Berg, Kevin and Lodha, Manivel and Delazer, Isabel and Bartosik, Karolina and Garcia, Yilliam Cruz and Hennig, Thomas and Wolf, Elmar and Doelken, Lars and Lusser, Alexandra and Prusty, Bhupesh K. and Erhard, Florian (2024) Correcting 4sU induced quantification bias in nucleotide conversion RNA-seq data. NUCLEIC ACIDS RESEARCH, 52 (7). e35. ISSN 0305-1048, 1362-4962
Full text not available from this repository. (Request a copy)Abstract
Nucleoside analogues like 4-thiouridine (4sU) are used to metabolically label newly synthesized RNA. Chemical conversion of 4sU before sequencing induces T-to-C mismatches in reads sequenced from labelled RNA, allowing to obtain total and labelled RNA expression profiles from a single sequencing library. Cytotoxicity due to extended periods of labelling or high 4sU concentrations has been described, but the effects of extensive 4sU labelling on expression estimates from nucleotide conversion RNA-seq have not been studied. Here, we performed nucleotide conversion RNA-seq with escalating doses of 4sU with short-term labelling (1h) and over a progressive time course (up to 2h) in different cell lines. With high concentrations or at later time points, expression estimates were biased in an RNA half-life dependent manner. We show that bias arose by a combination of reduced mappability of reads carrying multiple conversions, and a global, unspecific underrepresentation of labelled RNA emerging during library preparation and potentially global reduction of RNA synthesis. We developed a computational tool to rescue unmappable reads, which performed favourably compared to previous read mappers, and a statistical method, which could fully remove remaining bias. All methods developed here are freely available as part of our GRAND-SLAM pipeline and grandR package. Graphical Abstract
| Item Type: | Article |
|---|---|
| Subjects: | 000 Computer science, information & general works > 004 Computer science 600 Technology > 610 Medical sciences Medicine |
| Divisions: | Informatics and Data Science > Department Computational Life Science > Chair of Computational Immunology (Prof. Dr. Florian Erhard) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 27 Jan 2026 09:56 |
| Last Modified: | 27 Jan 2026 09:56 |
| URI: | https://pred.uni-regensburg.de/id/eprint/64457 |
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