Observation
Possible cause
Recommended action
Extra peaks are present in the
sequencing traces
(continued)
Primer
‑
dimer has occurred.
You can often diagnose primer-dimer by
looking at the raw trace data for questionable
sequences. When primer-dimer exists, the 5
′
sequence signal may be significantly higher for
a region of bases spanning the length of the
forward and reverse gene-specific primers.
Primer-dimer is the annealing of the 3
′
end of
primers during PCR. The resulting short
annealed fragment may amplify more
efficiently than fully extended template.
Primer-dimer fragments amplified during PCR
can display increased 5
′
signal and extra peaks
when multiple PCR products are sequenced
simultaneously. In some instances, the
secondary or extra peaks can be read as the
reverse compliment of the PCR primers in this
noisy 5
′
region. The secondary sequence or
multiple PCR product sequences appear as far
as 100–200 bp into the sequence, then
suddenly disappear.
The PCR amplification primers
do not have specificity and are
sequencing two different
regions of the genome. The
analyzed trace shows extra
peaks throughout the entire
length of the trace.
Redesign the primers or increase the
amplification temperature.
You accidentally contaminated
the DNA and are sequencing
two templates at the same
time. The analyzed trace shows
extra peaks throughout the
entire length of the trace.
Repeat the amplification and sequencing
reactions with uncontaminated DNA.
There were impure or
contaminated primers.
Primer stocks may have inadvertently had
other primer solution introduced. For best
results, use HPLC to purify the primers.
There was a contaminated
sample well.
Use a new sample plate and buffer/wash septa
whenever possible.
To avoid getting sample into adjacent wells,
centrifuge the plates before you remove the
adhesive seal.
There are pull-up or pull-down
peaks in the data
The manual dye calibration is
not current or is not matched to
the samples, or a high-quality
sample was not run with the
dye set for the first time in the
absence of a manual
calibration.
If the dye set is a fragment analysis dye set
that is being used for the first time, run a
manual dye calibration.
Alternatively, if a high-quality sample is run
with an uncalibrated dye set, and the
automated spectral calibration successfully
generates an optimized matrix, manual
calibration is not absolutely required. See
“Determine if manual calibration is
Appendix A
Troubleshooting
Sample and data troubleshooting
A
SeqStudio
™
Genetic Analyzer Instrument and Software User Guide
179
