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本研究提供了基于牛津纳米孔（Oxford Nanopore, ONT）和Illumina测序读段构建的食蟹猴疟原虫（Plasmodium cynomolgi）Berok与Ceylon菌株的参考基因组。食蟹猴疟原虫是旧世界猴的寄生原虫，亦是间日疟原虫（Plasmodium vivax）研究的重要模式生物。食蟹猴疟原虫Berok株的无性血液阶段已适配长期体外培养，正愈发广泛地用于间日疟原虫型生物学的相关研究¹。本研究构建的基因组组装结果可作为实验遗传学研究的宝贵资源。 食蟹猴疟原虫感染的红细胞（RBC）分别来源于两部分：一是体外培养的无性血液阶段寄生虫（Berok株：源自K2的2C7克隆）；二是直接取自感染的恒河猴（Ceylon株：该感染过程为另一项研究的一部分）。采用皂素裂解步骤后，依照试剂盒说明书的血液高分子量（High Molecular Weight, HMW）DNA提取流程，使用Monarch细胞与血液HMW DNA提取试剂盒（纽英伦生物科技，New England Biolabs）分离高分子量DNA。后续通过离心与AMPure XP磁珠纯化（贝克曼库尔特，Beckman Coulter）进一步纯化DNA。采用分光光度法（Denovix）与荧光定量法（Qubit）检测DNA的浓度与纯度。依照制造商说明书，使用SQK-LSK114试剂盒（牛津纳米孔科技公司，Oxford Nanopore Technologies）制备ONT测序文库。将文库加载至MinION R10.4.1测序流动池。测序运行时长设置为100小时，最小读段长度设为200 bp。Berok株与Ceylon株分别获得17.43 Gb与1.51 Gb的原始ONT测序数据。原始数据使用Dorado（v4.3 SUP）进行碱基识别。Illumina测序由GeneWiz（亚诺生命科学，Azenta Life Sciences）完成，每个样本获得10 Gb测序数据。 组装前，使用Kraken2（v2.1.6）与Krakentools（v1.2.1）对ONT读段进行过滤，以去除灵长类来源的读段³。使用过滤后的ONT读段，通过hifiasm（v0.25.0-r726）进行基因组组装，随后使用Illumina读段，通过Pilon（v1.24）进行两轮短读段校正⁴,⁵。使用Minimap2（v2.30-r1287）将ONT读段比对回组装结果，以便通过Geneious（2026.0.2）进行人工检查以识别组装错误。随后以食蟹猴疟原虫Mulligan（M）菌株为参考基因组，使用Companion（v2.2.11）对基因组进行注释⁶。 Berok株与Ceylon株的组装结果分别包含17条与18条重叠群（contig），涵盖14条完整的核染色体，以及顶质体与线粒体序列，无间隙区域。Berok株的14条组装核染色体中，13条在两端均带有端粒重复序列，仅1条染色体仅在单端带有端粒重复序列。Ceylon株的组装结果中，11条染色体两端均带有端粒重复序列，另有2条染色体仅在单端带有端粒重复序列。 参考文献 1. Chua, A. C. Y. 等. (2019). 食蟹猴疟原虫红细胞阶段的稳定连续体外培养. 《自然-通讯》, 10(1), 3635. https://doi.org/10.1038/s41467-019-11332-4 2. Dissanaike A. S. (1965). 锡兰的猴类疟原虫. 《世界卫生组织公报》, 32(4), 593-597 3. Wood, D. E. 等. (2019). 基于Kraken 2的宏基因组学分析优化. 《基因组生物学》, 20(1), 257. https://doi.org/10.1186/s13059-019-1891-0 4. Cheng, H. 等. (2026). 纳米孔单端读段的高效端粒到端粒组装. 《自然》, 10.1038/s41586-026-10105-6. 提前在线发表. https://doi.org/10.1038/s41586-026-10105-6 5. Walker, B. J. 等. (2014). Pilon：用于微生物变异检测与基因组组装优化的整合工具. 《公共科学图书馆·综合》, 9(11), e112963. https://doi.org/10.1371/journal.pone.0112963 6. Haese-Hill, W. 等. (2024). 利用Companion注释与可视化寄生虫、真菌及节肢动物基因组. 《核酸研究》, 52(W1), W39–W44. https://doi.org/10.1093/nar/gkae378