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

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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. 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.

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          Author and article information

          Journal
          J. Clin. Microbiol.
          Journal of clinical microbiology
          1098-660X
          0095-1137
          Feb 2015
          : 53
          : 2
          Affiliations
          [1 ] 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.
          [2 ] State Key Laboratory of Bioreactor Engineering, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai, China.
          [3 ] Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
          [4 ] Division of Scientific Resources, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.
          [5 ] 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.
          [6 ] 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.
          Article
          JCM.02962-14
          10.1128/JCM.02962-14
          4298554
          25520441
          7a87c9c5-5e02-4dcf-a25e-989a4ed38cdf
          Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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