Abstract
BackgroundCyclospora 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.ResultsOocysts 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%.ConclusionsDensity 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.
Highlights
Cyclospora cayetanensis is a food-borne intestinal human parasite that causes outbreaks of diarrhea
Discontinuous density gradient purification of oocysts The addition of Alconox to the gradient purification steps resulted in considerably less contamination (Fig. 1)
The addition of Alconox benefited the purification of oocysts from stool preserved in potassium dichromate, fixed in a zinc–polyvinyl alcohol (Zn–PVA)
Summary
Cyclospora cayetanensis is a food-borne intestinal human parasite that causes outbreaks of diarrhea. Genomic DNA must be extracted from oocysts purified from human stool. Generation sequencing (NGS) has recently been used to obtain draft assemblies of the genome of C. cayetanensis from two different geographic regions [13, 14] These studies were based on genomic sequences obtained from oocysts purified by density gradients and flow cytometry sorting. The focus of these publications was on the analysis of the genome sequence data; the descriptions of the laboratory methods to purify the oocysts and obtain genomic DNA were necessarily brief. The present study provides a detailed description of the laboratory methods involved in the genomic sequencing of C. cayetanensis We applied these methods to stool samples from different countries and U.S outbreaks, collected in three different stool preservatives or transport media, to ensure reproducibility
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