Miravet Verde, Samuel, 1992-Mazzolini, RoccoSegura-Morales, CarolinaBroto, AliciaLluch-Senar, Maria 1982-Serrano Pubull, Luis, 1982-2024-07-312024-07-312024Miravet-Verde S, Mazzolini R, Segura-Morales C, Broto A, Lluch-Senar M, Serrano L. ProTInSeq: transposon insertion tracking by ultra-deep DNA sequencing to identify translated large and small ORFs. Nat Commun. 2024 Mar 7;15(1):2091. DOI: 10.1038/s41467-024-46112-22041-1723http://hdl.handle.net/10230/60870Identifying open reading frames (ORFs) being translated is not a trivial task. ProTInSeq is a technique designed to characterize proteomes by sequencing transposon insertions engineered to express a selection marker when they occur in-frame within a protein-coding gene. In the bacterium Mycoplasma pneumoniae, ProTInSeq identifies 83% of its annotated proteins, along with 5 proteins and 153 small ORF-encoded proteins (SEPs; ≤100 aa) that were not previously annotated. Moreover, ProTInSeq can be utilized for detecting translational noise, as well as for relative quantification and transmembrane topology estimation of fitness and non-essential proteins. By integrating various identification approaches, the number of initially annotated SEPs in this bacterium increases from 27 to 329, with a quarter of them predicted to possess antimicrobial potential. Herein, we describe a methodology complementary to Ribo-Seq and mass spectroscopy that can identify SEPs while providing other insights in a proteome with a flexible and cost-effective DNA ultra-deep sequencing approach.application/pdfeng© The Author(s) 2024, corrected publication 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.info:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s41467-024-46112-2Bacterial geneticsNext-generation sequencingOpen reading framesProteinsinfo:eu-repo/semantics/openAccess