Epigenomics studies genome-wide patterns of DNA methylation, histone modifications, and chromatin accessibility. Two of the most widely used epigenomic techniques are ATAC-seq and bisulfite sequencing.
ATAC-Seq
ATAC-seq (Assay for Transposase-Accessible Chromatin) maps regions of open chromatin — nucleosome-depleted DNA where transcription factors and other regulatory proteins bind. It uses the hyperactive Tn5 transposase, which simultaneously fragments DNA and inserts sequencing adapters into accessible chromatin regions.
The protocol is remarkably simple:
- Collect 50,000 cells (fresh or cryopreserved).
- Lyse the cell membranes to release nuclei.
- Incubate nuclei with Tn5 transposase loaded with sequencing adapters for 30 minutes at 37 °C.
- Purify the tagmented DNA, amplify by PCR with barcoded primers, and sequence paired-end on an Illumina platform.
Reads are aligned to the reference genome, and peaks of transposase activity indicate open chromatin. These peaks are enriched at promoters, enhancers, and other regulatory elements. ATAC-seq can also infer nucleosome positioning and transcription factor binding footprints from the pattern of fragment lengths.
The main advantages are speed (the entire library preparation takes 2–3 hours) and low input requirements (down to 500 cells, and even single-cell ATAC-seq is now routine).
Bisulfite Sequencing
Bisulfite sequencing detects 5-methylcytosine (5mC) in DNA. Treatment with sodium bisulfite converts unmethylated cytosines to uracil (which is read as thymine after PCR), while methylated cytosines remain unchanged. The resulting sequence is compared to a reference to determine methylation status at single-base resolution.
Whole-genome bisulfite sequencing (WGBS) provides a comprehensive methylation map but is expensive. Reduced representation bisulfite sequencing (RRBS) uses MspI digestion to enrich CpG-rich regions, reducing the sequencing cost. Targeted bisulfite sequencing amplifies specific regions of interest, such as promoter CpG islands.
Key considerations:
- Bisulfite treatment degrades DNA. Start with 100–500 ng of high-quality DNA.
- Conversion efficiency should be >99%. Spike-in unmethylated lambda DNA controls allow calculating the conversion rate.
- Alignment requires a bisulfite-aware aligner (Bismark, BSMAP, or BWA-meth) that maps reads to a bisulfite-converted reference.
- Methylation is reported as β-values (0 to 1) or M-values for each CpG site.
Data Analysis Commonalities
Both ATAC-seq and bisulfite sequencing produce FASTQ files that are aligned to the reference genome. For ATAC-seq, peak calling uses MACS2 or Genrich; for bisulfite data, methylation calling uses scripts within the Bismark pipeline. Quality metrics include fraction of reads in peaks (FRiP) for ATAC-seq and bisulfite conversion rate for methylation data.
