title: A robust and cost-effective approach to sequence and analyze complete genomes of small RNA viruses.
abstract: Next-generation sequencing (NGS) allows ultra-deep sequencing of nucleic acids. The use of sequence-independent amplification of viral nucleic acids without utilization of target-specific primers provides advantages over traditional sequencing methods and allows detection of unsuspected variants and co-infecting agents. However, NGS is not widely used for small RNA viruses because of incorrectly perceived cost estimates and inefficient utilization of freely available bioinformatics tools.
  
  
  
  In this study, we have utilized NGS-based random sequencing of total RNA combined with barcode multiplexing of libraries to quickly, effectively and simultaneously characterize the genomic sequences of multiple avian paramyxoviruses. Thirty libraries were prepared from diagnostic samples amplified in allantoic fluids and their total RNAs were sequenced in a single flow cell on an Illumina MiSeq instrument. After digital normalization, data were assembled using the MIRA assembler within a customized workflow on the Galaxy platform.
  
  
  
  Twenty-eight avian paramyxovirus 1 (APMV-1), one APMV-13, four avian influenza and two infectious bronchitis virus complete or nearly complete genome sequences were obtained from the single run. The 29 avian paramyxovirus genomes displayed 99.6% mean coverage based on bases with Phred quality scores of 30 or more. The lower and upper quartiles of sample median depth per position for those 29 samples were 2984 and 6894, respectively, indicating coverage across samples sufficient for deep variant analysis. Sample processing and library preparation took approximately 25-30 h, the sequencing run took 39 h, and processing through the Galaxy workflow took approximately 2-3 h. The cost of all steps, excluding labor, was estimated to be 106 USD per sample.
  
  
  
  This work describes an efficient multiplexing NGS approach, a detailed analysis workflow, and customized tools for the characterization of the genomes of RNA viruses. The combination of multiplexing NGS technology with the Galaxy workflow platform resulted in a fast, user-friendly, and cost-efficient protocol for the simultaneous characterization of multiple full-length viral genomes. Twenty-nine full-length or near-full-length APMV genomes with a high median depth were successfully sequenced out of 30 samples. The applied de novo assembly approach also allowed identification of mixed viral populations in some of the samples.
authors.affiliation: Exotic and Emerging Avian Viral Diseases Research Unit, Southeast Poultry Research Laboratory, US National Poultry Research Center, Agricultural Research Service, USDA, 934 College Station Road, Athens, GA, 30605, USA.
authors.forename: Kiril M
authors.initials: KM
authors.lastname: Dimitrov
authors.affiliation: Exotic and Emerging Avian Viral Diseases Research Unit, Southeast Poultry Research Laboratory, US National Poultry Research Center, Agricultural Research Service, USDA, 934 College Station Road, Athens, GA, 30605, USA.
authors.forename: Poonam
authors.initials: P
authors.lastname: Sharma
authors.affiliation: BASE2BIO, 1945 Arlington Drive, Oshkosh, WI, 54904, USA.
authors.forename: Jeremy D
authors.initials: JD
authors.lastname: Volkening
authors.affiliation: Exotic and Emerging Avian Viral Diseases Research Unit, Southeast Poultry Research Laboratory, US National Poultry Research Center, Agricultural Research Service, USDA, 934 College Station Road, Athens, GA, 30605, USA.|National Scientific Center Institute of Experimental and Clinical Veterinary Medicine, 83 Pushkinskaya Street, Kharkiv, 61023, Ukraine.
authors.forename: Iryna V
authors.initials: IV
authors.lastname: Goraichuk
authors.affiliation: Quality Operations Laboratory (QOL), University of Veterinary and Animal Sciences, Syed Abdul Qadir Jilani Road, Lahore, 54000, Pakistan.|Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Syed Abdul Qadir Jilani Road, Lahore, 54000, Pakistan.
authors.forename: Abdul
authors.initials: A
authors.lastname: Wajid
authors.affiliation: Quality Operations Laboratory (QOL), University of Veterinary and Animal Sciences, Syed Abdul Qadir Jilani Road, Lahore, 54000, Pakistan.
authors.forename: Shafqat Fatima
authors.initials: SF
authors.lastname: Rehmani
authors.affiliation: Quality Operations Laboratory (QOL), University of Veterinary and Animal Sciences, Syed Abdul Qadir Jilani Road, Lahore, 54000, Pakistan.
authors.forename: Asma
authors.initials: A
authors.lastname: Basharat
authors.affiliation: Regional Laboratory for Animal Influenza and other Transboundary Animal Diseases, National Veterinary Research Institute, PMB01, Vom, 930010, Plateau State, Nigeria.
authors.forename: Ismaila
authors.initials: I
authors.lastname: Shittu
authors.affiliation: Regional Laboratory for Animal Influenza and other Transboundary Animal Diseases, National Veterinary Research Institute, PMB01, Vom, 930010, Plateau State, Nigeria.
authors.forename: Tony M
authors.initials: TM
authors.lastname: Joannis
authors.affiliation: Exotic and Emerging Avian Viral Diseases Research Unit, Southeast Poultry Research Laboratory, US National Poultry Research Center, Agricultural Research Service, USDA, 934 College Station Road, Athens, GA, 30605, USA.
authors.forename: Patti J
authors.initials: PJ
authors.lastname: Miller
authors.affiliation: Exotic and Emerging Avian Viral Diseases Research Unit, Southeast Poultry Research Laboratory, US National Poultry Research Center, Agricultural Research Service, USDA, 934 College Station Road, Athens, GA, 30605, USA. Claudio.Afonso@ars.usda.gov.
authors.forename: Claudio L
authors.initials: CL
authors.lastname: Afonso
mesh_terms: D000818:Animals; D001717:Birds; D019295:Computational Biology / Q000191:economics / Q000379:methods; D003362:Cost-Benefit Analysis; D023281:Genomics / Q000191:economics* / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000379:methods; D012328:RNA Viruses / Q000235:genetics*; D014773:Virology / Q000191:economics* / Q000379:methods*
keywords: Avian paramyxovirus; Complete genomes; De novo assembly; Galaxy; Mixed infection; Multiplexing; Newcastle disease virus; Next-generation sequencing