A hybrid sequencing and assembly strategy for generating culture free genomes.

is a pathogenic intestinal protozoan parasite of humans and many other animals. is found throughout the world, and infection is known to have adverse health consequences for human and other mammalian hosts. Yet, many aspects of the biology of this ubiquitous parasite remain unresolved. Whole genome sequencing and comparative genomics can provide insight into the biology of by helping to reveal traits that are shared by all assemblages or unique to an individual assemblage or strain. However, these types of analyses are currently hindered by the lack of available genomes, due, in part, to the difficulty in obtaining the genetic material needed to perform whole genome sequencing. In this study, a novel approach using a multistep cleaning procedure coupled with a hybrid sequencing and assembly strategy was assessed for use in producing high quality genomes directly from cysts obtained from feces of two naturally infected hosts, a cat and dog infected with assemblage A and D, respectively. Cysts were cleaned and concentrated using cesium chloride gradient centrifugation followed by immunomagnetic separation. Whole genome sequencing was performed using both Illumina MiSeq and Oxford Nanopore MinION platforms. A hybrid assembly strategy was found to produce higher quality genomes than assemblies from either platform alone. The hybrid genomes obtained from fecal isolates (cysts) in this study compare favorably for quality and completeness against reference genomes of from cultured isolates. The whole genome assembly for assemblage D is the most contiguous genome available for this assemblage and is an important reference genome for future comparative studies. The data presented here support a hybrid sequencing and assembly strategy as a suitable method to produce whole genome sequences from DNA obtained from cysts which can be used to produce novel reference genomes necessary to perform comparative genomics studies of this parasite.