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.

Updates in Paracoccidioides Biology and Genetic Advances in Fungus Manipulation

“…The first mitochondrial genome assemblies of P. brasiliensis have 71,334 bp [203], while that of P. americana (Pb03) has 75 kb [201]. A more recent assembly constructed using MinION sequencing indicated substantial differences in the genome sizes of P. brasiliensis (117,664 bp) and P. americana (112,887 bp) regarding the first assemblies [204]. All these genetic studies have contributed to a better understanding of the biology of the fungus, but advances still need to be accomplished in the basic genetics of the Paracoccidioides genus.…”

Molecular Tools for Detection and Identification of Paracoccidioides Species: Current Status and Future Perspectives

“…The GP43 gene has been discussed for use as a secondary barcode marker [ 90 , 156 ]. Perhaps a better answer lies in mitochondrial genes with larger copy numbers, increasing sensitivity in molecular assays, which could help circumvent the low fungal DNA content of samples [ 16 , 157 ]. Additionally, the average time between the discovery of a given molecular-based technique (e.g., PCR in 1985) and its application in the molecular diagnosis of PCM exceeds a decade (~15 years), revealing the slow pace of incorporating technology in the diagnosis of this important mycosis.…”

Molecular epidemiology of Paracoccidiodes spp. recovered from patients with paracoccidioidomycosis in a teaching hospital from Minas Gerais State of Brazil

“…[ 50 ], P . lutzii [ 58 , 67 ] and S1/ P . brasiliensis [ 11 , 55 ], while ED01 was characterized as P .…”

“…However the MS1 has already been defined as Paracoccidioides sp. [50], P. lutzii [58,67] and S1/P. brasiliensis [11,55], while ED01 was characterized as P. lutzii [11,58].…”