Cryptosporidium as a testbed for single cell genome characterization of unicellular eukaryotes

Abstract

Background: Infectious 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.

Results: Cells 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.

Conclusions: As 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.

Keywords: Apicomplexa; Cryptosporidium; FACS; Multiple infection; Single cell genomics; Whole genome amplification.

Fig. 1

Conceptual graph of single cell genomics workflow applied to Cryptosporidium parvum. a Scattergram of side scatter and antigen-based fluorescence used for sorting individual cells. The gate for sorting is indicated by a circle and individual analyzed cells are marked by color. Panel with antibody stained oocysts to the left. b. Real-time MDA kinetics for individual amplified genomes color coded as in (a). Blanks are given in brown. c Small subunit rRNAscreening of amplified cells with gel electrophoreses and sequencing. Negative control and positive control with 10 cells are included for comparison

Fig. 2

Coverage statistics for single cell genomes of Cryptosporidium parvum. a Color coded heat map for the eight chromosomes of individual cells where red is < 1 × coverage (sequence missing), orange is 1–5 × coverage, yellow is 5–20 × coverage and green is >20 × coverage. Positions with ambiguity characters in the reference genome are colored gray. The combined data for all 10 single cell genomes (all cells combined), the full parent metagenome and the same metagenome subsampled to equal sequencing depth as an average single cell genome (1.3 Gbp) are included for reference. b Density profiles showing more detailed view of the sequencing depth for chromosome 3 with the ten single cell genomes given in color according to (a). “All cells combined” are shown with gray shading. Similar plots for all chromosomes can be found in Additional file 4. The bold black line represents the metagenome

Fig. 3

Fractions of genome shared among single amplified genomes (0 to 10 cells) from Cryptosporidium sp. The “fraction of genome accounted for by N cells” at 0 “number of cells above threshold” represents the portion of the reference genome not accounted for in any of the cells at the indicated coverage. In contrast, the portion of the genome accounted for in all cells is found at 10 cells on the X-axis. Observed shared fraction of the genome is given at coverage cutoffs of 1, 5 and 20 ×