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Sequencing human rhinoviruses: direct sequencing versus plasmid cloning
Jodell E Linder1, Tatyana E Plachco2, Romina Libster3
1Department of Pediatrics, Vanderbilt University School of Medicine, Medical Center North, Nashville, TN 37232, United States.
Journal of Virological Methods
|October 7, 2014
Summary
Direct sequencing offers a cost-effective and faster alternative to plasmid-cloning for human rhinovirus (RV) strain sequencing. This method achieves high sequence identity, making it suitable for many research applications.
Area of Science:
- Virology
- Molecular Biology
- Genomics
Background:
- Human rhinoviruses (RV) are a leading cause of viral respiratory illnesses across all age groups.
- Investigating RV strains aids in understanding disease severity.
- Traditional sequencing preparation involves plasmid-cloning, which is time-consuming and costly.
Purpose of the Study:
- To compare and contrast direct Sanger sequencing with plasmid-cloning for RV strain sequencing.
- To evaluate the efficiency and accuracy of direct sequencing for RV VP4/VP2 region analysis.
Main Methods:
- PCR amplification of the RV VP4/VP2 region (approximately 500 base pairs).
- Sequencing using both plasmid-cloning and direct Sanger sequencing methods.
- Comparative analysis of sequence length, similarity, and success rates.
Main Results:
- Plasmid-cloning yielded slightly longer sequences (average 65 bp extra), with 86% initial similarity to direct sequencing.
- After trimming, cloned and direct sequences showed 99.7% identity.
- Both methods had similar overall success rates, with an average of 5% failure.
Conclusions:
- Direct Sanger sequencing is a viable and efficient alternative to plasmid-cloning for RV strain sequencing.
- Direct sequencing reduces costs and time, making it preferable for many research scenarios.
- The high sequence identity supports the use of direct sequencing for RV genetic studies.
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