Guo Yaqiong, Li Na, Lysén Colleen, Frace Michael, Tang Kevin, Sammons Scott, Roellig Dawn M, Feng Yaoyu, Xiao Lihua
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.
State Key Laboratory of Bioreactor Engineering, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai, China.
J Clin Microbiol. 2015 Feb;53(2):641-7. doi: 10.1128/JCM.02962-14. Epub 2014 Dec 17.
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.
隐孢子虫属的全基因组测序受到从临床标本中获取足够的、高纯度基因组DNA的困难的阻碍。在本研究中，我们开发了从粪便标本中分离和富集隐孢子虫基因组DNA以及验证用于全基因组测序的DNA纯度的程序。基因组DNA的分离和富集是通过三个卵囊纯化步骤和对纯化卵囊的DNA进行全基因组扩增（WGA）相结合来实现的。WGA产物的定量PCR（qPCR）分析被用作扩增基因组DNA的初始质量评估。WGA产物的纯度通过克隆产物的桑格测序进行评估。下一代测序工具用于基因组覆盖范围和污染程度的最终评估。总共用这些程序处理了24份小隐孢子虫、人隐孢子虫、安氏隐孢子虫、泛在隐孢子虫、泰泽隐孢子虫和花鼠隐孢子虫基因型I的粪便标本。正如预期的那样，阈值循环（CT）值低（<16.0）的WGA产物在桑格测序中大多产生隐孢子虫序列。然而，克隆测序分析显示5个WGA产物存在显著污染（源自隐孢子虫基因组DNA的阳性菌落比例≤25%）。按照该策略，将来自6种隐孢子虫物种或基因型且CT值低（大多<14.0）的20个WGA产物提交进行全基因组测序，生成的序列数据覆盖了隐孢子虫基因组的94.5%至99.7%，主要存在少量来自细菌、真菌和宿主DNA的污染。这些结果表明，所描述的策略可有效地用于从粪便标本中分离和富集隐孢子虫DNA以进行全基因组测序。
