10, 2026

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'
08:31

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Published on: May 26, 2013

Sequencing viral genomes from a single isolated plaque.

Jessica Depew1, Bin Zhou, Jamison M McCorrison

  • 1Department of Genomic Medicine, The J. Craig Venter Institute (JCVI), 9704 Medical Center Drive, Rockville, MD 20850, USA.

Virology Journal
|June 8, 2013
PubMed
Summary

This study presents an optimized Sequence Independent Single Primer Amplification (SISPA) protocol for rapid whole genome sequencing of viruses. The method requires minimal viral material, enabling efficient de novo assembly and identification during outbreaks.

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Last Updated: May 10, 2026

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Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Published on: May 26, 2013

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Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems
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Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Published on: November 4, 2014

Area of Science:

  • Virology
  • Genomics
  • Molecular Biology

Background:

  • Whole genome sequencing of viruses and bacteriophages is often limited by the requirement for substantial amounts of genomic material.
  • Current methods necessitate large-scale viral stock production and purification, hindering rapid analysis.

Purpose of the Study:

  • To describe an optimized Sequence Independent Single Primer Amplification (SISPA) method for de novo whole genome sequencing of viruses.
  • To enable next-generation sequencing using minimal viral input, such as a single plaque or picogram quantities of DNA.

Main Methods:

  • An optimized SISPA protocol was applied to a single plaque of Influenza A virus.
  • The protocol's sensitivity was assessed using bacteriophage DNA, with input as low as 10 pg.
  • Amplified products were sequenced using 454 and Illumina HiSeq platforms, followed by mapping and de novo assembly.

Main Results:

  • Sequencing a single Influenza A plaque yielded a mapping assembly covering 100% of the genome with 3590-fold average coverage.
  • De novo assembly of Influenza A data produced contigs representing 96.5% of the genome with 30-fold average coverage.
  • Using 10 pg of bacteriophage DNA resulted in 99.9% genome coverage via mapping assembly and 98.1% via de novo assembly.

Conclusions:

  • The optimized SISPA protocol generates high-quality sequencing data suitable for de novo assembly.
  • This method significantly reduces the required viral genetic material, facilitating rapid virus identification during outbreaks.
  • The protocol is advantageous for sequencing viruses that are challenging to propagate in culture.