Assembly Of Mitochondrial Genomes Research Articles

Sequencing and Reconstructing Helminth Mitochondrial Genomes Directly from Genomic Next-Generation Sequencing Data.

We present a detailed method for extraction of high-molecular weight genomic DNA suitable for numerous DNA sequencing applications, and a straightforward in silico approach for reconstructing novel mitochondrial (mt) genomes directly from total genomic DNA extracts derived from next-generation sequencing (NGS) data sets. The in silico post-sequencing pipeline described is fast, accurate, and highly efficient, with modest memory requirements that can be performed using a standard desktop computer. The approach is particularly effective for obtaining mitochondrial genomes for species with little or no mitochondrial sequence information currently available and overcomes many of the limitations of traditional strategies. The described methodologies are also applicable for metagenomics sequencing from mixed or pooled samples containing multiple species and subsequent specific assembly of specific mitochondrial genomes.

Assembly of Satsuma mandarin mitochondrial genome and identification of cytoplasmic male sterility–specific ORFs in a somatic cybrid of pummelo

Cytoplasmic male sterility (CMS) and seedlessness are highly desirable traits for citrus. However, a high-quality reference mitochondrial genome of citrus is unavailable, and the mechanisms of CMS remain to be elucidated in citrus. We previously generated a male sterile somatic cybrid of pummelo (G1+HBP) by fusing protoplasts from CMS ‘Guoqing No. 1’ Satsuma mandarin (G1) and fertile ‘Hirado Buntan’ pummelo (HBP). Here, we sequenced and assembled the mitochondrial genome of G1, G1+HBP, and HBP into circular molecules of 521.6 kb, 538.4 kb, and 518.3 kb, respectively. Mitochondrial genome of citrus encodes 35–36 proteins, three ribosomal RNAs, and 21 tRNAs. Comparative analysis indicated that nuclear genome and chloroplast genome of the cybrid are from HBP, whereas the mitochondrial genome sequences of the cybrid and G1 have 96.9% synteny, and that the cybrid has an extra recombinant mitochondrial DNA fragment of only 16.8 kb. Compared with the fertile HBP mitochondrial genome, 21 and 20 CMS-specific mitotype-specific sequences (MSSs), and 89 and 70 CMS-specific ORFs were identified in the cybrid and G1 mitochondrial genomes, respectively. Among them, 57 ORFs that co-transcribed with the same mitochondrial genes in the cybrid and G1 were identified as the CMS-specific ORFs. The assembly of mitochondrial genomes and the identification of the CMS-specific ORFs provide basis for determination of the CMS-associated gene and elucidation of CMS mechanisms in citrus.

Genomics and Virulence of Fonsecaea pugnacius, Agent of Disseminated Chromoblastomycosis.

Among agents of chromoblastomycosis, Fonsecaea pugnacius presents a unique type of infection because of its secondary neurotropic dissemination from a chronic cutaneous case in an immunocompetent patient. Neurotropism occurs with remarkable frequency in the fungal family Herpotrichiellaceae, possibly associated with the ability of some species to metabolize aromatic hydrocarbons. In an attempt to understand this new disease pattern, were conducted genomic analysis of Fonsecaea pugnacius (CBS 139214) performed with de novo assembly, gene prediction, annotation and mitochondrial genome assembly, supplemented with animal infection models performed with Tenebrio molitor in Mus musculus lineages BALB/c and C57BL/6. The genome draft of 34.8 Mb was assembled with a total of 12,217 protein-coding genes. Several proteins, enzymes and metabolic pathways related to extremotolerance and virulence were recognized. The enzyme profiles of black fungi involved in chromoblastomycosis and brain infection were analyzed with the Carbohydrate-Active Enzymes (CAZY) and peptidases database (MEROPS). The capacity of the fungus to survive inside Tenebrio molitor animal model was confirmed by histopathological analysis and by presence of melanin and hyphae in host tissue. Although F. pugnacius was isolated from brain in a murine model following intraperitoneal infection, cytokine levels were not statistically significant, indicating a profile of an opportunistic agent. A dual ecological ability can be concluded from presence of metabolic pathways for nutrient scavenging and extremotolerance, combined with a capacity to infect human hosts.

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

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.

New genomic resources for three exploited Mediterranean fishes

Extensive fishing has led to fish stock declines throughout the last decades. While clear stock identification is required for designing management schemes, stock delineation is problematic due to generally low levels of genetic structure in marine species. The development of genomic resources can help to solve this issue. Here, we present the first mitochondrial and nuclear draft genome assemblies of three economically important Mediterranean fishes, the white seabream, the striped red mullet, and the comber. The assemblies are between 613 and 785 Mbp long and contain between 27,222 and 32,375 predicted genes. They were used as references to map Restriction-site Associated DNA markers, which were developed with a single-digest approach. This approach provided between 15,710 and 21,101 Single Nucleotide Polymorphism markers per species. These genomic resources will allow uncovering subtle genetic structure, identifying stocks, assigning catches to populations and assessing connectivity. Furthermore, the annotated genomes will help to characterize adaptive divergence.

The Mitochondrial Genome Assembly of Fennel (Foeniculum vulgare) Reveals Two Different atp6 Gene Sequences in Cytoplasmic Male Sterile Accessions.

Cytoplasmic male sterility (CMS) has always aroused interest among researchers and breeders, being a valuable resource widely exploited not only to breed F1 hybrid varieties but also to investigate genes that control stamen and pollen development. With the aim of identifying candidate genes for CMS in fennel, we adopted an effective strategy relying on the comparison between mitochondrial genomes (mtDNA) of both fertile and sterile genotypes. mtDNA raw reads derived from a CMS genotype were assembled in a single molecule (296,483 bp), while a draft mtDNA assembly (166,124 nucleotides, 94 contigs) was performed using male fertile sample (MF) sequences. From their annotation and alignment, two atp6-like sequences were identified. atp6−, the putative mutant copy with a 300 bp truncation at the 5’-end, was found only in the mtDNA of CMS samples, while the wild type copy (atp6+) was detected only in the MF mtDNA. Further analyses (i.e., reads mapping and Sanger sequencing), revealed an atp6+ copy also in CMS samples, probably in the nuclear DNA. However, qPCRs performed on different tissues proved that, despite its availability, atp6+ is expressed only in MF samples, while apt6− mRNA was always detected in CMS individuals. In the light of these findings, the energy deficiency model could explain the pollen deficiency observed in male sterile flower. atp6− could represent a gene whose mRNA is translated into a not-fully functional protein leading to suboptimal ATP production that guarantees essential cellular processes but not a high energy demand process such as pollen development. Our study provides novel insights into the fennel mtDNA genome and its atp6 genes, and paves the way for further studies aimed at understanding their functional roles in the determination of male sterility.

Mitochondrial genomes of Columbicola feather lice are highly fragmented, indicating repeated evolution of minicircle-type genomes in parasitic lice.

Most animals have a conserved mitochondrial genome structure composed of a single chromosome. However, some organisms have their mitochondrial genes separated on several smaller circular or linear chromosomes. Highly fragmented circular chromosomes (“minicircles”) are especially prevalent in parasitic lice (Insecta: Phthiraptera), with 16 species known to have between nine and 20 mitochondrial minicircles per genome. All of these species belong to the same clade (mammalian lice), suggesting a single origin of drastic fragmentation. Nevertheless, other work indicates a lesser degree of fragmentation (2–3 chromosomes/genome) is present in some avian feather lice (Ischnocera: Philopteridae). In this study, we tested for minicircles in four species of the feather louse genus Columbicola (Philopteridae). Using whole genome shotgun sequence data, we applied three different bioinformatic approaches for assembling the Columbicola mitochondrial genome. We further confirmed these approaches by assembling the mitochondrial genome of Pediculus humanus from shotgun sequencing reads, a species known to have minicircles. Columbicola spp. genomes are highly fragmented into 15–17 minicircles between ∼1,100 and ∼3,100 bp in length, with 1–4 genes per minicircle. Subsequent annotation of the minicircles indicated that tRNA arrangements of minicircles varied substantially between species. These mitochondrial minicircles for species of Columbicola represent the first feather lice (Philopteridae) for which minicircles have been found in a full mitochondrial genome assembly. Combined with recent phylogenetic studies of parasitic lice, our results provide strong evidence that highly fragmented mitochondrial genomes, which are otherwise rare across the Tree of Life, evolved multiple times within parasitic lice.

Statistical Mitogenome Assembly with RepeaTs.

By using next-generation sequencing technologies, it is possible to quickly and inexpensively generate large numbers of relatively short reads from both the nuclear and mitochondrial DNA (mtDNA) contained in a biological sample. Unfortunately, assembling such whole-genome sequencing (WGS) data with standard de novo assemblers often fails to generate high-quality mitochondrial genome sequences due to the large difference in copy number (and hence sequencing depth) between the mitochondrial and nuclear genomes. Assembly of complete mitochondrial genome sequences is further complicated by the fact that many de novo assemblers are not designed for circular genomes and by the presence of repeats in the mitochondrial genomes of some species. In this article, we describe the Statistical Mitogenome Assembly with RepeaTs (SMART) pipeline for automated assembly of mitochondrial genomes from WGS data. SMART uses an efficient coverage-based filter to first select a subset of reads enriched in mtDNA sequences. Contigs produced by an initial assembly step are filtered using the Basic Local Alignment Search Tool searches against a comprehensive mitochondrial genome database and are used as "baits" for an alignment-based filter that produces the set of reads used in a second de novo assembly and scaffolding step. In the presence of repeats, the possible paths through the assembly graph are evaluated using a maximum likelihood model. Additionally, the assembly process is repeated for a user-specified number of times on resampled subsets of reads to select for annotation of the reconstructed sequences with highest bootstrap support. Experiments on WGS data sets from a variety of species show that the SMART pipeline produces complete circular mitochondrial genome sequences with a higher success rate than current state-of-the-art tools, particularly for low-coverage WGS data sets.

The Mitochondrial Genome of Eleusine indica and Characterization of Gene Content within Poaceae.

Plant mitochondrial (mt) genome assembly provides baseline data on size, structure, and gene content, but resolving the sequence of these large and complex organelle genomes remains challenging due to fragmentation, frequent recombination, and transfers of DNA from neighboring plastids. The mt genome for Eleusine indica (Poaceae: goosegrass) is comprehensibly analyzed here, providing key reference data for an economically significant invasive species that is also the maternal parent of the allotetraploid crop Finger millet (Eleusine coracana). The assembled E. indica genome contains 33 protein coding genes, 6 rRNA subunits, 24 tRNA, 8 large repetitive regions 15 kb of transposable elements across a total of 520,691 bp. Evidence of RNA editing and loss of rpl2, rpl5, rps14, rps11, sdh4, and sdh3 genes is evaluated in the context of an updated survey of mt genomic gene content across the grasses through an analysis of publicly available data. Hypothesized patterns of Poaceae mt gene loss are examined in a phylogenetic context to clarify timing, showing that rpl2 was transferred to the nucleus from the mitochondrion prior to the origin of the PACMAD clade.

Mitochondrial genome in Hypsizygus marmoreus and its evolution in Dikarya

BackgroundHypsizygus marmoreus, a high value commercialized edible mushroom is widely cultivated in East Asia, and has become one of the most popular edible mushrooms because of its rich nutritional and medicinal value. Mitochondria are vital organelles, and play various essential roles in eukaryotic cells.ResultsIn this study, we provide the Hypsizygus marmoreus mitochondrial (mt) genome assembly: the circular sequence is 102,752 bp in size and contains 15 putative protein-coding genes, 2 ribosomal RNAs subunits and 28 tRNAs. We compared the mt genomes of the 27 fungal species in the Pezizomycotina and Basidiomycotina subphyla, with the results revealing that H. marmoreus is a sister to Tricholoma matsutake and the phylogenetic distribution of this fungus based on the mt genome. Phylogenetic analysis shows that Ascomycetes mitochondria started to diverge earlier than that of Basidiomycetes and supported the robustness of the hyper metric tree. The fungal sequences are highly polymorphic and gene order varies significantly in the dikarya data set, suggesting a correlation between the gene order and divergence time in the fungi mt genome. To detect the mt genome variations in H. marmoreus, we analyzed the mtDNA sequences of 48 strains. The phylogeny and variation sited type statistics of H. marmoreus provide clear-cut evidence for the existence of four well-defined cultivations isolated lineages, suggesting female ancestor origin of H. marmoreus. Furthermore, variations on two loci were further identified to be molecular markers for distinguishing the subgroup containing 32 strains of other strains. Fifteen conserved protein-coding genes of mtDNAs were analyzed, with fourteen revealed to be under purifying selection in the examined fungal species, suggesting the rapid evolution was caused by positive selection of this gene.ConclusionsOur studies have provided new reference mt genomes and comparisons between species and intraspecies with other strains, and provided future perspectives for assessing diversity and origin of H. marmoreus.

Mitochondrial Genome Sequences of Diorhabda carinata and Diorhabda carinulata, Two Beetle Species Introduced to North America for Biological Control.

We announce the complete circularized mitochondrial genome assemblies of Diorhabda carinata and Diorhabda carinulata, beetle species introduced to North America for the biological control of invasive shrubs of the genus Tamarix L. (Tamaricaceae). The assemblies (16,232 and 16,298 bp, respectively) each comprise 13 protein-coding genes, 22 tRNAs, two rRNAs, and a noncoding region.

Whole mitochondrial genome of a Geoffroy’s Rousette, Rousettus amplexicaudatus (Pteropodidae)

The whole mitochondrial genome assembly of Rousettus amplexicaudatus belonging to the Pteropodidae found in the Philippines was sequenced and characterised. De novo sequence assembly yielded a 16,509bp sequence with an overall base composition of 32.43% A, 25.39% T, 26.17% C, and 14.02% G. The mitochondrial genome is composed 22 tRNA genes, 13 protein-coding genes, and two rRNA genes. Molecular phylogeny of the order Chiroptera based on the maximum likelihood and Bayesian inference trees supports the classification of R. amplexicaudatus to genus Rousettus and family Pteropodidae. Furthermore, both trees support the modern division of order Chiroptera into the Yinpterochiroptera and Yangochiroptera clades.

Trimitomics: An efficient pipeline for mitochondrial assembly from transcriptomic reads in nonmodel species.

Mitochondrial resources are of known utility to many fields of phylogenetic, population and molecular biology. Their combination of faster and slower-evolving regions and high copy number enables them to be used in many situations where other loci are unsuitable, with degraded samples and after recent speciation events.The advent of next-generation sequencing technologies (and notably the Illumina platform) has led to an explosion in the number of samples that can be studied at transcriptomic level, at relatively low cost. Here we describe a robust pipeline for the recovery of mitochondrial genomes from these RNA-sequencing resources. This pipeline can be used on sequencing of a variety of depths, and reliably recovers the protein coding and ribosomal gene complements of mitochondria from almost any transcriptomic sequencing experiment. The complete sequence of the mitochondrial genome can also be recovered when sequencing is performed in sufficient depth. We show the efficacy of our pipeline using data from eight nonmodel invertebrates of six disparate phyla. Interestingly, among our poriferan data, where microbiological symbionts are known empirically to make mitochondrial assembly difficult, this pipeline proved especially useful. Our pipeline will allow the recovery of mitochondrial data from a variety of previously sequenced samples, and add an additional angle of enquiry to future RNA-sequencing efforts, simplifying the process of mitochondrial genome assembly for even the most recalcitrant clades and adding these data to the scientific record for a range of future uses.

Analyses of mitochondrial genomes of the genus Ammopiptanthus provide new insights into the evolution of legume plants

The genus Ammopiptanthus is mainly distributed in the semi-arid regions of northwest China. It only contains two species: A. mongolicus and A. nanus. They exhibit similar morphological characteristics, but little is known about their genetic differences. We performed de novo sequencing and mitochondrial genome assembly and found that both species contain 36 protein-coding genes, 3 rRNA, and 16 tRNA genes. The comparison of A. mongolicus and A. nanus mitogenomes revealed a 6.5-kb region loss in A. nanus mitogenome, which was replaced by a 1.5-kb different sequence element. Other small variations between the two species include 25 SNPs, 4 deletions, and 2 insertions. Meta-analyses indicated that 34% of the A. mongolicus mitogenome was unique to Ammopiptanthus and has no similarity with any other known legume mitogenomes. These unique sequences are mainly distributed in intergenic regions. Gene gain and loss analysis showed that this mitogenome gained two genes, rps7 and sdh4, during evolution, with rps7 transferred from the chloroplast genome. Selective pressure analysis implied that the atp8 gene was under positive selection. Our study established high-quality reference mitogenomes for the genus Ammopiptanthus, revealed novel genetic differences between A. mongolicus and A. nanus, and provided new insights into the evolution of legume plants.

Mitochondrial and chloroplast genomes provide insights into the evolutionary origins of quinoa (Chenopodium quinoa Willd.)

Quinoa has recently gained international attention because of its nutritious seeds, prompting the expansion of its cultivation into new areas in which it was not originally selected as a crop. Improving quinoa production in these areas will benefit from the introduction of advantageous traits from free-living relatives that are native to these, or similar, environments. As part of an ongoing effort to characterize the primary and secondary germplasm pools for quinoa, we report the complete mitochondrial and chloroplast genome sequences of quinoa accession PI 614886 and the identification of sequence variants in additional accessions from quinoa and related species. This is the first reported mitochondrial genome assembly in the genus Chenopodium. Inference of phylogenetic relationships among Chenopodium species based on mitochondrial and chloroplast variants supports the hypotheses that 1) the A-genome ancestor was the cytoplasmic donor in the original tetraploidization event, and 2) highland and coastal quinoas were independently domesticated.

Assembly of Ancient Mitochondrial Genomes Without a Closely Related Reference Sequence.

Recent methodological advances have transformed the field of ancient DNA (aDNA). Basic bioinformatics skills are becoming essential requirements to process and analyze the sheer amounts of data generated by current aDNA studies and in biomedical research in general. This chapter is intended as a practical guide to the assembly of ancient mitochondrial genomes, directly from genomic DNA-derived next-generation sequencing (NGS) data, specifically in the absence of closely related reference genomes. In a hands-on tutorial suitable for readers with little to no prior bioinformatics experience, we reconstruct the mitochondrial genome of a woolly mammoth deposited ~45,000years ago. We introduce key software tools and outline general strategies for mitogenome assembly, including the critical quality assessment of assembly results without a reference genome.

Mind the gap! The mitochondrial control region and its power as a phylogenetic marker in echinoids

BackgroundIn Metazoa, mitochondrial markers are the most commonly used targets for inferring species-level molecular phylogenies due to their extremely low rate of recombination, maternal inheritance, ease of use and fast substitution rate in comparison to nuclear DNA. The mitochondrial control region (CR) is the main non-coding area of the mitochondrial genome and contains the mitochondrial origin of replication and transcription.While sequences of the cytochrome oxidase subunit 1 (COI) and 16S rRNA genes are the prime mitochondrial markers in phylogenetic studies, the highly variable CR is typically ignored and not targeted in such analyses. However, the higher substitution rate of the CR can be harnessed to infer the phylogeny of closely related species, and the use of a non-coding region alleviates biases resulting from both directional and purifying selection. Additionally, complete mitochondrial genome assemblies utilizing next generation sequencing (NGS) data often show exceptionally low coverage at specific regions, including the CR. This can only be resolved by targeted sequencing of this region.ResultsHere we provide novel sequence data for the echinoid mitochondrial control region in over 40 species across the echinoid phylogenetic tree. We demonstrate the advantages of directly targeting the CR and adjacent tRNAs to facilitate complementing low coverage NGS data from complete mitochondrial genome assemblies. Finally, we test the performance of this region as a phylogenetic marker both in the lab and in phylogenetic analyses, and demonstrate its superior performance over the other available mitochondrial markers in echinoids.ConclusionsOur target region of the mitochondrial CR (1) facilitates the first thorough investigation of this region across a wide range of echinoid taxa, (2) provides a tool for complementing missing data in NGS experiments, and (3) identifies the CR as a powerful, novel marker for phylogenetic inference in echinoids due to its high variability, lack of selection, and high compatibility across the entire class, outperforming conventional mitochondrial markers.

De novo assembly of mitochondrial genomes provides insights into genetic diversity and molecular evolution in wild boars and domestic pigs

Up to date, the scarcity of publicly available complete mitochondrial sequences for European wild pigs hampers deeper understanding about the genetic changes following domestication. Here, we have assembled 26 de novo mtDNA sequences of European wild boars from next generation sequencing (NGS) data and downloaded 174 complete mtDNA sequences to assess the genetic relationship, nucleotide diversity, and selection. The Bayesian consensus tree reveals the clear divergence between the European and Asian clade and a very small portion (10 out of 200 samples) of maternal introgression. The overall nucleotides diversities of the mtDNA sequences have been reduced following domestication. Interestingly, the selection efficiencies in both European and Asian domestic pigs are reduced, probably caused by changes in both selection constraints and maternal population size following domestication. This study suggests that de novo assembled mitogenomes can be a great boon to uncover the genetic turnover following domestication. Further investigation is warranted to include more samples from the ever-increasing amounts of NGS data to help us to better understand the process of domestication.

Genomic insights into the mitochondria of 11 eastern North American species of Cladonia

Cladonia is among the most species-rich genera of lichens globally. Species in this lineage, commonly referred to as reindeer lichens, are ecologically important in numerous regions worldwide. In some locations, species of Cladonia can comprise the dominant groundcover, and are a major food source for caribou and other mammals. Additionally, many species are known to produce substances with antimicrobial properties or other characteristics with potentially important medical applications. This exceptional morphological and ecological variation contrasts sharply with the limited molecular divergence often observed among species. As a new resource to facilitate ongoing and future studies of these important species, we analyse here the sequences of 11 Cladonia mitochondrial genomes, including new mitochondrial genome assemblies and annotations representing nine species: C. apodocarpa, C. caroliniana, C. furcata, C. leporina, C. petrophila, C. peziziformis, C. robbinsii, C. stipitata, and C. subtenuis. These 11 genomes varied in size, intron content, and complement of tRNAs. Genes annotated within these mitochondrial genomes include 15 protein-coding genes, the large and small ribosomal subunits (mtLSU and mtSSU), and 23–26 tRNAs. All Cladonia mitochondrial genomes contained atp9, an important energy transport gene that has been lost evolutionarily in some lichen mycobiont mitochondria. Using a concatenated alignment of five mitochondrial genes (nad2, nad4, cox1, cox2, and cox3), a Bayesian phylogeny of relationships among species was inferred and was consistent with previously published phylogenetic relationships, highlighting the utility of these regions in reconstructing phylogenetic history.

A hybrid reference-guided de novo assembly approach for generating Cyclospora mitochondrion genomes

Cyclospora cayetanensis is a coccidian parasite associated with large and complex foodborne outbreaks worldwide. Linking samples from cyclosporiasis patients during foodborne outbreaks with suspected contaminated food sources, using conventional epidemiological methods, has been a persistent challenge. To address this issue, development of new methods based on potential genomically-derived markers for strain-level identification has been a priority for the food safety research community. The absence of reference genomes to identify nucleotide and structural variants with a high degree of confidence has limited the application of using sequencing data for source tracking during outbreak investigations. In this work, we determined the quality of a high resolution, curated, public mitochondrial genome assembly to be used as a reference genome by applying bioinformatic analyses. Using this reference genome, three new mitochondrial genome assemblies were built starting with metagenomic reads generated by sequencing DNA extracted from oocysts present in stool samples from cyclosporiasis patients. Nucleotide variants were identified in the new and other publicly available genomes in comparison with the mitochondrial reference genome. A consolidated workflow, presented here, to generate new mitochondrion genomes using our reference-guided de novo assembly approach could be useful in facilitating the generation of other mitochondrion sequences, and in their application for subtyping C. cayetanensis strains during foodborne outbreak investigations.