Sanger Sequencing Support—Troubleshooting

For data run on capillary electrophoresis platforms, the troubleshooting process consists of the following steps:

• Identify the problem, i.e., low signal intensity, broad peaks, noisy data etc. 
• Check for proper storage of reagents and expiry dates.
• Run the controls. The use of controls will assist in simplifying troubleshooting:

Control DNA template (pGEM) and -21 M13 Primer are provided in the BigDye™ Terminator kit and can help you determine whether failed reactions are caused by poor template quality or sequencing reaction failure. See the table below for setting up the pGEM control. 

• BigDye™ Terminator Sequencing Standards. This standard is dried down sequenced product that gets reconstituted in HiDi™ Formamide and is loaded directly on to the instrument. It can help distinguish between chemistry problems and instrument problems.
• Use of HiDi™ Formamide only injection (optional). Results can help distinguish sample problems and instrument problems related to contamination.
• Laboratory internal DNA quality control standards. These are standards from your lab that have worked and can be used to determine if the problem is with the template or the primers you are using. 

Troubleshooting can be a time-consuming process but a methodical approach can save both time and money in finding the root problem and addressing it correctly. The Q/As below are some of the more common DNA Sequencing issues. However, additional troubleshooting can be found in the DNA Sequencing by Capillary Electrophoresis Guide.

Shoulders on all the peaks can be caused by the following:

• Capillary array needs to be replaced.
• Overloaded sample. The amount of template in the sequencing reaction needs to be reduced or the injection time on the instrument can be shortened.
• Sequencing primers contain n+1 or n-1 bases. Try primer with another template and if issue persists, resynthesize primers ensuring that they are HPLC purified. 
• A repeat region is present and stutter is occurring during the PCR amplification or cycle sequencing. If the stutter occurs during PCR, the use of anchored primer may help in the sequencing. Some customers have found that they can get past poly(A) regions using a mixture of oligo dT18 primers with either a C, A, or G as the 3’ terminal dinucleotide or 2-base anchors. 

If either G or C shouldering is occurring and the A and T peaks do not exhibit shouldering, it is possible that dye degradation is occurring. The G or C dye degradation may be due to photobleaching, oxidation, arcing event, or changes in pH. To prevent dye degradation, ensure the following:

• Protect the reactions from light.
• Load the samples in fresh Hi-Di™ Formamide, BigDye™ XTerminator™ Solution, or 0.1 mM EDTA, pH 8.0. 
• Check the expiration dates on all reagents and replace any reagents that have expired. 
• Avoid prolonged exposure to air. Heat-seal any plates that have to sit in loading solution more than 6 hours without running. Remove the seal prior to running, unless running on an Applied Biosystems™ 3730 Series DNA Analyzer, which can pierce heat-sealed plates. 
• If you have had an arcing event, clear the air bubbles and clean the system with warm water (<42 degrees C). If you cannot clear the discoloration caused by the arcing event or continue to get “Unstable current detected” errors, a service call may be required. 
• Reusing septa seals may cause this issue. Try using new septa on the water, buffer, and waste containers as well as on the sample plates.

Peaks observed within the first 100 bp can be due to excess dye terminators (ddNTPs) remaining in the sample, also known as dye blobs. These dye blobs can occur in several positions within the first 100 bp and are typically seen as broad C, G, or T peaks and can impact basecalling. Additional optimization of the DNA sequencing cleanup will be needed. Please see our suggestions below:

• If using spin columns or spin plates, ensure transfer of the sample to the center of the purification material without the pipet tip touching the purification material. Sample dispensed along the walls of the cleanup column may bypass the purification material. Use a single channel pipet and/or position the tip directly above the spin column/plate while dispensing at low speed.
• If using ethanol precipitation, the ethanol or salt concentration may be too high, leading to unincorporated dye terminators and salts precipitating with the sequencing product. Repeat procedure with the recommended ethanol concentration. 
• If using the BigDye™ XTerminator™ Kit, dye blobs in the data can be caused by the following factors:
  • Insufficient mixing of the sample during vortexing. The vortexing portion of the protocol is a critical step. The recommended vortexers (Digital Vortex-Genie™ 2, IKA MS3 Digital, IKA Vortex 3, Taitec MicroMixer E-36, or Union Scientific Vertical Shaker) have been qualified for use with the BigDye™ XTerminator™ Purification Kit. Use of one of the recommended vortexers is strongly advised. Any vortexer employed for use with the BigDye™ XTerminator™ Purification Kit should be capable of sustained operation at 2,000 RPM and have a maximum orbital diameter of 4 mm. After 30 minutes of vortexing, the plates should not be warm to the touch.
  • Incorrect ratio of BigDye™ XTerminator™ reagents to reaction volume. The precise ratio of liquid phase and insoluble phase in the BigDye™ XTerminator™ reagents is critical to achieving specified performance. 
  • Insufficient template in the reaction. We suggest that you use the recommended template concentration for this protocol.
  • Please see Page 28 of the BigDye™ Xterminator™ Purification Kit manual for other suggestions on dye blob troubleshooting.

Flat peaks are typically an indication of data with too high of a signal, also known as offscale data. Please check the raw data of the sample file and determine the signal intensity on the Y-axis. The maximum signal thresholds for raw data are 32,000 rfu for the Applied Biosystems™ 3730/3730xl DNA Analyzers and 3500/3500xL Genetic Analyzers, and 8,000 rfu for the Applied Biosystems™ 310 Genetic Analyzer and Applied Biosystems™ 3130/3130xl Genetic Analyzers. 

There are several options for reducing the signal, please see our suggestions below:

• Re-do the sequencing reaction using lesser template.
• If the sample has been on the instrument <24 hours, reduce the injection time in the run module, and then re‑inject the sample.
• If the sample is purified with the BigDye™ XTerminator Purification Kit and has been on the instrument <24 hours, carefully remove 10 µL of sample off the BigDye™ XTerminator beads in the plate, and then add 10 µL of 0.1 mM EDTA to dilute the sample. Re-inject the sample using a standard run module (non-BigDye™ XTerminator run module). Decrease the injection voltage and injection time to match the BDX run module.
• Ensure that either Hi‑Di™ Formamide or a 0.1 mM EDTA injection solution is used for samples.

Offscale sequencing data can be prevented by optimizing the amount of template and primer in the reaction. The recommended amount of primer is 3.2 pmol and the amount of template is dependent on the template type:

* The BigDye™ XTerminator™ Purification Kit is intended to purify the DNA sequencing reaction after thermal cycling by sequestering cycle-sequencing reaction components such as salt ions, unincorporated dye terminators, and dNTPs, to prevent their co-injection with dye-labeled extension products into a capillary electrophoresis DNA analyzer. 

If, after using the recommended amount of template and primer, offscale data is still present, it may be necessary to further decrease the amount of template used in the sequencing reaction.

Our capillary electrophoresis systems allow the editing of the injection time. The injection time can be reduced and the sample can be re-injected. Depending on the signal intensity, the sample may also require serial dilution to reduce the signal. Please refer to the appropriate instrument user guide for instructions on editing the injection time. 

It is common to observe noisy data after a homopolymer repeat due to stutter. Stutter can occur during the PCR amplification or cycle sequencing and is due to polymerase slippage. If the stutter occurs during PCR, the use of anchored primer may help in the sequencing. Some customers have found that they can get past poly(A) regions using a mixture of oligo dT18 primers with either a C, A, or G as the 3’ terminal dinucleotide or 2-base anchors. Sequencing in both directions can also assist with obtaining sequencing before and after the homopolymer repeat.

Low signal intensity can be caused by many factors including thermal cycler malfunction (in the case of an entire plate failure), poor cleanup, and insufficient sequencing template quantity/quality.

• Use the pGEM control and -21 M13 primer provided in the sequencing kit to isolate template quality/quality issues or sequencing reaction failure. Increasing template and primer in the sequencing reaction can improve signal if the DNA quality, PCR purification, and sequencing reaction purification steps have been performed properly. For amount of template DNA to use in the sequencing reaction, view this information. 
• Check the quality of the template. If needed, prepare fresh DNA and repeat the reaction. You can visit our Plasmid DNA Purification Support Center page for tips on purification. In general, we would recommend that you:
  • Ensure that fresh, unexpired reagents are used in the sequencing workflow.
  • Check the thermal cycler, set the ramp rate to 1 degree C/second, use correct thermal cycling parameters and check the thermal cycler calibration. 
  • Prepare a fresh working stock of primer or order new primer. Use 3.2 pmol in the final reaction.
  • If less than the recommended amount of the BigDye™ Terminator Ready Reaction Mix is used in the sequencing reaction, please use the recommended amount of 8 µL in a 20 µL final reaction volume, or 4 µL in a 10 µL final reaction volume. 
  • If dye blobs are present, please check the DNA Sequencing by Capillary Electrophoresis Guide or with the cleanup kit manufacturer for more troubleshooting recommendations to remove the dye blobs. 

Control DNA template (pGEM) and -21 M13 Primer are provided in the BigDye™ Terminator kit and can help you determine whether failed reactions are caused by poor template quality or sequencing reaction failure. See the table below for setting up the pGEM control. 

Sequence composition, such as hairpins or secondary structure, may be preventing the DNA polymerase from sequencing through the region. Redesigning the primers around the region may help. Some customers have reported that the use of additives such as 5-10% DMSO, betaine, or glycerol, or increasing the initial incubation from 1-10 mins may help denature the template, but we have not tested these in-house.

Some customers have reported that the use of additives such as 5-10% DMSO, betaine, or glycerol or increasing the initial incubation from 1-10 mins may help denature the template, but we have not tested these in-house.

Here are possible causes and solutions for a missing C peak:

• The samples are bisulfite converted and the unmethylated Cs have been converted to Ts
• A severe arcing event has elevated the blue baseline offscale and is masking the C peaks. Perform the water wash wizard with warm (42 degrees C) water several times (for the Applied Biosystems™ 3500/3500xL Genetic Analyzers, use the conditioning pouch). If the blue baseline has not returned, a service call may be required. 
• C dye degradation has occurred. This may be due to photobleaching, oxidation, an arcing event, or changes in pH. To prevent dye degradation, ensure the following:
  • Protect the reactions from light.
  • Load the samples in fresh Hi-Di™ Formamide, BigDye™ XTerminator™ Solution, or 0.1 mM EDTA, pH 8.0. 
  • Check the expiration dates on all reagents and replace any reagents that have expired. 
  • Avoid prolonged exposure to air. Heat-seal any plates that have to sit in loading solution for more than 6 hours without running. Remove the seal prior to running, unless running on an Applied Biosystems™ 3730 Series DNA Analyzer, which can pierce heat-sealed plates. 
  • If you have had an arcing event, clear the air bubbles and clean the system with warm water (< 42 degrees C). 

If the raw data shows good signals and a clean baseline, the issue is most likely the setting in the Sequencing Analysis Software. Please check the Analysis Protocol used to analyze the data. This can be done in Sequencing Analysis. Go to the Analysis menu > select Analysis Protocol Manager > highlight the Analysis Protocol in use > Select Edit > go to the Basecalling Tab and select ‘At PCR Stop.’ Then click OK. Apply the edited Analysis Protocol to the sample and re-analyze. Ideally a minimum of 100 bp is recommended to ensure successful basecalling. 

Lack of signal can be determined by looking at the scale of signal produced in the raw data view. Lack of signal can be caused by many factors which include problems in the sequencing reaction (template quantity/quality), thermal cycler malfunction (plate failure) and capillary electrophoresis failure (failing laser, air bubbles in lines, etc.):

• Template - check the quantity and quality of the DNA template. 
• Primer - check the quality and quantity of the primer. Also, review the primer design and confirm that the primer has an annealing site on the template. 
• Reagents - confirm that the reagents have not expired and have been stored properly. 
• Thermal cycler - calibrate the thermal cycler, set the ramp rate to 1 degree C/second, use correct thermal cycling parameters, and repeat reactions on another thermal cycler, if available. 
• The sequencing products are lost during purification - use the correct centrifuge speeds and times for the precipitation procedures and the spin column or spin plate procedures. Check that the ethanol concentration is correct for the precipitation protocol. Open a new bottle of ethanol if the age is unknown. 
• Fully resuspend the samples in Hi‑Di™ Formamide. 
• To eliminate instrument issues, run the sequencing standard to confirm the performance of the instrument and capillary array. Confirm that the BDX run module is being used for BigDye™ XTerminator™ Kit–purified samples. Confirm that the correct volume is in the well and that the sample plate does not have a bubble at the bottom of the well. Briefly centrifuge to remove the bubble.

The BigDye™ XTerminator™ Solution is expected to perform to specification if left out for no more than one 24-hour period. We cannot guarantee performance for reagent left out for a longer period of time. 

Yes. The precipitate should be resuspended by warming the SAM™ Solution (remember, never heat the BigDye™ XTerminator™ Solution) to 30-37 degrees C with gentle vortexing. Bubbles will form on the surface of the SAM™ Solution when vortexed. These will dissipate within a few minutes. Allow the SAM™ Solution to cool to ambient temperature before use. Do not use it while warm as using warm SAM™ Solution will produce poor results.

Dye blobs in the sequencing data can be caused by the following factors:

• Insufficient mixing of the sample during vortexing. The vortexing portion of the protocol is a critical step. The recommended vortexers (Digital Vortex-Genie™ 2, IKA MS3 Digital, IKA Vortex 3,Taitec MicroMixer E-36, or Union Scientific Vertical Shaker) have been qualified for use with the BigDye™ XTerminator™ Purification Kit. Use of one of the recommended vortexers is strongly advised. Any vortexer employed for use with the BigDye™ XTerminator™ Purification Kit should be capable of sustained operation at 2,000 RPM and have a maximum orbital diameter of 4 mm. After 30 minutes of vortexing, the plates should not be warm to the touch.
• Incorrect ratio of BigDye™ XTerminator™ reagents to reaction volume. The precise ratio of liquid phase and insoluble phase in the BigDye™ XTerminator™ reagents is critical to achieving specified performance. 
• Insufficient template in the reaction. We suggest that you use the recommended template concentration for this protocol.

Please see page 28 of the BigDye™ Xterminator™ Purification Kit manual for other troubleshooting tips.

Some possible reasons for reduced signal for smaller fragments may be:

• BigDye™ XTerminator™ Solution or premix was stored for >24 hours at ambient temperature.
• BigDye™ XTerminator™ Solution or premix was exposed to temperature in excess of 25 degrees C, even briefly.
• Reaction plate was inadvertently warmed during the mixture step or before transfer to the instrument. 
• BigDye™ XTerminator™ reagents are past their expiration date.
• The SAM™ Solution was used while warm (higher than ambient temperature) 
• The vortexing resulted in too much friction heat.

Please see page 28 of the BigDye™ Xterminator™ Purification Kit manual for other troubleshooting tips. 

A possible reason for no signal when using the BigDye™ XTerminator™ Purification Kit is that the capillaries are going into the BigDye™ XTerminator™ Solution. To alleviate this issue, please check the run module that is being used on the Genetic Analyzer. The template should have “BDX” in the run module name. If it does not, it means that the capillaries are going into the BigDye™ XTerminator™ Solution, and this will prevent the injection of the sample into the capillaries. 

Some possible reasons for low signal when using the BigDye™ XTerminator™ Purification Kit are:

• Incomplete desalting due to insufficient BigDye™ XTerminator™ Solution. Ensure that the wide bore tips are used for pipetting the BigDye™ XTerminator™ Solution and mix the solution prior to pipetting as the BigDye™ XTerminator™ Solution will settle. 
• Incomplete mixing. Please follow the mixing guidelines in Appendix D of the BigDye™ Xterminator™ Purification Kit manual.
• Incorrect ratio of BigDye™ XTerminator™ reagents to reaction volume. The precise ratio of liquid phase and insoluble phase in the BigDye™ XTerminator™ reagents are critical to achieving specified performance.

This is most likely due to incomplete conversion of the DNA template. Please see below for possible reasons why conversion may be incomplete:

• Too much DNA template may have been added to the bisulfite conversion reactions. The optimal amounts of template are the following:
• Purified gDNA: 100 ng to 1 µg
• Cultured cells: 5,000 cells to 10⁵ cells
• Blood: 2.5 µL
• Temperature variability may have occurred during the bisulfite conversion reactions. Verify the performance of your thermal cycler and repeat the conversion. 
• Insufficient amounts of denaturing or conversion reagents were added to the samples. Repeat the denaturation or the bisulfite conversion using harsher conversion conditions. Please refer to the conversion conditions in the Cells-to-CpG™ Bisulfite Conversion Kit manual.

There are several reasons why conversion may be incomplete:

• Too much DNA template may have been added to the bisulfite conversion reactions. The optimal amounts of template are the following: 
  • Purified gDNA: 100ng to 1µg
  • Cultured cells: 5000 cells to 10⁵ cells 
  • Blood: 2.5 µL
• Temperature variability may have occurred during the bisulfite conversion reactions. Verify the performance of your thermal cycler and repeat the conversion. 
• Insufficient amounts of denaturing or conversion reagents were added to the samples. Repeat the denaturation or the bisulfite conversion using harsher conversion conditions. Please refer to the conversion conditions in the Cells-to-CpG™ Bisulfite Conversion Kit manual.

If the DNA template was bisulfite converted successfully and the PCR amplification was successful, the issue may be due to long stretches of T, due to the C>T conversion, resulting in slippage and stutter peaks. Additionally, there may be secondary sequences or primer dimer sequences interfering with the sequencing reaction. The presence of these types of shorter amplicons can contribute to offscale signals at the beginning of the sequence trace, and depletion of the sequencing components, which causes rapid drop-off of signal intensity for the remaining sequence. If sequencing the PCR amplicon directly, the following list of recommendation may help with improving the sequencing reaction:

• PCR primer may not be able to anneal to the intended primer site due to converted Cs in that region. Use Methyl Primer Express to design primers that will work for amplification post conversion. 
• Quantification of PCR amplicons prior to sequencing.
• Use of M13-tailed primers attached to the 5’ end of the PCR primers. 
• Use of full-strength BigDye™ Terminator™ Ready Reaction Mix.
• Use of BigDye™ XTerminator™ Solution for the clean-up of sequencing reactions.