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

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…

• FD
  Florida Department of Health
• GC
  Georgia Clinical and Translational Science Alliance
• NI
  National Institute for Health and Care Research
• SR
  Surgical Reconstruction and Microbiology Research Centre
• HH
  Hamilton Health Sciences Foundation
• NI
  National Institutes of Health

  Awards: P51OD011107, UL1TR001114, R01AI099210, 1R21AI129479-01, HHSN272201400048C, U19AI135995, 5T32AI007244-33
• CF
  Centers for Disease Control and Prevention

  Award: 1U01CK000516
• UO
  University of California, Davis

  Award: 1R21AI129479-01
• MR
  Medical Research Council

  Awards: MR/M501621/1, P51OD011107, MR/L015080/1, MC_PC_15100
• NI
  National Institute of Allergy and Infectious Diseases

  Awards: HHSN272201400048C, R01AI099210, R21AI137690, U19AI135995
• NC
  National Center for Advancing Translational Sciences

  Awards: UL1TR002550, UL1TR001114
• CN
  California National Primate Research Center

  Award: P51OD011107
• OO
  Office of Research Infrastructure Programs, National Institutes of Health

  Award: P51OD011107