This is the project page for NIH grant 1R01AI148667-01A1 Capturing The Genomic Variation Present In Cryptosporidium and Cryptosporidiosis. This project represents a large community effort to survey the population genetic structure of human-infecting Cryptosporidia. A hybrid capture approach will be used to enrich Cryptosporidium DNA from fecal DNA or oocysts samples and generate sequence. The figure below shows results from SureSelect baits based on a previous release of the C. parvum genome (Khan et al. in prep). These baits performed quite well, except for mixtures of very different species of Cryptosporidium (III.B.Mixed; the green, yellow, and red should be equal in size). Thus, we are developing a version 2 of the whole genome bait set that takes advantage of the more complete C. parvum genome, as well as adding baits to better capture diverse species and strains. The new data will be compared to improved reference genome sequences and annotation that will be generated as a part of this project. The hybrid capture baits developed will be made available to the community via arborbiosci.com.
Figure. Hybridization-based capture enrichment whole genome sequencing of Cryptosporidium using SureSelect RNA baits. I) Sensitivity and specificity of capture enrichment sequencing (CES) for Cryptosporidium I A) Workflow of SureSelect CES. The whole genome of Cryptosporidium was captured by hybridization with genome wide uniformly distributed biotinylated baits. I B) and I C) Specificity and coverage map of Cryptosporidium sequences from serially diluted samples. II) CES of distantly related species of Cryptosporidium. II A) Experimental setup to test 3 distantly related species of Cryptosporidium. II B) Phylogenetic analysis of Cryptosporidium spp. II C) Bottlebrush representation of genome wide distribution of CES reads using capture enrichment. III) Detection of mixed infection using capture enrichment sequencing. III A) Diagram to generate an artificial mixed sample. III B) Estimation of multiplicity of infection by genome wide abundance of heterozygous genotypes. III C) Genome wide heterozygosity plot of 3 Cryptosporidium species and a mixed sample (red = heterozygous, blue = homozygous).
