Abstract
BackgroundInfectious disease involving multiple genetically distinct populations of pathogens is frequently concurrent, but difficult to detect or describe with current routine methodology. Cryptosporidium sp. is a widespread gastrointestinal protozoan of global significance in both animals and humans.It cannot be easily maintained in culture and infections of multiple strains have been reported.To explore the potential use of single cell genomics methodology for revealing genome-level variation in clinical samples from Cryptosporidium-infected hosts, we sorted individual oocysts for subsequent genome amplification and full-genome sequencing.ResultsCells were identified with fluorescent antibodies with an 80 % success rate for the entire single cell genomics workflow, demonstrating that the methodology can be applied directly to purified fecal samples. Ten amplified genomes from sorted single cells were selected for genome sequencing and compared both to the original population and a reference genome in order to evaluate the accuracy and performance of the method. Single cell genome coverage was on average 81 % even with a moderate sequencing effort and by combining the 10 single cell genomes, the full genome was accounted for. By a comparison to the original sample, biological variation could be distinguished and separated from noise introduced in the amplification.ConclusionsAs a proof of principle, we have demonstrated the power of applying single cell genomics to dissect infectious disease caused by closely related parasite species or subtypes. The workflow can easily be expanded and adapted to target other protozoans, and potential applications include mapping genome-encoded traits, virulence, pathogenicity, host specificity and resistance at the level of cells as truly meaningful biological units.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2815-y) contains supplementary material, which is available to authorized users.
Highlights
Infectious disease involving multiple genetically distinct populations of pathogens is frequently concurrent, but difficult to detect or describe with current routine methodology
To optimize the single cell genome workflow for protozoan parasites, 50 Cryptosporidium sp. cells tagged with fluorescent antibodies were individually fluorescence activated cell sorting (FACS) sorted (Fig. 1) and subject to alkaline heat lysis and subsequent whole genome amplification
On average 99.8 % of the sequence data mapped to the Cryptosporidium parvum IowaII reference genome (Table 1)
Summary
Infectious disease involving multiple genetically distinct populations of pathogens is frequently concurrent, but difficult to detect or describe with current routine methodology. Is a widespread gastrointestinal protozoan of global significance in both animals and humans. It cannot be maintained in culture and infections of multiple strains have been reported. To explore the potential use of single cell genomics methodology for revealing genome-level variation in clinical samples from Cryptosporidium-infected hosts, we sorted individual oocysts for subsequent genome amplification and full-genome sequencing. The result, an oocyst, is passed through feces and the only external life form (as well as post meiosis) and is a suitable target for detection and further genomic studies
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
