Genomic Variation in IbA10G2 and Other Patient-Derived Cryptosporidium hominis Subtypes

Abstract

In order to improve genotyping and epidemiological analysis of <i>Cryptosporidium</i> spp., genomic data need to be generated directly from a broad range of clinical specimens. Utilizing a robust method that we developed for the purification and generation of amplified target DNA, we present its application for the successful isolation and whole-genome sequencing of 14 different <i>Cryptosporidium hominis</i> patient specimens. Six isolates of subtype IbA10G2 were analyzed together with a single representative each of 8 other subtypes: IaA20R3, IaA23R3, IbA9G3, IbA13G3, IdA14, IeA11G3T3, IfA12G1, and IkA18G1. Parasite burden was measured over a range of more than 2 orders of magnitude for all samples, while the genomes were sequenced to mean depths of between 17× and 490× coverage. Sequence homology-based functional annotation identified several genes of interest, including the gene encoding <i>Cryptosporidium</i> oocyst wall protein 9 (COWP9), which presented a predicted loss-of-function mutation in all the sequence subtypes, except for that seen with IbA10G2, which has a sequence identical to the <i>Cryptosporidium parvum</i> reference Iowa II sequence. Furthermore, phylogenetic analysis showed that all the IbA10G2 genomes form a monophyletic clade in the <i>C. hominis</i> tree as expected and yet display some heterogeneity within the IbA10G2 subtype. The current report validates the aforementioned method for isolating and sequencing <i>Cryptosporidium</i> directly from clinical stool samples. In addition, the analysis demonstrates the potential in mining data generated from sequencing multiple whole genomes of <i>Cryptosporidium</i> from human fecal samples, while alluding to the potential for a higher degree of genotyping within <i>Cryptosporidium</i> epidemiology.