Understanding how genomes work depends on knowing where genes are and what they produce. While protein-coding genes are relatively well annotated, long non-coding RNAs, or lncRNAs, have remained one of the least understood parts of the genome. These RNAs do not encode proteins, but they play important roles in regulating gene activity, development, and disease. Many lncRNAs are expressed only in specific cell types or developmental stages, which makes them difficult to detect using traditional approaches.

Researchers from the GENCODE consortium and the Centre for Genomic Regulation (CRG), Spain set out to address this gap by building the most comprehensive catalog of lncRNAs in humans and mice to date. Their work is part of the GENCODE CLS project, which focuses on improving genome annotations using advanced sequencing strategies.

The team used targeted capture combined with long-read RNA sequencing to read full-length RNA molecules from matched embryonic and adult tissues in both species. Unlike short-read methods, long-read RNA sequencing can capture complete RNA transcripts, making it much easier to define transcript boundaries, splice variants, and gene structure. Importantly, the annotations were generated using carefully supervised computational analysis, helping ensure high confidence in the final gene models.

Targeting and sequencing the long non-coding transcriptome with CapTrap- CLS

A) Representation of the capture panel; each bar reports the number of targeted regions per catalog, for the human and mouse experiments, organized by the class of elements in focus. B) Application of CapTrap-CLS in matched adult and embryonic tissues from human and mouse. Samples were sequenced using long-read platforms from PacBio and Oxford Nanopore Technologies (ONT). Short reads were sequenced with Illumina and highlighted by an asterisk when available. An outline of CLS transcripts and their integration to GENCODE is shown for C) human and D) mouse. Top panel: final set of CLS transcripts categorized based on the novelty status with respect to GENCODE v27 (human) and vM16 (mouse). Bottom panel: CLS transcript models added to GENCODE v47 (human) and vM36 (mouse) See Figure S6 for a more detailed description E) Representation of GENCODE annotation history to releases v47 and vM36 Number of transcripts on primary assembly chromosomes in every year’s last GENCODE release, in human (left) and mouse (right), broken down by broad biotype. IG/TR genes excluded.

This effort dramatically expanded the known lncRNA landscape. Nearly 18,000 new human genes and more than 22,000 new mouse genes were added, representing the largest increase in annotated human genes since the original human genome was sequenced. The number of annotated transcripts increased even more sharply, reflecting the complex isoform structure of lncRNAs.

A major advance from this work is the improved ability to match lncRNAs between humans and mice. By assigning orthologous relationships at much higher rates than before, the researchers tripled the number of human disease associated lncRNAs with known mouse counterparts. This is especially important because mouse models are widely used to study human disease mechanisms.

Although many of these newly annotated lncRNAs are expressed at low levels in bulk samples, the data show that they can be highly active in specific cell populations. This suggests that lncRNAs may play key roles in defining cell identity and regulating specialized biological functions that are invisible in averaged measurements.

By expanding and refining lncRNA annotations, the GENCODE CLS project provides a stronger foundation for interpreting RNA sequencing data, understanding gene regulation, and linking non-coding RNAs to human disease. These updated reference annotations represent a major step toward fully decoding how the human and mouse genomes function.

Availability – The GENCODE Project