Semi-quantitative Detection of Pseudouridine Modifications and Type I/II I/Ii Hypermodifications in Human mRNAs Using Direct Long-Read Sequencing
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.
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.
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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.