Multiple Signals

This is caused by one of two reasons. The first cause is that there are multiple primers present. DNA sequencing is a single strand PCR reaction with dye terminators. If there are two or more primers present in your sample, it will create more than one signal that will cause multiple peaks in the same position. This creates confusion for the base calling software that will result in multiple N’s seen in your sequence.

The second reason is that there are multiple priming sites for the same primer. Double priming will also cause more than one signal that will interfere with the base calling software.

Top-Heavy Signals

This is a phenomenon where the shorter fragments at the beginning of the sequence are more intense than the larger fragments towards the end of the sequence. Overloading the reaction with too much DNA, template or primer will result in top-heavy signals. Underestimation of your DNA concentration will lead to sample overloading.

The other cause of this phenomenon is salt contamination. Salt contamination will cause the polymerase used for DNA sequencing to lose some of its efficiency. The result is a creation of more of the smaller fragments and less of the larger ones.

Low Signals

This is almost always a result of too little template being added to the reaction. If the template concentration is overestimated, insufficient DNA will be added to the reaction. Contaminants will also cause a loss of signal strength. There are several contaminants that will interfere with the extension step of the PCR reaction. This will result in low signals and even failures if the concentration of the contaminants is high enough. The Qiagen guide described above has an excellent section of the effect of different contaminants on DNA sequencing.

Poor primer design will also result in low signals. If the priming step of the PCR reaction is not efficient the polymerase will not create enough sequencing fragments, resulting in low signals.

GC rich DNA is also a cause of low signals, if your DNA is GC rich please indicate this with your sample submission (there is an option to indicate GC rich DNA in dnaTools).

No Readable Sequence Data

Reaction failures are more difficult to troubleshoot, mainly because there is no signal to help with the diagnosis of the problem. The most common cause of this is the lack of a priming site in the template. It is very common to see clients submit a template for sequencing with one of our universal supplied primers, and then see a failure because the wrong primers were selected. Another likely cause is the lack of a priming site inside your insert. Internally nested primers are often used to verify sequence inside of your insert. If for some reason the template submitted does not contain the insert of interest, it will also not contain your priming site. This is a very common cause failures, please be careful to select the correct primers and always verify that your insert of interest has been inserted into your template.

The other less common cause of failures is due to contaminants, mainly EDTA. EDTA is a chelating agent for Mg2+ which is a co-factor for the polymerase used in DNA sequencing. If EDTA is present it will make the Mg unavailable to the polymerase causing failures. TE contains EDTA, and therefore should not be used to resuspend your DNA.

Secondary Structures

Finally, it is sometimes possible for the DNA to contain secondary structures that interfere with the PCR reaction. This is mainly caused by inverted sequences forming hairpin loops. Hairpin loops that form around the priming site will usually prevent binding and cause the reaction to fail. Hairpin loops further downstream of your priming site will usually result in a sudden loss of signal in your sequence. This is similar to a top-heavy signal except that in this case, the larger fragments are not being created at all due to the structure interfering with the extension of the polymerase. This is mostly likely the case if there is an unexpected termination of signal in your analysis.