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


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          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.

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          The online version of this article (10.1186/s13059-018-1618-7) contains supplementary material, which is available to authorized users.

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          Most cited references69

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          Trimmomatic: a flexible trimmer for Illumina sequence data

          Motivation: Although many next-generation sequencing (NGS) read preprocessing tools already existed, we could not find any tool or combination of tools that met our requirements in terms of flexibility, correct handling of paired-end data and high performance. We have developed Trimmomatic as a more flexible and efficient preprocessing tool, which could correctly handle paired-end data. Results: The value of NGS read preprocessing is demonstrated for both reference-based and reference-free tasks. Trimmomatic is shown to produce output that is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested. Availability and implementation: Trimmomatic is licensed under GPL V3. It is cross-platform (Java 1.5+ required) and available at http://www.usadellab.org/cms/index.php?page=trimmomatic Contact: usadel@bio1.rwth-aachen.de Supplementary information: Supplementary data are available at Bioinformatics online.
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            The Sequence Alignment/Map format and SAMtools

            Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments. Availability: http://samtools.sourceforge.net Contact: rd@sanger.ac.uk
              • Record: found
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              Fast and accurate short read alignment with Burrows–Wheeler transform

              Motivation: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals. Results: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is ∼10–20× faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package. Availability: http://maq.sourceforge.net Contact: rd@sanger.ac.uk

                Author and article information

                Genome Biol
                Genome Biol
                Genome Biology
                BioMed Central (London )
                8 January 2019
                8 January 2019
                : 20
                : 8
                [1 ]ISNI 0000000122199231, GRID grid.214007.0, Department of Immunology and Microbiology, , The Scripps Research Institute, ; La Jolla, CA 92037 USA
                [2 ]ISNI 0000000419368710, GRID grid.47100.32, Department of Epidemiology of Microbial Diseases, , Yale School of Public Health, ; New Haven, CT 06510 USA
                [3 ]ISNI 0000 0004 1936 7486, GRID grid.6572.6, Institute of Microbiology and Infection, , University of Birmingham, ; Birmingham, B15 2TT UK
                [4 ]ISNI 0000 0001 0723 0931, GRID grid.418068.3, Laboratory of Experimental Pathology, Gonçalo Moniz Institute, , Oswaldo Cruz Foundation, ; Salvador, Bahia Brazil
                [5 ]ISNI 0000 0004 1936 9684, GRID grid.27860.3b, Department of Pathology, Microbiology and Immunology, , University of California, ; Davis, CA 95616 USA
                [6 ]ISNI 0000 0001 0647 2963, GRID grid.255962.f, Department of Biological Sciences, College of Arts and Sciences, , Florida Gulf Coast University, ; Fort Myers, FL 33965 USA
                [7 ]ISNI 0000 0000 8788 3977, GRID grid.421470.4, Department of Environmental Sciences, , The Connecticut Agricultural Experiment Station, ; New Haven, CT 06504 USA
                [8 ]Department of Environmental Health, San Diego County Vector Control Program, San Diego, CA 92123 USA
                [9 ]ISNI 0000 0004 1936 9684, GRID grid.27860.3b, California National Primate Research Center and Department of Pathology, Microbiology and Immunology, , University of California, ; Davis, CA 95616 USA
                [10 ]Scripps Research Translational Institute, La Jolla, CA 92037 USA
                Author information
                © The Author(s). 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                : 4 August 2018
                : 26 December 2018
                Funded by: FundRef http://dx.doi.org/10.13039/100000060, National Institute of Allergy and Infectious Diseases;
                Award ID: U19AI135995
                Award ID: R21AI137690
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100006108, National Center for Advancing Translational Sciences;
                Award ID: UL1TR002550
                Award Recipient :
                Custom metadata
                © The Author(s) 2019

                viral sequencing,amplicon sequencing,intrahost evolution,zika,west nile,snp calling
                viral sequencing, amplicon sequencing, intrahost evolution, zika, west nile, snp calling


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