BID-Seq: Quantitative Pseudouridine Mapping at Single-Base Resolution Without Antibodies

Uncover transcriptome-wide pseudouridine (Ψ) modifications with base-level precision using CD Genomics' BID-Seq platform. This bisulfite-induced deletion sequencing method offers unmatched sensitivity, single-base resolution, and absolute quantification—without antibodies or enzymes. Whether you're investigating mRNA stability, stop codon readthrough, or writer protein dynamics, BID-Seq empowers your RNA epitranscriptomics research with clarity and confidence.

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Introduction

Why Understanding Pseudouridine (Ψ) Modifications Requires BID-Seq

Pseudouridine (Ψ) is one of the most abundant and evolutionarily conserved RNA modifications, long studied in rRNA and tRNA. Yet in messenger RNA (mRNA), where its role may be equally critical, it remains poorly characterized—not due to lack of biological importance, but because of the limitations of existing detection technologies.

Standard pseudouridine mapping methods such as Ψ-seq and CeU-seq rely on indirect chemistry or antibody enrichment, providing low resolution, high background, or incomplete coverage. They fail to answer two essential questions for RNA biologists and transcriptomic researchers:

Where exactly is pseudouridine located in mRNA, at single-nucleotide resolution?
How much of a transcript is modified at a given site, and how does this vary by condition or cell type?

Without answers to these, understanding the functional consequences of Ψ on mRNA splicing, translation, decay, or stop codon dynamics remains speculative.

BID-Seq Enables Base-Level Clarity, Quantification, and Confidence

BID-Seq—short for Bisulfite-Induced Deletion Sequencing—is the first technology to overcome these barriers. Instead of relying on RT termination or enrichment, BID-Seq exploits a chemically precise conversion of Ψ into a reverse transcription–induced deletion signal, producing clear, quantifiable, and highly reproducible markers of pseudouridine at single-base resolution.

Scientific infographic comparing traditional Ψ detection methods with BID-Seq. On the left, low-resolution, noisy results from antibody-based or CMC methods. On the right, BID-Seq enables precise pseudouridine mapping with clear deletion signals, quantitative stoichiometry, and high confidence at single-nucleotide resolution. Central focus on mRNA structure and detection accuracy.

This enables researchers to:

Quantify modification stoichiometry across conditions and tissues
Detect dynamic changes in pseudouridylation in response to stress, disease, or differentiation
Map endogenous Ψ in critical regions, including CDS, 3′ UTRs, and stop codons
Associate specific PUS enzymes with modification profiles using knockdown or knockout models

Why It Matters Now

As RNA modification research shifts from static catalogs to functional regulation, the ability to resolve pseudouridine quantitatively and spatially becomes essential. With BID-Seq, researchers are finally equipped to explore Ψ as a true regulatory mark—on par with m6A—in both basic and translational transcriptomics.

How BID-Seq Works – From Chemistry to Confidence

At the heart of BID-Seq is a chemically precise transformation of pseudouridine (Ψ) into a detectable signal during reverse transcription—without compromising sequence complexity or requiring any enrichment.

Vertical infographic illustrating the three-step process of BID-Seq. Step 1 shows selective pseudouridine (Ψ) conversion to a Ψ–BS adduct at neutral pH. Step 2 depicts reverse transcription where the Ψ–BS adduct causes a deletion signal. Step 3 visualizes base-resolution detection and quantification of deletion frequencies.

Step 1: Selective Ψ Conversion at Neutral pH

Unlike conventional bisulfite protocols that aggressively deaminate cytosine (C→U), BID-Seq employs neutral pH bisulfite chemistry that selectively reacts with pseudouridine to form a Ψ–BS adduct. This optimized environment preserves unmodified bases, maintaining the full complexity of the RNA sequence for accurate downstream analysis.

Step 2: Deletion Signal During Reverse Transcription

The Ψ–BS adduct causes reverse transcriptase to skip a base during cDNA synthesis—creating a reproducible single-nucleotide deletion at the modification site. This deletion event forms the molecular "signature" of Ψ, uniquely distinguishable from sequencing errors or structural noise.

Step 3: Base-Resolution Detection and Quantification

Using calibrated synthetic RNA libraries and motif-aware models, deletion frequencies are interpreted as quantitative measures of pseudouridylation stoichiometry—not just presence/absence. The approach supports:

Absolute modification fraction calculations
Detection of dynamic changes across conditions
Motif enrichment and writer protein assignment
Mapping Ψ in CDS, UTRs, and stop codons with high confidence

How BID-Seq Compares to Other Pseudouridine Sequencing Methods

Understanding what sets BID-Seq apart requires a look at the current limitations in the field. Conventional Ψ mapping methods fall into two categories: termination-based detection (e.g., Ψ-seq, Pseudo-seq) and enrichment-based detection (e.g., CeU-seq). Each has critical trade-offs.

Method	Resolution	Quantitative	Background Interference	Enrichment Required	Limitation
Ψ-seq / Pseudo-seq	Low	❌	High (RT noise, false stops)	Yes (CMC)	Inconsistent between datasets
CeU-seq	Medium	❌	Moderate	Yes (biotin enrichment)	No stoichiometry or base-level mapping
Bisulfite-Seq (standard)	N/A	❌	High (C→U dominates)	No	Fails to detect Ψ reliably
BID-Seq	Single-base	✅	Minimal	No

Why BID-Seq Is the Preferred Choice

Truly quantitative: Deletion signal reflects actual modification fraction
Unambiguous detection: Base-resolution calling, not motif-based inference
No antibody dependency: Eliminates variability from commercial reagents
High sample compatibility: Works with low-input RNA and diverse tissues
Designed for publication: Based on peer-reviewed protocol (Nat Biotechnol)

Workflow

Service Workflow – From Sample to Results

Our BID-Seq service is designed as a full-scope, researcher-friendly workflow—from sample submission to bioinformatic insights—so you can focus on interpreting your findings, not managing technical details.

Our team remains available to support any downstream analysis, publication preparation, or experimental follow-up.

Bioinformatics

Bioinformatics & Data Analysis – From Signal to Scientific Insight

BID-Seq doesn't stop at sequencing—it transforms chemical deletion signals into high-resolution, interpretable RNA modification maps. Our integrated bioinformatics pipeline ensures your data is not only clean and accurate, but also biologically meaningful.

Core Data Outputs

Ψ site identification at single-nucleotide resolution
Deletion-based quantification of modification fraction
Transcriptome-wide Ψ profiling: including CDS, UTRs, and stop codons
Comparison across conditions, tissues, or knockdown models

Annotation & Contextualization

Each pseudouridine site is annotated with:

Transcript features (CDS, 3′UTR, exon/intron boundaries)
Associated motif context (e.g., GUΨCN, poly-U, ΨGA near stop codons)
Predicted or validated PUS writer enzymes based on existing models or user-provided knockdown data
Ψ fraction (stoichiometry) at site and transcript levels

Visualizations Included

We provide clean, publication-ready outputs to help you interpret and present your results:

IGV-compatible tracks for Ψ-site overlay on genome browsers
Heatmaps of modification fraction across samples or conditions
Metagene plots showing regional Ψ distribution
Motif enrichment plots and position weight matrices
Scatter and volcano plots for condition-specific Ψ dynamics

Optional Add-On Analyses

Correlation with gene expression (RNA-seq) or mRNA half-life
Ψ modification clustering for cell type or tissue classification
Integration with functional annotations: GO, KEGG, or pathway mapping
Support for custom writer–target validation and experimental design

Whether you're profiling Ψ under stress, mapping dynamic regulation, or validating the function of a PUS enzyme, our analytical workflows help you move from data to discovery—with clarity, speed, and confidence.

Applications

Applications – Where BID-Seq Delivers Scientific Value

BID-Seq is more than a sequencing solution—it's a platform for discovery. Whether you're exploring the landscape of RNA modifications or dissecting their functional impact on gene regulation, BID-Seq empowers you with actionable insights at single-base precision.

✧ Transcriptome-Wide Ψ Mapping

Build comprehensive pseudouridine atlases in various biological systems:

Human or mouse cell lines
Primary tissues across organs or developmental stages
Model organisms or engineered cell types

Quantify and localize Ψ in CDS, 3′UTRs, and stop codons—regions often linked to RNA stability and translation efficiency.

✧ Comparative Epitranscriptomics

Use BID-Seq to study Ψ modification dynamics under diverse biological conditions:

Cellular stress (e.g., heat shock, oxidative stress)
Immune activation and cytokine exposure
Differentiation, senescence, or tumor transformation
Environmental or chemical perturbations

Track condition-specific shifts in Ψ stoichiometry with base-level resolution.

✧ Writer Enzyme Functional Validation

Validate pseudouridine synthase (PUS) targets via:

RNA profiling in PUS knockout or knockdown models
Assigning Ψ sites to specific enzymes (e.g., TRUB1, PUS7, PUS1)
Linking motifs (e.g., GUΨCN, poly-U) to enzyme activity

Correlate enzyme loss with quantitative changes in Ψ at defined positions.

✧ mRNA Stability & Translational Control

Link Ψ modification with post-transcriptional regulation:

Analyze mRNA half-life changes associated with Ψ-enriched transcripts
Study how Ψ influences mRNA decay rates, translation efficiency, or localization
Integrate BID-Seq with RNA-seq to infer Ψ-dependent expression dynamics

✧ Readthrough at Stop Codons

Investigate a unique regulatory role of pseudouridine:

Identify Ψ-modified stop codons (ΨGA, ΨAA, ΨAG motifs)
Quantify readthrough efficiency under natural or engineered conditions
Assess implications for protein isoform diversity or coding potential

✧ Tissue-Specific & Disease-Associated Ψ Signatures

Use BID-Seq to uncover Ψ-based biomarkers:

Map tissue-enriched or cell-type–specific Ψ patterns
Discover altered Ψ profiles in disease models (e.g., cancer, neurodegeneration)
Explore mitochondrial mRNA pseudouridylation and its functional implications

Sample Requirements

Sample Requirements – What You Need to Get Started

Sample Type	Minimum Amount	Notes
Whole blood	> 2 mL	Use EDTA tubes only. Heparin is not compatible.
Cultured cells	> 5 × 10⁶ cells	Cell pellets preferred.
Tissue	100 mg	Fresh or frozen. Avoid necrotic material.
Total RNA	> 10 µg	OD260/280: 1.6–2.3; No visible degradation.

Shipping Instructions

Place sample in a 1.5 mL RNase-free microcentrifuge tube
Seal with parafilm or cap lock
Ship on dry ice with adequate insulation

Storage Guidelines

Cells / tissues: Preserve in TRIzol or RNA stabilization solution; snap freeze in liquid nitrogen and store at –80 °C
RNA: Resuspend in ethanol or RNase-free ultrapure water; store at –80 °C; avoid freeze–thaw cycles

For other sample types or low-input projects, please contact us for custom recommendations.

Advantages

Why Choose CD Genomics

At CD Genomics, we understand that RNA modification research demands more than just sequencing—it requires precision, reproducibility, and domain-specific insight. That's why we've built our BID-Seq platform not just as a technology offering, but as a research-enabling partnership.

Based on Peer-Reviewed Science

Our BID-Seq workflow is grounded in the original protocol published in Nature Biotechnology—recognized for enabling the first base-resolution, quantitative mapping of mRNA pseudouridines. We've optimized it for service scalability without compromising data integrity.

Full-Stack Expertise: Wet Lab + Bioinformatics

From sample QC and bisulfite chemistry to deletion-calling pipelines and transcriptomic annotation, every step is performed in-house by teams who specialize in RNA modification biology. This ensures end-to-end control and consistent quality.

Designed for Biological Interpretation

We go beyond raw site calling—our analysis highlights transcript-region specificity, motif context, writer enzyme association, and condition-dependent Ψ dynamics. That means your data is ready to support hypothesis generation, figure preparation, or publication.

Compatible with Low-Input and Diverse Samples

Whether working with rare primary cells, low-yield RNA, or custom model systems, BID-Seq adapts. We offer protocol adjustments and expert consultation to accommodate your study design.

Responsive Scientific Support

We understand that your project doesn't end with data delivery. Our team is available to answer follow-up questions, assist with downstream interpretation, or help design validation strategies. You're never left decoding your results alone.

Case Study

Uncovering Pseudouridine in Mammalian mRNA

Title: Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution

Published in: Nature Biotechnology, 2022 (Dai et al.)

DOI: https://doi.org/10.1038/s41587-022-01505-w

Overview

This foundational study demonstrated the power of BID-Seq to achieve single-nucleotide resolution and quantitative mapping of pseudouridine (Ψ) in mammalian mRNA. Using just 10–20 ng of total RNA, the authors profiled Ψ sites in multiple human cell lines and across 12 mouse tissues, identifying thousands of previously unannotated modifications.

Key Findings

Transcriptome-wide Ψ detection: Hundreds of Ψ sites were mapped in HeLa, HEK293T, and A549 cells; mouse tissues showed extensive tissue-specific Ψ signatures.
Writer enzyme assignments: The study linked Ψ deposition to enzymes like TRUB1, PUS7, and PUS1, demonstrating that TRUB1-mediated pseudouridylation stabilizes mRNAs.
Ψ at stop codons: Endogenous pseudouridine was found at stop codons in dozens of transcripts. Functional assays confirmed this modification promotes stop codon readthrough, expanding translational potential.

Significance

This work established BID-Seq as the first method to: