What Is Bisulfite Sequencing?

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  • Written By: Jessica Susan Reuter
  • Edited By: Shereen Skola
  • Last Modified Date: 10 September 2019
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Bisulfite sequencing is a method in which different regions of DNA are analyzed using methylation. Methylation is the process of adding a specific molecule, called a methyl group, to a nucleotide, in this case usually a cytosine. Inactive nucleotides are often methylated, so this method can be used for a variety of purposes, from determining active regions of a genome to identifying gene-rich regions. In bisulfite sequencing, methylated cytosines are not affected by the sequencing process, while non-methylated cytosines are converted into uracil, a nucleotide not usually found in the genetic material, deoxyribonucleic acid (DNA.)

This method is very sensitive to changes in methylation, so small changes in binding can give researchers specific information about particular nucleotides. Sodium bisulfite converts cytosine to uracil, but the conversion happens in an environment where methylated cytosine will not undergo this change. When bisulfite sequencing is complete, the original DNA has been converted into a significantly different molecule. Cytosines will be greatly depleted or potentially absent. If a cytosine is still found in this converted molecule, it represents a naturally methylated cytosine in the genome under consideration.


Like all experimental protocols, bisulfite sequencing has drawbacks. Its most significant drawback is that it requires a very high salt concentration in order to work properly. The salt encourages annealing of single-stranded DNA into its more natural double helix, and the sodium bisulfite can't always reach cytosines when they're part of double-stranded DNA. If the salt concentration is too high, a number of cytosines may not be converted into uracil, resulting in false identification of methylated cytosines within a genome. Denaturing agents may be necessary to minimize the number of false positive identifications.

Large amounts of genomic data are not necessary for bisulfite sequencing, so the method has a useful application analyzing clinical samples. The original nucleic acid source doesn't matter, but the source must be DNA. In theory, ribonucleic acid (RNA) could be sequenced using this method, since most RNA is single stranded and wouldn't be as susceptible to false positives because of blocked nucleotides. When put into practice, however, bisulfite sequencing is not useful for RNA, because RNA naturally has uracil in it. Without some sort of external marking or addition to the protocol, converted cytosines would be indistinguishable from natural uracil.

When undertaking any type of sequencing methodology, accuracy and precision are essential. Sensitive methods like bisulfite sequencing offer a reliable means of sequence analysis, which in turn allows for gene analysis and identification of targets for drugs and therapies. Although this method cannot be used on living people, it can still be of great help with only the tiniest of tissue samples to work with.


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