How is DNA methylation status is determined using bisulfite treatment?
How is DNA methylation status is determined using bisulfite treatment?
Following bisulfite conversion the unmethylated cytosines are converted into uracil while the methylated cytosines remain as cytosine. The methylation profile of the DNA can then be determined by PCR amplification followed by DNA sequencing, where the uracils will be detected as thymine.
How does bisulfite sequencing work and what is it used for?
Whole genome bisulfite sequencing (WGBS) is one such next generation sequencing technique that allows users to analyze DNA methylation at single base resolution. DNA methylation is an epigenetic mechanism that regulates gene expression and has applications in several biological fields, such as diseases and cancer.
What does bisulfite sequencing tell you?
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.
What type of sequencing must be used to detect DNA methylation?
Genome-wide detection of 5mC by bisulfite sequencing is regarded as the current gold standard for DNA methylation detection [5, 7, 9, 10].
How does whole-genome bisulfite sequencing work?
The bisulfite-treated DNA is purified on a spin column and is used to prepare the sequencing library using the EpiGnome™ Kit (Epicentre). In this procedure, bisulfite-treated single-stranded DNA is random- primed using a polymerase able to read uracil nucleotides, to synthesize DNA containing a specific sequence tag.
Why is bisulfite seq is a reliable method to detect DNA methylation at genome wide?
Bisulfite genomic sequencing is regarded as a gold-standard technology for detection of DNA methylation because it provides a qualitative, quantitative and efficient approach to identify 5-methylcytosine at single base-pair resolution.
How does whole genome bisulfite sequencing work?
Why is bisulfite seq is a reliable method to detect DNA methylation at genome-wide?
How much DNA is needed for bisulfite sequencing?
Commonly 10–20 samples are sufficient, but for some applications as many as 50 may be necessary.
How many reads for bisulfite sequencing?
Reduced Representation Bisulfite Sequencing (RRBS) enriches for CpG rich regions and greatly reduces the representation of the genome. Generally AGRF recommends a sequencing depth of 30 million reads (100bp single read) to provide high coverage of up to 4 million CpG loci.
What is long read bisulfite sequencing?
Detection of base modifications by bisulfite and long-read sequencing. Whole-genome bisulfite sequencing (WGBS) provides accurate binary calls of cytosine methylation status at nucleotide resolution, but cannot distinguish between 5mC and 5hmC or detect other oxidised forms without additional techniques.
How long is bisulfite sequencing?
Following bisulfite treatment and purification, PCR amplification along with PCR product purification requires about 3 hours. Samples are then prepared for sequencing. If sending the samples to a sequencing core, time requirements may vary, but 3 days should be allotted for sequencing by a core facility.
