title: Cryptosporidium as a testbed for single cell genome characterization of unicellular eukaryotes.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Microbiology, National Veterinary Institute, Uppsala, Sweden. karin.troell@sva.se.
authors.forename: Karin
authors.initials: K
authors.lastname: Troell
authors.affiliation: Department of Microbiology, Public Health Agency of Sweden, Solna, Sweden.
authors.forename: Björn
authors.initials: B
authors.lastname: Hallström
authors.affiliation: Microbial Single Cell Genomics Facility, Department of Cell and Molecular Biology and Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Anna-Maria
authors.initials: AM
authors.lastname: Divne
authors.affiliation: Department of Microbiology, National Veterinary Institute, Uppsala, Sweden.|Division of Pharmacognosy, Department of Medicinal Chemistry, Biomedical Center, Uppsala University, Uppsala, Sweden.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Alsmark
authors.affiliation: Department of Microbiology, Public Health Agency of Sweden, Solna, Sweden.
authors.forename: Romanico
authors.initials: R
authors.lastname: Arrighi
authors.affiliation: Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Solna, Sweden.
authors.forename: Mikael
authors.initials: M
authors.lastname: Huss
authors.affiliation: Department of Microbiology, Public Health Agency of Sweden, Solna, Sweden.
authors.forename: Jessica
authors.initials: J
authors.lastname: Beser
authors.affiliation: Department of Ecology and Genetics, Limnology and Science for Life Laboratory, Uppsala University, Uppsala, Sweden. stebe@ebc.uu.se.
authors.forename: Stefan
authors.initials: S
authors.lastname: Bertilsson
mesh_terms: D000483:Alleles; D003458:Cryptosporidium / Q000235:genetics*; D056890:Eukaryota / Q000235:genetics*; D014644:Genetic Variation; D016678:Genome; D023281:Genomics / Q000379:methods; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D009154:Mutation; D033661:Oocysts; D020641:Polymorphism, Single Nucleotide
keywords: Apicomplexa; Cryptosporidium; FACS; Multiple infection; Single cell genomics; Whole genome amplification