Choosing the most suitable NGS technology to combine with a standardized viral enrichment protocol for obtaining complete avian orthoreovirus genomes from metagenomic samples
Since viruses are obligate intracellular pathogens, sequencing their genomes results in metagenomic data from both the virus and the host. Virology researchers are constantly seeking new, cost-effective strategies and bioinformatic pipelines for the retrieval of complete viral genomes from these met...
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Frontiers Media S.A.
2025-02-01
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbinf.2025.1498921/full |
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| author | Sonsiray Álvarez-Narváez Sonsiray Álvarez-Narváez Telvin L. Harrell Islam Nour Sujit K. Mohanty Steven J. Conrad |
| author_facet | Sonsiray Álvarez-Narváez Sonsiray Álvarez-Narváez Telvin L. Harrell Islam Nour Sujit K. Mohanty Steven J. Conrad |
| author_sort | Sonsiray Álvarez-Narváez |
| collection | DOAJ |
| description | Since viruses are obligate intracellular pathogens, sequencing their genomes results in metagenomic data from both the virus and the host. 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. This study compares the effectiveness of long-read and short-read sequencing technologies for obtaining ARV complete genomes. We used eight clinical isolates of ARV, each previously processed using our published viral genome enrichment protocol. Additionally, we evaluate 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 caused some fragmentation of the viral 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. |
| format | Article |
| id | doaj-art-e56b9bf4dc1541b2b6ea3b57e7116f42 |
| institution | Kabale University |
| issn | 2673-7647 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioinformatics |
| spelling | doaj-art-e56b9bf4dc1541b2b6ea3b57e7116f422025-02-04T10:53:23ZengFrontiers Media S.A.Frontiers in Bioinformatics2673-76472025-02-01510.3389/fbinf.2025.14989211498921Choosing the most suitable NGS technology to combine with a standardized viral enrichment protocol for obtaining complete avian orthoreovirus genomes from metagenomic samplesSonsiray Álvarez-Narváez0Sonsiray Álvarez-Narváez1Telvin L. Harrell2Islam Nour3Sujit K. Mohanty4Steven J. Conrad5US National Poultry Research Center, United States Department of Agriculture, Agricultural Research Service, Athens, GA, United StatesDepartment of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, United StatesUS National Poultry Research Center, United States Department of Agriculture, Agricultural Research Service, Athens, GA, United StatesUS National Poultry Research Center, United States Department of Agriculture, Agricultural Research Service, Athens, GA, United StatesUS National Poultry Research Center, United States Department of Agriculture, Agricultural Research Service, Athens, GA, United StatesUS National Poultry Research Center, United States Department of Agriculture, Agricultural Research Service, Athens, GA, United StatesSince viruses are obligate intracellular pathogens, sequencing their genomes results in metagenomic data from both the virus and the host. 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. This study compares the effectiveness of long-read and short-read sequencing technologies for obtaining ARV complete genomes. We used eight clinical isolates of ARV, each previously processed using our published viral genome enrichment protocol. Additionally, we evaluate 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 caused some fragmentation of the viral 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.https://www.frontiersin.org/articles/10.3389/fbinf.2025.1498921/fullavian orthoreovirusARVwhole genome sequencingWGSshort-read sequencinglong-read sequencing |
| spellingShingle | Sonsiray Álvarez-Narváez Sonsiray Álvarez-Narváez Telvin L. Harrell Islam Nour Sujit K. Mohanty Steven J. Conrad 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 avian orthoreovirus ARV whole genome sequencing WGS short-read sequencing long-read sequencing |
| title | Choosing the most suitable NGS technology to combine with a standardized viral enrichment protocol for obtaining complete avian orthoreovirus genomes from metagenomic samples |
| title_full | Choosing the most suitable NGS technology to combine with a standardized viral enrichment protocol for obtaining complete avian orthoreovirus genomes from metagenomic samples |
| title_fullStr | Choosing the most suitable NGS technology to combine with a standardized viral enrichment protocol for obtaining complete avian orthoreovirus genomes from metagenomic samples |
| title_full_unstemmed | Choosing the most suitable NGS technology to combine with a standardized viral enrichment protocol for obtaining complete avian orthoreovirus genomes from metagenomic samples |
| title_short | Choosing the most suitable NGS technology to combine with a standardized viral enrichment protocol for obtaining complete avian orthoreovirus genomes from metagenomic samples |
| title_sort | choosing the most suitable ngs technology to combine with a standardized viral enrichment protocol for obtaining complete avian orthoreovirus genomes from metagenomic samples |
| topic | avian orthoreovirus ARV whole genome sequencing WGS short-read sequencing long-read sequencing |
| url | https://www.frontiersin.org/articles/10.3389/fbinf.2025.1498921/full |
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