Whole genome amplification (WGA) for archiving and genotyping of clinical isolates of<i>Cryptosporidium</i>species

Bouzid, Maha; Heavens, Darren; Elwin, Kristin; Chalmers, Rachel M.; Hadfield, Stephen J.; Hunter, Paul R.

doi:10.1017/s0031182009991132

Cited by 14 publications

(12 citation statements)

References 42 publications

(51 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The yield of amplified DNA, quantified by PicoGreen, was lower than the manufacturer's claim (10 μg per reaction), ranging from 0.8 to 1.2 μg in a 50 μL final volume corresponding to a 115–170‐fold amplification. This is coherent with the 50–160‐fold amplification obtained for bacteria with the REPLI‐g Mini kit (Bouzid et al , 2009; Woyke et al , 2009).…”

Section: Discussionsupporting

confidence: 83%

Whole-genome amplification (WGA) of marine photosynthetic eukaryote populations

Lepère

Demura

Kawachi

et al. 2011

FEMS Microbiology Ecology

View full text Add to dashboard Cite

Metagenomics approaches have been developing rapidly in marine sciences. However, the application of these approaches to marine eukaryotes, and in particular to the smallest ones, is challenging because marine microbial communities are dominated by prokaryotes. One way to circumvent this problem is to separate eukaryotic cells using techniques such as single-cell pipetting or flow cytometry sorting. However, the number of cells that can be recovered by such techniques remains low and genetic material needs to be amplified before metagenomic sequencing can be undertaken. In this methodological study, we tested the application of whole-genome amplification (WGA) to photosynthetic eukaryotes. We performed various optimization steps both on a mixture of known microalgal strains and on natural photosynthetic eukaryote populations sorted by flow cytometry. rRNA genes were used as markers for assessing the efficiency of different protocols. Our data indicate that WGA is suitable for the amplification of photosynthetic eukaryote genomes, but that biases are induced, reducing the diversity of the initial population. Nonetheless, this approach appears to be suitable for obtaining metagenomics data on microbial eukaryotic communities.

show abstract

Section: Discussionsupporting

confidence: 83%

Whole-genome amplification (WGA) of marine photosynthetic eukaryote populations

Lepère

Demura

Kawachi

et al. 2011

FEMS Microbiology Ecology

View full text Add to dashboard Cite

show abstract

“…In addition, reference primers Cry15 and Cry9 amplifying a 555 bp of the COWP gene [23] were used as a positive control. PCR conditions were carried out as described previously [40]. PCR screening of putative species genes was performed by testing a panel of DNA clinical samples isolated as described previously [41] and archived in the national collection at the UK Cryptosporidium Reference Unit (CRU) [42].…”

Section: Methodsmentioning

confidence: 99%

Multi-locus analysis of human infective Cryptosporidium species and subtypes using ten novel genetic loci

et al. 2010

Self Cite

View full text Add to dashboard Cite

BackgroundCryptosporidium is a protozoan parasite that causes diarrheal illness in a wide range of hosts including humans. Two species, C. parvum and C. hominis are of primary public health relevance. Genome sequences of these two species are available and show only 3-5% sequence divergence. We investigated this sequence variability, which could correspond either to sequence gaps in the published genome sequences or to the presence of species-specific genes. Comparative genomic tools were used to identify putative species-specific genes and a subset of these genes was tested by PCR in a collection of Cryptosporidium clinical isolates and reference strains.ResultsThe majority of the putative species-specific genes examined were in fact common to C. parvum and C. hominis. PCR product sequence analysis revealed interesting SNPs, the majority of which were species-specific. These genetic loci allowed us to construct a robust and multi-locus analysis. The Neighbour-Joining phylogenetic tree constructed clearly discriminated the previously described lineages of Cryptosporidium species and subtypes.ConclusionsMost of the genes identified as being species specific during bioinformatics in Cryptosporidium sp. are in fact present in multiple species and only appear species specific because of gaps in published genome sequences. Nevertheless SNPs may offer a promising approach to studying the taxonomy of closely related species of Cryptosporidia.

show abstract

“…Consequently, the amount of genomic DNA extracted from the purified oocysts is limited. To increase DNA concentrations for NGS library construction, we have used WGA to amplify extracted DNA, as already done for archiving Cryptosporidium DNA from clinical specimens (19). Although the visualization of genomic DNA of high molecular weight by electrophoresis is a direct demonstration of successes and failures in genome amplification, we used ssrRNA gene-based qPCR to assess the quality and quality of the Cryptosporidium DNA amplified, indicated by the C T values generated (Fig.…”

Section: Figmentioning

confidence: 99%

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

Guo

Lysen

et al. 2015

J Clin Microbiol

View full text Add to dashboard Cite

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...

show abstract

Whole genome amplification (WGA) for archiving and genotyping of clinical isolates ofCryptosporidiumspecies

Cited by 14 publications

References 42 publications

Whole-genome amplification (WGA) of marine photosynthetic eukaryote populations

Whole-genome amplification (WGA) of marine photosynthetic eukaryote populations

Multi-locus analysis of human infective Cryptosporidium species and subtypes using ten novel genetic loci

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

Contact Info

Product

Resources

About