Enzymatic Conversion for DNA Methylation Analysis

Enzymatic conversion generates more accurate data on DNA methylation than bisulfite conversion. The approach is utilized in basic epigenetics research and also offers technical advantages in key quality metrics for developing methyl-seq based diagnostic assays.

Enzymatic Methyl -seq (EM-seq™) minimizes DNA damage to enable generation of methylome libraries with superior quality that provide accurate methylation information from fewer sequencing reads. Lower inputs of genomic, cell-free, or formalin fixed paraffin embedded (FFPE) DNA samples can be used than with bisulfite converted DNA samples. EM-seq data is conveniently compatible with bioinformatic analysis tools for bisulfite sequencing.  Notably, a recent advance by NEB scientists that utilizes enzymatic conversion for direct detection of 5hmC called Enzymatic 5hmC-seq when combined with EM-seq data can facilitate discrimination of methylation and hydroxymethylation.

Enzymatic conversion is superior to sodium bisulfite conversion for sequencing library preparation

NEB offers enzymatic conversion modules, next generation sequencing library preparation workflows with enzymatic conversion integrated, and specialized primer sets for multiplexing.

• NEBNext^® Enzymatic Methyl-seq Conversion Module (NEB # E7125) enables enzymatic conversion for downstream detection of 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC) jointly.
  
  
• NEBNext Enzymatic Methyl-seq v2 Conversion Module (NEB #E8020) enables enzymatic conversion for downstream detection of 5mC and 5hmC with a faster workflow and wider input range (as low as 100 pg) than the original EM-seq Conversion Module.
  
  
• NEBNext Enzymatic Methyl-seq Kit (NEB #E7120) enables enzymatic conversion and includes high-efficiency Ultra II library prep reagents and the EM-seq Adaptor optimized for high performance using Illumina Sequencing.
  
  
• NEBNext Enzymatic Methyl-seq v2 Kit (NEB #E8015) enables enzymatic conversion and includes high-efficiency Ultra II library prep reagents and the EM-seq Adaptor optimized for high performance using Illumina Sequencing using a faster workflow and wider input range (as low as 100 pg) than the original EM-seq Kit for joint detection of 5mC and 5hmC.
  
  
• NEBNext Enzymatic 5hmC-seq (E5hmC-seq™) (NEB #E3350) enables enzyme-based specific detection of hydroxymethylcytosines only at the single base level with Illumina^® sequencing.
  
  
• NEBNext Enzymatic 5hmC-seq Conversion Module (NEB #E3365)
  
  
• NEBNext Multiplex Oligos for Enzymatic Methyl-seq (Unique Dual Index Primer Pairs) (NEB #E7140) include the NEBNext EM-seq™ Adaptor, and NEBNext PCR primers that enable incorporation of unique index pairs during library amplification.
  
  
• NEBNext Primers for Epigenetics sets (NEB #E3392, NEB #E3404) increase sample specificity and address the “barcode hopping” issue seen with some Illumina sequencing instruments to enable multiplexing and minimized index hopping.
• NEBNext UltraShear (NEB #M7634) has been optimized for the specific requirements for enzymatic fragmentation of DNA for EM-seq workflows.
  
  
• T4 Phage β-glucosyltransferase (T4-BGT) (NEB #M0357) protects 5-mC and 5-hmC from subsequent deamination in enzymatic conversion reactions.
  
  

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