Overview of DNA Methylation Capture Sequencing

DNA Methylation and CpG sites

DNA methylation stands as a prominent area of investigation within the realm of epigenetics. This phenomenon involves the conversion of cytosine at the 5' end of dinucleotides within CpG islands of genomic DNA sequences into 5' methylcytosine (5mC), facilitated by the enzymatic activity of DNA methyltransferase (DNMT). This modification does not induce alterations in gene sequences; however, it exerts a regulatory influence on gene expression, thereby shaping biological functions.

The distinctive characteristics of DNA methylation involve the selective methylation of cytosine, a process that is reversible in nature. In mammalian genomes, DNA methylation is notably prevalent at CpG sites, where cytosine residues are targeted. These CpG sites exhibit a dual nature: they are scattered throughout the genomic sequence, with approximately 70-80% undergoing methylation, and they can also coalesce to form highly concentrated regions known as CpG islands. This intricate interplay of methylation dynamics at both dispersed and aggregated CpG sites plays a crucial role in regulating gene expression, influencing various biological functions without altering the underlying genetic code.

DNA Methylation Capture Sequencing

Advancing the field of DNA methylation sequencing, scientists have seamlessly integrated next-generation sequencing technology. This innovative approach represents a significant leap from Sanger sequencing platform, offering customers a tailored solution marked by enhanced throughput and efficiency. Leveraging bisulfite conversion and NGS platform, our proprietary amplicon and probe capture sequencing technology facilitates the detection of specific methylation sites after capture. This targeted sequencing method, grounded in bisulfite conversion, proves versatile across biological samples from diverse species. Furthermore, the NGS-based sequencing service ensures single-base resolution, enabling the precise identification of C-methylation in each DNA molecule. This level of resolution empowers accurate mutation analysis and digital quantification of the percentage of methylated DNA molecules, showcasing a distinct advantage in achieving high detection accuracy.

Types of DNA Methylation Capture Sequencing

• Multiplex PCR Strategy

The program employs a Multiplex PCR strategy utilizing methylated Bis-specific primers. Targeted amplified libraries undergo sequencing through a specific multiplex PCR process with methylated primers. This approach utilizes multiple pairs of bisulfite-converted sequence-specific primers to amplify the DNA's target region, followed by next generation sequencing (NGS).

Advantages: This method allows for swift and precise methylation status analysis across several or dozens of target regions, achieving single-base resolution. The accurate calculation of the proportion of methylated DNA molecules adds to the program's strengths.

• Semi-Library Strategy

This innovative approach enables targeted sequencing of methylated regions through multiple specific primer extensions. Leveraging a semi-library and specific primer extension enrichment technology, users gain the ability to sequence specific regions, significantly enhancing sequencing depth and sample throughput while minimizing costs.

Advantages: The method boasts high throughput, enabling swift and accurate single-base resolution analysis of methylation status across tens and hundreds of target regions. Users can also achieve precise calculations of the proportion of methylated DNA molecules, further solidifying the technology's advantages.

• Probe-based Hybridization Capture Sequencing

Targeted library sequencing is realized through the hybridization capture of specific target sequences utilizing a pool of probes designed for methylated bisulfite sequences. This method involves the pre-amplification of bisulfite-transformed sequences, followed by the capture of the target DNA template through hybridization with a biotin probe. Subsequently, the biotin-labeled sequences are immobilized on magnetic beads and subjected to sequencing via next generation sequencing (NGS).

Advantages: This approach offers higher throughput, facilitating the rapid analysis of methylation status across hundreds or thousands of target regions with single-base resolution. The method excels in the accurate calculation of the content of methylated DNA molecules, underscoring its efficiency and precision.