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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Endemic Poultry Viral Diseases Research » Research » Publications at this Location » Publication #419001
Research Project: Systems Biology Approaches to Develop Medical Countermeasures to Detect, Prevent, and Control Poultry Production Viral Diseases

Location: Endemic Poultry Viral Diseases Research

Title: Choosing the most suitable NGS technology to combine with a standardized viral enrichment protocol for obtaining complete avian orthoreovirus genomes from metagenomic samples

Author
item Alvarez Narvaez, Sonsiray
item HARRELL, TELVIN - Orise Fellow
item MOHAMED, ISLAM - Orise Fellow
item Mohanty, Sujit
item Conrad, Steven

Submitted to: Frontiers in Bioinformatics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/2025
Publication Date: 2/4/2025
Citation: Alvarez Narvaez, S., Harrell, T., Mohamed, I., Mohanty, S.K., Conrad, S.J. 2025. Choosing the most suitable NGS technology to combine with a standardized viral enrichment protocol for obtaining complete avian orthoreovirus genomes from metagenomic samples. Frontiers in Bioinformatics. 5:e1498921. https://doi.org/10.3389/fbinf.2025.1498921.
DOI: https://doi.org/10.3389/fbinf.2025.1498921

Interpretive Summary: In this paper we compare the costs and benefits of whole genome sequencing of avian reovirus strains using two different sequencing technologies. The two most common long- and short- read sequencing methods are compared for their ability to produce contiguous segments over the entire genome and for their ability to produce acceptable depth of sequencing (how many times each base pair is successfully identified). We also compared various methods of mapping and assembling the raw read data. We conclude that short-read sequencing is the most suitable technology when used with our previously-published with our ARV genome sample enrichment protocol.

Technical Abstract: Because viruses are obligate intracellular pathogens, the sequencing of their genomes results in metagenomic data of both host cells and virus. Virology researchers are constantly seeking new, cost-effective strategies and bioinformatic pipelines for the retrieval of complete viral genomes from these metagenomic samples. Avian orthoreoviruses (ARVs) pose a significant and growing threat to the poultry industry and frequently cause economic losses associated with disease in production birds. Currently available commercial vaccines are ineffective against new ARV variants and ARV outbreaks are increasing worldwide, requiring whole genome sequencing (WGS) to characterize strains that evade vaccines. In this study we use eight ARV clinical isolates to compare the performance of long-readand short-read sequencing technologies for the sequencing of ARV after our previously published viral genome enrichment protocol has been applied to the sample. Additionally, we compared three assembly methods to determine which provided the most complete and reliable whole genomes: De novo, reference-guided or hybrid. The results suggest that our ARV genome enrichment protocol causes some fragmentation of the viral RNA or cDNA that impacted the length of the long reads but not the short reads) and, as a result, caused a failure to produce complete genomes via de novo assembly. Overall, we observed that regardless of the sequencing technology, the best quality assemblies were generated by mapping quality-trimmed reads to a custom reference genome. The custom reference genomes were in turn constructed with the publicly available ARV genomic segments that shared the highest sequence similarity with the contigs from short-read de novo assemblies. Hence, we conclude that short-read sequencing is the most suitable technology to combine with our ARV genome enrichment protocol.