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Microbiology JoVE (Open Access) Citable · DOI

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

DOI: 10.3791/54117-v
What you'll learn
  • ✓ Perform unbiased deep sequencing of RNA viruses from clinical samples
  • ✓ Deplete ribosomal and carrier RNA to enrich viral sequences
  • ✓ Construct cDNA libraries and prepare samples for next-generation sequencing
  • ✓ Interpret RNA virus sequencing results from complex clinical isolates
Protocol

This protocol describes a rapid and broadly applicable method for unbiased RNA-sequencing of viral samples from human clinical isolates.

Difficulty
advanced
Total time
~4–6 hours per sample (library prep) plus sequencing run time
Biosafety
BSL-2

Steps

1
Selectively deplete ribosomal and carrier RNA

Remove abundant non-viral RNA species from clinical viral samples using selective depletion methods to enrich for viral genetic material prior to sequencing.

▶ 01:06
2
Synthesize complementary DNA from viral RNA

Convert enriched viral RNA into cDNA using reverse transcriptase to generate a stable DNA template suitable for library construction and sequencing.

▶ 03:48
3
Construct DNA library for sequencing

Prepare cDNA library with appropriate adapters and fragments for next-generation sequencing platform compatibility.

▶ 05:41
4
Analyze RNA virus sequencing results

Interpret and evaluate sequencing output to identify and characterize viral sequences from clinical samples with unbiased coverage.

▶ 07:32
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