Long-read assembly and comparative evidence-based reanalysis of Cryptosporidium genome sequences reveal expanded transporter repertoire and duplication of entire chromosome ends including subtelomeric regions

Abstract

Cryptosporidiosis is a leading cause of waterborne diarrheal disease globally and an important contributor to mortality in infants and the immunosuppressed. Despite its importance, the Cryptosporidium community has only had access to a good, but incomplete, Cryptosporidium parvum IOWA reference genome sequence. Incomplete reference sequences hamper annotation, experimental design, and interpretation. We have generated a new C. parvum IOWA genome assembly supported by Pacific Biosciences (PacBio) and Oxford Nanopore long-read technologies and a new comparative and consistent genome annotation for three closely related species: C. parvum, Cryptosporidium hominis, and Cryptosporidium tyzzeri We made 1926 C. parvum annotation updates based on experimental evidence. They include new transporters, ncRNAs, introns, and altered gene structures. The new assembly and annotation revealed a complete Dnmt2 methylase ortholog. Comparative annotation between C. parvum, C. hominis, and C. tyzzeri revealed that most "missing" orthologs are found, suggesting that the biological differences between the species must result from gene copy number variation, differences in gene regulation, and single-nucleotide variants (SNVs). Using the new assembly and annotation as reference, 190 genes are identified as evolving under positive selection, including many not detected previously. The new C. parvum IOWA reference genome assembly is larger, gap free, and lacks ambiguous bases. This chromosomal assembly recovers all 16 chromosome ends, 13 of which are contiguously assembled. The three remaining chromosome ends are provisionally placed. These ends represent duplication of entire chromosome ends including subtelomeric regions revealing a new level of genome plasticity that will both inform and impact future research.

Figure 1.

Syntenic relationships between select Cryptosporidium chromosome assemblies. (A) Synteny between Chromosomes 2, 4, and 5. Vertical black lines within a chromosome represent known physical gaps. Synteny between chromosomes is shown in pink and inversions in blue. (B) PCR validation using C. parvum KSU-1 DNA (Supplemental Table S4). Lanes 1, 2, and 3 in all gels are 1-kb ladder, positive control Dnmt2 gene, and no template control, respectively. The remaining lanes test each orientation of the left (L) and right (R) inversion boundaries. Red stars indicate the location of primers designed based on the CpIA assembly, and gray stars indicate the same on the CpIRef assembly.

Figure 2.

Ortholog distribution of protein-coding genes reveals few differences between the species. (A) Venn diagram of automated protein orthology assignment between CpIA, Ch30976, and CtUGA55. (B) Venn diagram of the same orthologous genes following manual investigation and removal of putative false positives. (*) The 139 genes shared between C. hominis and C. tyzzeri in A are in complex regions with repeats and gaps and do not have enough evidence to prove their uniqueness at this stage (Supplemental Table S7). (C) Example of a false positive paralog count caused by gene fragments on different scaffolds. (D) Putative unique uncharacterized gene found on Chr 8 in CtUGA55.

Figure 3.

CpIA assembly and annotation reveal new transporters. The numbers of transporters correspond to the counts of genes encoding each type of transporter protein: (ABC) ATP-binding cassette transporter; (MFS) major facilitator superfamily; (DMT) divalent metal transporter; (AAAP) amino acid/auxin permease; (MC) mitochondrial carrier; (ZIP) zinc transporter protein; (CPA) cation/proton antiporter; (SulP) sulfate transporter; and (PUP) purine permeases.

Figure 4.

Resolution of repetitive subtelomeric regions found on Chr 1 identifies missing telomeres on Chromosomes 7 and 8. (A) Illumina reads from CpIA are mapped to the CpIA Chr 1 long-read assembly subtelomeric region to identify read pileups and estimates of sequence copy number by normalizing against the average genomics Illumina read depth. Vertical gray areas indicate regions with annotated genes. Annotated genes are represented below the shaded regions, the 5.8S rRNA is present but not indicated. (B) Subtelomeric variaton observed on different CpIA chromosomes is supported by CpBGF ONT long reads. Individual ONT long reads provide evidence of at least four different yet related subtelomeric regions that extend into the chromosomes that were missing telomeres in CpIA (Chr 7 and Chr 8) in addition to Chr 1. The white and black reference bar above each collection of annotated ONT reads identify the resolved subtelomeric regions (white) and linkage to existing assembly (black). The penultimate read on the Chr 7 3′ end panel indicates a unique region of insertion (nucleotide positions 1,191,705–1,217,462). This region contains mostly uncharacterized proteins and two transferases. Each ONT read is annotated as indicated in the key shown in A.