Whole Genome Bisulfite Sequencing (WGBS)

Whole Genome Bisulfite Sequencing is the most comprehensive DNA-methylation analysis method, enabling single-nucleotide-resolution analysis of methylation patterns across the entire genome. It combines chemical bisulfite conversion of DNA with next-generation sequencing, providing the highest resolution for epigenetic studies.

The method identifies methylated cytosines in both genic and intergenic regions, providing a complete view of epigenetic changes in the biological material under study.

WGBS workflow

Applications of WGBS

WGBS is used in a broad range of basic and applied epigenetic studies.

Cancer research

• identification of epigenetic biomarkers,
• analysis of cancer-development mechanisms,
• study of tumour heterogeneity,
• monitoring response to therapy.

Translational medicine and diagnostics

• discovery of diagnostic and prognostic markers,
• research on genetic and epigenetic diseases,
• development of personalised medicine.

Developmental studies

• analysis of epigenetic changes during cell differentiation,
• embryogenesis studies,
• assessment of organismal ageing processes.

Plant and animal biology

• analysis of gene expression regulation,
• studies of environmental adaptation,
• breeding projects and functional genomics.

Environmental and toxicological studies

• assessment of environmental-factor effects on the epigenome,
• studies of organismal responses to environmental stress,
• analysis of the effects of chemical exposure.

Technical specifications of WGBS

• Sequencing mode: 2x150 bp
• Sequencers: Illumina platform, NovaSeq 6000 and NovaSeq X sequencers.
• Recommended genome coverage:
  • 30x per sample - at least 3 biological replicates and an available high-quality reference genome
  • Other configurations are also available depending on the client's needs.
• Recommended Sample Requirements - DNA/RNA:
  • Sample type: genomic DNA
  • Amount: >100 ng
  • Concentration (NanoDrop): >2 ng/μL
  • Volume: >20 μL
  • No degradation or contamination
• Also available: DNA isolation from submitted samples. Details here.

WGBS bioinformatics analysis

Bioinformatics analysis of WGBS data includes multi-stage processing and interpretation of sequencing results, leading to comprehensive characterisation of the organism's methylome.

1. 01Data quality control:The first stage assesses raw sequencing-data quality. Parameters include read quality, adapter content, duplication level and bisulfite-conversion efficiency. Data are then filtered and cleaned before further analysis.
2. 02Mapping to the reference genome.Filtered reads are mapped to the appropriate reference genome using algorithms designed for bisulfite-converted data. The report includes:
   • read-mapping statistics,
   • genome-alignment efficiency,
   • sequencing depth,
   • coverage of analysed cytosine positions,
   • assessment of data completeness for individual genomic regions.
3. 03DNA-methylation identification.Mapped reads are used to identify DNA-methylation sites at single-nucleotide resolution. The analysis includes:
   • identification of methylated cytosines (mC),
   • determination of methylation level at each site,
   • characterisation of methylation contexts:
     • mCG (CpG),
     • mCHG,
     • mCHH (where H = A, T or C),
   • analysis of methylated-site distribution across the genome,
   • identification of sequence motifs associated with methylation.
4. 04Genome-wide methylation profilingThe next stage comprehensively characterises the epigenetic landscape of the genome. The analysis generates:
   • genome-wide methylation-density and distribution profiles,
   • methylation levels in functional regions (promoters, exons, introns and intergenic regions),
   • methylation profiles in gene upstream and downstream regions,
   • analysis of CpG-island methylation, CpG shores and adjacent regions,
   • annotation of genes associated with observed methylation patterns.
5. 05Inter-group comparison.Comparative projects include differential-methylation analysis between study groups or experimental conditions. This includes:
   • identification of differentially methylated regions (DMRs),
   • annotation of DMRs relative to genes and regulatory elements,
   • identification of genes associated with differential methylation,
   • GO (Gene Ontology) functional enrichment analysis,,
   • KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis,,
   • biological interpretation of epigenetic changes in the context of the process or phenotype studied.

The final output is a detailed report containing methylation profiles, statistical-analysis results, identification of differentially methylated regions and functional interpretation of the results.