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...

Descripción completa

Detalles Bibliográficos
Autores principales: Narvaez, Sonsiray Alvarez, Harrell, Telvin L., Oluwayinka, Olatunde, Sellers, Holly S., Khalid, Zubair, Hauck, Ruediger, Chowdhury, Erfan U., Conrad, Steven J.
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