An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar

Nathan D. Grubaugh, Karthik Gangavarapu, Joshua Quick, Nathaniel L. Matteson, Jaqueline Goes De Jesus, Bradley Main, Amanda L. Tan, Lauren M. Paul, Doug E. Brackney, Saran Grewal, Nikos Gurfield, Koen K.A. Van Rompay, Sharon Isern, Scott F. Michael, Lark L Schneider, Nicholas J. Loman, Kristian G. Andersen

Research output: Contribution to journalArticle

25 Scopus citations

Abstract

How viruses evolve within hosts can dictate infection outcomes; however, reconstructing this process is challenging. We evaluate 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 develop an experimental protocol and computational tool, iVar, for using PrimalSeq to measure virus diversity using Illumina and compare 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.

Original languageEnglish (US)
Article number8
JournalGenome Biology
Volume20
Issue number1
DOIs
StatePublished - Jan 8 2019

Keywords

  • Amplicon sequencing
  • Intrahost evolution
  • SNP calling
  • Viral sequencing
  • West Nile
  • Zika

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Genetics
  • Cell Biology

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    Grubaugh, N. D., Gangavarapu, K., Quick, J., Matteson, N. L., De Jesus, J. G., Main, B., Tan, A. L., Paul, L. M., Brackney, D. E., Grewal, S., Gurfield, N., Van Rompay, K. K. A., Isern, S., Michael, S. F., Schneider, L. L., Loman, N. J., & Andersen, K. G. (2019). An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar. Genome Biology, 20(1), [8]. https://doi.org/10.1186/s13059-018-1618-7