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Genome Biol. 2019 Jan 8;20(1):8. doi: 10.1186/s13059-018-1618-7.

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

Author information

1
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, 92037, USA. nathan.grubaugh@yale.edu.
2
Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, 06510, USA. nathan.grubaugh@yale.edu.
3
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, 92037, USA. gkarthik@scripps.edu.
4
Institute of Microbiology and Infection, University of Birmingham, Birmingham, B15 2TT, UK.
5
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, 92037, USA.
6
Laboratory of Experimental Pathology, Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Bahia, Brazil.
7
Department of Pathology, Microbiology and Immunology, University of California, Davis, CA, 95616, USA.
8
Department of Biological Sciences, College of Arts and Sciences, Florida Gulf Coast University, Fort Myers, FL, 33965, USA.
9
Department of Environmental Sciences, The Connecticut Agricultural Experiment Station, New Haven, CT, 06504, USA.
10
Department of Environmental Health, San Diego County Vector Control Program, San Diego, CA, 92123, USA.
11
California National Primate Research Center and Department of Pathology, Microbiology and Immunology, University of California, Davis, CA, 95616, USA.
12
Scripps Research Translational Institute, La Jolla, CA, 92037, USA.

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.

KEYWORDS:

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

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