More than 2,500 coding genes in the human reference gene set still have unsettled status.

In 2018 we analysed the three main repositories for the human proteome, Ensembl/GENCODE, RefSeq and UniProtKB. They disagreed on the coding status of one of every eight annotated coding genes. The analysis inspired bilateral collaborations between annotation groups.

The rapid degradation of translated upstream regions points to an inefficient translation initiation process.

Large-scale experimental analyses find ever more abundant evidence of translation from start codons upstream of the canonical start site. This translation either generates entirely new proteins (from novel upstream open reading frames) or produces isoforms with extended N-terminals when the novel start codon is in frame Most extended N-terminals are likely to just add a disordered region to the canonical protein isoform, but some may also block the recognition of the signal peptide causing the isoform to accumulate in the incorrect cellular compartment. This analysis finds evidence that upstream translations that would interfere with signal peptides are detected in expected quantities in ribosome profiling experiments, but that the equivalent N-terminally extended protein isoforms are significantly reduced in multiple proteomics experiments.

A deep audit of the PeptideAtlas database uncovers evidence for unannotated coding genes and aberrant translation.

The human genome has been the subject of intense scrutiny by experimental and manual curation projects for more than two decades. Novel coding genes have been proposed from large-scale RNASeq, ribosome profiling and proteomics experiments. Here we carry out an in-depth analysis of an entire proteomics database.

GENCODE 2025: reference gene annotation for human and mouse.

GENCODE produces comprehensive reference gene annotation for human and mouse. Entering its twentieth year, the project remains highly active as new technologies and methodologies allow us to catalog the genome at ever-increasing granularity. In particular, long-read transcriptome sequencing enables us to identify large numbers of missing transcripts and to substantially improve existing models, and our long non-coding RNA catalogs have undergone a dramatic expansion and reconfiguration as a result.

Evidence for widespread translation of 5' untranslated regions.

Ribosome profiling experiments support the translation of a range of novel human open reading frames. By contrast, most peptides from large-scale proteomics experiments derive from just one source, 5' untranslated regions. Across the human genome we find evidence for 192 translated upstream regions, most of which would produce protein isoforms with extended N-terminal ends.

The T2T-CHM13 reference assembly uncovers essential WASH1 and GPRIN2 paralogues.

Summary: The recently published T2T-CHM13 reference assembly completed the annotation of the final 8% of the human genome. It introduced 1956 genes, close to 100 of which are predicted to be coding because they have a protein coding parent gene. Here, we confirm the coding status and functional relevance of two of these genes, paralogues of and .

Lost in the WASH. The functional human WASH complex 1 gene is on chromosome 20.

The WASH1 gene produces a protein that forms part of the developmentally important WASH complex. The WASH complex activates the Arp2/3 complex to initiate branched actin networks at the surface of endosomes. As a curiosity, the human reference gene set includes nine WASH1 genes.

GENCODE: reference annotation for the human and mouse genomes in 2023.

GENCODE produces high quality gene and transcript annotation for the human and mouse genomes. All GENCODE annotation is supported by experimental data and serves as a reference for genome biology and clinical genomics. The GENCODE consortium generates targeted experimental data, develops bioinformatic tools and carries out analyses that, along with externally produced data and methods, support the identification and annotation of transcript structures and the determination of their function.

Origins and Evolution of Human Tandem Duplicated Exon Substitution Events.

The mutually exclusive splicing of tandem duplicated exons produces protein isoforms that are identical save for a homologous region that allows for the fine tuning of protein function. Tandem duplicated exon substitution events are rare, yet highly important alternative splicing events. Most events are ancient, their isoforms are highly expressed, and they have significantly more pathogenic mutations than other splice events.

Clinical variant interpretation and biologically relevant reference transcripts.

Clinical variant interpretation is highly dependent on the choice of reference transcript. Although the longest transcript has traditionally been chosen as the reference, APPRIS principal and MANE Select transcripts, biologically supported reference sequences, are now available. In this study, we show that MANE Select and APPRIS principal transcripts are the best reference transcripts for clinical variation.

APPRIS principal isoforms and MANE Select transcripts define reference splice variants.

Motivation: Selecting the splice variant that best represents a coding gene is a crucial first step in many experimental analyses, and vital for mapping clinically relevant variants. This study compares the longest isoforms, MANE Select transcripts, APPRIS principal isoforms, and expression data, and aims to determine which method is best for selecting biological important reference splice variants for large-scale analyses.

Results: Proteomics analyses and human genetic variation data suggest that most coding genes have a single main protein isoform.

APPRIS: selecting functionally important isoforms.

APPRIS (https://appris.bioinfo.cnio.

The clinical importance of tandem exon duplication-derived substitutions.

Most coding genes in the human genome are annotated with multiple alternative transcripts. However, clear evidence for the functional relevance of the protein isoforms produced by these alternative transcripts is often hard to find. Alternative isoforms generated from tandem exon duplication-derived substitutions are an exception.

Assessing the functional relevance of splice isoforms.

Alternative splicing of messenger RNA can generate an array of mature transcripts, but it is not clear how many go on to produce functionally relevant protein isoforms. There is only limited evidence for alternative proteins in proteomics analyses and data from population genetic variation studies indicate that most alternative exons are evolving neutrally. Determining which transcripts produce biologically important isoforms is key to understanding isoform function and to interpreting the real impact of somatic mutations and germline variations.

GENCODE 2021.

The GENCODE project annotates human and mouse genes and transcripts supported by experimental data with high accuracy, providing a foundational resource that supports genome biology and clinical genomics. GENCODE annotation processes make use of primary data and bioinformatic tools and analysis generated both within the consortium and externally to support the creation of transcript structures and the determination of their function. Here, we present improvements to our annotation infrastructure, bioinformatics tools, and analysis, and the advances they support in the annotation of the human and mouse genomes including: the completion of first pass manual annotation for the mouse reference genome; targeted improvements to the annotation of genes associated with SARS-CoV-2 infection; collaborative projects to achieve convergence across reference annotation databases for the annotation of human and mouse protein-coding genes; and the first GENCODE manually supervised automated annotation of lncRNAs.

An analysis of tissue-specific alternative splicing at the protein level.

The role of alternative splicing is one of the great unanswered questions in cellular biology. There is strong evidence for alternative splicing at the transcript level, and transcriptomics experiments show that many splice events are tissue specific. It has been suggested that alternative splicing evolved in order to remodel tissue-specific protein-protein networks.

Few SINEs of life: Alu elements have little evidence for biological relevance despite elevated translation.

Transposable elements colonize genomes and with time may end up being incorporated into functional regions. SINE Alu elements, which appeared in the primate lineage, are ubiquitous in the human genome and more than a thousand overlap annotated coding exons. Although almost all Alu-derived coding exons appear to be in alternative transcripts, they have been incorporated into the main coding transcript in at least 11 genes.

The CAFA challenge reports improved protein function prediction and new functional annotations for hundreds of genes through experimental screens.

Background: The Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function.

Results: Here, we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes.

GENCODE reference annotation for the human and mouse genomes.

The accurate identification and description of the genes in the human and mouse genomes is a fundamental requirement for high quality analysis of data informing both genome biology and clinical genomics. Over the last 15 years, the GENCODE consortium has been producing reference quality gene annotations to provide this foundational resource. The GENCODE consortium includes both experimental and computational biology groups who work together to improve and extend the GENCODE gene annotation.

Loose ends: almost one in five human genes still have unresolved coding status.

Seventeen years after the sequencing of the human genome, the human proteome is still under revision. One in eight of the 22 210 coding genes listed by the Ensembl/GENCODE, RefSeq and UniProtKB reference databases are annotated differently across the three sets. We have carried out an in-depth investigation on the 2764 genes classified as coding by one or more sets of manual curators and not coding by others.

SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification.

High-throughput sequencing of full-length transcripts using long reads has paved the way for the discovery of thousands of novel transcripts, even in well-annotated mammalian species. The advances in sequencing technology have created a need for studies and tools that can characterize these novel variants. Here, we present SQANTI, an automated pipeline for the classification of long-read transcripts that can assess the quality of data and the preprocessing pipeline using 47 unique descriptors.