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Related Experiment Video

Updated: Jun 9, 2025

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples
09:36

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A single-stranded based library preparation method for virome characterization.

Xichuan Zhai1, Alex Gobbi2,3, Witold Kot2

  • 1Section for Food Microbiology, Gut Health and Fermentation, Department of Food Science, University of Copenhagen, Rolighedsvej 26, Frederiksberg C, 1958, Denmark.

Microbiome
|October 25, 2024
PubMed
Summary
This summary is machine-generated.

A new ligation-based single-stranded library preparation method enhances gut virome studies by accurately quantifying diverse viral genomes. This efficient technique improves reproducibility and comparability in gut microbiome research.

Keywords:
DsDNA viromeGut viromeModified nucleotidesPhage mock communityRNA viromeSingle-stranded librarySsDNA virome

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Area of Science:

  • Microbiology
  • Virology
  • Bioinformatics

Background:

  • The gut virome, a key part of the gut microbiome, is vital for gut health.
  • Accurate analysis of the gut virome is hindered by diverse viral genome types and topologies.
  • Current sequencing library preparation methods introduce biases, limiting study reproducibility and comparability.

Purpose of the Study:

  • To develop and validate a universal and standardized library preparation method for gut virome sequencing.
  • To overcome biases associated with existing library preparation techniques.
  • To improve the accuracy, reproducibility, and comparability of gut virome studies.

Main Methods:

  • Repurposing and optimizing a ligation-based single-stranded library preparation method (SSLR).
  • Evaluating SSLR efficiency for quantifying viral DNA genomes (dsDNA and ssDNA).
  • Comparing SSLR performance against existing methods like Nextera, xGen, and MDA+Nextera.

Main Results:

  • The SSLR method demonstrated exceptional efficiency in quantifying viral DNA genomes.
  • SSLR outperformed other methods in minimal amplification bias, coverage evenness, and genome assembly integrity.
  • SSLR enables simultaneous preparation of DNA and RNA viral libraries and captures highly modified phage genomes.

Conclusions:

  • A fast, simple, and efficient ligation-based single-stranded DNA library preparation method for gut virome studies has been developed.
  • This method is compatible with Illumina sequencing and offers a rapid 3-hour preparation time.
  • The SSLR method has the potential to significantly enhance the comparability and reproducibility of gut virome research.