Purification of Cyclospora cayetanensis oocysts obtained from human stool specimens for whole genome sequencing
Yvonne Qvarnstrom,
Yuping Wei-Pridgeon,
Erik Van Roey,
Subin Park,
Ganesh Srinivasamoorthy,
Fernanda S. Nascimento,
Delynn M. Moss,
Eldin Talundzic,
Michael J. Arrowood
Affiliations
Yvonne Qvarnstrom
Parasitic Disease Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention
Yuping Wei-Pridgeon
Parasitic Disease Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention
Erik Van Roey
Parasitic Disease Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention
Subin Park
Parasitic Disease Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention
Ganesh Srinivasamoorthy
SRA International
Fernanda S. Nascimento
Parasitic Disease Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention
Delynn M. Moss
Waterborne Disease Prevention Branch, Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Enteric and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention
Eldin Talundzic
Malaria Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention
Michael J. Arrowood
Waterborne Disease Prevention Branch, Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Enteric and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention
Abstract Background Cyclospora cayetanensis is a food-borne intestinal human parasite that causes outbreaks of diarrhea. There is a need for efficient laboratory methods for strain-level characterization to assist in outbreak investigations. By using next generation sequencing, genomic sequences can be obtained and compared to identify potential genotyping markers. However, there is no method available to propagate this parasite in the laboratory. Therefore, genomic DNA must be extracted from oocysts purified from human stool. The objective of this study was to apply optimized methods to purify C. cayetanensis oocysts and extract DNA in order to obtain high-quality whole genome sequences with minimum contamination of DNA from other organisms. Results Oocysts from 21 human stool specimens were separated from other stool components using discontinuous density gradient centrifugation and purified further by flow cytometry. Genomic DNA was used to construct Ovation Ultralow libraries for Illumina sequencing. MiSeq sequencing reads were taxonomically profiled for contamination, de novo assembled, and mapped to a draft genome available in GenBank to assess the quality of the resulting genomic sequences. Following all purification steps, the majority (81–99%) of sequencing reads were from C. cayetanensis. They could be assembled into draft genomes of around 45 MB in length with GC-content of 52%. Conclusions Density gradients performed in the presence of a detergent followed by flow cytometry sorting of oocysts yielded sufficient genomic DNA largely free from contamination and suitable for whole genome sequencing of C. cayetanensis. The methods described here will facilitate the accumulation of genomic sequences from various samples, which is a prerequisite for the development of typing tools to aid in outbreak investigations.
