Isolation and enrichment of Cryptosporidium DNA and verification of DNA purity for whole genome sequencing.

Guo Y1, Li N2, Lysén C3, Frace M4, Tang K4, Sammons S4, Roellig DM3, Feng Y5, Xiao L6.

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Abstract

Whole genome sequencing of Cryptosporidium spp. is hampered by difficulties in obtaining sufficient, highly pure genomic DNA from clinical specimens. In this study, we developed procedures for the isolation and enrichment of Cryptosporidium genomic DNA from fecal specimens and verification of DNA purity for whole genome sequencing. The isolation and enrichment of genomic DNA were achieved by a combination of three oocyst purification steps and whole genome amplification (WGA) of DNA from purified oocysts. qPCR analysis of WGA products was used as an initial quality assessment of amplified genomic DNA. The purity of WGA products was assessed by Sanger sequencing of cloned products. Next generation sequencing tools were used in final evaluations of genome coverage and extent of contamination. Altogether, 24 fecal specimens of Cryptosporidium parvum, C. hominis, C. andersoni, C. ubiquitum, C. tyzzeri, and Cryptosporidium chipmunk genotype I were processed with the procedures. As expected, WGA products with low Ct values (<16.0) yielded mostly Cryptosporidium sequences in Sanger sequencing. The cloning-sequencing analysis, however, showed significant contamination in 5 WGA products (percentage of positive colonies derived from Cryptosporidium genomic DNA ≤ 25%). Following this strategy, 20 WGA products from six Cryptosporidium species/genotypes with low Ct values (mostly <14.0) were submitted to whole genome sequencing, generating sequence data covering 94.5-99.7% Cryptosporidium genomes, with mostly minor contamination from bacterial, fungal, and host DNA. These results suggest that the described strategy can be used effectively for the isolation and enrichment of Cryptosporidium DNA from fecal specimens for whole genome sequencing.