Purpose: The apicoplast is a non-photosynthetic plastid with an independent genome found in most apicomplexan parasites including C. cayetanensis. Genetic markers identified in apicoplast genomes of other parasites have been useful for detection and traceback analysis. Distinct differences in the apicoplast genomes of C. cayetanensis could be potentially useful to design advanced rapid molecular methods for rapid detection, subtyping and geographical source attribution, being applicable to outbreak investigations and surveillance.
Methods: We sequenced the C. cayetanensis genomic DNA extracted from stool samples from patients with cyclosporiasis using the Illumina MiSeq platform. Bioinformatic workflow included tools like Mulan, Bowtie2, Geneious, CLC workbench, RATT, MAKER2, and NCBI Blast++. The draft genome was manually curated using NGS data from C. cayetanensis in our collections. Raw reads from many samples originated from Nepal, New York, Texas, Indonesia and elsewhere were mapped to the apicoplast reference. Multiple alignment of apicoplast genomes with the reference assembly was carried out using MEGA6.
Results: Comparative analysis using curated and annotated circular 34146 bp reference genome resulted in assembly 20+ SNPs and some small indels spanning the reference genome, and a 31 bp-sequence repeat at the terminal spacer region unique to some Nepalese samples. Phylogenetic analysis of apicoplast genomes from C. cayetanensis displayed a familiar pattern of tight clustering with Eimeria.
Significance: This is the first report of end-sequence curated and annotated complete reference genome for the C. cayetanensis apicoplast. SNPs and sequence-repeats from this study can be used as genetic markers for geographic differentiation applicable to traceback investigations.