Journal: bioRxiv

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

doi: 10.1101/2020.03.10.985549

Figure Lengend Snippet: (A) Bivariate plot showing a comparison of the aligned read length with the sequencing read length. (B) Bivariate plot showing a comparison of the aligned corrected read length (log10 transformed) with the percent identity. In this case corrected reads refer to the method deployed by Canu using read overlap. (C) Histogram showing comparison of chromosome size between ToxoDB-48_TgGT1 genome and TgRH88 initial long-read assembly. (D) Interchromosomal Hi-C contact-count heat map plotted using the TgRH88 initial long-read assembly sequence showing 13 chromosomes in the assembly. (E) Intrachromosomal Hi-C contact-count heat map plotted using the sequence of TgRH88_tig00000001 in TgRH88 initial long-read assembly showing no aberrant signal along the contig

Article Snippet: In recent years single molecule sequencing approaches (developed by Oxford Nanopore and PacBio) have revolutionized de novo sequence assembly by enabling high-throughput generation of kilobase-sized sequence reads.

Techniques: Comparison, Sequencing, Transformation Assay, Hi-C

Journal: bioRxiv

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

doi: 10.1101/2020.03.10.985549

Figure Lengend Snippet: (A) Inversion in the RH88 long-read assembly on chromosome III relative to the ToxoDB-48-TgGT1 assembly. (B) Inversion in the ME49 long-read assembly on chromosome XII relative to the ToxoDB_44-TgME49 genome. (C) Dot plot comparison of the TgRH88 long-read assembly and the ToxoDB-48_TgGT1 genome showing a 429.3-Kb inversion at 2,096,529-2,525,795 bp on chrIV. (D) Intrachromosomal Hi-C contact-count heat map plotted using the sequence of contig_14 in TgRH88 long-read assembly showing a clear centromere signal at position 2.2-2.3 Mb. (E) ChIP-on-chip signal of centromeric histone 3 variant (CenH3) plotted using the TgRH88 long-read assembly as coordinate.

Article Snippet: In recent years single molecule sequencing approaches (developed by Oxford Nanopore and PacBio) have revolutionized de novo sequence assembly by enabling high-throughput generation of kilobase-sized sequence reads.

Techniques: Comparison, Hi-C, Sequencing, Variant Assay

Journal: bioRxiv

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

doi: 10.1101/2020.03.10.985549

Figure Lengend Snippet: (A-D) Estimated copy number for Nanopore assemblies and existing genome assemblies on ToxoDB (“v48”) for T. gondii strain types 1, 2 and 3 and IIxIII F1 progeny. In all cases, Nanopore assemblies identified higher numbers of each repeat locus. In the F1 progeny, B1 gene copy number tracked directly with the genotype (type 2 or 3) at that locus (A), while these same F1 progeny harbored unique numbers of 529 bp copies, all of which were not only distinct from their respective genotypes of origin but distinct from one another (B). The TgIRE and SAT350 repeats also were better resolved in our Nanopore assemblies (C,D) although determining genotype of the corresponding region is not possible since these repeats are found at multiple locations throughout the genome. (E) Whole chromosome alignment focused on the 529 bp repeat region for the v48 ToxoDB assembly (bottom) and our Nanopore-based assembly (top). Expansion of the known genome sequence at this locus in the Nanopore sequence compared to the ToxoDB assembly is clear, and consistent with our identification of ∼140 previously unknown 529 bp repeats in the ME49 genome. (F) Alignment and annotation of repeat sequences of ME49 v48 ToxoDB chromosome IV and that from our polished Nanopore assembly. Grey bars with red borders indicate mapping regions ≥10,000 bp determined using nucmer, while orange boxes with blue borders indicate tandem repeats with period sizes ≥ 500 bp and at least 2 copies. Bars that appear orange are larger than those that are only blue. Incorrect inversion on the right arm of chromosome IV in the ToxoDB assembly is evident, as is the more accurately resolve 529 bp repeat locus that was likely a cause for the inversion in standard assemblies from multiple strains.

Article Snippet: In recent years single molecule sequencing approaches (developed by Oxford Nanopore and PacBio) have revolutionized de novo sequence assembly by enabling high-throughput generation of kilobase-sized sequence reads.

Techniques: Sequencing

Journal: bioRxiv

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

doi: 10.1101/2020.03.10.985549

Figure Lengend Snippet: (A) Two unresolved scaffold gaps on chrIa in ToxoDB-48_TgME49 genome span a 17.5-Kb tandem repeat containing multiple copies of ROP4 and ROP7. The ROP4/7 gaps are closed by the TgME49 long-read assembly (TgME49_tig00000028), revealing a tandem array of 5 copies of this gene in the order shown. (B) BLASTN alignment of the ROP4/ROP7 coding sequence in the ToxoDB-48_TgME49 genome (upper panel) and the TgME49 long-read assembly (lower panel). (C) Copy number determination at 6 canonical tandem gene arrays across 8 T. gondii strains and 1 N. caninum strain. Data from CL13, S27, S21 and S26 show that copy number can change during sexual recombination since copy number in these F1 progeny clones do not match copy number in either parent. (D-F) Whole chromosome alignments between ME49ToxoDB-48 and our Nanopore assemblies at loci harboring tandem gene arrays. Grey boxes with red borders indicate 1-to-1 mapping regions ≥ 10,000 bp determined by nucmer and orange/blue boxes are as described in . Black bars indicate size of select tandem repeats in the ToxoDB and Nanopore assemblies.

Article Snippet: In recent years single molecule sequencing approaches (developed by Oxford Nanopore and PacBio) have revolutionized de novo sequence assembly by enabling high-throughput generation of kilobase-sized sequence reads.

Techniques: Sequencing, Clone Assay

Journal: bioRxiv

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

doi: 10.1101/2020.03.10.985549

Figure Lengend Snippet: (A) We identified 1121 single exon genes with predicted proteins that mapped with 100% identity and 100% coverage using TBLASTN against the ToxoDB-48 ME49 genome. Then, we mapped these against the raw assembly generated by Canu, the polished assembly generated by 4 rounds of Pilon, and after 4 rounds of supplemental error correction. Pilon error correction was sufficient for perfect mapping of 94% of the query single exon genes (compared to only 0.4% for the raw Canu assembly), and supplemental error correction only increased this mapping percentage slightly. (B) Plots representing TBLASTN analysis of protein sequences from two single copy genes showing the improved mapping achieved by Pilon-based error correction. Mapping identity is indicated by the color of the box representing the alignment. (C-D) Plots representing protein sequence from the ROP5A (C) or ROP38 (D) gene mapped using TBLASTN against the raw Canu-only assembly, the Pilon-corrected assembly and the region corrected using our supplemental approach tailored to tandem gene arrays. Both loci have multiple pseudogenes in the Canu-only and Canu-plus Pilon assemblies, but many of these errors are removed upon supplemental correction. The presence of a pseudogene in the ME49 ROP5 locus has been predicted before based on direct sequencing, suggesting that this may represent the most accurate version of the ME49 ROP5 locus sequenced to date.

Article Snippet: In recent years single molecule sequencing approaches (developed by Oxford Nanopore and PacBio) have revolutionized de novo sequence assembly by enabling high-throughput generation of kilobase-sized sequence reads.

Techniques: Generated, Sequencing