New England Biolabs, E8015L, NEBNext® Enzymatic Methyl-seq v2 Kit

NEBNext Enzymatic Methyl-seq (EM-seq 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 original NEBNext Enzymatic Methyl-seq Kit (NEB #E7120)? A: The NEBNext Enzymatic Methyl-seq v2 Kit has a broader input range (100 pg - 200 ng) and a faster, more streamlined workflow that reduces consumables use. Overall data quality is improved, with higher yields and lower duplication rates resulting in increased CpG coverage levels across a wider input range. 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 is the expected size of an EM-seq™ v2 (NEB #E8015) library? A: EM-seq v2 libraries are approximately 470-550 bp. Q: How should EM-seq libraries be sequenced? A: For Illumina sequencing, read length is ultimately determined by library size. Standard sized libraries can be sequenced using 2x 76 base or 2x 100 base reads. 2x 150 base reads can be used for longer insert libraries. Q: How should EM-seq™ sequencing data be analyzed? A: Sequenced EM-seq libraries give the same output as bisulfite libraries, with cytosine sequenced as thymine and 5mC or 5hmC sequenced as cytosine. Established pipelines for bisulfite data analysis can be used. We also have a demo pipeline and example data on the GitHub library: https://github.com/nebiolabs/EM-seq/ Q: Can the EM-seq Adaptor be substituted with another adaptor? A: This is not recommended. The EM-seq Adaptor has been optimized for use with the NEBNext Enzymatic Methyl-seq Kit, and substitution with another adaptor will likely result in reduced performance. Q: Are EM-seq libraries directional or non-directional? A: EM-seq libraries are directional. Q: Can other buffers be used in place of the supplied EM-seq Elution Buffer? A: The EM-seq Elution Buffer should be used where stated in the protocol, except in the case of long term storage of PCR-amplified EM-seq libraries, where other options (specified in the manual) are possible. Q: How are EM-seq™ libraries prepared from cell-free DNA (cfDNA)? A: cfDNA does not require fragmentation before library preparation. As outlined in the product manual, 0.1 – 200 ng of cfDNA can move directly into the EM-seq v2 workflow. Pre-sheared Control DNA, Unmethylated Lambda, and CpG methylated pUC19 can be added to cfDNA going into the End Prep step. Q: Can libraries be prepared from FFPE DNA using the NEBNext® EM-seq v2 Kit? A: Yes, libraries can be prepared using 0.1-200 ng FFPE DNA inputs. The DNA should be sheared, and the libraries should be constructed according to the EM-seq v2 Manual. PCR cycles may need to be optimized, but we recommend increasing by at least 2 cycles. Note that libraries prepared from FFPE DNA may have higher methylation backgrounds in the CHH and CHG contexts. However, using a 3C filter (available in the Bismark pipeline) or similar reduces background. The filter removes reads that contain 3 or more consecutive unconverted Cs in the non-CpG context (i.e., CHG/CHH). 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 enzymatically fragmented DNA be used as input material for EM-seq™ v2? A: NEBNext UltraShear® is the only enzymatic fragmentation reagent we recommend upstream of the EM-Seq workflow. We do not recommend other enzyme-based fragmentation methods as they may affect DNA methylation marks. To use NEBNext UltraShear with EM-seq v2, please follow the recommendation provided in the UltraShear manual. Q: Are Sample Sheets available with the NEBNext® LV Unique Dual Index Primers? A: Sample sheets can be found on the Usage Guidelines section of the Product Page. 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: Are dual indexed libraries compatible with single end sequencing? A: Yes, the NEBNext Oligos for Illumina, including dual index oligos, are compatible with single read sequencing. Please refer to Illumina’s Indexed Sequencing Overview Guide (current version) for details on dual indexed sequencing workflows on a single-read flow cell or a paired-end flow cell. 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: What percentage of PhiX is needed for EM-seq™ libraries? A: Check recommendations from Illumina®. We have had success with the following PhiX percentages: NovaSeq®: 5% PhiX NextSeq® 2000: 5% PhiX NextSeq 500/550: 35% PhiX MiSeq®: 5% PhiX 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: What should I do if I observe adaptor-dimers in my EM-seq™ v2 libraries? A: Occasionally you may observe adaptor-dimer for lower input samples. An additional 0.8X cleanup of the individual libraries or the pool of libraries can be performed. 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. Q: Does the kit include primers? A: No. The kit can be used with the NEBNext LV Unique Dual Index Primers. For a complete list of low volume (LV) unique dual index primers, please see the third column of NEBNext® Multiplex Oligos Selection Chart. For use with NEBNext library prep kits, consult the library prep kit manual. The NEBNext LV Unique Dual Index Primers manuals also contain tips for setting up ligation reactions. For color balancing options, please see NEBNext® Index Oligo Selector for valid barcode combinations.