Document Type
Article
Publication Date
1-19-2023
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.
Recommended Citation
Tavakoli, Sepideh; Nabizadeh, Mohammad; Makhamreh, Amr; Gamper, Howard; McCormick, Caroline A; Rezapour, Neda K; Hou, Ya-Ming; Wanunu, Meni; and Rouhanifard, Sara H, "Semi-quantitative Detection of Pseudouridine Modifications and Type I/II I/Ii Hypermodifications in Human mRNAs Using Direct Long-Read Sequencing" (2023). Department of Biochemistry and Molecular Biology Faculty Papers. Paper 233.
https://jdc.jefferson.edu/bmpfp/233
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.
PubMed ID
36658122
Language
English
Comments
This article is the author’s final published version in Nature Communications, Volume 14, Issue 1, December 2023, Article number 334.
The published version is available at https://doi.org/10.1038/s41467-023-35858-w. Copyright © Tavakoli et al.