bioRxivEvolutionary Biology
An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar
N. D. Grubaugh, K. Gangavarapu, J. Quick, N. L. Matteson, J. Goes De Jesus, B. J. Main, A. L. Tan, L. M. Paul, D. E. Brackney, S. Grewal, N. Gurfield, K. K. Van Rompay, S. Isern, S. F. Michael, L. L. Coffey, N. J. Loman, K. G. Andersen
Abstract
How viruses evolve within hosts can dictate infection outcomes; however, reconstructing this process is challenging. We evaluated our multiplexed amplicon approach - PrimalSeq - to demonstrate how virus concentration, sequencing coverage, primer mismatches, and replicates influence the accuracy of measuring intrahost virus diversity. We developed an experimental protocol and computational tool (iVar) for using PrimalSeq to measure virus diversity using Illumina and compared the results to Oxford Nanopore sequencing. We demonstrate the utility of PrimalSeq by measuring Zika and West Nile virus diversity from varied sample types and show that the accumulation of genetic diversity is influenced by experimental and biological systems.
- assay: bulk RNA-seq
- research focus: variant calling
- research focus: differential expression
- approach: experimental assay
- approach: statistical model
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