Brush Up: What Is Bisulfite Sequencing and How Do Researchers Use It to Research DNA Methylation?

What’s DNA Methylation?
DNA methylation is a basic epigenetic mechanism wherein a methyl group is added onto a nucleotide, generally cytosine. DNA methylation impacts many organic processes, together with gene expression, embryonic growth, irritation, and mobile proliferation and differentiation. A number of advanced ailments have aberrant DNA methylation patterns, similar to various kinds of most cancers and neurodegenerative problems, which are sometimes related to genomic instability and lack of DNA homeostasis.^1–3

Researchers want efficient strategies with excessive sensitivity and reliability to discover the significance of DNA methylation. The gold customary know-how that scientists use to detect DNA methylation is bisulfite genomic sequencing. It’s a qualitative, quantitative, and environment friendly strategy to establish methylated cytosine at single base-pair decision.³

Bisulfite Conversion of Methylated Cytosine
Cytosine methylation happens when a methyl group binds to the fifth carbon of a cytosine to kind 5-methylcytosine (5mC). Researchers can not detect 5mC with conventional molecular methods similar to PCR or cloning strategies as a result of methyl teams will not be copied throughout PCR amplification. As an alternative, researchers should deal with the methylated DNA samples with bisulfite previous to detection, to differentiate between methylated and unmethylated cytosines.^1,2

Bisulfite conversion is the chemical response that happens when scientists deal with DNA with sodium bisulfite. In 1992, researchers discovered that the amination response of sodium bisulfite with unmethylated cytosine is completely different than the response of sodium bisulfite with 5mC. Due to this distinction, unmethylated cytosines in single-stranded DNA grow to be uracil residues after publicity to sodium bisulfite, whereas 5mCs stay cytosines. Pretreatment with sodium bisulfite is the premise of many methylation detection and evaluation methods.¹ Following bisulfite conversion, researchers decide the methylation standing in loci of curiosity as a result of unmethylated cytosines are acknowledged as thymine after PCR amplification and sequencing.³ 

Challenges of Treating DNA with Bisulfite
Incomplete bisulfite therapy is a serious problem in methylation detection strategies. Bisulfite conversion depends on the chemical modification of cytosine in single-stranded DNA, so scientists should be sure that their DNA pattern is totally denatured previous to sodium bisulfite therapy. As such, the standard and amount of purified DNA and the pH of the response are basic pattern parameters in full bisulfite conversion.¹ Incomplete conversion usually leads researchers to overestimate the quantity of methylation in a pattern.² A further problem to bisulfite-based methylation analyses is the sensitivity of the DNA pattern to degradation throughout lengthy incubation steps.¹

Kinds of Bisulfite Sequencing
Entire Genome Bisulfite Subsequent-Era Sequencing
Methylation detection strategies are numerous in accuracy, sensitivity, pace, simplicity, and value. Researchers examine the methylation of a whole genome, additionally known as a methylome, with complete genome bisulfite sequencing (WGBS). WGBS consists of library preparation with bisulfite therapy, next-generation sequencing (NGS), and excessive throughput evaluation wherein researchers discover the organic processes related to the noticed methylome.^1,2,5  Challenges throughout WGBS come up from protocol adjustments that trigger discrepancies in protection depth, learn high quality, duplication charges, mapping effectivity, and methylation estimation. Moreover, the usual WGBS protocol requires a considerable amount of DNA, which can be a roadblock in lots of methylome research.^2,5

Diminished Illustration Bisulfite Sequencing
Another strategy to WGBS is lowered illustration bisulfite sequencing (RRBS). This technique reduces the fee and complexity of methylome evaluation. With RRBS, scientists enrich for GC-rich elements of the genome by digesting the DNA with an enzyme that generates fragments with CpG dinucleotides at each ends, unbiased of methylation standing. This enrichment is informative as a result of DNA methylation exists primarily within the context of symmetric CpG dinucleotides. After digestion, researchers use dimension choice to isolate brief DNA fragments that correspond to GC-rich areas. Bigger fragments that aren’t GC-rich and include fewer potential methylation websites are discarded. This digestion and choice step basically excludes uninformative DNA previous to bisulfite therapy, whereas capturing the vast majority of related genomic areas of the methylome. RRBS reduces the variety of reads wanted to acquire excessive protection and requires a lot much less DNA than WGBS, however researchers can solely look at a fraction of the methylome with this method because of the quantity of excluded DNA.^4–7

Focused Bisulfite Sequencing
In distinction to the broad enrichment of GC-rich areas with RRBS, focused bisulfite sequencing permits researchers to analyze the methylation standing of every cytosine in a particular genomic area. Scientists apply focused strategies to validate differentially-methylated areas or analyze candidate areas slightly than the whole methylome. Focused bisulfite sequencing depends on two PCR amplification steps after bisulfite conversion, adopted by deep sequencing. The PCR steps enhance copy quantity and introduce adaptors required for sequencing in addition to sample-specific identifiers. These adaptors and identifiers enable researchers to course of a lot of samples concurrently on a high-throughput instrument.^7,8

References

1. R. Halabian et al., “Laboratory strategies to decipher epigenetic signatures: a comparative assessment,” Cell Mol Biol Lett, 26:1-30, 2021.
2. T. Gong et al., “Evaluation and efficiency evaluation of the entire genome bisulfite sequencing information workflow: presently out there instruments and a sensible information to advance DNA methylation research,” Small Strategies, 6:e2101251, 2022.
3. Y. Li, T.O. Tollefsbol, “DNA methylation detection: bisulfite genomic sequencing evaluation,” Strategies Mol Biol, 791:11-21, 2011.
4. Y. Zhang, A. Jeltsch, “The applying of subsequent technology sequencing in DNA methylation evaluation,” Genes (Basel), 1:85-101, 2010.
5. Q. Wang et al., “Tagmentation-based whole-genome bisulfite sequencing,” Nat Protoc, 8:2022-32, 2013.
6. H. Gu et al., “Preparation of lowered illustration bisulfite sequencing libraries for genome-scale DNA methylation profiling,” Nat Protoc, 6:468-81, 2011.
7. D.A. Moser et al., “Focused bisulfite sequencing: A novel software for the evaluation of DNA methylation with excessive sensitivity and elevated protection,” Psychoneuroendocrinology, 120:1-8, 2020.
8. E. Leitão et al., “Locus-specific DNA methylation evaluation by focused deep bisulfite sequencing,” Strategies Mol Biol, 1767:351-66, 2018.