title: Isolation and enrichment of Cryptosporidium DNA and verification of DNA purity for whole-genome sequencing.
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. 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 Cryptosporidium chipmunk genotype I were processed with the procedures. As expected, WGA products with low (<16.0) threshold cycle (CT) values yielded mostly Cryptosporidium sequences in Sanger sequencing. The cloning-sequencing analysis, however, showed significant contamination in 5 WGA products (proportion of positive colonies derived from Cryptosporidium genomic DNA, ≤25%). Following this strategy, 20 WGA products from six Cryptosporidium species or genotypes with low (mostly <14.0) CT values were submitted to whole-genome sequencing, generating sequence data covering 94.5% to 99.7% of 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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai, China Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Yaqiong
authors.initials: Y
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Bioreactor Engineering, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.affiliation: Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Colleen
authors.initials: C
authors.lastname: Lysén
authors.affiliation: Division of Scientific Resources, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Frace
authors.affiliation: Division of Scientific Resources, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Tang
authors.affiliation: Division of Scientific Resources, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Sammons
authors.affiliation: Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: Dawn M
authors.initials: DM
authors.lastname: Roellig
authors.affiliation: State Key Laboratory of Bioreactor Engineering, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai, China yyfeng@ecust.edu.cn lxiao@cdc.gov.
authors.forename: Yaoyu
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA yyfeng@ecust.edu.cn lxiao@cdc.gov.
authors.forename: Lihua
authors.initials: L
authors.lastname: Xiao
mesh_terms: D000818:Animals; D003458:Cryptosporidium / Q000235:genetics* / Q000302:isolation & purification; D016054:DNA, Protozoan / Q000302:isolation & purification*; D005243:Feces / Q000469:parasitology*; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods; D013048:Specimen Handling / Q000379:methods*
chemical_list: D016054:DNA, Protozoan