Reference genomes for the Plasmodium cynomolgi Berok and Ceylon strains

Here we present reference genomes for the Plasmodium cynomolgi Berok and Ceylon strains generated with Oxford Nanopore (ONT) and Illumina reads. P. cynomolgi is a parasite of old-world monkeys and serves as an important model for P. vivax. The asexual blood stages of P. cynomolgi Berok have been adapted to long-term culture and are increasingly being used to study vivax-type biology1. Our genome assemblies should serve as useful resources for experimental genetic studies. Plasmodium cynomolgi infected RBCs were obtained either from cultured asexual blood stage parasites (Berok – K2 derived 2C7 clone) or directly from an infected rhesus macaque (Ceylon – infected as part of another study). High molecular weight (HMW) DNA was isolated using the Monarch HMW DNA Extraction Kit for Cells & Blood (New England Biolabs) according to the manufacturer’s instructions for HMW DNA Extraction from Blood, preceded by saponin lysis. DNA was further cleaned through centrifugation and AMPure XP bead clean-up (Beckman Coulter). DNA concentration and purity was measured using both spectrophotometry (Denovix) and fluorescence (Qubit). Samples for ONT sequencing were prepared using the SQK-LSK114 kit (Oxford Nanopore Technologies) following the manufacturer’s instructions. Samples were loaded onto a MinION R10.4.1 flow cell. The sequencing run was set at 100 hours, with a minimum read length of 200 bp. 17.43 and 1.51 Gb of raw ONT data were obtained for Berok and Ceylon respectively. The raw data was basecalled with Dorado (v4.3 SUP). Illumina sequencing was done by GeneWiz (Azenta Life Sciences) obtaining 10 Gb per sample. Prior to assembly, ONT reads were filtered using Kraken2 (v2.1.6) and Krakentools (v1.2.1) to remove primate reads3. The genomes were assembled using the filtered ONT reads with hifiasm (v0.25.0-r726) followed by two rounds of short-read correction with Pilon (v1.24) using the Illumina reads4,5. The ONT reads were mapped back to the assemblies using Minimap2 (v2.30-r1287) to allow detection of misassemblies by manual inspection using Geneious (2026.0.2). The genomes were then annotated with Companion (v2.2.11) using the Plasmodium cynomolgi Mulligan (M) strain as reference6. Our Berok and Ceylon assemblies are comprised of 17 and 18 contigs respectively, with 14 clear nuclear chromosomes, as well as sequences for the apicoplast and mitochondria and no gaps. Thirteen of the fourteen assembled nuclear chromosomes for Berok feature telomeric repeats at both ends, with a single chromosome featuring telomeric repeats at only one end. The Ceylon assembly features eleven chromosomes with telomeric repeats at both ends, as well as two featuring telomeric repeats at one end. References 1. Chua, A. C. Y. et al. (2019). Robust continuous in vitro culture of the Plasmodium cynomolgi erythrocytic stages. Nature communications, 10(1), 3635. https://doi.org/10.1038/s41467-019-11332-4 2. Dissanaike A. S. (1965). Simian malaria parasites of Ceylon. Bulletin of the WHO, 32(4), 593-597 3. Wood, D. E. et al. (2019). Improved metagenomic analysis with Kraken 2. Genome biology, 20(1), 257. https://doi.org/10.1186/s13059-019-1891-0 4. Cheng, H. et al. (2026). Efficient near-telomere-to-telomere assembly of nanopore simplex reads. Nature, 10.1038/s41586-026-10105-6. Advance online publication. https://doi.org/10.1038/s41586-026-10105-6 5. Walker, B. J. et al. (2014). Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PloS one, 9(11), e112963. https://doi.org/10.1371/journal.pone.0112963 6. Haese-Hill, W. et al. (2024). Annotation and visualization of parasite, fungi and arthropod genomes with Companion. Nucleic acids research, 52(W1), W39–W44. https://doi.org/10.1093/nar/gkae378