Madry, T. | Search bioRxiv

bioRxiv2025

CapTrap-seq: Advancing zebrafish transcriptomic research through high-fidelity full-length RNA sequencing

Zebrafish is a valuable model organism thanks to its genetic and anatomical similarities to humans, offering a more relevant system for studying human biology and disease than in vitro or non-vertebrate models. However, its use in large-scale transcriptomic research is still limited. Most zebrafish studies focus on global expression profiling in specific developmental contexts, contributing little toward improving and expanding gene annotations. These gaps lead to inaccuracies in gene quantification and downstream functional analyses, ultimately reducing the effectiveness of zebrafish as a model system. Further challenges include ineffective ribodepletion methods and limited resources for validate transcript boundaries and splicing patterns. To overcome these challenges, we applied CapTrap-seq, a long-read sequencing method that combines Cap-trapping with oligo(dT) priming to identify 5-capped, full-length transcripts from both developmental and adult tissue samples. To promote detection of longer RNA molecules, we introduced a size-selection step into CapTrap-seq protocol, which further improved detection of transcript ends without compromising its quantitative accuracy. Our results highlight the genome-agnostic nature of CapTrap-seq, enabling generation of accurate transcript models in non-mammalian systems at high-throughput. CapTrap-seq also facilitates functional annotation of zebrafish genes by uncovering novel, spliced transcript isoforms with potential biological significance.

genomics↗

bioRxiv2024

GENCODE: massively expanding the lncRNA catalog through capture long-read RNA sequencing

Accurate and complete gene annotations are indispensable for understanding how genome sequences encode biological functions. For more than twenty years, the GENCODE consortium has developed reference annotations for the human and mouse genomes, becoming a foundation for biomedical and genomics communities worldwide. Nevertheless, collections of important yet poorly-understood gene classes like long non-coding RNAs (lncRNAs) remain incomplete and scattered across multiple, uncoordinated catalogs. To address this, GENCODE has undertaken the most comprehensive lncRNA annotation effort to date. This is founded on the manually supervised computational annotation of full-length targeted long-read sequencing, on matched embryonic and adult tissues, of orthologous regions in human and mouse. Altogether 17,931 human genes (140,268 transcripts) and 22,784 mouse genes (136,169 transcripts) have been added to the GENCODE catalog representing a 2-fold and 6-fold growth in transcripts, respectively - the greatest increase in the number of annotated human genes since the sequencing of the human genome. Our targeted design assigned human-mouse orthologs at a rate beyond previous studies, tripling the number of human disease-associated lncRNAs that have mouse orthologs. Novel lncRNA genes consistently exhibit biological signals of functionality, and they greatly enhance the functional interpretability of the human genome. While poorly expressed in bulk RNA-Seq samples, many of them are highly expressed in specific cell populations, maybe even contributing to cell-type determination. The expanded GENCODE lncRNA annotations mark a critical step toward deciphering the human and mouse genomes.

genomics↗