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Next-generation fungal identification using target enrichment and Nanopore sequencing

BACKGROUND: Rapid and accurate pathogen identification is required for disease management. Compared to sequencing entire genomes, targeted sequencing may be used to direct sequencing resources to genes of interest for microbe identification and mitigate the low resolution that single-locus molecular...

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Autores principales: Yu, Pei-Ling, Fulton, James C., Hudson, Owen H., Huguet-Tapia, Jose C., Brawner, Jeremy T.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10544392/
https://www.ncbi.nlm.nih.gov/pubmed/37784013
http://dx.doi.org/10.1186/s12864-023-09691-w
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author Yu, Pei-Ling
Fulton, James C.
Hudson, Owen H.
Huguet-Tapia, Jose C.
Brawner, Jeremy T.
author_facet Yu, Pei-Ling
Fulton, James C.
Hudson, Owen H.
Huguet-Tapia, Jose C.
Brawner, Jeremy T.
author_sort Yu, Pei-Ling
collection PubMed
description BACKGROUND: Rapid and accurate pathogen identification is required for disease management. Compared to sequencing entire genomes, targeted sequencing may be used to direct sequencing resources to genes of interest for microbe identification and mitigate the low resolution that single-locus molecular identification provides. This work describes a broad-spectrum fungal identification tool developed to focus high-throughput Nanopore sequencing on genes commonly employed for disease diagnostics and phylogenetic inference. RESULTS: Orthologs of targeted genes were extracted from 386 reference genomes of fungal species spanning six phyla to identify homologous regions that were used to design the baits used for enrichment. To reduce the cost of producing probes without diminishing the phylogenetic power, DNA sequences were first clustered, and then consensus sequences within each cluster were identified to produce 26,000 probes that targeted 114 genes. To test the efficacy of our probes, we applied the technique to three species representing Ascomycota and Basidiomycota fungi. The efficiency of enrichment, quantified as mean target coverage over the mean genome-wide coverage, ranged from 200 to 300. Furthermore, enrichment of long reads increased the depth of coverage across the targeted genes and into non-coding flanking sequence. The assemblies generated from enriched samples provided well-resolved phylogenetic trees for taxonomic assignment and molecular identification. CONCLUSIONS: Our work provides data to support the utility of targeted Nanopore sequencing for fungal identification and provides a platform that may be extended for use with other phytopathogens. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-023-09691-w.
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spelling pubmed-105443922023-10-03 Next-generation fungal identification using target enrichment and Nanopore sequencing Yu, Pei-Ling Fulton, James C. Hudson, Owen H. Huguet-Tapia, Jose C. Brawner, Jeremy T. BMC Genomics Research BACKGROUND: Rapid and accurate pathogen identification is required for disease management. Compared to sequencing entire genomes, targeted sequencing may be used to direct sequencing resources to genes of interest for microbe identification and mitigate the low resolution that single-locus molecular identification provides. This work describes a broad-spectrum fungal identification tool developed to focus high-throughput Nanopore sequencing on genes commonly employed for disease diagnostics and phylogenetic inference. RESULTS: Orthologs of targeted genes were extracted from 386 reference genomes of fungal species spanning six phyla to identify homologous regions that were used to design the baits used for enrichment. To reduce the cost of producing probes without diminishing the phylogenetic power, DNA sequences were first clustered, and then consensus sequences within each cluster were identified to produce 26,000 probes that targeted 114 genes. To test the efficacy of our probes, we applied the technique to three species representing Ascomycota and Basidiomycota fungi. The efficiency of enrichment, quantified as mean target coverage over the mean genome-wide coverage, ranged from 200 to 300. Furthermore, enrichment of long reads increased the depth of coverage across the targeted genes and into non-coding flanking sequence. The assemblies generated from enriched samples provided well-resolved phylogenetic trees for taxonomic assignment and molecular identification. CONCLUSIONS: Our work provides data to support the utility of targeted Nanopore sequencing for fungal identification and provides a platform that may be extended for use with other phytopathogens. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-023-09691-w. BioMed Central 2023-10-02 /pmc/articles/PMC10544392/ /pubmed/37784013 http://dx.doi.org/10.1186/s12864-023-09691-w Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Yu, Pei-Ling
Fulton, James C.
Hudson, Owen H.
Huguet-Tapia, Jose C.
Brawner, Jeremy T.
Next-generation fungal identification using target enrichment and Nanopore sequencing
title Next-generation fungal identification using target enrichment and Nanopore sequencing
title_full Next-generation fungal identification using target enrichment and Nanopore sequencing
title_fullStr Next-generation fungal identification using target enrichment and Nanopore sequencing
title_full_unstemmed Next-generation fungal identification using target enrichment and Nanopore sequencing
title_short Next-generation fungal identification using target enrichment and Nanopore sequencing
title_sort next-generation fungal identification using target enrichment and nanopore sequencing
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10544392/
https://www.ncbi.nlm.nih.gov/pubmed/37784013
http://dx.doi.org/10.1186/s12864-023-09691-w
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