|CHRZASTEK, KLAUDIA - ORISE FELLOW|
|LEE, DONGHUN - ORISE FELLOW|
|SHARMA, POONAM - ORISE FELLOW|
Submitted to: International Poultry Scientific Forum
Publication Type: Abstract Only
Publication Acceptance Date: 12/20/2016
Publication Date: N/A
Technical Abstract: Over the past decade, Next Generation Sequencing (NGS) technologies, also called deep sequencing, have continued to evolve, increasing capacity and lower the cost necessary for large genome sequencing projects. The one of the advantage of NGS platforms is the possibility to sequence the samples without any prior knowledge of what is within the sample. NGS can also be a valuable tool to monitor the changes in viral genomes, or for virus discovery, including diagnostics. However, the samples used in such studies are often available in limited quantities. Here, we present a simplified, Sequence Independent Single Primer Amplification (SISPA) in combination with NGS which provided the viral genome sequences of avian influenza virus (AIV), Newcastle disease virus (NDV) and Infections bronchitis virus (IBV), representing negative- and positive-sense single-stranded RNA viruses. The second goal was to assess feasibility of virus identification using SISPA – NGS and to get an estimate of sensitivity. The simplified SISPA protocol produces amplified product that when sequenced give high quality data that can be used for de novo assembly or reference mapping. This method allowed us successfully assembly sequences into full or near full length AIV, NDV, IBV viral genomes. The genome coverage ranged from 94.9% to 100% for all three viruses. Moreover, analysis of the sequence data demonstrated that from a single clinical sample, a mapping assembly representing 98.2 % of NDV and 99.7% of IBV genomes could be produced. The detection limit depended on viral load of samples. Based on our findings, a minimum of 105 virus particles per RT-PCR reaction is needed to obtain the full or nearly full genome sequence. This application has potential for rapid sequencing, diagnosis and virus discovery and because of the nonspecific nature of the amplification should be adaptable to the other RNA and DNA viruses.