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Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing

Author

Listed:
  • Sepideh Tavakoli

    (Northeastern University)

  • Mohammad Nabizadeh

    (Northeastern University)

  • Amr Makhamreh

    (Northeastern University)

  • Howard Gamper

    (Thomas Jefferson University)

  • Caroline A. McCormick

    (Northeastern University)

  • Neda K. Rezapour

    (Northeastern University)

  • Ya-Ming Hou

    (Thomas Jefferson University)

  • Meni Wanunu

    (Northeastern University
    Northeastern University)

  • Sara H. Rouhanifard

    (Northeastern University)

Abstract

Here, we develop and apply a semi-quantitative method for the high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identify many known sites of pseudouridylation and uncover previously unreported uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases. Identified sites are validated using 1000-mer synthetic RNA controls bearing a single pseudouridine in the center position, demonstrating systematic under-calling using our approach. We identify mRNAs with up to 7 unique modification sites. Our workflow allows direct detection of low-, medium-, and high-occupancy pseudouridine modifications on native RNA molecules from nanopore sequencing data and multiple modifications on the same strand.

Suggested Citation

  • Sepideh Tavakoli & Mohammad Nabizadeh & Amr Makhamreh & Howard Gamper & Caroline A. McCormick & Neda K. Rezapour & Ya-Ming Hou & Meni Wanunu & Sara H. Rouhanifard, 2023. "Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing," Nature Communications, Nature, vol. 14(1), pages 1-12, December.
  • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-35858-w
    DOI: 10.1038/s41467-023-35858-w
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