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

Samuel Miravet-Verde (), Rocco Mazzolini, Carolina Segura-Morales, Alicia Broto, Maria Lluch-Senar () and Luis Serrano ()
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Samuel Miravet-Verde: The Barcelona Institute of Science and Technology, Dr Aiguader 88
Rocco Mazzolini: Pulmobiotics, Dr Aiguader 88
Carolina Segura-Morales: The Barcelona Institute of Science and Technology, Dr Aiguader 88
Alicia Broto: The Barcelona Institute of Science and Technology, Dr Aiguader 88
Maria Lluch-Senar: Pulmobiotics, Dr Aiguader 88
Luis Serrano: The Barcelona Institute of Science and Technology, Dr Aiguader 88

Nature Communications, 2024, vol. 15, issue 1, 1-17

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-46112-2

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