文献类型：期刊文献

英文题名：Isolation and Enrichment of Cryptosporidium DNA and Verification of DNA Purity for Whole-Genome Sequencing

作者：Guo, Yaqiong[1,2];Li, Na[1];Lysen, Colleen[2];Frace, Michael[3];Tang, Kevin[3];Sammons, Scott[3];Roellig, Dawn M.[2];Feng, Yaoyu[1];Xiao, Lihua[2]

机构：[1]E China Univ Sci & Technol, Sch Resources & Environm Engn, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Ctr Dis Control & Prevent, Natl Ctr Emerging & Zoonot Infect Dis, Div Foodborne Waterborne & Environm Dis, Atlanta, GA USA;[3]Ctr Dis Control & Prevent, Natl Ctr Emerging & Zoonot Infect Dis, Div Sci Resources, Atlanta, GA USA

年份：2015

卷号：53

期号：2

起止页码：641

外文期刊名：JOURNAL OF CLINICAL MICROBIOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000348568500036)】；

基金：This work was supported by the National Natural Science Foundation of China (31229005 and 31110103901) and Centers for Disease Control and Prevention, USA.

语种：英文

摘要：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.