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Third generation sequencing revises the molecular karyotype for Toxoplasma gondii and identifies emerging copy number variants in sexual recombinants

Jing Xia, Aarthi Venkat, View ORCID ProfileMichael L. Reese, View ORCID ProfileKarine Le Roch, View ORCID ProfileFerhat Ay, View ORCID ProfileJon P. Boyle
doi: https://doi.org/10.1101/2020.03.10.985549
Jing Xia
1Department of Biological Sciences, Dietrich School of Arts and Sciences, University of Pittsburgh
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Aarthi Venkat
2Computational Biology and Bioinformatics, Yale University, New Haven, CT, 06520
3La Jolla Institute for Immunology, La Jolla, CA, 92037
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Michael L. Reese
4University of Texas-Southwestern, Dallas, TX. 75390
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Karine Le Roch
5Department of Molecular, Cell and Systems Biology, College of Agricultural and Life Sciences, University of California-Riverside
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Ferhat Ay
3La Jolla Institute for Immunology, La Jolla, CA, 92037
5Department of Pediatrics, University of California - San Diego, La Jolla, CA, 92093.
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Jon P. Boyle
1Department of Biological Sciences, Dietrich School of Arts and Sciences, University of Pittsburgh
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  • For correspondence: boylej@pitt.edu
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ABSTRACT

Toxoplasma gondii is an obligate intracellular parasite that has a significant impact on human health, especially in the immunocompromised. This parasite is also a useful genetic model for intracellular parasitism given its ease of culture in the laboratory and relevant animal models. However, as for many other eukaryotes, the T. gondii genome is incomplete, containing hundreds of sequence gaps due to the presence of repetitive and/or uncloneable sequences that prevent complete telomere-to-telomere de novo chromosome assembly. Here, we report the first use of single molecule DNA sequencing to generate near complete de novo genome assemblies for T. gondii and its near relative, N. caninum. Using the Oxford Nanopore Minion platform, we dramatically improved the contiguity of the T. gondii genome (N50 of ∼6.6Mb) and increased overall assembled sequence compared to current reference sequences by ∼2 Mb. Multiple complete chromosomes were fully assembled as evidenced by clear telomeric repeats on the end of each contig. Interestingly, for all of the Toxoplasma gondii strains that we sequenced (RH, CTG, II×III F1 progeny clones CL13, S27, S21, and S26), the largest contig ranged in size between 11.9 and 12.1 Mb in size, which is larger than any previously reported T. gondii chromosome. This was due to a repeatable and consistent fusion of chromosomes VIIb and VIII. These data were further validated by mapping existing T. gondii ME49 Hi-C data to our assembly, providing parallel lines of evidence that the T. gondii karyotype consists of 13, rather than 14, chromosomes. In addition revising the molecular karyotype we were also able to resolve hundreds of repeats derived from both coding and non-coding tandem sequence expansions. For well-known host-targeting effector loci like rhoptry protein 5 (ROP5) and ROP38, we were also able to accurately determine the precise gene count, order and orientation using established assembly approaches and the most likely primary sequence of each using our own assembly correction scripts tailored to correcting homopolymeric run errors in tandem sequence arrays. Finally, when we compared the T. gondii and N. caninum assemblies we found that while the 13 chromosome karyotype was conserved, we determined that previously unidentified large scale translocation events occurred in T. gondii and N. caninum since their most recent common ancestry.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • We have improved the description of repetitive sequences (both coding and non-coding), added an additional level of error correction specifically for tandem gene expansions that greatly improves our assessment of gene content and sequence at these loci, and finalized submission of the genome assemblies to Genbank. This was in response to reviewer comments and critiques.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted October 12, 2020.
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Third generation sequencing revises the molecular karyotype for Toxoplasma gondii and identifies emerging copy number variants in sexual recombinants
Jing Xia, Aarthi Venkat, Michael L. Reese, Karine Le Roch, Ferhat Ay, Jon P. Boyle
bioRxiv 2020.03.10.985549; doi: https://doi.org/10.1101/2020.03.10.985549
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Third generation sequencing revises the molecular karyotype for Toxoplasma gondii and identifies emerging copy number variants in sexual recombinants
Jing Xia, Aarthi Venkat, Michael L. Reese, Karine Le Roch, Ferhat Ay, Jon P. Boyle
bioRxiv 2020.03.10.985549; doi: https://doi.org/10.1101/2020.03.10.985549

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