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 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 1,926 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.

Fully resolved assembly of Cryptosporidium parvum

“… : Assembly comparisons. (A) The Canu assembly shows a high concordance with the previously published C. parvum assembly (GCA_015245375.1) [ 27 ] (dot plots) and agreements in length (bottom). Nevertheless, clear assembly differences are visual when comparing it to GCA_000165345.1 [ 13 ].…”

“…The dot plot from a MUMmer (MUMmer, RRID:SCR_018171 ) alignment analysis indicates that the GCA_015245375.1 [ 27 ] and Canu genome assemblies are largely collinear (Fig. 3A ).…”

“…The present work highlights how next-generation sequencing, including third-generation long-read sequencing, can be used to generate a high-quality genome assembly complete with centromeric regions and numerous telomeres. The genome assembly generated provides a gapless reference compared to the previously published GCA_000165345.1 [ 13 ] and extends into some telomeric regions over GCA_015245375.1 [ 27 ]. Telomeric regions added to those from GCA_000165345.1, which is a hybrid assembly based on two different subtypes of Cryptosporidium spp .…”

The Long and Short of Next Generation Sequencing for Cryptosporidium Research

“…The Cryptosporidium genome is very compact at ~9 Mb and the parasite scavenges nutrients from the host, obscuring metabolic pathways. The parasite also uses alternative splice forms, making it a challenge to validate and annotate genes and their functions ( Baptista et al., 2022 ). As a result, many genome assemblies contain large percentages of hypothetical or uncharacterised proteins ( Table 1 ).…”

“…For many Cryptosporidium genomes significant time has elapsed since they were initially sequenced and assembled. These references may require updated sequencing with next generation technologies, or genome re–annotation and polishing to ensure that as reference genomes they do not mislead interpretations during genomic or molecular studies ( Baptista et al., 2022 ). A greater understanding of the parasite genome in silico will complement recent in vivo and in vitro advances that have increased our understanding of parasite and infection biology ( Marzook and Sateriale, 2020 ).…”