Troubleshooting Poor or Failed Sequencing Results

If you received a readme.seq file in your results folder, it was to inform you one or a number of your sequence samples failed to produce any sequence data. Also, some samples that you do receive may be of poor quality. Either scenario may be attributed to the following:

• Incorrect template or primer concentration
• Poor quality or poor purification of template DNA
• Custom primers binding to multiple sites
• Failure of custom primers to bind the template DNA
• Inherent difficulties within the template DNA
• Contaminants within your template DNA
• Primer melting temperature is incorrect
• Template does not contain a sequence complementary to the primer
• Primer doesn't work in fluorescent sequencing
• Repetitive regions

Sharp peaks characterize DNA sequence of high quality with little to no background as seen below.

Incorrect Template or Primer Concentration

This is one of the main reasons samples fail to generate little or no data. You may want to try a richer growth media such as Terrific Broth or a more robust mini-prep. The primer concentration should be at 10ng/µl and template concentration should be at 200ng/ml. If the concentration of the DNA is too high, strong peak signals may be seen at the beginning of the sequence and then drop off rapidly by 100 or 200 bases.

The following is a chromatogram of a sequence where the concentration of the sample is too high.

Poor Quality or Poor Purification of Template DNA

Next to insufficient template concentration, poor quality DNA is the primary cause of poor or no results. Please refer to the Information page for recommended protocols and host strains. Please note that template DNA that has successfully generated sequence data by manual P32 sequencing is generally not pure enough for fluorescent sequencing. The AmpliTaq polymerase used for fluorescent sequencing is much more sensitive than standard Taq and hence requires much cleaner template DNA. The following sequence is dirty and many peaks are not distinguishable from the background.

Custom Primers Binding to Multiple Sites

If your results appear to have multiple peaks per base pair, there may have been multiple binding. You may need to re-design your primer and utilize commercially available software to design primers. It is vital to ensure that the primer sequence will not self-hybridize. Also, primers that fail to bind to the template due to an insufficient concentration will not yield sequence data.

The following is an example of a sample of a template containing multiple priming sites.

Inherent Difficulties Within the Template DNA

Highly repetitive regions, regions with hairpin loops or secondary structures, or other factors inherent with the template DNA may also lead to poor or no data.

Contaminants Within Your Template DNA

The presence of EDTA in your template DNA will inhibit the cycle sequencing reaction, as will the presence of high salt concentrations. Capillary electrophoresis is much less forgiving of salt present in DNA as compared to other sequencing methods. Cellular debris, RNA, and phenol also inhibit this reaction. Please be certain to submit your samples in water, not TE, and refrain from protocols calling for phenol: chloroform extractions.

The following chromatogram shows a sequencing sample with a moderate amount of contaminants, which can be seen running along the base of the peaks.

Prime Melting Temperature Is Incorrect

All primers should have a melting temperature between 50-65 °C. Several methods and all commercially available primer designing programs can determine the correct Tm.

Template Does Not Contain a Sequence Complementary to the Primer or the Primer Does Not Work with Flourescent Sequencing

Occasionally one of the primers used to generate the PCR product does not work in fluorescent cycle sequencing. It is likely that such a primer, although sufficiently competent in the exponential PCR process, is very inefficient in the linear amplification of cycle sequencing.

Repetitive Regions

Sometimes a loss of signal will occur after a poly nucleotide region (especially G's or T's). There are two options. First, you could use a primer that sets on the poly N region. If you get through and know the final base, a primer can be designed with 17 like bases coupled with the known base, or make it a mixed base site if it is unknown (GGGGGGGGGGGGGGGGGN). The second option is to try sequencing from the reverse direction.

Below are two samples that have a repetitive poly T region with poor sequence that follows it.