RT Journal Article
SR Electronic
T1 Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct and long-read sequencing
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.11.03.467190
DO 10.1101/2021.11.03.467190
A1 Sepideh Tavakoli
A1 Mohammad Nabizadehmashhadtoroghi
A1 Amr Makhamreh
A1 Howard Gamper
A1 Caroline A. McCormick
A1 Neda K. Rezapour
A1 Ya-Ming Hou
A1 Meni Wanunu
A1 Sara H. Rouhanifard
YR 2022
UL http://biorxiv.org/content/early/2022/11/10/2021.11.03.467190.abstract
AB We developed and applied a semi-quantitative method for 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 identified many known sites of pseudouridylation and uncovered new uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases. Identified sites were validated using 1,000-mer synthetic RNA controls bearing a single pseudouridine in the center position which demonstrate systematical under-calling using our approach. We identify mRNAs with up to 7 unique modification sites. Our pipeline allows direct detection of low-, medium-, and high-occupancy pseudouridine modifications on native RNA molecules from nanopore sequencing data as well as multiple modifications on the same strand.Competing Interest StatementThe authors have declared no competing interest.