A robust and cost-effective approach to sequence and analyze complete genomes of small RNA viruses

Authors: DIMITROV, KIRIL - CONSULTANT; SHARMA, POONAM - ORISE FELLOW; VOLKENING, JEREMY - BASE2BIO; GORAICHUK, IRYNA - CONSULTANT; WAJID, ABDUL - UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES; REHMANI, SHAFQAT FATIMA - UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES; ASMA, BASHARAT - UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES; SHITTU, ISMAILA - NATIONAL VETERINARY RESEARCH INSTITUTE; JOANNIS, TONY - NATIONAL VETERINARY RESEARCH INSTITUTE; MILLER, PATTI; AFONSO, CLAUDIO

Submitted to: Virology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/29/2017
Publication Date: 4/7/2017
Publication URL: https://handle.nal.usda.gov/10113/5723705
Citation: Dimitrov, K.M., Sharma, P., Volkening, J.D., Goraichuk, I.V., Wajid, A., Rehmani, S., Asma, B., Shittu, I., Joannis, T.M., Miller, P.J., Afonso, C.L. 2017. A robust and cost-effective approach to sequence and analyze complete genomes of small RNA viruses. Virology Journal. 14(2017):72. doi: 10.1186/s12985-017-0741-5.

Interpretive Summary: Newcastle Disease is a devastating disease of poultry caused by a negative stranded RNA virus. The virus naturally evolve into new genetic forms and occasionally cause disease when it infects chickens together with other RNA viruses. Effective detection of Newcastle disease and other contaminant viruses is important for effective control of the disease. Next-generation sequencing (NGS) allows ultra-deep sequencing of viruses. In this study, we have utilized NGS-based random sequencing of total RNA to quickly, effectively and simultaneously characterize the genomic sequences of multiple avian RNA viruses. Here describe the rapid characterization of the complete genomes of twenty-nine Newcastle disease viruses (NDV), one avian paramyxovirus 13, four avian influenza and two infectious bronchitis. This technology allowed the simultaneously identification of mixed viral populations in some of the samples.

Technical Abstract: Background: 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, the use of NGS for small RNA viruses is not widely used because of incorrectly perceived cost estimates and inefficient utilization of freely available bioinformatics tools. Methods: 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 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. Results: Twenty-nine Newcastle disease viruses (NDV), one avian paramyxovirus 13, four avian influenza and two infectious bronchitis viruses nearly complete or near complete viral genomes were obtained from one single run. The 29 NDV genomes displayed 99,6% mean coverage based on bases with Phred quality of scores of 30. The lower and upper quartiles of sample median depth per position for those 29 samples were 3077 and 6270, respectively indicating high coverage sufficient for variant analysis. Sample processing and library preparation took approximately 25-30 hours, the sequencing run took 39 hours, and processing through the Galaxy workflow took approximately 2-3 hours. The cost of all steps, excluding labor, was estimated to be 106 USD per sample. Conclusions: This work describes an efficient multiplexing NGS approach and analysis workflow for the characterization of the genomes of RNA viruses. The combination of multiplexing NGS technology with the Galaxy workflow platform enabled a user friendly quick turnaround time and cost-efficient methodology for simultaneous characterization of multiple full-length viral genomes. Twenty-nine full-length or near-full-length Newcastle Disease virus genomes with a high median depth were successfully sequenced out of 30 samples. The applied de novo approach allowed simultaneously identification of mixed viral populations in some of the samples.