Cargando…
Optimizing the Conditions for Whole-Genome Sequencing of Avian Reoviruses
Whole-genome sequencing (WGS) is becoming an essential tool to characterize the genomes of avian reovirus (ARV), a viral disease of economic significance to poultry producers. The current strategies and procedures used to obtain the complete genome sequences of ARV isolates are not cost-effective be...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10536876/ https://www.ncbi.nlm.nih.gov/pubmed/37766345 http://dx.doi.org/10.3390/v15091938 |
_version_ | 1785112971726290944 |
---|---|
author | Narvaez, Sonsiray Alvarez Harrell, Telvin L. Oluwayinka, Olatunde Sellers, Holly S. Khalid, Zubair Hauck, Ruediger Chowdhury, Erfan U. Conrad, Steven J. |
author_facet | Narvaez, Sonsiray Alvarez Harrell, Telvin L. Oluwayinka, Olatunde Sellers, Holly S. Khalid, Zubair Hauck, Ruediger Chowdhury, Erfan U. Conrad, Steven J. |
author_sort | Narvaez, Sonsiray Alvarez |
collection | PubMed |
description | Whole-genome sequencing (WGS) is becoming an essential tool to characterize the genomes of avian reovirus (ARV), a viral disease of economic significance to poultry producers. The current strategies and procedures used to obtain the complete genome sequences of ARV isolates are not cost-effective because most of the genetic material data resulting from next-generation sequencing belong to the host and cannot be used to assemble the viral genome. The purpose of this study was to develop a workflow to enrich the ARV genomic content in a sample before subjecting it to next-generation sequencing (NGS). Herein, we compare four different ARV purification and enrichment approaches at the virion, RNA and cDNA levels to determine which treatment or treatment combination would provide a higher proportion of ARV-specific reads after WGS. Seven ARV isolates were subjected to different combinations of virion purification via ultracentrifugation in sucrose density gradient or Capto Core 700 resin with or without a subsequent Benzonase treatment, followed by a chicken rRNA depletion step after RNA extraction and a final ARV cDNA amplification step using a single-primer amplification assay. Our results show that the combination of Capto Core 700 resin, Chicken rRNA depletion and cDNA amplification is the most cost-effective strategy to obtain ARV whole genomes after short-read sequencing. |
format | Online Article Text |
id | pubmed-10536876 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-105368762023-09-29 Optimizing the Conditions for Whole-Genome Sequencing of Avian Reoviruses Narvaez, Sonsiray Alvarez Harrell, Telvin L. Oluwayinka, Olatunde Sellers, Holly S. Khalid, Zubair Hauck, Ruediger Chowdhury, Erfan U. Conrad, Steven J. Viruses Article Whole-genome sequencing (WGS) is becoming an essential tool to characterize the genomes of avian reovirus (ARV), a viral disease of economic significance to poultry producers. The current strategies and procedures used to obtain the complete genome sequences of ARV isolates are not cost-effective because most of the genetic material data resulting from next-generation sequencing belong to the host and cannot be used to assemble the viral genome. The purpose of this study was to develop a workflow to enrich the ARV genomic content in a sample before subjecting it to next-generation sequencing (NGS). Herein, we compare four different ARV purification and enrichment approaches at the virion, RNA and cDNA levels to determine which treatment or treatment combination would provide a higher proportion of ARV-specific reads after WGS. Seven ARV isolates were subjected to different combinations of virion purification via ultracentrifugation in sucrose density gradient or Capto Core 700 resin with or without a subsequent Benzonase treatment, followed by a chicken rRNA depletion step after RNA extraction and a final ARV cDNA amplification step using a single-primer amplification assay. Our results show that the combination of Capto Core 700 resin, Chicken rRNA depletion and cDNA amplification is the most cost-effective strategy to obtain ARV whole genomes after short-read sequencing. MDPI 2023-09-16 /pmc/articles/PMC10536876/ /pubmed/37766345 http://dx.doi.org/10.3390/v15091938 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Narvaez, Sonsiray Alvarez Harrell, Telvin L. Oluwayinka, Olatunde Sellers, Holly S. Khalid, Zubair Hauck, Ruediger Chowdhury, Erfan U. Conrad, Steven J. Optimizing the Conditions for Whole-Genome Sequencing of Avian Reoviruses |
title | Optimizing the Conditions for Whole-Genome Sequencing of Avian Reoviruses |
title_full | Optimizing the Conditions for Whole-Genome Sequencing of Avian Reoviruses |
title_fullStr | Optimizing the Conditions for Whole-Genome Sequencing of Avian Reoviruses |
title_full_unstemmed | Optimizing the Conditions for Whole-Genome Sequencing of Avian Reoviruses |
title_short | Optimizing the Conditions for Whole-Genome Sequencing of Avian Reoviruses |
title_sort | optimizing the conditions for whole-genome sequencing of avian reoviruses |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10536876/ https://www.ncbi.nlm.nih.gov/pubmed/37766345 http://dx.doi.org/10.3390/v15091938 |
work_keys_str_mv | AT narvaezsonsirayalvarez optimizingtheconditionsforwholegenomesequencingofavianreoviruses AT harrelltelvinl optimizingtheconditionsforwholegenomesequencingofavianreoviruses AT oluwayinkaolatunde optimizingtheconditionsforwholegenomesequencingofavianreoviruses AT sellershollys optimizingtheconditionsforwholegenomesequencingofavianreoviruses AT khalidzubair optimizingtheconditionsforwholegenomesequencingofavianreoviruses AT hauckruediger optimizingtheconditionsforwholegenomesequencingofavianreoviruses AT chowdhuryerfanu optimizingtheconditionsforwholegenomesequencingofavianreoviruses AT conradstevenj optimizingtheconditionsforwholegenomesequencingofavianreoviruses |