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Troubleshooting

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Troubleshooting guide

Always check raw signals and sequence tracing when evaluating sequencing results.
The following images were generated by ABI Sequence Scanner, which is available for free download on ABI website.

Click here for software installation instructions and operations.

Result:


Normal read

Long read for plasmid

Long read for plasmid chromatogram

Long read for plasmid Raw signal plot
 
 
  • Good quality read till 800-1000 bases
  • Clean/flat baseline
  • Relatively even raw signal level across the whole read length, gradual drop is normal


 

Short read for PCR product

Short read for PCR product chromatogram

Short read for PCR product Raw signal plot
 
 
  • Good quality read stopped after reaching the end of template
  • Clean/flat baseline
  • Even signal level across the whole read length

 

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Short read

Signal dropped soon after read start

Signal dropped soon after read start chromatogram

Signal dropped soon after read start Raw signal plot


 

Possible Causes
  • Primer-dimer formation
  • Secondary structure
  • High GC-content
  • Inappropriate template/primer ratio


Possible Solutions
  • Re-design sequencing primer to avoid primer-dimer formation
  • Re-design sequencing primer to sequence the opposite strand
  • Optimize cycle sequencing conditions and submit purification & run samples
  • Optimize template/primer concentration

 

Signal dropped after homopolymer region
Poly C/T

Signal dropped after homopolymer region chromatogram

Signal dropped after homopolymer region Raw signal plot
 

Poly G/T

Signal dropped after homopolymer region chromatogram

Signal dropped after homopolymer region Raw signal plot
 

Possible Causes
  • Replication slippage


Possible Solutions
  • Optimize cycle sequencing conditions and submit purification & run samples
  • Re-design sequencing primer to avoid homopolymer region
  • Re-design sequencing primer to sequence the opposite strand

 

“Top heavy” Signal

“Top heavy” Signal

“Top heavy” Signal
 

Possible Causes
  • Too much DNA template/Too much primer
  • Salt or ethanol contamination
  • High concentration of EB, EDTA


Possible Solutions
  • Quantify DNA and follow our sample submission guidelines to prepare premixed samples
  • Perform extra ethanol wash when purifying DNA
  • Dry spin column/DNA pellet longer before eluting DNA
  • Elute DNA in Elution buffer (10mM Tris-Cl, pH 8.5) or nuclease free H2O

 

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Noisy read

Overlapping sequences after a short clean read

Overlapping sequences after a short clean read chromatogram

Overlapping sequences after a short clean read Raw signal plot
 

Possible Causes
  • Non-homogeneous template, e.g. more than one plasmid


Possible Solutions
  • Sub-clone again and ensure a single colony is picked

 

Overall noisy sequence with weak raw signals

Overall noisy sequence with weak raw signals chromatogram

Overall noisy sequence with weak raw signals Raw signal plot
 

Possible Causes
  • Not enough DNA template
  • Presence of inhibitors
  • Low priming efficiency
  • Sub-optimal cycle sequencing annealing temperature


Possible Solutions
  • Quantify DNA and follow our sample submission guidelines to prepare premixed samples
  • Purify DNA before adding into premixed sample
  • Use new aliquot of primer or re-design sequencing primer
  • Optimize cycle sequencing conditions and submit purification & run samples

 

Overall noisy sequence with strong signal

Overall noisy sequence with strong signal chromatogram

Overall noisy sequence with strong signal Raw signal plot
 

Possible Causes
  • Multiple templates
  • Non-specific priming sites
  • Contaminated template/primer
  • Multiple primers
  • Primer with N-1 contamination


Possible Solutions
  • Resubmit pure sample
  • Re-design sequencing primer
  • Use new aliquot of template/primer
  • Ensure only one primer is used at a time, either forward or reverse, NOT both
  • Order quality guaranteed primers from CGS Oligo Ordering Services

 

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No read

No sequence read with raw signal below 100

No sequence read with raw signal below 100 chromatogram

No sequence read with raw signal below 100 Raw signal plot
 

Possible Causes
  • Insufficient DNA template
  • Insufficient Primer
  • Poor/No primer annealing
  • Inhibition by contaminants


Possible Solutions
  • Quantify DNA and follow our sample submission guidelines to prepare premixed samples
  • QC DNA on an agarose gel
  • Re-design sequencing primer
  • Clean the sample before submission

 

Delayed read start with initial irregular spacing and/or presence of large peaks before sequence read

Delayed read start with initial irregular spacing and/or presence of large peaks before sequence read chromatogram

Delayed read start with initial irregular spacing and/or presence of large peaks before sequence read Raw signal plot
 

Possible Causes
  • Capillary blockage
  • Capillary overloaded with DNA/protein/salt or other contaminants


Possible Solutions
  • Contact platform specialist to check capillary performance
  • Quantify DNA and follow our sample submission guidelines to prepare premixed samples
  • Purify DNA before adding into premixed sample
  • QC DNA with a Nano-Drop Spectrophotometer

 

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Cases for reruns

Large raw signal spike

Large raw signal spike chromatogram

Large raw signal spike Raw signal plot

Possible Causes
  • Bubbles forming in the capillaries during electrophoresis


Possible Solutions
  • Request sample rerun

Unexpected read beyond expected length or noisy baseline

Unexpected read beyond expected length or noisy baseline chromatogram

Unexpected read beyond expected length or noisy baseline Raw signal plot

Unexpected read beyond expected length or noisy baseline sequence text
 

Possible Causes
  • Cross-talk from neighbouring capillary fluorescense

Illustrated is the effect of cross-talk for a template of 180bp PCR product.  Sequence reads beyond 180bp are signals emitted from the neighboring capillary.



Possible Solutions
  • Request sample rerun

Cross talk will not affect samples with strong signal, but may affect those with weak or no signal.

Since cross talk is hard to determine without knowing the neighbouring sample intensity,  please feel free to consult platform staff for inspection if necessary.


 

Unexpected board peak starting early in the sequence

Unexpected board peak starting early in the sequence chromatogram

Unexpected board peak starting early in the sequence Raw signal plot
 

Possible Causes
  • Capillary deterioration


Possible Solutions
  • Request sample rerun

Capillary deterioration is hard to detect without comparing different runs.  Please be assured that CGS staffs are constantly monitoring every run to detect possible capillary deterioration as early as possible.  Affected clients will be contacted and samples will be rerun at no cost as soon as possible.


 

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