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Large-scale genomic initiatives, such as the Earth BioGenome Project, require efficient methods for eukaryotic genome annotation. Here we present an automatic gene finder, GeneMark-ETP, integrating genomic-, transcriptomic-, and protein-derived evidence that has been developed with a focus on large plant and animal genomes. GeneMark-ETP first identifies genomic loci where extrinsic data are sufficient for making gene predictions with "high confidence." The genes situated in the genomic space between the high-confidence genes are predicted in the next stage. The set of high-confidence genes serves as an initial training set for the statistical model. Further on, the model parameters are iteratively updated in the rounds of gene prediction and parameter re-estimation. Upon reaching convergence, GeneMark-ETP makes the final predictions and delivers the whole complement of predicted genes. GeneMark-ETP outperforms gene finders using a single type of extrinsic evidence. Comparisons with gene finders MAKER2 and TSEBRA, those that use both transcript- and protein-derived extrinsic evidence, show that GeneMark-ETP delivers state-of-the-art gene-prediction accuracy, with the margin of outperforming existing approaches increasing in its application to larger and more complex eukaryotic genomes., (© 2024 Brůna et al.; Published by Cold Spring Harbor Laboratory Press.)

Gene prediction has remained an active area of bioinformatics research for a long time. Still, gene prediction in large eukaryotic genomes presents a challenge that must be addressed by new algorithms. The amount and significance of the evidence available from transcriptomes and proteomes vary across genomes, between genes, and even along a single gene. User-friendly and accurate annotation pipelines that can cope with such data heterogeneity are needed. The previously developed annotation pipelines BRAKER1 and BRAKER2 use RNA-seq or protein data, respectively, but not both. A further significant performance improvement integrating all three data types was made by the recently released GeneMark-ETP. We here present the BRAKER3 pipeline that builds on GeneMark-ETP and AUGUSTUS, and further improves accuracy using the TSEBRA combiner. BRAKER3 annotates protein-coding genes in eukaryotic genomes using both short-read RNA-seq and a large protein database, along with statistical models learned iteratively and specifically for the target genome. We benchmarked the new pipeline on genomes of 11 species under an assumed level of relatedness of the target species proteome to available proteomes. BRAKER3 outperforms BRAKER1 and BRAKER2. The average transcript-level F1-score is increased by about 20 percentage points on average, whereas the difference is most pronounced for species with large and complex genomes. BRAKER3 also outperforms other existing tools, MAKER2, Funannotate, and FINDER. The code of BRAKER3 is available on GitHub and as a ready-to-run Docker container for execution with Docker or Singularity. Overall, BRAKER3 is an accurate, easy-to-use tool for eukaryotic genome annotation., (© 2024 Gabriel et al.; Published by Cold Spring Harbor Laboratory Press.)