- Telomere-to-telomere assembly of the. - Results: Here, we present a chromosome-scale genome assembly (OKI2018_I69) of the Okinawan O. - 99% of the assembly is contained within five megabase-scale scaffolds.. - We found telomeres on both ends of the two largest scaffolds, which represent assemblies of two fully contiguous autosomal chromosomes. - Each of the other three large scaffolds have telomeres at one end only and we propose that they correspond to sex chromosomes split into a pseudo-autosomal region and X-specific or Y-specific regions.. - At the sequence level, multiple genomic features such as GC content and repetitive elements are distributed differently along the short and long arms of the same chromosome.. - Full list of author information is available at the end of the article. - As a member of the tunicates, a sister taxonomic group to vertebrates, O.. - dioica’ s genome size is 65 – 70 Mbp [8, 9], making it one of the smallest among all sequenced animals. - Here, we present a chromosome-length assembly of the Okinawan O. - Based on k-mer counting of the Illumina reads, the genome was estimated to contain ~ 50 Mbp (Fig. - We corrected sequencing errors and local misas- semblies of the draft contigs with Nanopore reads using. - More than 99% of the Hi-C reads could be mapped to the contig assembly. - The resulting assembly consisted of 8 megabase-scale scaffolds containing 99% of the total se- quence (Fig. - One of the small scaffolds is a draft assembly of the mitochondrial genome that we discuss below.. - Most of the other smaller scaffolds are highly repetitive and might represent unplaced fragments of centromeric or telomeric regions. - We annotated telomeres by search- ing for the TTAGGG repeat sequence and found that most of the megabase-scale scaffolds have single telo- meric regions: therefore, we reasoned that they represent chromosome arms. - Table 1) comprises telomere-to- telomere assemblies of the autosomal chromosomes 1 (chr 1) and 2 (chr 2). - We assume that the sex-specific regions belong to the long arm of the PAR, as the long arm does not contain any telomeric repeats (Fig. - Alignment of the Illumina polishing reads to the OKI2018_I69 assembly estimated an error rate of 1.3% showing high sequence accuracy.. - 3c) shows bright, off-diagonal spots that suggest spatial clustering of the telomeres and centromeres both within the same and across different chromosomes [18].. - The two sex-specific regions have lower apparent contact frequencies compared with the rest of the assembly which is consistent with their. - The chromosome arms them- selves show few interactions between each other, even when they are part of the same chromosome.. - b Estimated total and repetitive genome size based on k-mer counting of the Illumina paired-end reads used for polishing the OKI2018_I69 assembly. - c Pairwise genome alignment of the contig assemblies of I69 and I28 O.. - 3 OKI2018_I69 assembly of the Okinawan O. - a Treemap comparison between the contig (left) and scaffold (right) assemblies of the O.. - Table 1 Comparison of the OKI2018_I69 assembly with the previously published O. - 4 Chromosome-level features of the Okinawan O. - It should be noted that the differences in GC contents affects the density of the GATC DpnII restric- tion enzyme recognition sites used for Hi-C library prep- aration. - Interspersed repeats make up 14.4% of the as- sembly (9.25 Mbp. - Of the annotated elements, the most abundant type is the long terminal repeat (LTRs;. - 4.6%) with Ty3/gypsy Oikopleura transposons (TORs) dominating 2.97 Mbp of the sequence. - interspersed nuclear elements (SINEs) make up a smaller portion of the OKI2018_I69 sequence (<. - Indeed, 44% of the predicted re- peats in the Okinawan O. - Annotation of the genome yielded 18,794 tran- script isoforms distributed among 17,260 protein-coding genes. - The rest of the genes are either lost from the Okinawan O.. - On the other hand, the higher number of genes might be artifacts of the OdB3 and OSKA2016 annotations. - The completeness of the annotation com- pares to the genome: BUSCO recovered 75.3% complete and 4.8% fragmented metazoan genes (Fig. - Indeed, we found a high frequency of the non-canonical (non-GT/AG) in- trons in the OKI2018_I69 (11. - reported that 12% of the introns were non- canonical in the OdB3 [9]. - However, more close examination is required to under- stand if it is the case for the rest of the genes. - The ribosomal DNA gene encoding the precursor of the 18S, 5.8S and 28S rRNAs occurs as long tandem re- peats that form specific chromatin domains in the nucle- olus. - We identified 4 full tandem copies of the rDNA gene at the tip of the PAR’s short arm, separated by 8738 bp (median distance). - the real number of the tandem rDNA copies could range between 20 (MiSeq) and 100 (Nanopore) copies. - 5 Quality assessment of the OKI2018_I69 genome assembly. - The synteny- based approach with OdB3’s linkage groups as a refer- ence was only required to guide final pairing of chromo- some arms into single scaffolds of chr 1, chr 2 and PAR, as we found that these scaffolds mostly align to one of the autosomal LGs or PAR. - The repeat landscape and proportions of various repeat classes in the genome are indicated and color- coded according to the classes shown on the right side of the figure. - The non-repetitive fraction of the genome is shown in black. - Table 2 Comparison of the annotations of the three O. - 7 Draft scaffold of the mitochondrial genome in the OKI2018_I69 assembly. - a Predicted gene annotation of the draft mitochondrial genome sequence. - b Self-similarity plot of the draft mitochondrial genome sequence. - A tandem repeat can be seen, which complicates the complete assembly of the mitochondrial genome from whole-genome sequencing data. - dioica populations that will elucidate the rela- tion of the Okinawan populations to the North Atlantic and North Pacific ones.. - By comparison, in flies and mosquitoes, the degree of contacts between two arms of the same chromosome appear to be reduced but nonetheless more frequent than between different chromosomes [18]. - As we prepared our Hi-C libraries from adult ani- mals, where polyploidy is high [38], we cannot rule out that it could be a possible cause of the low inter-arm in- teractions in our contact matrix. - The view of the OKI2018_I69 genome assem- bly can be found here:. - We believe that the current version of the assembly will serve as an essential resource for a broad range of biological studies, including genome- wide comparative studies of Oikopleura and other spe- cies, and provides insights into chromosomal evolution.. - Q32850), and the integrity of the genomic DNA was val- idated using Agilent 4200 TapeStation (Agilent, 5067–. - The quality of the reads before and after filtering were checked with FASTQC v0.11.5 [44]. - Read pairs that lacked one of the reads after the filtering were discarded in order to preserve paired-end information.. - Next, one round of the HaploMerger2 processing pipeline [48] was applied to eliminate redundancy in contigs and to merge haplotypes.. - 2 (“Draft chromosome scale assembly based on scaffolds of the reference genome sequence”) in Denoeud et al. - 500×) than the rest of the assembly, and were therefore removed from the final assembly.. - The completeness and quality of the assembly were checked with QUAST v5.0.2 [52] and by searching for the set of 978 highly conserved metazoan genes (OrthoDB version 9.1) [23] using BUSCO v . - Staged embryos were initiated by gently mixing 10 μl of the spawned male sperm to the awaiting eggs in FASW at 23 °C. - Further processing for mRNA selection was performed with Oligo-d(T)25 Magnetic Beads (NEB, E7490) and the integrity of the RNA was validated once more with Agilent 4200 TapeStation (Agilent . - Adapters for the creation of DNA libraries for the Illu- mina platform were added per manufacturer’s guidance (NEB, E7805) as were unique indexed oligonucleotides (NEB, E7600) to each of the three staged samples. - The quality and completeness of the transcriptome assembly was verified with rna- QUAST v1.5.1 [61] and BUSCO.. - The quality of the predicted gene models was assessed with BUSCO.. - A draft annotation of the mitochondrial genome was obtained by submitting the corresponding scaffold (chr_. - For visualization of the results, we converted the alignments to GFF3 format and collated the colinear “match_part” alignment blocks in “match”. - For each of GC content, sequencing depth, repeat content, gene count, and DpnII restriction sites, the significance of the differences between long and short arms was assessed with Welch’s two-sided T test as well as a nonparametric Mann-Whitney test imple- mented in R (Suppl. - The results of the two tests were largely in agreement, but groups were only in- dicated as significantly different if they both produced significance values below 0.05 ( p <. - Contami- nations found in smaller scaffold of the OKI2018_I69 assembly. - We would like to thank the DNA Sequencing Section and the Scientific Computing and Data Analysis Section of the Research Support Division at OIST for their support, Danny Miller for advices on Nanopore sequencing, Dan Rokhsar, Gene Myers, Ferdinand Marlétaz and Konstantin Khalturin for critical comments, Takeshi Onuma and Hiroki Nishida for sharing the OSKA2016 genome sequence prior publication, Simon Henriet for the technical advice on the DNA extraction protocol, and Lin Zhang for helping with the culture.. - MJM received funding as an International Research Fellow of the Japan Society for the Promotion of Science. - Streamlined sampling and cultivation of the pelagic cosmopolitan larvacean, Oikopleura dioica.. - Anatomy and functional morphology of the Appendicularia. - https://doi.org/10.1038/nature04336.. - https://doi.org/10.1016/. - https://doi.. - https://doi.org/10.1007/s . - A genome database for a Japanese population of the larvacean Oikopleura dioica. - Comprehensive mapping of long-range interactions reveals folding principles of the human genome. - De novo assembly of the Aedes aegypti genome using hi-C yields chromosome-length scaffolds.. - https://doi.org/10.1101/. - De novo draft assembly of the Botrylloides leachii genome provides further insight into tunicate evolution. - Shedding light on the expansion and diversification of the Cdc48 protein family during the rise of the eukaryotic cell. - https://doi.org/10.1186/s . - https://doi.org/10.1128/. - Widespread use of the “ ascidian ” mitochondrial genetic code in tunicates. - Patterning through differential endoreduplication in epithelial organogenesis of the chordate, Oikopleura dioica. - https://doi.org/10.1093/bioinformatics/. - https://doi.org/10.1016/j.. - In: Proceedings of the 2018 ACM International Conference on Bioinformatics, Computational Biology, and Health Informatics. - Phosphorylation of the histone H3. - 3 variant in mitosis and meiosis of the urochordate Oikopleura dioica.. - https://doi.org
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