Whole-genome sequencing of dog-specific assemblages C and D of Giardia duodenalis from single and pooled cysts indicates host-associated genes

Giardia duodenalis (syn. Giardia intestinalis or Giardia lamblia) infSAects over 280 million people each year and numerous animals. G. duodenalis can be subdivided into eight assemblages with different host specificity. Unculturable assemblages have so far resisted genome sequencing efforts. In this study, we isolated single and pooled cysts of assemblages C and D from dog faeces by FACS, and sequenced them using multiple displacement amplification and Illumina paired-end sequencing. The genomes of assemblages C and D were compared with genomes of assemblages A and B from humans and assemblage E from ruminants and pigs. The genomes obtained from the pooled cysts and from the single cysts were considered complete (>99 % marker genes observed) and the allelic sequence heterozygosity (ASH) values of assemblages C and D were 0.89 and 0.74 %, respectively. These ASH values were slightly higher than for assemblage B (>0.43 %) and much higher than for assemblages A and E, which ranged from 0.002 to 0.037 %. The flavohaemoglobin and 4Fe-4S binding domain family encoding genes involved in O(2) and NO detoxification were only present in assemblages A, B and E. Cathepsin B orthologs were found in all genomes. Six clades of cathepsin B orthologs contained one gene of each genome, while in three clades not all assemblages were represented. We conclude that whole-genome sequencing from a single Giardia cyst results in complete draft genomes, making the genomes of unculturable Giardia assemblages accessible. Observed differences between the genomes of assemblages C and D on one hand and the assemblages A, B and E on the other hand are possibly associated with host specificity.