Description

Cause

Solution

-Broad unresolved peaks throughout read.

-Degraded reagents on capillary instrument.
-Excess salts.

-Request sample be re-run.
-Request dilution of sample and rerun.

Description

Cause

Solution

-Broad florescent signal spanning multiple sequencing peaks

-Sephadex clean-up failed to remove unincorporated dye terminators.
-Fluorescent contaminant in sample.

-Request sample be cleaned-up and re-run.

Description

Cause

Solution

-Intense fluorescent signal obscuring  few sequencing peaks

-Air bubble or contaminant.

-Request sample be re-run.

Description

Cause

Solution

-Excessive signal intensity followed by an early drop-off of sequence read.

-Too much template or primer resulting excess of short fragments.
-PCR product highly concentrated

-Request sample be diluted and resequenced or rerun.

Description

Cause

Solution

-Low peak height and signal strength. High background noise.

-Template and/or primer concentration too low.
-Poor quality of template and/or primer.
-Excess salts or contaminant present.
-Capillary failed top pickup sample.

-Double check template and primer concentration.
-Clean-up template.
-Double check primer design.
-Request samples be rerun.

Description

Cause

Solution

-Peak annotation is missing, or incorrect, or represented with an "N"

-Limitation of software algorithm to recognize base correctly. Sometimes due to overlapping peaks, compressed peaks - poor peak spacing, weak signal, poor resolution of peak.

Description

Cause

Solution

-No peaks detected.  Signal strength <25 for all bases.

-Template and/or primer concentration too low or absent.
-Priming site not present or wrong primer used.
-Excess salts or contaminants present.
-Capillary failed to pickup sample.

-Double check template and primer concentration.
-Double check primer site is present and correct primer was added.
-Clean-up template.
-Request sample be rerun.

Description

Cause

Solution

-Sequence drop-off  or double peaks after homopolymer region

-Polymerase slippage

-Sequence in opposite direction.
-Redesign primers closer to homopolymer region.
-Redesign polyN primer with wobble base.
-Request sequencing with alternative chemistry.

Description

Cause

Solution

-Sequence drop-off after repeat region

-Depletion of dNTPs or presence of secondary structure.

-Request sequencing with dGTP chemistry
-Sequence from opposite direction.
-Redesign primers closer to repeat region.

Description

Cause

Solution

-Abrupt decrease or loss of sequencing

-Secondary structure/hairpin restricting the polymerase from moving through the region. 

-Request sequencing with dGTP chemistry.
-Sequence from opposite direction.

Description

Cause

Solution

-Overlapping peaks after stretch of good sequence

-Plasmid prep contaminated: with a clean sequence, vector regions with two different inserts can cause overlapping sequences.
-Human error: two templates mixed together.

-Pick new single clone and reprep.
-Request sample be resequenced. Possibly provide fresh aliquot of sample.

-Overlapping peaks from the very beginning

-Human error: two different templates mixed together.
-Two primers added to sample.
-Two priming sites on template.

-Request sample be resequenced.
-Submit new sample with one primer
-Redesign primer with specific binding site.