Updates and Comparative Analysis of the Mitochondrial Genomes of Paracoccidioides spp. Using Oxford Nanopore MinION Sequencing

Elizabeth Misas; Oscar M Gómez; Vanessa Botero; José F Muñoz; Marcus M Teixeira; Juan E Gallo; Oliver K Clay; Juan G McEwen

doi:10.3389/fmicb.2020.01751

Updates and Comparative Analysis of the Mitochondrial Genomes of Paracoccidioides spp. Using Oxford Nanopore MinION Sequencing

Título traducido de la contribución: Actualizaciones y análisis comparativo de los genomas mitocondriales de Paracoccidioides spp. mediante secuenciación Oxford Nanopore MinION

Elizabeth Misas, Oscar M Gómez, Vanessa Botero, José F Muñoz, Marcus M Teixeira, Juan E Gallo, Oliver K Clay, Juan G McEwen

Producción científica: Contribución a una revista › Artículo de Investigación › revisión exhaustiva

6 Citas (Scopus)

Resumen

El genoma mitocondrial del aislado de referencia Pb18 de Paracoccidioides brasiliensis fue secuenciado y descrito por primera vez por Cardoso et al. (2007), como un genoma circular con un tamaño de 71,3 kb y que contiene 14 genes codificadores de proteínas, 25 ARNt y las subunidades grande y pequeña del ARN ribosómico. Más tarde, en 2011, Desjardins et al. (2011) obtuvieron ensamblajes parciales de genomas mitocondriales de P. lutzii (Pb01), P. americana (Pb03) y P. brasiliensis sensu stricto (Pb18), aunque con un tamaño de solo 43,1 kb para Pb18. Los errores de secuenciación u otras limitaciones resultantes de tecnologías anteriores, y las ventajas de la NGS (lecturas cortas y largas), nos impulsaron a mejorar y actualizar las secuencias y anotaciones de ADNmt de dos especies de Paracoccidioides. Utilizando la secuenciación de lecturas de Oxford Nanopore e Illumina, generamos ensamblajes y anotaciones de genomas mitocondriales de novo completos de alta calidad para P. brasiliensis (Pb18) y P. americana (Pb03). Ambos ensamblajes se caracterizaron por una región espaciadora o intrón inusualmente larga (>50 kb) entre los exones 2 y 3 del gen nad5, que se conservó moderadamente entre Pb03 y Pb18 pero no de manera similar a otras secuencias informadas, excepto por un contig no asignado en el ensamblaje de 2011 de Pb03. La confiabilidad del inserto que faltaba en los ensamblajes de genomas de ADNmt anteriores se confirmó mediante la inspección de las secuencias de lecturas de Nanopore individuales que contenían ADN codificante de nad5, y experimentalmente mediante PCR para Pb18. Proponemos que el inserto puede ayudar a la iniciación de la replicación y puede ser escindido para producir una variante estructural más pequeña. Los genomas de mtADN actualizados deberían permitir análisis de SNP y otros análisis comparativos o evolutivos más precisos y diseños de cebadores/sondas. Un análisis comparativo del mtADN de 32 aislamientos de Paracoccidioides spp., utilizando los SNP de los genomas mitocondriales alineados, mostró agrupaciones dentro del complejo de especies brasiliensis que eran en gran medida consistentes con hallazgos previos de solo cinco loci mitocondriales.

Título traducido de la contribución	Actualizaciones y análisis comparativo de los genomas mitocondriales de Paracoccidioides spp. mediante secuenciación Oxford Nanopore MinION
Idioma original	Inglés estadounidense
Páginas (desde-hasta)	1751
Publicación	Frontiers in Microbiology
Volumen	11
DOI	https://doi.org/10.3389/fmicb.2020.01751
Estado	Publicada - 2020

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Microbiología (médica)

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@article{788eb31be2bc455cb076af4f68748afa,

title = "Updates and Comparative Analysis of the Mitochondrial Genomes of Paracoccidioides spp. Using Oxford Nanopore MinION Sequencing",

abstract = "The mitochondrial genome of the Paracoccidioides brasiliensis reference isolate Pb18 was first sequenced and described by Cardoso et al. (2007), as a circular genome with a size of 71.3 kb and containing 14 protein coding genes, 25 tRNAs, and the large and small subunits of ribosomal RNA. Later in 2011, Desjardins et al. (2011) obtained partial assemblies of mitochondrial genomes of P. lutzii (Pb01), P. americana (Pb03), and P. brasiliensis sensu stricto (Pb18), although with a size of only 43.1 kb for Pb18. Sequencing errors or other limitations resulting from earlier technologies, and the advantages of NGS (short and long reads), prompted us to improve and update the mtDNA sequences and annotations of two Paracoccidioides species. Using Oxford Nanopore and Illumina read sequencing, we generated high-quality complete de novo mitochondrial genome assemblies and annotations for P. brasiliensis (Pb18) and P. americana (Pb03). Both assemblies were characterized by an unusually long spacer or intron region (>50 kb) between exons 2 and 3 of the nad5 gene, which was moderately conserved between Pb03 and Pb18 but not similar to other reported sequences, except for an unassigned contig in the 2011 assembly of Pb03. The reliability of the insert missing from previous mtDNA genome assemblies was confirmed by inspection of the individual Nanopore read sequences containing nad5 coding DNA, and experimentally by PCR for Pb18. We propose that the insert may aid replication initiation and may be excised to produce a smaller structural variant. The updated mtDNA genomes should enable more accurate SNP and other comparative or evolutionary analyses and primer/probe designs. A comparative analysis of the mtDNA from 32 isolates of Paracoccidioides spp., using the SNPs of the aligned mitochondrial genomes, showed groupings within the brasiliensis species complex that were largely consistent with previous findings from only five mitochondrial loci.",

author = "Elizabeth Misas and G{\'o}mez, {Oscar M} and Vanessa Botero and Mu{\~n}oz, {Jos{\'e} F} and Teixeira, {Marcus M} and Gallo, {Juan E} and Clay, {Oliver K} and McEwen, {Juan G}",

note = "Copyright {\textcopyright} 2020 Misas, G{\'o}mez, Botero, Mu{\~n}oz, Teixeira, Gallo, Clay and McEwen.",

year = "2020",

doi = "10.3389/fmicb.2020.01751",

language = "English (US)",

volume = "11",

pages = "1751",

journal = "Frontiers in Microbiology",

issn = "1664-302X",

publisher = "Frontiers in Bioscience",

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TY - JOUR

T1 - Updates and Comparative Analysis of the Mitochondrial Genomes of Paracoccidioides spp. Using Oxford Nanopore MinION Sequencing

AU - Misas, Elizabeth

AU - Gómez, Oscar M

AU - Botero, Vanessa

AU - Muñoz, José F

AU - Teixeira, Marcus M

AU - Gallo, Juan E

AU - Clay, Oliver K

AU - McEwen, Juan G

PY - 2020

Y1 - 2020

N2 - The mitochondrial genome of the Paracoccidioides brasiliensis reference isolate Pb18 was first sequenced and described by Cardoso et al. (2007), as a circular genome with a size of 71.3 kb and containing 14 protein coding genes, 25 tRNAs, and the large and small subunits of ribosomal RNA. Later in 2011, Desjardins et al. (2011) obtained partial assemblies of mitochondrial genomes of P. lutzii (Pb01), P. americana (Pb03), and P. brasiliensis sensu stricto (Pb18), although with a size of only 43.1 kb for Pb18. Sequencing errors or other limitations resulting from earlier technologies, and the advantages of NGS (short and long reads), prompted us to improve and update the mtDNA sequences and annotations of two Paracoccidioides species. Using Oxford Nanopore and Illumina read sequencing, we generated high-quality complete de novo mitochondrial genome assemblies and annotations for P. brasiliensis (Pb18) and P. americana (Pb03). Both assemblies were characterized by an unusually long spacer or intron region (>50 kb) between exons 2 and 3 of the nad5 gene, which was moderately conserved between Pb03 and Pb18 but not similar to other reported sequences, except for an unassigned contig in the 2011 assembly of Pb03. The reliability of the insert missing from previous mtDNA genome assemblies was confirmed by inspection of the individual Nanopore read sequences containing nad5 coding DNA, and experimentally by PCR for Pb18. We propose that the insert may aid replication initiation and may be excised to produce a smaller structural variant. The updated mtDNA genomes should enable more accurate SNP and other comparative or evolutionary analyses and primer/probe designs. A comparative analysis of the mtDNA from 32 isolates of Paracoccidioides spp., using the SNPs of the aligned mitochondrial genomes, showed groupings within the brasiliensis species complex that were largely consistent with previous findings from only five mitochondrial loci.

AB - The mitochondrial genome of the Paracoccidioides brasiliensis reference isolate Pb18 was first sequenced and described by Cardoso et al. (2007), as a circular genome with a size of 71.3 kb and containing 14 protein coding genes, 25 tRNAs, and the large and small subunits of ribosomal RNA. Later in 2011, Desjardins et al. (2011) obtained partial assemblies of mitochondrial genomes of P. lutzii (Pb01), P. americana (Pb03), and P. brasiliensis sensu stricto (Pb18), although with a size of only 43.1 kb for Pb18. Sequencing errors or other limitations resulting from earlier technologies, and the advantages of NGS (short and long reads), prompted us to improve and update the mtDNA sequences and annotations of two Paracoccidioides species. Using Oxford Nanopore and Illumina read sequencing, we generated high-quality complete de novo mitochondrial genome assemblies and annotations for P. brasiliensis (Pb18) and P. americana (Pb03). Both assemblies were characterized by an unusually long spacer or intron region (>50 kb) between exons 2 and 3 of the nad5 gene, which was moderately conserved between Pb03 and Pb18 but not similar to other reported sequences, except for an unassigned contig in the 2011 assembly of Pb03. The reliability of the insert missing from previous mtDNA genome assemblies was confirmed by inspection of the individual Nanopore read sequences containing nad5 coding DNA, and experimentally by PCR for Pb18. We propose that the insert may aid replication initiation and may be excised to produce a smaller structural variant. The updated mtDNA genomes should enable more accurate SNP and other comparative or evolutionary analyses and primer/probe designs. A comparative analysis of the mtDNA from 32 isolates of Paracoccidioides spp., using the SNPs of the aligned mitochondrial genomes, showed groupings within the brasiliensis species complex that were largely consistent with previous findings from only five mitochondrial loci.

U2 - 10.3389/fmicb.2020.01751

DO - 10.3389/fmicb.2020.01751

M3 - Research Article

C2 - 32849380

SN - 1664-302X

VL - 11

SP - 1751

JO - Frontiers in Microbiology

JF - Frontiers in Microbiology

ER -

Updates and Comparative Analysis of the Mitochondrial Genomes of Paracoccidioides spp. Using Oxford Nanopore MinION Sequencing

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