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Within the same strain, model organisms are typically assumed to be isogenic; for\n                      <jats:italic>T. gondii<\/jats:italic>\n                      , 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\n                      <jats:italic>T. gondii<\/jats:italic>\n                      ME49 isolates: 2015T and 2020T (obtained from ATCC and propagated in cell culture), and 2000B (propagated in mice).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      We\n                      <jats:italic>de novo<\/jats:italic>\n                      assembled a new\n                      <jats:italic>T. gondii<\/jats:italic>\n                      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\u2009=\u20096.68\u00a0Mb versus 1.2\u00a0Mb 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\n                      <jats:italic>T. gondii<\/jats:italic>\n                      life cycle (AP2X-8) and virulence in vivo (6-phosphogluconate dehydrogenase). A 13\u00a0kb expansion in the ROP8-ROP2A virulence locus increased the copy number of ROP2A-ROP8 genes in isolates 2000B and 2020T from three to six.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      We provide an improved\n                      <jats:italic>T. gondii<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12864-025-12372-5","type":"journal-article","created":{"date-parts":[[2025,12,3]],"date-time":"2025-12-03T13:55:30Z","timestamp":1764770130000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Intra-strain genetic heterogeneity in Toxoplasma gondii ME49: Oxford Nanopore long-read sequencing reveals copy number variation in the ROP8-ROP2A locus"],"prefix":"10.1186","volume":"26","author":[{"given":"Yomna","family":"Gohar","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marie","family":"Neumann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lisanna","family":"H\u00fclse","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Wind","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julia","family":"Mock","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karin","family":"Buchholz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marcel","family":"Helle","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ursula R.","family":"Sorg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Degrandi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Klaus","family":"Pfeffer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Dilthey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,12,3]]},"reference":[{"key":"12372_CR1","doi-asserted-by":"publisher","DOI":"10.1103\/PhysRevLett.118.028102","volume":"118","author":"P Bittihn","year":"2017","unstructured":"Bittihn P, Hasty J, Tsimring LS. 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Obtaining of cysts for infection studies was also approved by the German authorities (LAVE, formerly known as LANUV) under permit numbers 81-02.04.2021.A060, 81-02.04.2020.A113, and 81-02.04.2018.A406.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"1094"}}