Why there are multiple overlapping peaks in my sequencing data.

Overlapping peaks are caused most likely by the presence of more than one template or by multiple primer binding sites.

The presence of multiple peaks within your sequence can be caused by numerous factors. To help determine the cause, look at two aspects: where the multiple peaks begin, and the overall signal strength of your sample. Samples with low signal strength can have artificially high background noise that can give the appearance of multiple peaks. However, if your average signal intensity is high, then you can rule out the possibility that background noise is the cause. We've broken down this section into two parts, based on where your multiple peaks begin.

Observation: Multiple Overlapping Peaks from Beginning
Pic 2-1: Overlapping peaks from beginning.

Possible Cause 1: Multiple priming sites involving vectors. Your primer may have a secondary priming site on the plasmid that may be identical or closely related, with different nucleotide sequences following each site, giving superimposed bands within your sequence. if the priming sites are identical (e.g. When more than one T7 promoter site is present), the double peaks will be strong from the outset. The fragments may also show shifted migration so that the double peaks are not directly on top of one another but will be offset to one side or the other due to the differing mobility patterns of the strands with dissimilar nucleotide composition. On other instances, a secondary priming site may not be exactly the same, but may differ by a few internal bases. In this case, the mismatched primer may not hybridize as efficiently but can still anneal and extend, and give rise to less intense fragments that can be seen underneath your peaks of interest.

Solution: In both cases, it's necessary to screen both your vector and insert carefully to look for sequences that may match or be similar to your proposed primer. You may need to choose another vector primer on the same end of the multiple cloning site or redesign your custom primer. when choosing another primer is difficult, such as when primer walking through a repetitive area, try to find a primer that has a 3'-base match specific to your area of interest which can help as an "anchor".

Possible Cause 2: Multiple priming sites in generating PCR products.

Solution: This may occur when one or both of the PCR primers hybridizes to more than one position on the template DNA, giving rise to multiple PCR products. Often this will be obvious when visualizing the PCR products on an agarose gel as there will be more than one band present. In this case, gel purification of the desired product will be necessary. One can run into difficulty, however, when the products are very similar in size, which may arise when amplifying related or repetitive DNA, and do not separate well on the gel. In this case, optimization of the PCR reaction may be necessary or redesign of the PCR primers in order to choose a more specific priming site.

Possible Cause 3: PCR primers acting as both forward and reverse primers.

Solution: Sometimes, a PCR product may be generated when one primer functions as both the forward and reverse primer in the PCR reaction, giving rise to an artificial product. This is fairly easy to detect when sequencing the PCR product as one primer will give double peaks from the start, while the other fails to give any sequence data. Redesign your set of PCR primers.

Possible Cause 4: Residual PCR primers and/or dNTPs

Solution: As two primers are present in the PCR reaction, incomplete removal of these primers can lead to double peaks within the sequencing data. Both primers will act as sequencing primers and lead to superimposed bands which correspond to the complementary strands from opposite orientations. It is critical to remove excess primers and dNTPs from the PCR reaction by purification. If attempting to do direct sequencing of PCR products without purification by diluting an aliquot of your PCR product with water to lower the concentration of residual primers and dNTPS (a method which we do not recommend), then it is imperative to optimize your PCR reaction so that primers and dNTPS are used in limiting amounts so that most are used up by the end of the PCR.

Possible Cause 5: Primers with high Tm

Solution: Primers that have a Tm much higher (>65ºC) than our suggested 57ºC-60ºC often do not function well as sequencing primers. When primers have a Tm that high, it is often a result of increased G-C content or because the primer is quite long, both factors that can increase the potential for primer secondary structure formation. In this case, redesign your primer with a lower Tm. The Tm of a primer is defined as the temperature at which 50% of the oligonucleotide and its perfect complement are in duplex. The Tm of an oligo can be roughly calculated by using the following formula:
Tm = 2°C( A+T ) + 4°C( G+C )