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Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
The Complete Mitochondrial Genome of the Foodborne Parasitic Pathogen Cyclospora cayetanensis
Hediye Nese Cinar1, Gopal Gopinath1, Karen Jarvis1
1Food and Drug Administration, Center for Food Safety and Nutrition, Office of Applied Research, Division of Virulence Assessment, Laurel, Maryland, United States of America.
Abstract:
Cyclospora cayetanensis is a human-specific coccidian parasite responsible for several food and water-related outbreaks around the world, including the most recent ones involving over 900 persons in 2013 and 2014 outbreaks in the USA. Multicopy organellar DNA such as mitochondrion genomes have been particularly informative for detection and genetic traceback analysis in other parasites. We sequenced the C. cayetanensis genomic DNA obtained from stool samples from patients infected with Cyclospora in Nepal using the Illumina MiSeq platform. By bioinformatically filtering out the metagenomic reads of non-coccidian origin sequences and concentrating the reads by targeted alignment, we were able to obtain contigs containing Eimeria-like mitochondrial, apicoplastic and some chromosomal genomic fragments. A mitochondrial genomic sequence was assembled and confirmed by cloning and sequencing targeted PCR products amplified from Cyclospora DNA using primers based on our draft assembly sequence. The results show that the C. cayetanensis mitochondrion genome is 6274 bp in length, with 33% GC content, and likely exists in concatemeric arrays as in Eimeria mitochondrial genomes. Phylogenetic analysis of the C. cayetanensis mitochondrial genome places this organism in a tight cluster with Eimeria species. The mitochondrial genome of C. cayetanensis contains three protein coding genes, cytochrome (cytb), cytochrome C oxidase subunit 1 (cox1), and cytochrome C oxidase subunit 3 (cox3), in addition to 14 large subunit (LSU) and nine small subunit (SSU) fragmented rRNA genes.
Insights
Cyclospora cayetanensis mitochondrial genome sequencing reveals its genetic relationship to Eimeria species. This study provides crucial genetic data for detecting and tracing Cyclospora outbreaks.
Area of Science:
- Parasitology
- Genomics
- Molecular Biology
Background:
- Cyclospora cayetanensis causes food and waterborne outbreaks globally.
- Organellar DNA, like mitochondrial genomes, aids parasite detection and traceback.
Purpose of the Study:
- To sequence and analyze the mitochondrial genome of Cyclospora cayetanensis.
- To understand the phylogenetic position of C. cayetanensis using mitochondrial DNA.
Main Methods:
- Genomic DNA sequencing of C. cayetanensis from patient stool samples using Illumina MiSeq.
- Bioinformatic analysis to isolate parasite DNA and assemble mitochondrial genome.
- PCR amplification and sequencing for confirmation.
Main Results:
- Assembled a 6274 bp mitochondrial genome for C. cayetanensis with 33% GC content.
- Phylogenetic analysis placed C. cayetanensis closely with Eimeria species.
- Identified three protein-coding genes (cytb, cox1, cox3) and fragmented rRNA genes in the mitochondrial genome.
Conclusions:
- The C. cayetanensis mitochondrial genome provides valuable genetic markers for parasite identification.
- Mitochondrial genome data supports the close evolutionary relationship between Cyclospora and Eimeria.
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