Failed DNA sequencing reaction
Identifying failed DNA sequencing reactions
- The trace chromatogram has noisy or “messy” sequence peaks with low quality scores. Can have peaks that look real and have high quality scores, especially when the trace has been base called using the KB base caller.
- No or little signal in the raw data channels except for leftover dye at the beginning of the trace.
- Signal strength in the raw channel usually below 100.
- The trace sequence does not match either the expected sequence or other sequences in GenBank.
Figure 1. Example of a failed reaction trace. (A) Processed channel data. (B) Raw channel data at the same base location.
Causes of failed DNA sequencing reactions
- Poor quality DNA. Very common when sequencing plasmid miniprep templates.
- Loss of the reaction during clean up. This can be a particular problem when using ethanol precipitation clean up protocols.
- Too much template DNA. Excess template can kill the sequencing reaction.
- Wrong primer used. More common than you might think!
- Bad water. The water used contains an sequencing inhibitor.
- Degraded or failed synthesis primer. Oligonucleotide synthesis is chemically complex and primer synthesis failures is fairly common.
- Dead sequencing chemistry. Can occur if the BigDye chemistry is stored under the wrong conditions or is freeze-thawed too many times. Either the Taq DNA polymerase or dye labeled nucleotides can have degraded.
- Blocked capillary. Every trace using a particular capillary fails. Can be identified by tracking trace quality on a trace by trace basis.
Solving DNA sequencing reaction failures
- Poor quality DNA. The best way of avoiding this problem is to not sequence plasmid DNA and sequence a PCR amplified fragment of the plasmid insert. If this is not possible then it is recommended that a plasmid miniprep kit is used. One tip is to perform a final ethanol precipitation on the kit purified plasmid DNA. This often solves problems with the quality of the template.
- Loss of sequencing reaction during clean up. This can be avoided by not using an ethanol precipitation protocol to clean up the sequencing reaction. There are a number of kits that work very well, unfortunately they can be very expensive. One tip for avoiding loss of the reaction DNA pellet when using the ethanol protocol is to add 1µl of a 20 mg/ml solution of glycogen (Sigma G-1508) to the sequencing reaction before adding the ethanol. This helps make the pellet visible and the glycogen does not seem to interfere with the injection of the sequencing fragments onto the sequencers capillaries.
- Too much template DNA was used. This can be avoid by checking the concentration of the template on an agarose gel before sequencing. This will also allow you to see the purity of the template DNA and if there is a significant amount of contaminating genomic DNA or RNA present. Do not rely on a spectrophotometer reading to calculate the template concentration.
- Wrong primer. This is simple to solve, but can be difficult to detect. Check the sequence of the primer and template to make sure that the primer binding site is present. This can be a particular problem with some "universal" forward and reverse primer sequences which do not work with some common plasmids. Do not trust other people's working stock solutions (especially those of your unreliable your lab colleagues!) and make your own. It might take 5 minutes longer, but it will save you a lot of future headaches.
- Bad water. Inhibitors can end up in lab water stocks that can kill DNA sequencing reactions. If you think this may be a problem then throw out the water and use a fresh stock - remember water is cheap.
- Degraded primer. Don't use old diluted primer stocks. Store the primers in 10mM Tris/ 0.1 mM EDTA (pH 8.5) rather than water. Don't use other peoples stocks. If you have any doubt about how the primer quality through it out and make up a fresh working solution from the primer stock.
- Failed oligonucleotide synthesis. If you suspect that the primer is poor quality either have it presbyters or check in a polymerase chain reaction (PCR). Alternatively if you have a control template that you know should work with the primer then this can be a good way of identifying primer problems.
- Dead sequencing chemistry. This is a relatively rare problem, however, if a batch of BigDye chemistry has not been used for some time, or there is any doubt about how it has been stored, then it is advisable to perform a control sequencing reaction before undertaking a large number of experimental reactions. Many problems with dead chemistry can be prevented by storing the BigDye chemistry in small aliquots and avoiding repeated freeze/thaw cycles.
- Blocked capillary. Can be identified by tracking trace quality on a trace by trace basis. While this can be done manually we recommend using our QualTrace software. If you suspect that a sequencer capillary is blocked then inform the operator.
For more information on automated QC tracking of sequencing traces please visit the QualTrace DNA sequencing analysis software page.
Return to the main DNA sequencing troubleshooting page.