Rather, scientists should treat the methylated DNA samples with bisulfite prior to detection, to identify in between methylated and unmethylated cytosines.1,2 Bisulfite conversion is the chemical response that happens when researchers treat DNA with sodium bisulfite. The quality and quantity of purified DNA and the pH of the response are fundamental sample criteria in complete bisulfite conversion.1 Incomplete conversion normally leads scientists to overstate the amount of methylation in a sample.2 An extra challenge to bisulfite-based methylation analyses is the level of sensitivity of the DNA sample to destruction throughout long incubation actions.1 After treatment with salt bisulfite, unmethylated cytosine residues become uracil residues, which are recognized as thymine after PCR amplification and sequencing analysis. RRBS lowers the number of reads needed to obtain high protection and requires much less DNA than WGBS, however scientists can just analyze a portion of the methylome with this strategy due to the amount of excluded DNA.4– 7Targeted Bisulfite SequencingIn contrast to the broad enrichment of GC-rich regions with RRBS, targeted bisulfite sequencing enables researchers to investigate the methylation status of each cytosine in a particular genomic area.
Instead, researchers need to treat the methylated DNA samples with bisulfite prior to detection, to differentiate in between methylated and unmethylated cytosines.1,2 Bisulfite conversion is the chemical reaction that occurs when researchers treat DNA with salt bisulfite. Bisulfite conversion relies on the chemical adjustment of cytosine in single-stranded DNA, so researchers should guarantee that their DNA sample is totally denatured prior to salt bisulfite treatment. The quality and amount of purified DNA and the pH of the reaction are fundamental sample parameters in total bisulfite conversion.1 Incomplete conversion usually leads researchers to overstate the quantity of methylation in a sample.2 An additional difficulty to bisulfite-based methylation analyses is the level of sensitivity of the DNA sample to destruction throughout long incubation actions.1 After treatment with sodium bisulfite, unmethylated cytosine residues become uracil residues, which are acknowledged as thymine after PCR amplification and sequencing analysis. In addition, the standard WGBS protocol requires a large amount of DNA, which might be a roadblock in numerous methylome research studies.2,5 Reduced Representation Bisulfite SequencingAn alternative approach to WGBS is minimized representation bisulfite sequencing (RRBS). RRBS decreases the number of reads needed to get high protection and requires much less DNA than WGBS, however researchers can just take a look at a fraction of the methylome with this strategy due to the amount of omitted DNA.4– 7Targeted Bisulfite SequencingIn contrast to the broad enrichment of GC-rich areas with RRBS, targeted bisulfite sequencing enables researchers to examine the methylation status of each cytosine in a specific genomic region.
