New England Biolabs, E7120L, NEBNext® Enzymatic Methyl-seq Kit

This kit is no longer supplied with sequencing primers. NEB recommends using Related Categories Enzymatic Conversion for DNA Methylation Analysis,, Automation for NEBNext® NGS Library Prep,, Methylome Analysis, Specification Materials Required but not Supplied Covaris® S2 instrument or other fragmentation equipment PCR strip tubes Formamide (Sigma #F9037-100 ml) or 0.1 N NaOH 80% Ethanol • 0.1X TE, pH 8.0 Nuclease-free Water Magnetic rack/stand, such as NEBNext Magnetic Separation Rack (NEB #S1515) PCR machine Bioanalyzer®, TapeStation® and associated consumables or other fragment analyzer FAQ Q: What types of sample can be processed using the NEBNext® Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module? 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 Kit and the Enzymatic Methyl-seq Conversion Module? A: The protocol has been optimized for inputs ranging between 10–200 ng. A modified protocol is also available for inputs up to 500 ng. Q: What is the difference between the NEBNext Enzymatic Methyl-seq Kit and the Enzymatic Methyl-seq Conversion Module? A: The NEBNext Enzymatic Methyl-seq Kit contains all of the components needed to make an EM-seq library, including reagents for 5-mC/5-hmC oxidation and cytosine deamination, library construction reagents and the EM-seq Adaptor, as well as the NEBNext Q5U Master Mix and dual unique primers for library amplification. The Enzymatic Methyl-seq Conversion Module does not contain reagents for library construction and amplification, or adaptors and primers. 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 EM-seq Index Primers? A: The EM-seq Adaptor is 15 µM and the EM-seq 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: Why, at some stages of the EM-seq protocol, do the NEBNext Sample Purification Beads behave differently when cleaning up the sample? A: The behavior of the beads does change depending on the clean-up and the previous EM-seq reaction step. Special care should be taken after APOBEC deamination as the beads tend to clump more easily. Do not over-dry the beads before DNA elution at this step as this can result in difficulties in resuspending the beads, and potentially reduced DNA recovery from the beads. Q: What is the expected size of an EM-seq library? A: Standard EM-seq libraries are approximately 370-420 bp. Larger inserts of 470-520 bp can be achieved using the modified protocol included in the manual. 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 libraries prepared from cell-free DNA (cfDNA) using the NEBNext® EM-seq Kit (NEB #E7120)? A: With cfDNA, there is no need to shear the DNA before library preparation. 10 – 200 ng of cfDNA can move directly into NEBNext Ultra II DNA library preparation, and then EM-seq enzymatic conversion and library amplification steps are followed as outlined in the product manual. Q: Can libraries be prepared from FFPE DNA using the NEBNext® EM-seq Kit (NEB #E7120)? A: Yes, libraries can be prepared using 10-200 ng FFPE DNA inputs. The DNA should be sheared and the libraries constructed according to the EM-seq Manual. PCR cycles may need to be optimized, but we recommend increasing by at least 2 cycles for the 10 ng and 50 ng inputs (i.e., for 10 ng use at least 10 PCR cycles and for 50 ng use at least 8 PCR cycles). Note that libraries prepared from FFPE DNA may have higher methylation backgrounds in the CHH and CHG contexts. However, use of a 3xC 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 levels of conversion are typical with the control DNAs supplied in the EM-seq kit? A: Two DNA controls are provided in the EM-seq kit: unmethylated lambda DNA and CpG-methylated pUC19 DNA. Up to 0.5% methylation is typically detected in unmethylated lambda, indicating a conversion efficiency of 99.5%. This is similar to the observed conversion efficiency with WGBS. For CpG-methylated pUC19, 96-98% CpG methylation is detected for both the EM-seq workflow and WGBS. As the pUC19 DNA is enzymatically methylated, it is likely that 100% methylation was not achieved and that not all CpGs are methylated. Q: Are Sample Sheets available for use with the NEBNext® Multiplex Oligos for EM-seq? A: Sample sheets can be found on the Usage Guidelines section of the Product Page. Q: Can enzymatically fragmented DNA be used as input material for EM-seq™? A: NEBNext UltraShear™ is the only enzymatic fragmentation reagent that we recommend upstream of the EM-Seq workflow. We do not recommend other enzyme-based fragmentation methods as they may affect DNA methylation marks. For EM-seq™ use with NEBNext UltraShear™, please follow the protocol in “Section 3” in the linked manual below. https://www.neb.com/-/media/nebus/files/manuals/manualm7634.pdf?rev=64e0e81d781748dba43702e4a78da971&hash=09F5E6499849C9045DFAE1A3AB3BDAA9 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 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: 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. 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, variation in color (colorless to yellow) has been observed. Testing has shown no difference in performance based on this variation. Q: Are EM-seq libraries directional or non-directional? A: EM-seq libraries are directional. 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: Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA prior to the deamination reaction? A: Yes, it is possible to use NaOH, although formamide is recommended since it is easier to handle. If using NaOH it is critical that the concentration of the NaOH solution is 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 prior to 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: Can the “active” TET2 Buffer be stored longer than 4 months? A: No. The “active” TET2 Buffer should be discarded after 4 months. Q: What buffers are recommended for shearing DNA in NEB’s enzymatic methyl-seq (EM-seq™) workflows? A: For use with the NEBNext® Enzymatic Methyl-seq Kit (NEB #E7120), 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. For use with the NEBNext® Enzymatic Methyl-seq Conversion Module (NEB #E7125), 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: 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/