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08:57
Amplicon Sequencing using the Long-Read Sequencing Technologies
Published on: August 29, 2025
525
An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar
Nathan D Grubaugh1,2, Karthik Gangavarapu3, Joshua Quick4
1Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, 92037, USA. nathan.grubaugh@yale.edu.
Genome Biology
|January 10, 2019
Summary
Understanding virus evolution within hosts is key to infection outcomes. Our PrimalSeq method accurately measures intrahost virus diversity, revealing factors influencing genetic variation in Zika and West Nile viruses.
Area of Science:
- Virology
- Genomics
- Bioinformatics
Background:
- Understanding virus evolution within hosts is crucial for predicting infection outcomes.
- Reconstructing intrahost virus evolution presents significant challenges.
- Accurate measurement of intrahost virus diversity is essential for studying viral adaptation.
Purpose of the Study:
- To evaluate the PrimalSeq multiplexed amplicon approach for measuring intrahost virus diversity.
- To identify factors influencing the accuracy of intrahost virus diversity measurements.
- To compare PrimalSeq performance with Illumina and Oxford Nanopore sequencing technologies.
Main Methods:
- Developed and validated the PrimalSeq experimental protocol and iVar computational tool.
- Assessed the impact of virus concentration, sequencing coverage, primer mismatches, and replicates on diversity measurements.
- Compared Illumina and Oxford Nanopore sequencing data generated using PrimalSeq.
- Quantified Zika and West Nile virus diversity in various sample types.
Main Results:
- PrimalSeq, coupled with iVar, accurately measures intrahost virus diversity.
- Virus concentration, sequencing coverage, primer mismatches, and replicates significantly influence measurement accuracy.
- Demonstrated PrimalSeq utility for analyzing Zika and West Nile virus diversity.
- Showed that experimental and biological systems impact the accumulation of viral genetic diversity.
Conclusions:
- PrimalSeq is a robust method for quantifying intrahost virus diversity.
- The accuracy of diversity measurements is sensitive to experimental parameters and biological context.
- This approach provides valuable insights into viral evolution dynamics within hosts.
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