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

Abstract

c 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 (C T ) 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) C T 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. C ryptosporidium spp. are an important cause of moderate to severe diarrhea in humans and various animals (1, 2). Over the past decade, great efforts have been made to understand the interaction between Cryptosporidium spp. and their hosts. Thus far, at least 26 species and more than 60 genotypes have been described (3), most with some host specificity. In contrast to the expanding knowledge of the taxonomic complexity of the genus Cryptosporidium, little progress has been made in understanding the molecular basis of phenotypic traits such as the host specificity. This is mainly due to the lack of whole-genome characterization of most Cryptosporidium species and genotypes.Thus far, the genomes of only four Cryptosporidium isolates have been sequenced. Sequence data of the whole genomes of C. parvum and C. hominis, the two species responsible for most human infections (4), were first published in 2004 (5, 6). Several years later, the genome of C. muris was sequenced, and data from the project are available on CryptoDB (http://cryptodb.org). More recently, the genome of anthroponotic subtype family IIc of C. parvum has been sequenced using the next-generation sequencing (NGS) tools (7). The availability of the whole-genome sequenc...

Genomic Variation in IbA10G2 and Other Patient-Derived Cryptosporidium hominis Subtypes

“…In recent years, several laboratories have published a number of strategies for the purification and enrichment of Cryptosporidium oocysts from fecal specimens, using filtration and salt flotation, as well as such methods as amplification of extracted DNA (19)(20)(21). The current study, using clinical C. hominis samples, confirms the proof-ofprinciple method previously published by our group (18) as an effective pipeline for processing Cryptosporidium species patient samples for genome sequencing.…”

“…However, the vast majority of such studies compare data between limited numbers of genomes, which makes the task of identifying new markers even more problematic. Cryptosporidium genomes are being sequenced at a pace (17)(18)(19)(20)(21)37) that is increasing our general knowledge of Cryptosporidium spp. Subsequent sequencing of additional genomes, especially from clinical samples, will increase the likelihood of facilitating the identification of new genetic markers in order to improve the classification of species status, as well as increasing the genotyping resolution of C. parvum and C. hominis subtype population genetics and epidemiology.…”

“…Recently, several studies have aimed to develop methods to purify and overcome the limitation of low quantities of target material in order to enable whole-genome sequencing of Cryptosporidium directly from fecal specimens (18)(19)(20)(21). This has involved removing as much of the contaminating material and microbe population as possible, thereby improving the target-specific enrichment (immunomagnetic separation [IMS], flotation, bleaching) as well as increasing the quantity of Cryptosporidium DNA available for sequencing (whole-genome amplification kits, PCR amplification) and the use of different sequencing platforms and analysis pipelines.…”

Evolution of mitosome metabolism and invasion-related proteins in Cryptosporidium

“…Among the three C. ubiquitum isolates, 39668 and 39726 belonged to the XIIb subtype family whereas 39725 belonged to the XIIc subtype family. Cryptosporidium oocysts were purified from the specimens using a combination of sucrose and cesium chloride gradient centrifugation and immunomagnetic separation [50]. Total genomic DNA was extracted from purified oocysts using the QIAamp®DNA Mini Kit (Qiagen Sciences, Germantown, Maryland), after the oocysts were subjected to five freeze-thaw cycles and overnight digestion with proteinase K. Extracted DNA was amplified using REPLI-g Midi Kit (Qiagen).…”

Comparative genomic analysis of the IId subtype family of Cryptosporidium parvum

“…Cryptosporidium oocysts were isolated from stool specimens by sucrose and cesium chloride gradient centrifugation, and immunomagnetic separation as previously described (Guo et al, 2015a). They were subjected to treatment with 10% commercial bleach on ice for 10 min and five freezing-and-thawing cycles.…”