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

Author

Listed:
  • Samuel Miravet-Verde

    (The Barcelona Institute of Science and Technology, Dr Aiguader 88
    Institute of Microbiology and Swiss Institute of Bioinformatics, ETH Zurich)

  • 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
    Universitat Autònoma de Barcelona)

  • Luis Serrano

    (The Barcelona Institute of Science and Technology, Dr Aiguader 88
    Universitat Pompeu Fabra (UPF)
    ICREA, Pg. Lluis Companys 23)

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.

Suggested Citation

  • Samuel Miravet-Verde & Rocco Mazzolini & Carolina Segura-Morales & Alicia Broto & Maria Lluch-Senar & Luis Serrano, 2024. "ProTInSeq: transposon insertion tracking by ultra-deep DNA sequencing to identify translated large and small ORFs," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-46112-2
    DOI: 10.1038/s41467-024-46112-2
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    References listed on IDEAS

    as
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