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BID-seq Chemistry-Based Pseudouridine Sequencing Without Cytosine Deamination

Lead Inventor: Chuan He

SUMMARY

More sensitive, quantitative mapping of RNA pseudouridine at single-base resolution is provided by a mild bisulfite-based sequencing method that converts the modification into a detectable deletion signature

The Unmet Need: Existing methods struggle to sensitively and quantitatively identify RNA pseudouridine sites

  • Pseudouridine is the most abundant RNA modification, yet established detection methods have limitations. CMC-based approaches can produce false-positive reverse-transcription stops and inconsistent site detection, while enrichment methods do not quantify modification levels and may rely on reagents with limited storage stability. Earlier bisulfite sequencing approaches produced weak signals and could convert cytidines, complicating sequence alignment.

  • Advances in RNA epitranscriptomics are increasing demand for methods that can map RNA modifications at single-base resolution and measure their prevalence across molecules. Sequencing workflows that preserve sequence complexity while supporting quantitative analysis offer an opportunity to improve RNA modification profiling.

The Proposed Solution: A mild bisulfite treatment converts pseudouridine into an adduct that generates a quantifiable deletion signature during sequencing

  • The faculty inventor developed BID-seq (bisulfite-induced deletion sequeining)- a technique using near-neutral, room-temperature bisulfite conditions to convert pseudouridine into a bisulfite adduct while avoiding detectable cytosine. During reverse transcription, the adduct produces a deletion that can be identified by amplification and sequencing; calibration with sequence-context-specific standards enables estimation of modification levels. The workflow differs from earlier methods by producing a strong, quantitative signal while preserving sequence complexity. BID-seq was also reported to work with approximately 10–20 ng of RNA and was applied to human cell lines and mouse tissues.

ADVANTAGES

  • Single-base pseudouridine resolution

  • Quantitative modification-level estimates

  • Improved sensitivity over earlier bisulfite sequencing

  • Preserved sequence complexity

  • Low RNA input requirements

  • Detection of RNA modifications across samples

APPLICATIONS

  • Transcriptome-wide pseudouridine profiling in human cell lines

  • Pseudouridine mapping in mouse tissues

  • 5-hydroxymethylcytosine profiling in DNA and RNA

PUBLICATIONS