title: Comparison of Illumina de novo assembled and Sanger sequenced viral genomes: A case study for RNA viruses recovered from the plant pathogenic fungus Sclerotinia sclerotiorum.
abstract: The advent of 'next generation sequencing' (NGS) technologies has led to the discovery of many novel mycoviruses, the majority of which are sufficiently different from previously sequenced viruses that there is no appropriate reference sequence on which to base the sequence assembly. Although many new genome sequences are generated by NGS, confirmation of the sequence by Sanger sequencing is still essential for formal classification by the International Committee for the Taxonomy of Viruses (ICTV), although this is currently under review. To empirically test the validity of de novo assembled mycovirus genomes from dsRNA extracts, we compared the results from Illumina sequencing with those from random cloning plus targeted PCR coupled with Sanger sequencing for viruses from five Sclerotinia sclerotiorum isolates. Through Sanger sequencing we detected nine viral genomes while through Illumina sequencing we detected the same nine viruses plus one additional virus from the same samples. Critically, the Illumina derived sequences share >99.3 % identity to those obtained by cloning and Sanger sequencing. Although, there is scope for errors in de novo assembled viral genomes, our results demonstrate that by maximising the proportion of viral sequence in the data and using sufficiently rigorous quality controls, it is possible to generate de novo genome sequences of comparable accuracy from Illumina sequencing to those obtained by Sanger sequencing.
authors.affiliation: School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand; Faculty of Sciences, Damietta University, Damietta, Egypt.
authors.forename: Mahmoud E
authors.initials: ME
authors.lastname: Khalifa
authors.affiliation: Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand; Structural Biology Research Unit, Department of Clinical Laboratory Sciences, University of Cape Town, Rondebosch, 7701 Cape Town, South Africa; Department of Plant Pathology and Emerging Pathogens Institute, University of Florida, Gainesville, Florida, FL 32611, USA.
authors.forename: Arvind
authors.initials: A
authors.lastname: Varsani
authors.affiliation: Institute of Natural and Mathematical Sciences, Massey University, Auckland, New Zealand.
authors.forename: Austen R D
authors.initials: ARD
authors.lastname: Ganley
authors.affiliation: School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand. Electronic address: m.pearson@auckland.ac.nz.
authors.forename: Michael N
authors.initials: MN
authors.lastname: Pearson
mesh_terms: D001203:Ascomycota / Q000821:virology; D019295:Computational Biology; D000067148:Fungal Viruses / Q000235:genetics; D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D010944:Plants / Q000382:microbiology; D012328:RNA Viruses / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods
keywords: De novo viral genome assembly; Illumina sequencing; Mycovirus; Next generation sequencing; Virus detection