Intra-strain genetic heterogeneity in Toxoplasma gondii ME49: Oxford Nanopore long-read sequencing reveals copy number variation in the ROP8-ROP2A locus

Toxoplasma gondii is an important pathogen and model organism for studying mechanisms of immune evasion and defense. Within the same strain, model organisms are typically assumed to be isogenic; for T. gondii, within-strain genetic divergence has been detected based on phenotypic changes and older molecular techniques but not characterized at the genomic level. We therefore used Oxford Nanopore long-read sequencing to characterize three independently maintained T. gondii ME49 isolates: 2015T and 2020T (obtained from ATCC and propagated in cell culture), and 2000B (propagated in mice). We de novo assembled a new T. gondii ME49 reference genome and, using state-of-the-art variant calling combined with pangenomic genotyping, detected variants between the sequenced isolates. Our new reference genome exceeded existing reference genomes in continuity (NG50 = 6.68 Mb versus 1.2 Mb in RefSeq) and structural accuracy, resolving all chromosomes except for a single break in the ribosomal DNA region. For isolates 2000B and 2020T, we identified 106 and 128 variants, respectively, across a final call set of 79 SNVs, 93 INDELs, and five structural variants; 18 small non-synonymous variants included genes associated with T. gondii life cycle (AP2X-8) and virulence in vivo (6-phosphogluconate dehydrogenase). A 13 kb expansion in the ROP8-ROP2A virulence locus increased the copy number of ROP2A-ROP8 genes in isolates 2000B and 2020T from three to six. We provide an improved T. gondii ME49 reference genome and demonstrate the potentially confounding effect of intra-strain genetic heterogeneity, highlighting the need for continuous genomic monitoring for long-term genetic identity.