The most common factors limiting sequence quality:
  • Impure template DNA
    • Contaminants: salts (EDTA, NaCl, NaAc, Kac, KCl), proteins, detergents (SDS, Triton X-100), RNA, chromosomal DNA, organic chemicals (ethanol, chloroform, phenol), divalent cations (Mg, Ca, Mn), and excess PCR primers, dNTPs, enzyme, and buffer components from PCR. Be sure to check the quality of the DNA before submitting for sequencing.
    • Incorrect template or primer concentration
    • Sub-optimal primer selection or annealing
    • Secondary structure of template

Good Sequencing

Example of good sequencing with evenly distributed waves
 
The sequencing electropherogram will show machine model and analysis software version in the left hand corner. The sample name and investigator will appear to the middle left. On the middle right you will see notation showing signal strength. Typical good signal will show GATC values 100-600.

Peaks are sharper for the first 500 bases, resolution of peaks drops off from that point, but are still readable out to 750 bases.

 

Bad and Not-So-Good Sequencing

The following shows some examples of problems in sequencing and what the possible reason for failure may be. This list is by no means complete or all-inclusive of reasons for failures. If you notice any obvious omissions or alternate solutions, please let us know.
 

  • Complete failure - No signal present at all

Example of failure sequencing with few if no spikes
 
Notice poly C from dye front and one to two spikes
See section below on low signal strength for suggested solutions
Insufficient template
Quantitate the double stranded DNA by fluorimeter, Double check gel quantitation. Increase the amount of DNA
Inhibitor contaminating template such as salt or ethanol
Clean up template. Spin columns
Insufficient primer
Make sure you diluted primer to correct concentration. 12 pmol primer per reaction (NOT 12 picomolar)
Primer has no annealing site, poor primer design (low melting temp) or incorrect primer sequence
Pick good sequencing area. If there are any mismatches between the primer and template the primer will not anneal correctly. Do not make primer in areas of ambiguity.
 

  • Low signal strength of G A T C values less than 50

Example of low signal strength sequencing with few waves
 
Insufficient template
Quantitate the double stranded DNA by fluorimeter, Double check gel quantitation. Increase the amount of DNA
Inhibitor contaminating template such as salt or ethanol
Clean up template. Spin columns
Insufficient primer
Make sure you diluted primer to correct concentration. 12 pmol primer per reaction (NOT 12 picomolar)
Primer has no annealing site, poor primer design (low melting temp) or incorrect primer sequence
Pick good sequencing area. If there are any mismatches between the primer and template the primer will not anneal correctly. Do not make primer in areas of ambiguity.
 

  • Primer-dimer

Example of Primer-dimer with large spikes only at the beginning
 
Primer dimer caused by self binding primer or primer binding to other primers present in template mixture (PCR). Short fragment is predominately amplified. There may or may not be any sequencing following such an artifact.
Analyze primer used for sequencing.

Could template be contaminated with other primers to cause primer dimers?

Is point of drop-off at the cloning site?
 

  • Abrupt signal loss

Example of abrupt Signal loss where waves cease
 
PCR product
This is normal
Plasmid insert with secondary structure
Sequence opposite strand, try a primer that anneals in a different position. Ask Core Labs staff to add DMSO or try other techniques
 

  • Noisy Sequencing

Example of Noisy Sequencing where spikes occur at random
 
Probable cause: Low signal strength due to low template/primer or impurities such as salt or ethanol
 
 
Example of Noisy Sequencing where spikes are not consistent
 
Probable cause: Multiple templates/primers/priming sites or high signal strength
 
 
Example of Noisy Sequencing caused by Mixed plasmid
 
Probable cause: Mixed plasmid prep, two inserts
 
low signal strength
Low signal strength may cause background to appear even higher. Check for impurities. Increase primer and/or template concentration
high signal strength (saturating detector)
Decrease template concentration
Contaminated template (impurities)
Purify template with spin columns
Mixed plasmid prep, vector portion is clean 2 inserts seen
streak for isolation 2X
Multiple templates/primers/priming sites
Streak plasmids for isolation 2X,

Are there possible multiple priming sites? N-1 primer?

If a PCR product make sure you removed original primers, also one primer may have generated both ends, or primers may amplify more than one product of DNA.
 

  • Slow drop-off read / Early signal loss

Example of Slow drop off similar to abrupt signal loss
 
Low template or primer concentration
Increase either primer or template concentration
Contamination with salt, ethanol, or other impurity
Purify template with spin columns
High GC content
See next section
High GT content
Talk to Core Facilities staff about what we can do
Secondary structure
Sequence opposite strand, try a primer that anneals in a different position. Ask Core Labs staff to add DMSO or try other techniques
 

  • High GC content with short read

    High GC content

    Talk to Core Facilities staff to determine alternate sequencing strategies such as adding DMSO, increasing denaturation temperatures, alternate sequencing chemistries
 
 
 
 
 
 
 
 
 
 
 
 
 
 

  • Waves due to enzyme slippage at poly T region

Waves due to enzyme slippage
 
The DNA polymerase may slip during sequencing reaction when it reaches a poly base region. A partially synthesized strand will rebind a base ahead or behind where it should and a series of products are generated with slightly different lengths of poly base regions. This generates a wave pattern as seen above.
Use an anchored primer or alternate sequencing chemistry such as dRhodamine.