Probe Capture-Seq    

While gene editing tools are designed to target specific sequences, they may induce unintended DNA modifications at similar sequences throughout the genome. These off-target effects can potentially disrupt normal gene function, impacting the safety and efficacy of gene editing. Creative Biogene introduces a comprehensive off-target detection service based on liquid-phase hybridization capture technology - Probe Capture-Seq, a versatile targeted NGS platform specifically designed for extensive genomic region characterization.

Probe Capture-Seq technology offers advantages including simplified probe design, high tolerance, and flexible coverage of target regions ranging from kilobases to megabases. Our service enables the accurate detection of low-frequency variants and achieves broad coverage across multiple genomic regions while maintaining cost-effective multiplexing capabilities through optimized workflows with reduced hands-on time.

Figure 1. Overview of the target enrichment process. (Munyuza C, et al., 2022)

Creative Biogene provides comprehensive service support from sample processing to data analysis, ensuring the highest quality standards at every step. Our expert team offers customized experimental design and analysis solutions to help you obtain the most accurate and comprehensive off-target analysis results.

Table 1. Sample Preparation Requirements and Quality

Sample Preparation	Requirements
Sample Type	Fresh tissue/cells
	High molecular weight genomic DNA
Sample Quantity	Tissue: ≥100 mg
	Cells: ≥2 × 10⁶ cells
	gDNA: ≥2 μg
Quality Control Indicators	DNA Integrity: >50 kb
	Purity: A260/280 = 1.8-2.0
	Concentration: >50 ng/μl

Workflow Steps

1. Library Construction: Utilizing standard DNA library preparation protocols, we fragment genomic DNA and perform adapter ligation using standard DNA library preparation protocols.

2. Library Pooling: Multiple indexed sample libraries are combined for efficient processing.

3. Hybridization Reaction: Biotinylated probes bind to target regions, ensuring optimal capture efficiency through solution-phase hybridization.

4. Capture Process: Streptavidin magnetic beads isolate specific target fragments by binding to the biotinylated probe-DNA complexes.

5. Washing Procedures: A series of washing steps, both hot and at room temperature, enhance capture specificity by removing non-specifically bound fragments.

6. PCR Amplification and Purification: Captured libraries are enriched and prepared for sequencing.

7. Bioinformatics Analysis: We detect variants such as SNVs, indels, and structural variations while also analyzing editing efficiency and modification patterns.

Figure 2. Targeted Capture Sequencing Detection Workflow

Service Advantages

1. Pre-designed and custom panels available: Quickly apply existing panels or support custom designs to meet specific research goals.

2. High sensitivity and low false positive rate: Using molecular tagging technology, detection limits as low as 0.1%, ensuring accuracy and reliability of results.

3. Reproducibility and reliability: Consistently cover the same regions with each sequencing run, with a target data missing rate of less than 1%, suitable for commercialization and agricultural research.

4. Fast turnaround and efficient analysis: Complete the entire process within 8 weeks, from tissue/DNA to Fastq data and variant analysis.

5. Compatible with historical data: Compatible with existing data, facilitating data integration and comparative analysis.