figshare
Browse
krnb_a_1462654_sm4833.pptx (254 kB)

Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing

Download (254 kB)
presentation
posted on 2018-04-23, 14:38 authored by Katherine I. Zhou, Wesley C. Clark, David W. Pan, Matthew J. Eckwahl, Qing Dai, Tao Pan

The abundant RNA modification pseudouridine (Ψ) has been mapped transcriptome-wide by chemically modifying pseudouridines with carbodiimide and detecting the resulting reverse transcription stops in high-throughput sequencing. However, these methods have limited sensitivity and specificity, in part due to the use of reverse transcription stops. We sought to use mutations rather than just stops in sequencing data to identify pseudouridine sites. Here, we identify reverse transcription conditions that allow read-through of carbodiimide-modified pseudouridine (CMC-Ψ), and we show that pseudouridines in carbodiimide-treated human ribosomal RNA have context-dependent mutation and stop rates in high-throughput sequencing libraries prepared under these conditions. Furthermore, accounting for the context-dependence of mutation and stop rates can enhance the detection of pseudouridine sites. Similar approaches could contribute to the sequencing-based detection of many RNA modifications.

Funding

This work was supported by the National Institutes of Health (RM1HG008935 to T.P., F30GM120917 to K.I.Z., and K01HG006699 to Q.D.), the National Institutes of Health Medical Scientist Training Program grant (T32GM007281), and the University of Chicago Biological Sciences Division and Frank Family Endowment (K.I.Z.). The University of Chicago Genomics Facility is supported by the Cancer Center Support Grant (P30CA014599).

History

Usage metrics

    RNA Biology

    Licence

    Exports

    RefWorks
    BibTeX
    Ref. manager
    Endnote
    DataCite
    NLM
    DC