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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 KA Van Rompay, Sharon Isern, Scott F Michael, Lark L Coffey, Nicholas J Loman, Kristian G Andersen
doi: https://doi.org/10.1101/383513
Nathan D Grubaugh
Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT;
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Karthik Gangavarapu
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA;
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Joshua Quick
Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK;
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Nathaniel L Matteson
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA;
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Jaqueline Goes De Jesus
Laboratory of Experimental Pathology, Goncalo Moniz Inst, Oswaldo Cruz Foundation, Salvador, Brazil;
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Bradley J Main
Department of Pathology, Microbiology and Immunology, University of California, Davis, CA 95616, USA;
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Amanda L Tan
Department of Biological Sciences, College of Arts and Sciences, Florida Gulf Coast University;
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Lauren M Paul
Department of Biological Sciences, College of Arts and Sciences, Florida Gulf Coast University;
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Doug E Brackney
Department of Environmental Sciences, The Connecticut Agricultural Experiment Station, New Haven, CT;
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Saran Grewal
Department of Environmental Health, San Diego County Vector Control Program, San Diego, CA;
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Nikos Gurfield
Department of Environmental Health, San Diego County Vector Control Program, San Diego, CA;
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Koen KA Van Rompay
California National Primate Research Center and Department of Pathology, Microbiology and Immunology
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Sharon Isern
Department of Biological Sciences, College of Arts and Sciences, Florida Gulf Coast University;
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Scott F Michael
Department of Biological Sciences, College of Arts and Sciences, Florida Gulf Coast University;
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Lark L Coffey
Department of Pathology, Microbiology and Immunology, University of California, Davis, CA 95616, USA;
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Nicholas J Loman
Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK;
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Kristian G Andersen
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA;
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  • For correspondence: kristian@andersen-lab.com
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Abstract

How viruses evolves within hosts can dictate infection outcomes; however, accurately reconstructing this process is challenging from samples with low virus concentrations. We evaluated our highly multiplexed amplicon approach - PrimalSeq - to enrich for viral RNA and demonstrate how virus concentration, sequencing coverage depth, primer mismatches, and technical replicates influence the accuracy of measuring intrahost virus diversity. Using this data, we developed an open source experimental protocol and computational tool (iVar; github.com/andersen-lab/ivar) for using PrimalSeq to measure intrahost virus diversity using Illumina and compared the results to Oxford Nanopore sequencing. Furthermore, we demonstrate the utility of our approach by measuring Zika and West Nile virus diversity from varied sample types, and show that the accumulation of genetic diversity is heavily influenced by experimental and biological systems.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted August 05, 2018.
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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 KA Van Rompay, Sharon Isern, Scott F Michael, Lark L Coffey, Nicholas J Loman, Kristian G Andersen
bioRxiv 383513; doi: https://doi.org/10.1101/383513
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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 KA Van Rompay, Sharon Isern, Scott F Michael, Lark L Coffey, Nicholas J Loman, Kristian G Andersen
bioRxiv 383513; doi: https://doi.org/10.1101/383513

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