TY - JOUR AU - Tavakoli, Sepideh AU - Nabizadeh, Mohammad AU - Makhamreh, Amr AU - Gamper, Howard AU - McCormick, Caroline A. AU - Rezapour, Neda K. AU - Hou, Ya-Ming AU - Wanunu, Meni AU - Rouhanifard, Sara H. PY - 2023 DA - 2023/01/19 TI - Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing JO - Nature Communications SP - 334 VL - 14 IS - 1 AB - 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. SN - 2041-1723 UR - https://doi.org/10.1038/s41467-023-35858-w DO - 10.1038/s41467-023-35858-w ID - Tavakoli2023 ER -