ProTInSeq: transposon insertion tracking by ultra-deep DNA sequencing to identify translated large and small ORFs

Miravet Verde, Samuel, 1992-; Mazzolini, Rocco; Segura-Morales, Carolina; Broto, Alicia; Lluch-Senar, Maria 1982-; Serrano Pubull, Luis, 1982-

doi:http://dx.doi.org/10.1038/s41467-024-46112-2

ProTInSeq: transposon insertion tracking by ultra-deep DNA sequencing to identify translated large and small ORFs

License

Citation

Miravet-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/s41467-024-46112-2

Abstract

Identifying 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.