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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
1Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
2Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06510, USA
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  • For correspondence: nathan.grubaugh@yale.edu gkarthik@scripps.edu
Karthik Gangavarapu
1Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
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  • For correspondence: nathan.grubaugh@yale.edu gkarthik@scripps.edu
Joshua Quick
3Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK
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Nathaniel L. Matteson
1Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
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Jaqueline Goes De Jesus
3Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK
4Laboratory of Experimental Pathology, Gonçalo Moniz Institute, Oswaldo Cruz Foundation, Salvador, Bahia, Brazil
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Bradley J Main
5Department of Pathology, Microbiology and Immunology, University of California, Davis, CA 95616, USA
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Amanda L Tan
6Department of Biological Sciences, College of Arts and Sciences, Florida Gulf Coast University, Fort Myers, FL 33965, USA
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Lauren M Paul
6Department of Biological Sciences, College of Arts and Sciences, Florida Gulf Coast University, Fort Myers, FL 33965, USA
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Doug E Brackney
7Department of Environmental Sciences, The Connecticut Agricultural Experiment Station, New Haven, CT 06504, USA
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Saran Grewal
8Department of Environmental Health, San Diego County Vector Control Program, San Diego, CA 92123, USA
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Nikos Gurfield
8Department of Environmental Health, San Diego County Vector Control Program, San Diego, CA 92123, USA
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Koen KA Van Rompay
9California National Primate Research Center and Department of Pathology, Microbiology and Immunology, University of California, Davis, CA 95616, USA
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Sharon Isern
6Department of Biological Sciences, College of Arts and Sciences, Florida Gulf Coast University, Fort Myers, FL 33965, USA
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Scott F Michael
6Department of Biological Sciences, College of Arts and Sciences, Florida Gulf Coast University, Fort Myers, FL 33965, USA
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Lark L Coffey
5Department of Pathology, Microbiology and Immunology, University of California, Davis, CA 95616, USA
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Nicholas J Loman
3Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK
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Kristian G Andersen
1Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
10Scripps Translational Science Institute, La Jolla, CA 92037, USA
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Abstract

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

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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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