Targeted next generation sequencing of Cyclospora cayetanensis mitochondrial genomes from seeded fresh produce and other seeded food samples

Cyclospora cayetanensis, a coccidian apicomplexan parasite, causes large outbreaks of foodborne diarrheal disease globally. Tracking the source of C. cayetanensis oocyst contamination in food items is essential to reduce, even prevent outbreaks. We previously showed that a genotyping method based on mitochondrial single nucleotide polymorphism (SNP) profiles had discriminatory power in classifying C. cayetanensis clinical isolates. In food specimens, low level contamination by oocysts and difficulties in DNA extraction present significant challenges in genotyping method development. Here, we report the development of a highly sensitive, custom-designed, targeted sequencing method based on the Illumina AmpliSeq platform; our method was capable of consistently generating near-complete mitochondrial genome sequences of C. cayetanensis from foods with low levels of contamination. To simulate environmentally observed contamination levels in foods, we seeded various food matrices, such as fresh produce and prepared dishes, with known quantities of oocysts, and isolated genomic DNA from washed food samples. Using the Ampliseq Targeted Sequencing method, we obtained near-complete mitochondrial genome sequences of C. cayetanensis from food samples seeded with as low as five to ten oocysts and used the data in downstream analysis. The flexibility of the AmpliSeq platform could potentially allow for more genomic targets to be added to achieve higher discriminatory power. This level of sensitivity in capturing high resolution genome data from contaminated food samples is a critical milestone towards the potential development of a comprehensive genotyping method for C. cayetanensis.