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AAT Bioquest

What are the limitations of reduced representation bisulfite sequencing (RRBS)?

Posted April 26, 2024


Answer

One main drawback is that not all CG regions in the genome may be covered due to restriction enzyme digestion, resulting in missed CpGs and lower coverage in certain regions. This results in lower resolution. This method also only measures 10-15% of all CpGs in the genome and is unable to distinguish between 5mC and 5hmC. Another con is that achieving complete bisulfite conversion of double-stranded DNA (dsDNA) requires a denaturation step because bisulfite sequencing exclusively targets single-stranded DNA (ssDNA). Additionally, potential PCR amplification errors may arise and non CpG rich areas are also not typically covered. Another con is that RRBS may not effectively detect methylation in repetitive elements. Additionally, its library preparation and sequencing are more complex compared to traditional bisulfite sequencing methods. RRBS requires high-quality DNA, as the reduced representation approach may lead to poor sequencing of degraded DNA. Lastly, since RRBS relies on PCR amplification, it may introduce bias in the data, especially in regions that are hard to amplify. 

Additional resources

Opportunities and limitations of reduced representation bisulfite sequencing in plant ecological epigenomics

Next Generation Sequencing (NGS)

MagaDye™ 535-ddGTP

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