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Bisulfite [1] sequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA before routine sequencing to determine the pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied.
The widespread use of whole genome bisulfite sequencing has been primarily limited by its excessive cost, complex data output, and minimal required coverage. Due to the high amount and subsequent cost of DNA input, many studies using whole genome bisulfite sequencing assays occur with few or no biological replicates. [15]
Reduced representation bisulfite sequencing (RRBS) is an efficient and high-throughput technique for analyzing the genome-wide methylation profiles on a single nucleotide level. It combines restriction enzymes and bisulfite sequencing to enrich for areas of the genome with a high CpG content.
Sodium bisulfite is a decoloration agent in purification procedures because it reduces strongly coloured oxidizing agents, conjugated alkenes and carbonyl compounds. Bisulfite is also the key ingredient in the Bucherer reaction. In this reaction an aromatic hydroxyl group is converted to the corresponding amine group. This is a reversible reaction.
This is similar to single cell genome sequencing, but with the addition of a bisulfite treatment before sequencing. Forms include whole genome bisulfite sequencing, [4] [5] and reduced representation bisulfite sequencing [6] [7] Comparison of single cell DNA methylation sequencing methods in terms of coverage as at 2015 on Mus musculus
The first few steps of COBRA, and the molecular changes caused by each step to methylated and unmethylated CpG sites. Combined Bisulfite Restriction Analysis (or COBRA) is a molecular biology technique that allows for the sensitive quantification of DNA methylation levels at a specific genomic locus on a DNA sequence in a small sample of genomic DNA. [1]
Bisulfite sequencing-based methods, despite possible single-nucleotide resolution, have a drawback: the conversion of unmethylated cytosine to uracil can be unstable. [19] In addition, when bisulfite conversion is coupled with DNA microarrays to detect bisulfite converted sites, the reduced sequence complexity of DNA is a problem.
This method is an extension of bisulfite sequencing, which is the gold standard for determining DNA methylation. [2] NOMe-seq relies on the methyltransferase M.CviPl, which methylates cytosines in GpC dinucleotides unbound by nucleosomes or other proteins, creating a nucleosome footprint.