PT  - JOURNAL ARTICLE
AU  - Kevin Berg
AU  - Manivel Lodha
AU  - Yilliam Cruz Garcia
AU  - Thomas Hennig
AU  - Elmar Wolf
AU  - Bhupesh K Prusty
AU  - Florian Erhard
TI  - Correcting 4sU induced quantification bias in nucleotide conversion RNA-seq data
AID  - 10.1101/2023.04.21.537786
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2023.04.21.537786
4099  - http://biorxiv.org/content/early/2023/04/21/2023.04.21.537786.short
4100  - http://biorxiv.org/content/early/2023/04/21/2023.04.21.537786.full
AB  - 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 due to impaired reverse transcription efficiency 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.Competing Interest StatementThe authors have declared no competing interest.