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Bisulfite sequencing (BS-Seq)/WGBS

Bisulfite sequencing using NGS technology for DNA methylation sequencing analysis

What is bisulfite sequencing?

Bisulfite sequencing (BS-Seq) or whole-genome bisulfite sequencing (WGBS) is a well-established protocol to detect methylated cytosines in genomic DNA. In this method, genomic DNA is treated with sodium bisulfite and then sequenced, providing single-base resolution of methylated cytosines in the genome. Upon treatment of DNA with sodium bisulfite, unmethylated cytosines deaminate into uracils and, after sequencing, are converted to thymines. Simultaneously, methylated cytosines resist deamination and are read as cytosines. The location of the methylated cytosines is determined by comparing treated and untreated sequences.1,2

On this page:

Whole-genome bisulfite sequencing workflow tools

Bisulfite sequencing methods to detect DNA methylation

Bisulfite sequencing advantages and disadvantages

Bisulfite sequencing limitations

The principle of bisulfite sequencing and FAQs

novaseq x dual run sequencing screen

Understanding DNA methylation and the power of BS-Seq and multiomics

DNA methylation eBook

Understanding DNA methylation is critical for the study of human diseases and other phenotypes. This eBook provides considerations for analyzing DNA methylation and performing epigenome-wide association studies, including study design, sample types, and data analysis.

Gene expression and regulation eBook

Gene regulation is a biological process that controls the temporal and spatial expression of gene products, including mRNA and noncoding RNA transcripts. Discover how modern transcriptomics and epigenetics methods, including BS-Seq and NGS technologies, are being used to make discoveries and better understand this biological process.

The power of multiomics eBook

DNA stores molecular information in both genetic and epigenetic bases, making it inherently multiomic. Combining transcriptomic information from RNA-Seq with methylation sequencing data enables a better understanding of cellular differentiation, mechanisms, and behaviors. Learn more and unlock the power of a multiomic approach.

Whole-genome bisulfite sequencing

workflow tools

1
Library preparation
2
Sequencing
3
Data analysis

Bisulfite sequencing methods to
detect DNA methylation

Reduced-representation bisulfite sequencing (RRBS-Seq)

Reduced-representation bisulfite sequencing (RRBS-Seq), or single-cell reduced-representation bisulfite sequencing (scRRBS), is a protocol that uses one or multiple restriction enzymes on the genomic DNA to produce sequence-specific fragmentation. The fragmented genomic DNA is then treated with bisulfite and sequenced. RRBS-Seq is the method of choice for studying specific regions of interest. It is particularly effective where methylation is high, such as in promoters and repeat regions.3,4

Oxidative bisulfite sequencing
(oxBS-Seq)

Oxidative bisulfite sequencing (oxBS-Seq) differentiates between 5mC and 5hmC. With oxBS, 5hmC is oxidized to 5-formylcytosine (5fC) with an oxidizing agent, while 5mC remains unchanged. Sodium bisulfite treatment of oxidized 5hmC results in its deamination to uracil, which, upon sequencing, is read as a thymine. Deep sequencing of oxBS-treated DNA and sequence comparison of treated vs. untreated can identify 5mC locations at base resolution.5,6

Tagmentation-based whole-genome bisulfite sequencing (T-WGBS)

Tagmentation-based whole-genome bisulfite sequencing (T-WGBS) is a protocol that uses Tn5 transposase and bisulfite conversion to study 5mC. Compared to standard WGBS methods, T-WGBS can sequence material with minimal DNA (~20 ng). Additionally, the Tn5 transposase provides the benefit of DNA fragmentation and attaching sequencing adapters in a single step. DNA is then treated with sodium bisulfite, PCR-amplified, and sequenced.7

Single-cell bisulfite sequencing (scBS-Seq)

Single-cell bisulfite sequencing (scBS-Seq) is a version of the well-established bisulfite sequencing (BS-Seq) and post-bisulfite adaptor tagging (PBAT) protocols, modified to detect methylated cytosines in genomic DNA from single cells. In this method, after single cells are isolated, genomic DNA is treated with sodium bisulfite, which fragments the DNA. The converted DNA then undergoes random priming several times and is PCR amplified for sequencing. Deep sequencing provides high-resolution single-nucleotide resolution of methylated cytosines from single cells.8

Bisulfite sequencing advantages

and disadvantages

Method

Bisulfite sequencing advantages

Bisulfite sequencing disadvantages
BS-Seq
  • CpG and non-CpG methylation throughout the genome is covered at single-base resolution
  • 5mC in dense, less dense, and repeat regions are covered
  • Bisulfite converts unmethylated cytosines to thymines, reducing sequence complexity, which can make it difficult to create alignments
  • Single nucleotide polymorphisms (SNPs) where a cytosine is converted to thymine will be missed upon bisulfite conversion
  • Bisulfite conversion does not distinguish between 5mC and 5hmC
RRBS-Seq / scRRBS
  • Genome-wide coverage of CpGs in islands at single-base resolution
  • Areas dense in CpG methylation are covered
  • Restriction enzymes cut at specific sites, providing biased sequence selection
  • Method measures 10–15% of all CpGs in genome
  • Cannot distinguish between 5mC and 5hmC
  • Does not cover non-CpG areas, genome-wide CpGs, and CpGs in areas without the enzyme restriction site
oxBS-Seq
  • CpG and non-CpG methylation throughout the genome is covered at single-base resolution
  • 5mC dense and less dense in repeat regions are covered
  • Method clearly differentiates between 5mC and 5hmC, precisely identifying 5mC
  • Bisulfite converts unmethylated cytosines to thymines, reducing sequence complexity, which can make it difficult to create alignments
  • SNPs where a cytosine is converted to thymine will be missed upon bisulfite conversion
T-WGBS
  • Can sequence samples with very limited starting material (~20 ng)
  • Fast protocol with few steps
  • Elimination of multiple steps prevents loss of DNA
  • Bisulfite converts unmethylated cytosines to thymines, reducing sequence complexity, which can make it difficult to create alignments
  • SNPs where a cytosine is converted to thymine will be missed upon bisulfite conversion
  • Bisulfite conversion does not distinguish between 5mC and 5hmC

Bisulfite sequencing limitations

Bisulfite sequencing is a commonly used method for detecting DNA methylation that uses bisulfite to convert unmethylated C to T in sequencing data. However, there are several limitations with this method. It is estimated that approximately 10% of the CpG sites in the genome will be hard to align after bisulfite conversion, and there is DNA degradation that can reach the level of 90%.9

An alternative to bisulfite sequencing, the Illumina 5-base solution, leverages novel chemistry and analyses to enable simultaneous genetic variant and methylation detection. Illumina 5-base chemistry directly converts only 5mC to T in a simple, single-step process that is nondamaging to DNA and retains library complexity. The Illumina 5-base solution can read unmodified bases (A, T, G, C) and 5mC in a single assay. 

Learn more about the 5-base solution

Illumina 5-base solution for genetic variant and methylation studies

Bisulfite or enzymatic conversion

Illumina 5-base conversion

The principle of bisulfite treatment and FAQs

Bisulfite conversion is the process by which sodium bisulfite selectively converts unmethylated cytosine to uracil, while methylated cytosines remain unconverted. Bisulfite treatment is commonly used in tandem with NGS to enable genome-wide DNA methylation profiling. 
Learn more about the basics of NGS. 

Bisulfite sequencing is a method commonly used to study DNA methylation. Prior to sequencing, unmethylated cytosines are converted to uracils via treatment with sodium bisulfite. Sequenced DNA from bisulfite-treated and untreated samples are then compared to identify the locations of the methylated and unmethylated cytosines.

The treatment of DNA with sodium bisulfite results in the conversion of unmethylated cytosines to uracil by chemical deamination, while methylated cytosines are protected from this process.

There are several limitations that arise from the bisulfite conversion process and subsequent sequencing, including DNA degradation, reduction in genome complexity, and additional complexity with the alignment process.10

Targeted BS-Seq has been studied for use in biomarker discovery and uses components, such as biotinylated RNA probes, to capture specific genomic fragments of interest. Conversely, WGBS results in sequencing and analysis of the entire genome.11

/ Results

Additional resources

Methylation sequencing

Learn about the advantages of methylation sequencing, including bisulfite conversion sequencing.

Multiomics

Unlock the power of a multiomic analyses to gain better insights into complex biological mechanisms.

References

  1. Frommer M, McDonald LE, Millar DS, et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A. 1992;89(5):1827-1831. doi:10.1073/pnas.89.5.1827
  2. Clark SJ, Harrison J, Paul CL, Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 1994;22(15):2990-2997. doi:10.1093/nar/22.15.2990
  3. Guo H, Zhu P, Wu X, Li X, Wen L, Tang F. Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res. 2013;23(12):2126-2135. doi:10.1101/gr.161679.113
  4. Guo H, Zhu P, Guo F, et al. Profiling DNA methylome landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing. Nat Protoc. 2015;10(5):645-659. doi:10.1038/nprot.2015.039
  5. Booth MJ, Branco MR, Ficz G, et al. Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science. 2012;336(6083):934-937. doi:10.1126/science.1220671
  6. Kisil O, Sergeev A, Bacheva A, Zvereva M. Methods for Detection and Mapping of Methylated and Hydroxymethylated Cytosine in DNA. Biomolecules. 2024;14(11):1346. doi:10.3390/biom14111346
  7. Wang Q, Gu L, Adey A, et al. Tagmentation-based whole-genome bisulfite sequencing. Nat Protoc. 2013;8(10):2022-2032. doi:10.1038/nprot.2013.118
  8. Smallwood SA, Lee HJ, Angermueller C, et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods. 2014;11(8):817-820. doi:10.1038/nmeth.3035
  9. Leontiou CA, Hadjidaniel MD, Mina P, Antoniou P, Ioannides M, Patsalis PC. Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing. PLoS One. 2015;10(8):e0135058. doi:10.1371/journal.pone.0135058
  10. Karimzadeh M, Ernst C, Kundaje A, Hoffman MM. Umap and Bismap: quantifying genome and methylome mappability. Nucleic Acids Res. 2018;46(20):e120. doi:10.1093/nar/gky677
  11. Morselli M, Farrell C, Rubbi L, Fehling HL, Henkhaus R, Pellegrini M. Targeted Bisulfite Sequencing for Biomarker Discovery. Methods. 2021;187:13-27. doi:10.1016/j.ymeth.2020.07.006

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