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Bisulfite sequencing applies routine sequencing methods on bisulfite-treated genomic DNA to determine methylation status at CpG dinucleotides. Other non-sequencing strategies are also employed to interrogate the methylation at specific loci or at a genome-wide level. All strategies assume that bisulfite-induced conversion of unmethylated ...
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.
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]
When the bisulfite-treated DNA is amplified via polymerase chain reaction, the uracil is amplified as thymine and the methylated cytosines are amplified as cytosine. DNA sequencing techniques are then used to read the sequence of the bisulfite-treated DNA. Those cytosines that are read as cytosines after sequencing represent methylated ...
For the Infinium I assay chemistry technology, the process is outlined in Figure 1. Bisulfite treatment Approximately 1 μg of genomic DNA is used in bisulfite conversion to convert the unmethylated cytosine into uracil. The product contains unconverted cytosine where they were previously methylated, but cytosine converted to uracil if they ...
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.
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.