High Resolution Annotation of Zebrafish Transcriptome Using Long-Read Sequencing

Abstract

With the emergence of zebrafish as an important model organism, a concerted effort has been made to study its transcriptome. This effort is limited, however, by gaps in zebrafish annotation, which are especially pronounced concerning transcripts dynamically expressed during zygotic genome activation (ZGA). To date, short read sequencing has been the principal technology for zebrafish transcriptome annotation. In part because these sequence reads are too short for assembly methods to resolve the full complexity of the transcriptome, the current annotation is rudimentary. By providing direct observation of full-length transcripts, recently refined long-read sequencing platforms can dramatically improve annotation coverage and accuracy. Here, we leveraged the SMRT platform to study transcriptome of zebrafish embryos before and after ZGA. Our analysis revealed additional novelty and complexity in the zebrafish transcriptome, identifying 2748 high confidence novel transcripts that originated from previously unannotated loci and 1835 high confidence new isoforms in previously annotated genes. We validated these findings using a suite of computational approaches including structural prediction, sequence homology and functional conservation analyses, as well as by confirmatory transcript quantification with short-read sequencing data. Our analyses provided insight into new homologs and paralogs of functionally important proteins and non-coding RNAs, isoform switching occurrences and different classes of novel splicing events. Several novel isoforms representing distinct splicing events were validated through PCR experiments, including the discovery and validation of a novel 8 kb transcript spanning multiple miR-430 elements, an important driver of early development. Our study provides a significantly improved zebrafish transcriptome annotation resource.

Dynamic transcriptional and chromatin accessibility landscape of medaka embryogenesis

“…The remaining 2657 (9%) transcripts are different from the Ensembl models in various ways. This low proportion of well-matched and the high proportion of novel isoforms were similar to a recent study using long-read RNA-seq technology to annotate the zebrafish genome (Nudelman et al 2018), indicating that the short-read based gene model construction is far insufficient to unveil the complex transcriptome structure.…”

“…For example, short reads often fail in resolving large structural features, including alternative splicing, alternative transcription initiation, or alternative transcription termination. In addition, short reads originating from transcriptional noise and often mapping to intronic or intergenic regions may produce false gene models (Goodwin et al 2016;Kuo et al 2017;Nudelman et al 2018). In contrast, with long-read RNA-sequencing technologies, it has become a reality that one read is one transcript (Wang et al 2019).…”

“…These technologies produce single reads covering the entire DNA or RNA molecules, independent of the error-prone computational assembly. Thus, these technologies could eliminate many common issues in data derived from short reads, and have become important tools to improve the gene annotation in multiple species (Sharon et al 2013;Zhang et al 2017;Kuo et al 2017;Nudelman et al 2018;Wang et al 2018).…”

The Spatio-Temporal Control of Zygotic Genome Activation

“…This indicates that rapid cell divisions do not necessarily restrict RNAPII elongation to short runs of ~1 kb, the average gene length among earliest activated genes. In fact, recent long-read sequencing of the early zebrafish transcriptome identified a zygotic 8-kb transcript spanning multiple pri-miR-430 elements (Nudelman et al, 2018). Thus, it remains to be determined why RNAPII, despite its abundance, is initially prevented from more wide-spread elongation.…”

An improved deyolking procedure facilitates the proteomics analysis of early developmental stages of zebrafish embryos

“…Zebrafish have become a prominent and broadly used model system to study developmental biology, neurogenetic disorders, genetics, toxicology, reproduction, pathology, and pharmacology [1][2][3][4][5]. The genome annotation is relatively well developed [6], and the embryonic transcriptome of zebrafish has been characterized in several studies [7][8][9][10][11]. However, knowledge about the comprehensive proteome dynamics during embryogenesis in zebrafish remains elusive.…”