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 J. 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. Coffey, Nicholas J. Loman, Kristian G. Andersen

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

140 Citations (Scopus)


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
Article number8
JournalGenome Biology
Issue number1
Publication statusPublished - 8 Jan 2019
Externally publishedYes

Bibliographical note

Funding Information:
NDG was supported by NIH training grant 5T32AI007244-33. JQ is supported by a grant from the NIHR Surgical Reconstruction and Microbiology Research Centre (SRMRC). KKAVR is supported by the Office of Research Infrastructure Programs/OD (P51OD011107) to CNPRC and NIH R21AI129479. SI and SFM are supported by NIH NIAID R01AI099210. LLC was supported by startup funds from the UC Davis Department of Pathology, Microbiology and Immunology and the Pacific Southwest Regional Center of Excellence for Vector-Borne Diseases funded by the U.S. Centers for Disease Control and Prevention (Cooperative Agreement 1U01CK000516). BJM was supported by Abt Associates and a consortium of vector control districts in California: Coachella Valley, Orange County, Greater Los Angeles County, San Gabriel Valley, West Valley, Kern, Butte County, Tulare, Sacramento-Yolo, Placer, and Turlock. The rhesus macaque studies were supported by NIH 1R21AI129479–01 & Supplement, California National Primate Research Center pilot research grant P51OD011107 and FDA HHS HHSF223201610542P. NJL is supported by a Medical Research Council Bioinformatics Fellowship as part of the Cloud Infrastructure for Microbial Bioinformatics (CLIMB) project. KGA is a Pew Biomedical Scholar, and is supported by NIH NCATS CTSA UL1TR001114, NIAID contract HHSN272201400048C, NIAID R21AI137690, NIAID U19AI135995, and The Ray Thomas Foundation.

Publisher Copyright:
© 2019 The Author(s).


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


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