In the fields of epigenetics research and early cancer screening, DNA methylation testing has become one of the core technologies. As a leader in domestic gene capture technology, iGeneTech has launched a complete solution for methylation hybridization capture based on independently developed probe design algorithms and experimental systems, covering the entire workflow from sample processing to data analysis.

This article will systematically introduce the technical architecture and product portfolio of iGeneTech’s platform.

Dual-Track Library Construction Solution: Flexible Options for Different Sample Types

For different sample types and input amounts, iGeneTech has developed two sets of methylation library construction kits (single-stranded and double-stranded), forming a complementary technical system.

Figure 1: Comparison of Single-stranded (a) vs. Double-stranded (b) Library Construction Workflows

1. Double-stranded Library Construction Solution: IGT® Methylation Fast Library Prep Kit v2.0

Technical Principle: For high-input and high-integrity genomic DNA (gDNA), the traditional double-stranded ligation method is adopted. Adapter ligation is completed prior to bisulfite conversion, followed by PCR amplification to generate libraries after conversion.

Double-stranded Library Construction Workflow: gDNA → Fragmentation → End Repair → Methylated Adapter Ligation → BS Conversion → PCR Amplification → Library

2. Single-stranded Library Construction Solution: IGT® ssDNA Library Prep Kit v2.0

Technical Principle: For low-input cell-free DNA (cfDNA) or highly degraded gDNA, the Post-BS (bisulfite conversion first, library construction later) strategy is employed. Using single-stranded adapter ligation technology, fragmented DNA segments treated with bisulfite can also be efficiently used for library construction, which greatly improves the utilization rate of original molecules and avoids information loss caused by DNA fragmentation in traditional double-stranded library construction.

Single-stranded Library Construction Workflow: cfDNA → BS Conversion → Single-stranded Adapter Ligation → Extension to Form Complementary Strands → Adapter Ligation on the Other End → PCR Amplification → Library

Core Advantages

l Maximized Template Utilization: Even 5'-end damaged single-stranded DNA can be efficiently converted into libraries.

l Outstanding Low-input Performance: Library construction can be achieved with as little as 1 ng of cfDNA.

l Superior Sequencing Depth: At the same input amount, the sequencing depth of single-stranded library construction is significantly higher than that of the double-stranded protocol.

Core Capture Technologies: BisCap® and MethCap® Dual Platforms for Full-scenario Applications

1. BisCap® Hybridization Capture Technology: A Dedicated Tool for CpG Methylation Detection

Technical Principle: Based on the BisCap® probe hybridization capture system, the capture-after-bisulfite-conversion strategy is adopted. After bisulfite treatment, unmethylated C residues in the genome are converted to U (ultimately T in sequencing). Probe design adopts a dual-state design targeting CG sites on both the positive and negative strands (matching methylated and unmethylated states respectively), ensuring efficient capture of DNA fragments with different methylation statuses.

Technical Advantages:

l Flexible Panel Size: Supports custom capture of any genomic region ranging from 1 kb to 100 Mb.

l High Multiplexing Capacity: Supports up to 8-plex pooled capture for cost efficiency.

l Application Scope: Suitable for CpG methylation research in mammals (e.g., humans, mice) with low CHG/CHH methylation levels.

2. MethCap® Technology Platform: A Versatile Tool for All Types of Methylation Research

Technical Principle: The capture-before-bisulfite-conversion strategy is adopted. It is species-independent and can cover three methylation types (CpG, CHG, CHH) across the entire genome, meeting diverse research needs.

Figure 2: Comparison of Experimental Workflows Between BisCap® and MethCap®

Customized Panel Service System: Semi-customized + Fully Customized Options Tailored to Specific Needs

iGeneTech offers two customization modes to match diverse research requirements, delivering a one-stop service from probe design to final product delivery.

1. Semi-customized Solution

Based on standard products (e.g., Human CpG Island Panel), this solution supports targeted region additions. It is ideal for supplementing unique loci on the basis of well-established panels.

2. Fully Customized Solution

Fully open for customization in full compliance with specific customer requirements.

3. Customization Workflow

l Requirement Assessment: Confirm target regions, sample types and sequencing depth.

l Probe Design: Optimized based on thermodynamic stability algorithms, balancing specificity and coverage.

l Synthesis and Quality Control: Guaranteed by iGeneTech’ s GMP-grade reagent production system, with NGS test data provided.

l Delivery and Application: Provision of supporting kits and experimental protocols.

Standard Product Portfolio: Four Methylation Panel Solutions

1. CGI Panel

Product Positioning: Human Genome-wide CpG Island Ultra-high Coverage Panel

Core Parameters:

l Coverage Range: 27,000 CpG Islands

l Target Region: 21.2 Mb

l Featured Regions: Added 13 clinical tumor methylation markers (GSTP1, SHOX2, APC, MGMT, etc.)

l Capture Performance of Regional Panel:

Capture Efficiency: Target Reads Capture Rate = 93.36%

Uniformity: Rmdup Target 20%× Rate = 96.93%

Figure 3: Reproducibility and Accuracy of the CGI Panel

2. Mouse CGI Panel

Product Positioning: Mouse Genome-wide CpG Island Ultra-high Coverage Panel

l Technical Specifications:

l Coverage Range: 17,017 CpG Islands

l Target Region: 10.5 Mb

l Panel Capture Performance:

Capture Efficiency: Target Reads Capture Rate = 93.07%

Uniformity: Rmdup Target 20%× Rate = 97.94%

Figure 4: Reproducibility and Accuracy of the Mouse CGI Panel

3. CpG Galaxy Panel

Product Features:

l CpG Site Coverage: 3,081,190 CpG sites, covering 98% of the sites on the Illumina 935K BeadChip

l Probe Coverage Region of the Panel: 100 Mb

l Panel Capture Performance:

Capture Efficiency: Flank Reads Capture Rate = 90.16%

Uniformity: Rmdup Target 20%× Rate = 97.75%

Figure 5: Reproducibility and Accuracy of the CpG Galaxy Panel

Figure 6: Consistency of Methylation Levels Between CpG Galaxy Panel and 935K Solid-Phase BeadChip

4. OmniTrait Methylation Panel

CpG Site Coverage: 940,314 CpG sites, covering 98.5% of the sites on the Illumina MSA BeadChip

Panel Probe Coverage Region: 30 Mb

Panel Capture Performance:

Capture Efficiency: Flank Reads Capture Rate = 78.83%

Uniformity: Rmdup Target 20%× Rate = 96.42%

Figure 7: Reproducibility and Accuracy of the OmniTrait Methylation Panel

Centered on a one-stop workflow solution, iGeneTech’s Methylation Technology Platform leverages complementary double-stranded and single-stranded library construction systems, along with dual-track BisCap® and MethCap® capture technologies, to cover diverse sample types and meet varied methylation research needs. The standardized Panel portfolio features high coverage, high capture efficiency and excellent uniformity, while the customized service flexibly adapts to multiple research scenarios, forming a one-stop closed-loop service from probe design to data analysis.

iGeneTech’s methylation technology solutions not only satisfy the needs of basic epigenetic research but also support clinical translational applications such as early cancer screening. Endowed with independent intellectual property rights and a mature production and quality control system, it has become a benchmark choice in the domestic methylation testing field, providing stable, efficient and flexible technical support for both scientific research and clinical users.

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