Downloads: 69

Files in This Item:
File Description SizeFormat 
j.ygeno.2022.110372.pdf2.11 MBAdobe PDFView/Open
Title: An informatics approach to distinguish RNA modifications in nanopore direct RNA sequencing
Authors: Ramasamy, Soundhar
Mishra, Shubham
Sharma, Surbhi
Parimalam, Sangamithirai Subramanian
Vaijayanthi, Thangavel
Fujita, Yoto
Kovi, Basavaraj
Sugiyama, Hiroshi  kyouindb  KAKEN_id  orcid (unconfirmed)
Pandian, Ganesh N
Author's alias: 藤田, 陽子
杉山, 弘
Keywords: RNA modifications
Nanopore sequencing
Chemical probe
Trace value
Issue Date: Apr-2022
Publisher: Elsevier BV
Journal title: Genomics
Volume: 114
Issue: 3
Thesis number: 110372
Abstract: Modifications in RNA can influence their structure, function, and stability and play essential roles in gene expression and regulation. Methods to detect RNA modifications rely on biophysical techniques such as chromatography or mass spectrometry, which are low throughput, or on high throughput short-read sequencing techniques based on selectively reactive chemical probes. Recent studies have utilized nanopore-based fourth-generation sequencing methods to detect modifications by directly sequencing RNA in its native state. However, these approaches are based on modification-associated mismatch errors that are liable to be confounded by SNPs. Also, there is a need to generate matched knockout controls for reference, which is laborious. In this work, we introduce an internal comparison strategy termed “IndoC, ” where features such as ‘trace’ and ‘current signal intensity’ of potentially modified sites are compared to similar sequence contexts on the same RNA molecule within the sample, alleviating the need for matched knockout controls. We first show that in an IVT model, ‘trace’ is able to distinguish between artificially generated SNPs and true pseudouridine (Ψ) modifications, both of which display highly similar mismatch profiles. We then apply IndoC on yeast and human ribosomal RNA to demonstrate that previously reported Ψ sites show marked changes in their trace and signal intensity profiles compared with their unmodified counterparts in the same dataset. Finally, we perform direct RNA sequencing of RNA containing Ψ intact with a chemical probe adduct (N-cyclohexyl-N′-β-(4-methylmorpholinium) ethylcarbodiimide [CMC]) and show that CMC reactivity also induces changes in trace and signal intensity distributions in a Ψ specific manner, allowing their separation from high mismatch sites that display SNP-like behavior.
Description: Reading RNA modifications more precisely in a pocket-sized device. 京都大学プレスリリース. 2022-08-24.
Rights: © 2022 The Authors. Published by Elsevier Inc.
This is an open access article under the Creative Commons Attribution 4.0 International license.
DOI(Published Version): 10.1016/j.ygeno.2022.110372
PubMed ID: 35460817
Related Link:
Appears in Collections:Journal Articles

Show full item record

Export to RefWorks

Export Format: 

This item is licensed under a Creative Commons License Creative Commons