New England Biolabs, E8020L, NEBNext® Enzymatic Methyl-seq v2 Conversion Module

NEBNext Enzymatic Methyl-seq (EM-seq™) is a high-performance enzyme-based alternative to bisulfite conversion for the identification of 5mC and 5hmC. Unlike bisulfite conversion, this highly efficient method minimizes DNA damage, resulting in superior detection of methylated cytosines, with fewer sequencing reads. Related Categories Enzymatic Conversion for DNA Methylation Analysis,, Methylome Analysis,, DNA Methylation Analysis, Specification Materials Required but not Supplied NEBNext UltraShear® (M7634) or Covaris® instrument and the required tubes or other fragmentation equipment PCR strip tubes or 96-well plates Cleanup beads: SPRIselect Reagent Kit (Beckman Coulter®, Inc. #B23317), AMPure® XP beads (Beckman Coulter, Inc. #A63881) or preferred bead manufacturer Hi-Di™ Formamide (Thermo Fisher Scientific® #4401457), Formamide (Sigma #F9037-100 ml), or 0.05 N NaOH. Formamide is preferred. If using NaOH, please see FAQ associated with NEB #E8015. 80% Ethanol 10 mM Tris-HCl pH 7.5 or 8.0 or low TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) Nuclease-free Water Magnetic rack/stand, such as NEBNext Magnetic Separation Rack (NEB #S1515) Metal cooling block, such as Diversified Biotech® (#CHAM-1000) PCR machine FAQ Q: What is the difference between the NEBNext® Enzymatic Methyl-seq v2 Kit (NEB #E8015) and the Enzymatic Methyl-seq v2 Conversion Module (NEB #E8020)? A: The NEBNext Enzymatic Methyl-seq v2 Kit contains the components needed to make an EM-seq™ library, including reagents for End prep, Adaptor ligation, 5mC/5hmC protection, cytosine deamination, and NEBNext Q5U Master Mix for amplification. The EM-seq v2 kit can be combined with any of the LV Unique Dual Index Primers. The Enzymatic Methyl-seq v2 Conversion Module does not contain reagents for library preparation, amplification, or adaptors and primers. Q: What types of samples can be processed using the NEBNext® Enzymatic Methyl-seq v2 Kit (NEB #E8015) and the Enzymatic Methyl-seq v2 Conversion Module (NEB #E8020)? A: NEBNext Enzymatic Methyl-seq can be used with genomic DNA, cell-free DNA and FFPE DNA. Q: What are the recommended inputs for the NEBNext® Enzymatic Methyl-seq v2 Kit (NEB #E8015) and the Enzymatic Methyl-seq v2 Conversion Module (NEB #E8020)? A: The protocol has been optimized for 0.1–200 ng inputs. Q: What buffers are recommended for shearing DNA in NEBNext® Enzymatic Methyl-seq (EM-seq™) workflows? A: For use with the NEBNext® Enzymatic Methyl-seq Kits (NEB #E7120, #E8015), DNA should be sheared in one of the following buffers: 10 mM Tris-HCl pH 7.5 or pH 8.0, 1X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA), or low TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA). We do not recommend shearing input DNA in 0.1X TE (1 mM Tris-HCl, 0.1 mM EDTA) or water. When using the NEBNext Enzymatic Methyl-seq Conversion Modules (NEB #E7125, #E8020), the DNA must not be in a buffer that contains EDTA before proceeding with the Oxidation Reaction. Therefore, it is recommended that the DNA is sheared in 10 mM Tris pH 8.0. Alternatively, if shearing in 1X TE or low TE, a column or bead cleanup followed by elution in 10 mM Tris-HCl pH 8.0 or nuclease-free water is required. Q: What is the concentration of the EM-seq™ Adaptor and the NEBNext® LV Unique Dual Index Primers? A: The EM-seq Adaptor is 15 µM and the the LV Unique Dual Index Primers are 10 µM. Q: Can the “active” TET2 Buffer be stored longer than 4 months? A: No. The “active” TET2 Buffer should be discarded after 4 months. Q: Can the freshly diluted T4-BGT be stored long term? A: No. The freshly diluted T4-BGT should be discarded immediately after use. Q: What conversion levels are typical with the control DNAs supplied in the EM-seq™ v2 kits? A: Two DNA controls are provided in the EM-seq v2 kit: Unmethylated Lambda DNA and CpG-methylated pUC19 DNA. For 200 ng inputs, we observe ≤ 0.5% methylation, and for 0.1 ng inputs, we observe ≤ 1% methylation for unmethylated lambda spike in, indicating a conversion efficiency of 99.5% and 99%, respectively. For CpG-methylated pUC19, 96-98% CpG methylation is detected across the input range of 0.1-200 ng. As the pUC19 DNA is enzymatically methylated, it is likely that 100% methylation may not be achieved and that not all CpGs are methylated. Q: Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA before the deamination reaction? A: Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA before the deamination reaction? Yes, it is possible to use NaOH, although formamide is recommended since it is easier to handle. If using NaOH, the concentration of the NaOH solution must be accurate, and the volume added to the reaction is exactly 4 μl. NaOH is highly corrosive. Local and institutional safety guidelines must be followed when working with NaOH. If you are making a dilution from 2 N NaOH, ensure the stock solution has at least a pH of 12.5. Verify the pH of the stock solution before use. Avoid inaccurate dilutions by making a large enough volume of diluted NaOH to minimize pipetting errors. We recommend making at least 1 ml. Prepare NaOH dilutions fresh or store diluted aliquots at -20°C for up to 6 months to avoid a change in concentration. Solid NaOH is deliquescent (it absorbs water from the atmosphere). It cannot be weighed accurately. Therefore, solutions prepared from NaOH pellets must be titrated to achieve the proper concentration. Q: Is it normal if the Fe(II) solution from the EM-seq™ product is yellow or a color change is observed? A: Yes, color variation (colorless to yellow) has been observed. Testing has shown no difference in performance based on this variation. Q: How can pre-shear spike-in controls be added to cfDNA or other pre-fragmented DNA (amplicons, restriction digested DNA, etc.) going into EM-seq™ workflow? A: Shear unmethylated lambda and CpG methylated pUC19 controls for addition to cfDNA or other pre-fragmented DNA (amplicons, restriction digested DNA, etc.) as follows: 10 µl of Control DNA Unmethylated Lambda (2ng/µl) 10 µl of Control DNA CpG methylated pUC19 (0.1ng/µl) 30 µl of 15 mM Tris-HCl pH 8.0 Shear to an average size of 350 bp The Control DNA (combined Control DNA CpG methylated pUC19 (0.1ng/µl) and Control DNA Unmethylated Lambda (2ng/µl)) sheared in a final volume of 50 µl is equivalent to a 1:5 dilution. Therefore, to further dilute these pre-fragmented controls, for example to a 1:10 dilution, a further 1:2 dilution is required. Note: Unmethylated Lambda and CpG methylated pUC19 control DNAs are provided in 1 mM Tris pH 7.5 and 0.1 mM EDTA-containing buffer. It is critical to have at least 10 mM Tris when fragmenting these controls using mechanical shearing methods like Covaris. Using a buffer with lower than 10 mM Tris during shearing results in altered conversion metrics, and the conversion efficiencies assessed might not be accurate. Q: How should controls be diluted for applications involving shallow sequencing? A: For applications where you are sequencing to a depth less than 10 M paired reads, we recommend the following control dilutions based on sample input amount: 200 ng: No dilution of control DNAs 10 ng: 1:10 dilution of control DNAs 1 ng: 1:25 dilution of control DNAs 0.1 ng: 1:25 dilution of control DNAs We recommend having at least 5000 reads mapping to Unmethylated Lambda genome and 500 reads mapping to CpG methylated pUC19 genome with a read length of 76 bases to have enough coverage to confidently call conversion efficiencies. Q: Can the freshly diluted Fe(II) Solution be stored long term? A: No. The freshly diluted iron solution should be discarded immediately after use.