Table 1.
Libraries utilized for transcriptome assembly.
Table 2.
Comparison of transcriptome assembly statistics.
Fig 1.
Improved transcriptome assembly with increased coverage and completeness.
(A) Percentage of non-normalized fragments per library that are properly paired to the transcriptome. Each bar represents an individual library. The black bars represent the percentage of proper alignments to the previously published transcriptome “Matsunami2019” [8], while the green bars show the improved alignment percentages to the newly assembled transcriptome from this study “New_assembly”. The average proper alignment percentage for all libraries is shown in black for the previously published transcriptome and in green for the new transcriptome (top right). The last 9 bars correspond to newly sequenced ventricle libraries (heart tissue), from left to right. (B) Full-length transcript analysis based on grouping multiple high-scoring segment pairs using BLAST+ against the Swiss-Prot database. The black bars represent the previously published transcriptome “Matsunami2019” [8], while the green bars represent the newly assembled transcriptome from this study. (C) BUSCO assessment for the previously published transcriptome “Matsunami2019” [8] and the newly assembled transcriptome from this study, evaluated against the Eukaryota, Metazoa, Vertebrata, and Tetrapoda datasets. The numbers within the bars represent BUSCO counts, where C stands for Complete, S for Single-copy, D for Duplicated, F for Fragmented, and M for Missing. (D) Principal Component Analysis of the 1000 most variable genes. (E) Heatmap depicting row-scaled gene expression (VST) for differentially expressed genes across various tissue types, highlighting significant patterns in gene expression. The order of the column annotations matches the corresponding legend text, from top to bottom.
Fig 2.
Differential expression testing and enrichment analysis during limb regeneration.
(A) Upset plot showing the number of differentially expressed genes in the various groups compared to adult limb stump (0dpa). (B) Principal Component Analysis for the top 1000 variable genes. (C) Heatmap depicting row-scaled gene expression (VST) for differentially expressed genes during limb regeneration and development, relative to adult limb stump (0dpa). (D, E) GSEA of Biological Processes and KEGG Orthology, respectively, on the different groups compared to adult limb stump (0dpa). (F) Normalized gene expression counts for key genes involved in immune response, muscle interaction, and cell cycle proliferation are presented. Pairwise comparisons between each time point and the adult limb stump (0dpa) were conducted using the DESeq2 R package, with p-values adjusted using the Benjamini-Hochberg method. Each box plot represents the following groups from left to right: 0 dpa, 3 dpa, 7 dpa, larvae, and late embryo. Asterisks denote statistical significance: ***P < 0.001, **P < 0.01, and *P < 0.05.
Fig 3.
Gene expression changes in signaling pathways during limb regeneration.
Normalized gene expression counts for key components of the WNT, FGF, TGF-β, BMP, and Notch signaling pathways are presented. Pairwise comparisons between each time point and the adult limb stump (0dpa) were conducted using the DESeq2 R package, with p-values adjusted using the Benjamini-Hochberg method. Each box plot represents the following groups from left to right: 0 dpa, 3 dpa, 7 dpa, larvae, and late embryo. Asterisks denote statistical significance: ***P < 0.001, **P < 0.01, and *P < 0.05.