Analysis of DNA Methylation by Bisulfite Conversion

Methylation analysis by use of bisulfite conversion of DNA poses technical challenges for amplification and next generation sequencing due to incomplete conversion, sample damage, and degradation. The impacts of this harsh treatment can be reduced to some degree with optimized DNA polymerases, high yielding library preparation, and targeted sequencing techniques. However, overall, enzymatic conversion is highly recommended over bisulfite conversion for DNA methylation analysis. Enzymatic conversion minimizes damage to DNA, resulting in high-quality libraries for superior detection of 5mC and/or 5hmC from fewer sequencing reads. The NEBNext^® Enzymatic Methyl-seq and NEBNext Enzymatic 5hmC-seq kits incorporate enzymatic conversion.

NGS library prep with sodium bisulfite conversion compared to the EM-seq method

Sodium bisulfite treatment of DNA deaminates unmethylated cytosine residues to uracil, leaving methylated cytosines (5mC) and hydroxymethylated cytosines (5-hmC) intact. Subsequent amplification requires DNA polymerases that tolerate uracil-containing DNA and high AT ratios of target DNA. Uracil is amplified as thymine, whereas 5mC or 5hmC residues are amplified as cytosine. Bisulfite sequencing methods produce biased coverage and are unable to distinguish between 5mC and 5hmC.

Whole-genome bisulfite sequencing (WGBS) compares sequences of bisulfite-treated DNA with reference genomes to reveal methylation profiles. Bisulfite treated samples benefit from library prep designed to achieve high yields that could compensate for sample loss. Multiplexing and targeted sequencing techniques can reduce the number of sequencing runs required to compensate for limited coverage. Similarly, the enrichment of methylated CpGs in samples before bisulfite conversion can mitigate the costs of low coverage. The NEBNext Multiplex Oligos Selection Chart indicates single, dual, and unique dual index oligos compatible for bisulfite sequencing.

• Q5U^® Hot Start High-Fidelity DNA Polymerase (NEB #M0515) achieves high fidelity amplification of bisulfite-converted DNA.
  
  
• NEBNext Q5U Master Mix (NEB #M0597) is formulated to require only the addition of primers and DNA template for robust, hot start, bisulfite sequencing library amplification.
  
  
• EpiMark^® Methylated DNA Enrichment Kit (NEB #E2600) is useful to enrich double-stranded methyl-CpG DNA from fragmented genomic DNA prior to bisulfite conversion
  
  
• NEBNext^® Multiplex Oligos for Enzymatic Methyl-seq (Unique Dual Index Primer Pairs) (NEB #E7140) are validated for bisulfite sequencing workflows.
  
  
• NEBNext Ultra™ II DNA Library Prep Kit for Illumina^® (NEB #E7645) generates libraries with robust yield and high quality from GC-rich targets using inputs as low as 500 pg.
  
  
• NEBNext Ultra™ II DNA Library Prep with Sample Purification Beads (NEB # E7103) generates libraries with robust yield and high quality from GC-rich targets and includes beads for size selection and enzyme reaction cleanup.