5caC Chemically Assisted Bisulfite Sequencing (caCAB-seq) Service

While traditional bisulfite sequencing is invaluable for DNA methylation analysis, it lacks the specificity to distinguish the final oxidative product of active demethylation, 5-carboxylcytosine (5caC), from other cytosine modifications. To address this, CD BioSciences provides a specialized 5caC chemically assisted bisulfite sequencing (caCAB-seq) service, offering researchers a powerful tool to directly map this rare but critical epigenetic mark with high precision.

Introduction to caCAB-seq

5-Carboxylcytosine (5caC) is the terminal oxidation product in the active DNA demethylation pathway initiated by TET enzymes. Its presence is transient and low in abundance, yet it serves as a key intermediate for subsequent base excision repair, making its precise localization crucial for understanding the final stages of demethylation.

Standard bisulfite sequencing fails to differentiate 5caC from 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), as all are read as cytosine. caCAB-seq overcomes this limitation through targeted chemical decarboxylation prior to bisulfite treatment. This chemical step specifically converts 5caC to 5-hydroxymethyluracil (5hmU), which is subsequently read as thymine (T) during sequencing. By comparing caCAB-seq data with conventional BS-seq or other oxidative bisulfite sequencing data, genuine 5caC sites can be uniquely identified at single-base resolution, providing direct insight into the endpoints of active DNA demethylation.

Fig.1 Deamination mechanism of 5caC under bisulfite treatment. (Lu X, et al., 2013)

Features of caCAB-seq

The caCAB-seq technique is engineered with distinct characteristics that make it uniquely suited for 5caC analysis:

1. Targeted Specificity: Employs selective chemical decarboxylation to convert only 5caC to a distinct sequencing signal, effectively isolating it from 5mC, 5hmC, and 5fC.
2. Single-Base Resolution: Enables precise, nucleotide-level mapping of 5caC sites across the entire genome.
3. High Sensitivity: Designed to detect the low-abundance 5caC modifications that are characteristic of this transient mark.
4. Genome-Wide Profiling: Provides an unbiased, comprehensive view of 5caC distribution in all genomic regions.
5. Direct Chemical Readout: Utilizes a defined chemical conversion, ensuring method robustness and reproducibility across experiments.

Our Services

At CD BioSciences, our comprehensive caCAB-seq service provides researchers with a direct pathway to explore the elusive 5-carboxylcytosine (5caC) landscape. By managing the intricate chemical conversion and sequencing workflow, we transform challenging samples into clear, actionable datasets. From precise experimental handling to insightful bioinformatics interpretation, we support your investigation into critical biological processes where 5caC plays a definitive role.

Workflow of caCAB-seq Service

Our caCAB-seq service follows a rigorously optimized, multi-stage workflow designed to ensure precision and reproducibility at every step. From initial consultation to final data delivery, each phase is executed by specialized teams and supported by stringent quality controls, guaranteeing results that meet the highest scientific standards.

Project Design

We initiate every project with a detailed consultation to understand your specific research objectives. Our epigenetic specialists then design a tailored experimental and bioinformatic plan, determining optimal sequencing depth, controls, and analysis parameters to maximize the relevance and impact of your data.

Sample Submission & QC

Following project confirmation, you receive clear protocols for sample preparation and shipment. Upon arrival, all samples undergo rigorous quality assessment, including quantification, purity analysis, and integrity checks, to ensure they meet the strict input requirements for the sensitive chemical conversion process.

Library Construction

This phase is performed by our experts using optimized protocols. Genomic DNA undergoes the specific chemical decarboxylation reaction to convert 5caC, followed by bisulfite treatment. High-quality sequencing libraries are then constructed from the converted DNA, with efficiency verified at multiple checkpoints.

Report Delivery

We deliver a complete project report containing all processed data, publication-ready figures, detailed methodology, and a clear summary of biological insights. Our scientists remain available for a results review session to help you interpret the findings and plan subsequent research steps.

Bioinformatics Analysis

Our dedicated bioinformatics team processes data through a validated pipeline for 5caC detection. Analysis includes precise read alignment, 5caC site calling at single-base resolution, genomic annotation, differential analysis between sample groups, and the generation of comprehensive visualizations for interpretation.

High-Throughput Sequencing

Libraries are sequenced on Illumina platforms to achieve the agreed-upon depth and coverage. We perform real-time monitoring of sequencing metrics and deliver a primary data quality control report, ensuring the raw data meets our high standards before proceeding to advanced analysis.

Supported Sample Types

1. Purified Genomic DNA
2. Cultured Cells (e.g., mammalian)
3. Fresh-Frozen Tissue

1. Blood and Blood Products
2. FFPE Tissue Sections
3. Other Types (Please Inquire)

Our Advantages

1. Specialized Protocol Expertise: We apply a meticulously optimized and validated caCAB-seq protocol to ensure high sensitivity and accuracy for detecting low-abundance 5caC.
2. Integrated Service Pipeline: We manage the entire project, providing a seamless turnkey solution from initial design to final data interpretation.
3. Dedicated Bioinformatics Support: Our analysis goes beyond standard calling, offering advanced customization and integration with other epigenetic datasets upon request.
4. End-to-End Quality Control: We maintain rigorous QC standards at every stage, from sample assessment to final data validation, guaranteeing reliable results.
5. Collaborative Scientific Partnership: You benefit from direct access to our scientific team for consultation and are assured of complete data security and confidentiality.

For comprehensive 5caC profiling beyond caCAB-seq, CD BioSciences provides a suite of complementary analytical solutions. These include global quantification via LC-MS/MS and ELISA, alongside advanced mapping through MAB-seq, enabling fully customized epigenetic investigation. If you are interested in our services, please feel free to contact us for more details and quotation information of related services.

Reference 1. Lu X, Song C X, Szulwach K, et al. Chemical modification-assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection in DNA[J]. Journal of the American Chemical Society, 2013, 135(25): 9315-9317.