An improved zebrafish transcriptome annotation for sensitive and comprehensive detection of cell type-specific genes

Abstract

The zebrafish is ideal for studying embryogenesis and is increasingly applied to model human disease. In these contexts, RNA-sequencing (RNA-seq) provides mechanistic insights by identifying transcriptome changes between experimental conditions. Application of RNA-seq relies on accurate transcript annotation for a genome of interest. Here, we find discrepancies in analysis from RNA-seq datasets quantified using Ensembl and RefSeq zebrafish annotations. These issues were due, in part, to variably annotated 3' untranslated regions and thousands of gene models missing from each annotation. Since these discrepancies could compromise downstream analyses and biological reproducibility, we built a more comprehensive zebrafish transcriptome annotation that addresses these deficiencies. Our annotation improves detection of cell type-specific genes in both bulk and single cell RNA-seq datasets, where it also improves resolution of cell clustering. Thus, we demonstrate that our new transcriptome annotation can outperform existing annotations, providing an important resource for zebrafish researchers.

A genomic portrait of zebrafish transposable elements and their spatiotemporal embryonic expression

“…C. Length of extended 3' UTR regions for Lawson et al 2020 and this study show similar distribution. Supplementary Figure 6.…”

“…RNA-seq track shows merged replicates at 20-25 somites. Ensembl GRCz11.98 and Lawson et al 2020 annotations are shown, together with extracted extended 3' UTR region from Lawson et al and this study. In this study, transcriptome assembly using StringTie was used to find extended 3' UTR regions using White et al 2017 RNA-seq data. B.…”

Vinculin controls endothelial cell junction dynamics during vascular lumen formation

“…Recent studies have established distinct roles for vcla and vclb during zebrafish heart development, which reflect their expression in the myocardium and epicardium, respectively (Cheng et al, 2016; Fukuda et al, 2019b; Han et al, 2017). In contrast, in the endothelium vcla and vclb are co-expressed (Lawson et al, 2020) and, consistent with this, we did not notice phenotypic variation in different mutant allele combinations (e.g. vcla -/- ;vclab +/- vs. vcla +/- ;vclab -/- ) with regard to the appearance of junctional fingers - in agreement with the notion that vcla and vclb mutants can genetically compensate for each other (El-Brolosy et al, 2018).…”

Conserved and context-dependent roles for Pdgfrb signaling during zebrafish vascular mural cell development

“…S2A) and both show enriched expression in RNA-seq from isolated kdrl:mcherry-positive cells, consistent with their recently described endothelial expression in zebrafish (Fig. 1I; Lawson et al, 2020;Stratman et al, 2020). By contrast, pdgfrb is enriched in pdgfrb:citrine cells from the same larvae (Fig.…”