Isolation and Enrichment of CryptosporidiumDNA and Verification of DNA Purity for Whole-Genome Sequencing

ABSTRACTWhole-genome sequencing of Cryptosporidiumspp. 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 Cryptosporidiumgenomic 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. Quantitative PCR (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 of the extent of contamination. Altogether, 24 fecal specimens of Cryptosporidium parvum, C. hominis, C. andersoni, C. ubiquitum, C. tyzzeri, and Cryptosporidiumchipmunk genotype I were processed with the procedures. As expected, WGA products with low (<16.0) threshold cycle (CT) values yielded mostly Cryptosporidiumsequences in Sanger sequencing. The cloning-sequencing analysis, however, showed significant contamination in 5 WGA products (proportion of positive colonies derived from Cryptosporidiumgenomic DNA, =25%). Following this strategy, 20 WGA products from six Cryptosporidiumspecies or genotypes with low (mostly <14.0) CTvalues were submitted to whole-genome sequencing, generating sequence data covering 94.5% to 99.7% of Cryptosporidiumgenomes, 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 CryptosporidiumDNA from fecal specimens for whole-genome sequencing.