EM-seq Troubleshooting Guide
Troubleshooting Guide for NEBNext® Enzymatic Methyl-seq v2 Kit NEB# E8015, NEBNext® Enzymatic Methyl-seq Kit NEB# E7120 and NEBNext® Enzymatic 5hmC-seq Kit NEB# E3320.
| Problem | Potential Cause | Solution |
|---|---|---|
| Oxidation Efficiency Low (pUC19 CpG methylation below 96%; minimum of 500 reads mapping to pUC19) | EDTA in DNA prior to TET2 step | Elute DNA in nuclease free water or NEBNext EM-seq Elution Buffer after ligation or perform a buffer exchange prior to TET2 reaction |
| Resuspended TET2 Reaction Buffer is older than 4 months | Resuspend a fresh vial of TET2 Reaction Buffer Supplement. Do not use resuspended TET2 Reaction Buffer Supplement longer than 4 months. | |
| No DTT added, DTT concentration too low | Ensure the correct volume of DTT is added and do not reuse old tubes of DTT. For each new library prep kit open a new tube of DTT and discard the DTT tube once the kit is used up. | |
| Reagents not made up correctly. | Prepare reagents (enzymes, buffers, other reagents) by thawing on ice and prior to using by vortex briefly and spin down. Ensure accurate pipetting of viscous reagents and sufficient mixing. | |
| Fe(II) solution old or not at the correct concentration | Pipette 1 µL of Fe(II) solution accurately using a P2 pipette tip and do not dip the tip too deep into the Fe(II) stock solution. Dilute Fe(II) solution and use within 15 minutes, discard the diluted Fe(II)) solution. Vortex or pipette mix or after addition of the Fe(II) solution. | |
| Fe(II) solution added to the TET2 master mix | Do not add Fe(II) to the TET2 master mix. The sample should be mixed with the oxidation reagents before addition of Fe(II). | |
| Mixing after addition of the TET2 master mix or Glucosylation master mix is skipped or inefficient | Mix the sample well after addition of TET2 or Glucosylation master mix. Vortex on high speed briefly or set a P 200 pipette to 80 or 90% of the total reaction volume and pipette up and down 10 times. | |
| Correct/ precise volumes of reagents added in TET2 or Glucosylation reaction | For best results consider making a master mix of the Oxidation Reagents (E7120, E8015) or the Glucosylation Reagents (E3320). Mix the master mix prior to addition to the samples. | |
| DNA input below minimum required input | Ensure that the input requirement are followed for each kit and that the appropriate protocol is followed for samples inputs 10ng or less (E8015, E3320) | |
| Reaction setup too warm | Keep all reagents on ice and set up samples on an aluminum chill block or on ice. | |
| Deamination Efficiency Low (Lambda CpG/CHG/CHH methylation above 1.0 %; minimum of 5000 reads mapping to lambda) | DNA too long, not properly fragmented preventing DNA from denaturing | Ensure proper fragmenting of the DNA sample prior to library prep. If DNA fragmentation may be incomplete optimize fragmentation conditions and visualize fragmented DNA on a fragment analyzer until fragmentation is reproducible |
| DNA input below minimum required input | Use minimum input of DNA. | |
| NaOH wrong concentration | Use formamide or be sure to handle NaOH according to these guidelines https://knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000006560 | |
| Reagents not made up correctly. | Prepare reagents (enzymes, buffers, other reagents) by thawing on ice and prior to using by vortex briefly and spin down. Ensure accurate pipetting of viscous reagents and sufficient mixing. | |
| Correct/ precise volumes of reagents added in APOBEC / deamination reaction | For best results consider making a master mix of the Deamination Reagents (E7120, E8015, E3320). Add the APOBEC enzyme last to the mastermix and mix prior to addition to the samples. | |
| Beads in APOBEC reaction | During the elution transfer, use a small tip, aspirate slowly holding the tip orifice away from the beads. Check the tips over a white piece of paper before dispensing. | |
| Insufficient mixing of the reaction. | Mix the sample well after addition of Deamination reaction components. Vortex on high speed briefly or set a P 200 pipette to 80 or 90% of the total reaction volume and pipette up and down 10 times. | |
| Traces of ethanol in eluate/ beads not dry enough | Remove last droplets of ethanol with a P 10 pipette prior to adding the elution buffer. | |
| Do not overdry the beads before elution or before adding ethanol . | Watch the beads after the last ethanol removal rather than relying on a timer, especially during the next bead cleanup after the APOBEC reaction. Remove supernatant/ethanol only from as many samples as can be comfortably processed prior to adding the next reagent. | |
| Samples not cool enough prior to adding APOBEC | Make sure sample cools after denaturing before adding the Deamination components. It is best to use an aluminum chill block. If not possible use ice + water to ensure contact between the cooling agent and the sample container. Cool quickly to prevent renaturation of the DNA. | |
| Library Low Yield Yields vary depending on sample type and input. Typically yields between 5 nM - 40 nM are observed. Note: lower library yields can still result in good sequencing data. |
Samples dried out during bead cleanup steps. | Observe the samples during the washes to ensure that the beads do not dry out. Remove supernatant/ethanol only from as many samples as can be comfortably processed prior to adding the next reagent. The beads dry out very quickly during the cleanup step after APOBEC in EM-seq E7120 and Post PCR cleanup in EM-seq v2 (E8015) and E5hmC-seq E3350 |
| Sample loss during bead cleanup. | Optimize bead cleanup step reagent addition, mixing, supernatant removal, elution mix, elution transfer. | |
| Bead loss during supernatant removal. | During the supernatant removal, aspirate slowly holding the tip orifice away from the beads. Check the tips over a white piece of paper before discarding supernatant. | |
| NaOH concentration incorrect. | Use formamide or be sure to handle NaOH according to these guidelines https://knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000006560 | |
| Issue with TET2 reaction buffer supplement | Use a fresh vial of TET2 Reaction Buffer Supplement, do not use resuspended TET2 Reaction Buffer Supplement longer than 4 months after initial resuspension. Ensure the correct volume gets into the TET2 reaction and samples are mixed well before and after adding Fe(II) solution. | |
| EDTA in DNA prior to TET2 step | Elute DNA in nuclease free water or NEBNext EM-seq Elution Buffer after ligation or perform a buffer exchange prior to TET2 reaction | |
| Fe(II) solution old or not at the correct concentration | Pipette 1 µL of Fe(II) solution accurately using a P2 pipette tip and do not dip the tip too deep into the Fe(II) stock solution. Mix dilution well by vortexing. Use diluted Fe(II) solution within 15 minutes and discard any remains of the diluted Fe(II)) solution. Vortex or pipette mix the sample after addition of the Fe(II) solution. | |
| Fe(II) solution added to the TET2 master mix | Do not add Fe(II) to the TET2 master mix | |
| Delay in workflow. | Use only the recommended stop points and avoid leaving samples too long between steps. | |
| Low efficiency of End Prep reaction | Sample contaminated, not enough enzyme added, problem during incubation. Other issues that are possible to inhibit End Prep. | |
| Low Ligation efficiency | Ligation master mix not mixed prior to using or mixed poorly after adding. Adaptor stored or thawed incorrectly. Problem during incubation. Other issues that are possible to inhibit ligation. | |
| Incorrect adaptor used |
Ensure that the EM-seq adaptor is used for EM-seq libraries and the E5hmC-seq adaptor is used for E5hmC-seq libraries |
|
| Low efficiency of PCR | PCR master mix not mixed prior to using or mixed poorly after adding. Incorrect primer amount added. Problem during thermal cycling. Other issues that are possible to inhibit PCR. | |
| Variable performance / results (oxidation, deamination or yield) | Reagent addition inconsistent. | Make a master mix whenever possible. Do not add Fe(II) or EM-seq adaptor to master mix. |
| DNA sample to sample variation | QC DNA input for concentration, presence of contaminants and fragmentation. Perform an extra cleanup of the DNA if contamination is suspected. Repeat same DNA sample and compare results. | |
| Processing issue. | Low Oxidation efficiency, low deamination efficiency or low library yield can also occur on a sample to sample basis due to variation mixing, carryover contamination, drying out, bead carryover, lack of cooling etc. Evaluate the process for potential causes. Reduce the batch size to a number of samples that can comfortably processed at the same time. |
