Automated prediction and annotation of small proteins in microbial genomes

Recent work performed by Sberro et al. (2019) revealed a vast unexplored space of small proteins existing within the human microbiome. At present, these small open reading frames (smORFs) are unannotated in existing reference genomes and standard genome annotation tools are not able to accurately predict them. In this study, we introduce an annotation tool named SmORFinder that predicts small proteins based on those identified by Sberro et al. This tool combines profile Hidden Markov models (pHMMs) of each small protein family and deep learning models that may better generalize to smORF families not seen in the training set. We find that combining predictions of both pHMM and deep learning models leads to more precise smORF predictions and that these predicted smORFs are enriched for Ribo-Seq or MetaRibo-Seq translation signals. Feature importance analysis reveals that the deep learning models learned to identify Shine-Dalgarno sequences, deprioritize the wobble position in each codon, and group codons in a way that strongly corresponds to the codon synonyms found in the codon table. We perform a core genome analysis of 26 bacterial species and identify many core smORFs of unknown function. We pre-compute small protein annotations for thousands of RefSeq isolate genomes and HMP metagenomes, and we make these data available through a web portal along with other useful tools for small protein annotation and analysis. The systematic identification and annotation of those important small proteins will help researchers to expand our understanding of this exciting field of biology.