Enzymatic Methyl-seq (EM-seq)
NEBNext EM-Seq kit is an alternative to whole-genome bisulfite sequencing (WGBS). The enzymatic conversion of unmethylated cytosines in EM-seq is more gentle to DNA than WGBS and results in libraries with more even genome coverage and better coverage of CpG sites across the genome.
At NGI, we offer genome-wide methylome sequencing using the NEBNext EM-seq kit from New England Biolabs (NEB), a two step enzymatic conversion process to detect modified cytosines
Sample Requirements
- Sample type: high-quality genomic DNA
- Amount of sample: minimum 500 ng
- Volume: 25-50 µL
- Amount of material needed for preparing one library: 10-200 ng.
How to evaluate the sample quality
We check your samples upon arrival, however we still require our users to do their own QC steps before sending samples.
Checking the concentration
Please use fluorometric measurements (e.g. Qubit, Quant-it), not absorbance measurements (Nanodrop, spectrophotometer).
Checking the quality and purity
The DNA should be in water or EB buffer, of high quality, and ideally have an OD260/280 of 1.8-2.
If you are not able to carry out these steps, or your samples are below the required thresholds, please get in touch.
What we do with your samples
Once your samples arrive at NGI, we start by performing a reception control step in which we make sure the samples meet our requirements.
If the samples fail this quality control step, we will contact you to discuss possible options. Should you choose to proceed with samples not fulfilling the criteria it will be at your own risk, NGI will only make one attempt at library preparation and if the library preparation does not work you will be charged for the library preparation regardless.
If the samples pass reception control, the samples will be queued for library preparation.
Library Preparation
DNA is fragmented using a Covaris sonicator followed by ligation of EM-seq adapters with sample-specific barcode sequences. The DNA is converted by a two step Oxidation/Deamination step using TET2 and APOBEC enzymes, and the final converted libraries are PCR amplified before sequencing.
Two DNA controls are provided in the EM-seq kit: unmethylated lambda DNA and CpG-methylated pUC19 DNA to verify the conversion.
Library QC and Sequencing
We evaluate the concentration of the libraries using qPCR and look at the fragment size distribution of the library molecules. Once the libraries have passed this QC step, they are normalised, pooled, and queued for sequencing.
The sequencing will be carried out following the setup stated in the agreement.
Last Updated: 28th April 2021