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. 2020 Aug 24;9:e55792. doi: 10.7554/eLife.55792

Figure 1. Comparison of Ensembl and RefSeq zebrafish transcriptome annotations for bulk RNA-seq analysis.

(A, B) Volcano plots showing differentially expressed genes from Tg(kdrl:HRAS-mCherry)s896-positive and negative (kdrlpos and kdrlneg) cells identified using RNA-seq reads quantified with (A) RefSeq or (B) Ensembl, version 95 (Ens95) transcript annotations. Genes with significant enrichment (padj <0.05) are shown as red or blue (log2 fold change >1 or <-1, respectively) across replicate samples (n = 3). Grey dots are genes that fall below statistical cutoffs. Green dots indicate selected genes previously found to be enriched in endothelial cells in zebrafish or other models. (C) Venn diagram illustrating the intersection of genes with a common NCBI ID in Ens95 and RefSeq found significantly enriched in kdrlpos cells using either annotation. (D, E) Plots of commonly annotated genes identified as kdrlpos-enriched only by Ens95 with indicated values from (D) Ens95 or (E) RefSeq. (F, G) Correlation of expression levels from indicated annotation for kdrlpos-enriched genes identified (F) selectively as such by Ens95 (maroon) or RefSeq (grey) only, or (G) both annotations. Data are not normally distributed, Spearman correlation, r values are indicated.

Figure 1—source data 1. DESeq2 output for kdrlpos and kdrlneg RNA-seq quantified with RefSeq (GCF_000002035.6_GRCz11; worksheet 1) or Ensembl, v95 (worksheet 2).
Gene expression levels were quantified using RSEM. Median ratio normalized expression values are shown for each replicate, along with adjusted p-value, and log2 fold change. Data used to generate plots in Figure 1A,B, and incorporated into source data tables indicated below.
Figure 1—source data 2. Intersection of kdrlpos-enriched genes from RefSeq and Ens95 commonly annotated by NCBI ID.
Gene symbol, along with matching Ensembl gene ID and NCBI ID, as well as differential annotation (i.e., identified as differentially expressed only in RefSeq, Ens95, or both) are indicated. Expression data are derived from Figure 1—source data 1. Data used to generate plots in Figure 1C–G.
elife-55792-fig1-data2.xlsx (416.1KB, xlsx)

Figure 1.

Figure 1—figure supplement 1. Comparison of Ens95 and RefSeq zebrafish transcriptome annotations for bulk RNA-seq analysis.

Figure 1—figure supplement 1.

(A) Log10 average expression (n = 3) for kdrlpos-enriched genes as quantified by each indicated annotation. Each separate plot shows genes identified as kdrlpos-enriched only using or Ens95 or RefSeq. Data are not normally distributed, Wilcoxon matched-pairs signed-rank test, p values are indicated. (B) Venn diagram of intersection for genes with a common NCBI ID in Ens95 and RefSeq found significantly enriched in pdgfrbpos cells (log2 fold change [pdgfrbpos/pdgfrbneg]>1,adjp <0.05) using each annotation. (C, D) Volcano plots showing differentially expressed genes from TgBAC(pdgfrb:citrine)s1010-positive and negative cells identified using RNA-seq reads quantified using (C) RefSeq or (D) Ens95 annotations. Genes with significant differences (padj <0.05) are shown as red (log2 fold change >1) or blue (log2 fold change<-1). Grey dots are genes that fall below these statistical cutoffs; n = 3. Green dots indicate selected genes previously identified as expressed in vascular smooth muscle cells or pericytes (see main text). (E) Plots of commonly annotated genes identified as pdgfrbpos-enriched only by Ens95 with indicated values from Ens95 or RefSeq. (F) Correlation of expression levels from indicated annotation for pdgfrbpos-enriched genes identified selectively as such by Ens95 (maroon) or RefSeq (grey) only (left plot) or in both annotations (right plot). Data are not normally distributed, Spearman correlation, r values are indicated. (G) Log10 average expression (n = 3) for pdgfrbpos-enriched genes as quantified by each indicated annotation. Each separate plot shows genes only identified as pdgfrbpos-enriched using Ens95 or RefSeq. Data are not normally distributed, Wilcoxon matched-pairs signed-rank test, p values are indicated.
Figure 1—figure supplement 1—source data 1. DESeq2 output for pdgfrbpos and pdgfrbneg RNA-seq quantified with RefSeq (GCF_000002035.6_GRCz11) or Ensembl, v95.
Gene expression levels were quantified using RSEM. Median ratio normalized expression values are shown for each replicate, along with adjusted p-value, and log2 fold change. Data used to generate plots in Figure 1—figure supplement 1C–E, and incorporated into source data tables indicated below.
Figure 1—figure supplement 1—source data 2. Intersection of pdgfrbpos-enriched genes from RefSeq and Ens95 commonly annotated by NCBI ID.
Gene symbol, along with matching Ensembl gene ID and NCBI ID, as well as differential annotation (i.e., identified as differentially expressed only in RefSeq, Ens95, or both) are indicated. Expression data are derived from Figure 1—source data 1. Data used to generate plots in Figure 1—figure supplement 1B,F–H.