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Iodixanol density gradients as an effective phytoplasma enrichment approach to improve genome sequencing

Obtaining complete phytoplasma genomes is difficult due to the lack of a culture system for these bacteria. To improve genome assembly, a non-ionic, low- and iso-osmotic iodixanol (Optiprep™) density gradient centrifugation method was developed to enrich for phytoplasma cells and deplete plant host...

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Autores principales: Rodrigues Jardim, Bianca, Tran-Nguyen, Lucy T. T., Gambley, Cherie, Rodoni, Brendan, Constable, Fiona E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9411968/
https://www.ncbi.nlm.nih.gov/pubmed/36033837
http://dx.doi.org/10.3389/fmicb.2022.937648
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author Rodrigues Jardim, Bianca
Tran-Nguyen, Lucy T. T.
Gambley, Cherie
Rodoni, Brendan
Constable, Fiona E.
author_facet Rodrigues Jardim, Bianca
Tran-Nguyen, Lucy T. T.
Gambley, Cherie
Rodoni, Brendan
Constable, Fiona E.
author_sort Rodrigues Jardim, Bianca
collection PubMed
description Obtaining complete phytoplasma genomes is difficult due to the lack of a culture system for these bacteria. To improve genome assembly, a non-ionic, low- and iso-osmotic iodixanol (Optiprep™) density gradient centrifugation method was developed to enrich for phytoplasma cells and deplete plant host tissues prior to deoxyribonucleic acid (DNA) extraction and high-throughput sequencing (HTS). After density gradient enrichment, potato infected with a ‘Candidatus Phytoplasma australasia’-related strain showed a ∼14-fold increase in phytoplasma HTS reads, with a ∼1.7-fold decrease in host genomic reads compared to the DNA extracted from the same sample without density gradient centrifugation enrichment. Additionally, phytoplasma genome assemblies from libraries equalized to 5 million reads were, on average, ∼15,000 bp larger and more contiguous (N50 ∼14,800 bp larger) than assemblies from the DNA extracted from the infected potato without enrichment. The method was repeated on capsicum infected with Sweet Potato Little Leaf phytoplasma (‘Ca. Phytoplasma australasia’-related strain) with a lower phytoplasma titer than the potato. In capsicum, ∼threefold more phytoplasma reads and ∼twofold less host genomic reads were obtained, with the genome assembly size and N50 values from libraries equalized to 3.4 million reads ∼137,000 and ∼4,000 bp larger, respectively, compared to the DNA extracted from infected capsicum without enrichment. Phytoplasmas from potato and capsicum were both enriched at a density of 1.049–1.058 g/ml. Finally, we present two highly contiguous ‘Ca. Phytoplasma australasia’ phytoplasma reference genomes sequenced from naturally infected Solanaceae hosts in Australia. Obtaining high-quality phytoplasma genomes from naturally infected hosts will improve insights into phytoplasma taxonomy, which will improve their detection and disease management.
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spelling pubmed-94119682022-08-27 Iodixanol density gradients as an effective phytoplasma enrichment approach to improve genome sequencing Rodrigues Jardim, Bianca Tran-Nguyen, Lucy T. T. Gambley, Cherie Rodoni, Brendan Constable, Fiona E. Front Microbiol Microbiology Obtaining complete phytoplasma genomes is difficult due to the lack of a culture system for these bacteria. To improve genome assembly, a non-ionic, low- and iso-osmotic iodixanol (Optiprep™) density gradient centrifugation method was developed to enrich for phytoplasma cells and deplete plant host tissues prior to deoxyribonucleic acid (DNA) extraction and high-throughput sequencing (HTS). After density gradient enrichment, potato infected with a ‘Candidatus Phytoplasma australasia’-related strain showed a ∼14-fold increase in phytoplasma HTS reads, with a ∼1.7-fold decrease in host genomic reads compared to the DNA extracted from the same sample without density gradient centrifugation enrichment. Additionally, phytoplasma genome assemblies from libraries equalized to 5 million reads were, on average, ∼15,000 bp larger and more contiguous (N50 ∼14,800 bp larger) than assemblies from the DNA extracted from the infected potato without enrichment. The method was repeated on capsicum infected with Sweet Potato Little Leaf phytoplasma (‘Ca. Phytoplasma australasia’-related strain) with a lower phytoplasma titer than the potato. In capsicum, ∼threefold more phytoplasma reads and ∼twofold less host genomic reads were obtained, with the genome assembly size and N50 values from libraries equalized to 3.4 million reads ∼137,000 and ∼4,000 bp larger, respectively, compared to the DNA extracted from infected capsicum without enrichment. Phytoplasmas from potato and capsicum were both enriched at a density of 1.049–1.058 g/ml. Finally, we present two highly contiguous ‘Ca. Phytoplasma australasia’ phytoplasma reference genomes sequenced from naturally infected Solanaceae hosts in Australia. Obtaining high-quality phytoplasma genomes from naturally infected hosts will improve insights into phytoplasma taxonomy, which will improve their detection and disease management. Frontiers Media S.A. 2022-08-12 /pmc/articles/PMC9411968/ /pubmed/36033837 http://dx.doi.org/10.3389/fmicb.2022.937648 Text en Copyright © 2022 Rodrigues Jardim, Tran-Nguyen, Gambley, Rodoni and Constable. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Rodrigues Jardim, Bianca
Tran-Nguyen, Lucy T. T.
Gambley, Cherie
Rodoni, Brendan
Constable, Fiona E.
Iodixanol density gradients as an effective phytoplasma enrichment approach to improve genome sequencing
title Iodixanol density gradients as an effective phytoplasma enrichment approach to improve genome sequencing
title_full Iodixanol density gradients as an effective phytoplasma enrichment approach to improve genome sequencing
title_fullStr Iodixanol density gradients as an effective phytoplasma enrichment approach to improve genome sequencing
title_full_unstemmed Iodixanol density gradients as an effective phytoplasma enrichment approach to improve genome sequencing
title_short Iodixanol density gradients as an effective phytoplasma enrichment approach to improve genome sequencing
title_sort iodixanol density gradients as an effective phytoplasma enrichment approach to improve genome sequencing
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9411968/
https://www.ncbi.nlm.nih.gov/pubmed/36033837
http://dx.doi.org/10.3389/fmicb.2022.937648
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