De novo genome assembly and comparative genomics for the colonial ascidian Botrylloides violaceus

Abstract

Ascidians have the potential to reveal fundamental biological insights related to coloniality, regeneration, immune function, and the evolution of these traits. This study implements a hybrid assembly technique to produce a genome assembly and annotation for the botryllid ascidian, Botrylloides violaceus. A hybrid genome assembly was produced using Illumina, Inc. short and Oxford Nanopore Technologies long-read sequencing technologies. The resulting assembly is comprised of 831 contigs, has a total length of 121 Mbp, N50 of 1 Mbp, and a BUSCO score of 96.1%. Genome annotation identified 13 K protein-coding genes. Comparative genomic analysis with other tunicates reveals patterns of conservation and divergence within orthologous gene families even among closely related species. Characterization of the Wnt gene family, encoding signaling ligands involved in development and regeneration, reveals conserved patterns of subfamily presence and gene copy number among botryllids. This supports the use of genomic data from nonmodel organisms in the investigation of biological phenomena.

Keywords: Illumina; Oxford Nanopore; Wnt; comparative genomics; genome assembly; tunicate; ‌Botrylloides violaceus.

Fig. 1.

Botrylloides violaceus imaged in Morro Bay, CA.

Fig. 2.

Using BUSCO score and the total number of scaffolds and contigs in genome assemblies to assess their quality and completeness. Botrylloides violaceus assemblies are compared to genomes of other botryllids (B. leachii, B. schlosseri), other selected tunicates (O. dioica, P. fumigata, C. savignyi, H. roretzi, M. oculata), and other selected invertebrates (S. purpuratus, A. californica, H. robusta). Triangular points are B. violaceus assemblies color coded by assembly type, while circular points represent other genomes color coded by grouping. BUSCO scores are the percentage of complete BUSCOs based on the Metazoa dataset.

Fig. 3.

Comparative genomic analysis using hierarchical orthogroups reveals patterns of conservation and divergence in the botryllid lineage. a,b) HOGBs clustered using variation in gene copy number per species. A species by HOGB copy number matrix was clustered to identify groups with similar patterns of gene family evolution in botryllids. The appropriate number of clusters (18) was chosen using the silhouette method to account for variation between clusters. c) HOGB clusters binned by duplication pattern similarity. d) Number of B. violaceus genes in each cluster that are members of developmentally relevant pathways according to KEGG annotation. Clusters ordered by general trends depicted in (c).

Fig. 4.

Relative position and orientation of the Wnt genes in B. violaceus. Distances between genes and gene sizes are to scale, with the exception of Wnt11 which was slightly enlarged for clarity. PS indicates a Wnt5 pseudogene. Gene identification number from GenSAS annotation is on top. Numbers at rightmost position of fragments refer to specific contig or scaffold${}^{†}$ in the hybrid assembly. Kb refers to genomic size; CDS bp refers to coding DNA sequence length; double-parallel lines indicate the intergenic region is >30 kb. ${}^{*}$Wnt1 gene identification number is from GeneMark-ES gene model predictions.

Fig. 5.

Wnt gene family analysis. a) Summary of Wnt repertoire for comparative analysis of selected tunicates and humans. Coloring of the boxes indicates the presence of the indicated Wnt subfamily, while a white box indicates the absence. Total copy number of the gene is provided in the respective box. Botryllids are highlighted in grey on the phylogeny. b) Maximum likelihood phylogeny of tunicate Wnt protein sequences. Branch support values derived from SH-like aLRT method. Species represented: Botrylloides violaceus (Bviol), Botrylloides leachii (Bleac), Botryllus schlosseri (Bsc), Ciona savignyi (Csa), Ciona robusta (Cro), Halocynthia roretzi (Hro), Halocynthia aurantium (Hau), Mogula occulta (Moccu), Mogula oculata (Mocul), Mogula occidentalis (Mocci), Phallusia fumigata (Pfu), Phallusia mammillata (Pma), Oikopleura dioica (Odi), and Homo sapiens (Hsa). Oikopleura dioica Wnt10 was removed from the Wnt10 cluster due to long branch length in order to increase visibility of other leaves. Scale bar represents amino acid substitutions.