Section III: Sequencing Troubleshooting Quick Reference Table
30
CEQ™ 8000 Series Genetic Analysis System
• Irreproducibility
• Short Read lengths
• Inaccurate base
calling;
• Data not analyzed
Jump in the Raw
Signal baseline
Some sort of carry over in the
sample from
- Salt
- Dirty Primer
- Too much primer in template
PCR reaction
Elute DNA template in sterile water or
10mM Tris pH 8.5 solution
Do not use a strong buffer (ionic
strength) for elution of DNA in
template purification
Use HPLC purified primers
Use correct concentration of primers
Do not add salts of any kind to the
sample
Reduce amount of primer in template
PCR reaction
• High raw signal but
wrong bases called;
• Sequence contains
inserted bases.
High Raw signal
(off-scale) and
normal current
profile.
Too many cycles
Cut down the number of cycles
Very efficient cycling causing
the raw signal to over-range
the detector
Reinject the sample for less time—7.5
seconds instead of 15 seconds
Poor quality primer (n-1
primer)
Improve quality of DNA synthesis
Degraded primer
• Effects of Formamide Loss of dyes
Overall reduction in
signal intensity
No signal and/or
current problems
Formamide breaks down into
formic acid and ammonia, and
these ionic products compete
significantly with the larger
DNA ions for injection and
thus cause reduction in signal
intensity. In addition they can
also cause degradation of the
sample
Use SLS for sample resuspension
Store and use the SLS solution
correctly
• Primer Artifacts
The analyzed data
contains a lot of
inserted bases.
Peaks under peaks
in the raw data and
also in the analyzed
data.
More than 8–10
bases per minute of
raw data.
Mis-priming
Mixed templates
Poor quality primer
Select annealing temperatures that limit
the annealing of mismatched primers
Unambiguous sequence regions should
be chosen for selecting the priming
sites
Prepare the DNA template in such a
manner to avoid mixed template
contamination
Use a primer that binds to a site internal
to the primers used for PCR
In some instances the contaminating
template is present in very small
quantities and dilution of the sample
helps to decrease the concentration of
the contaminant even further
• Salt mobility
Compression of
bases in the region
of 20–70 bases from
the primer
Caused by the migration of
salts in the gel
Only seen in software v1.1
Not present in v2.X or greater
Check if denaturation has been set to 0
time length
PROBLEM
RAW DATA
SIGNAL /
CURRENT
PROFILE
POTENTIAL SOURCE OF
PROBLEM
POSSIBLE SOLUTIONS
