Thermo Scientific appliedbiosystems SeqStudio Flex Series User Manual page 518

Appendix A Troubleshooting
A
Sample and data troubleshooting
Observation
Extra peaks are present in the
sequencing traces
(continued)
518
Possible cause
There is low signal. With very
low signal, the peaks are barely
visible in the baseline noise.
There is a heterozygous
insertion-deletion (het indel)
that is causing multiple peaks
to appear at the same basecall
position. The sequence can
appear "clean" for some
number of bases until the het
indel is encountered.
Primer‑dimer has occurred.
SeqStudio ™ Flex Series Genetic Analyzer with Instrument Software v1.0 User Guide
Recommended action
Check the analyzed data, the raw data, the
raw data signal intensity, and average raw
signal‑to‑noise ratio, then:
• Increase the injection time and re-inject.
Or
• Remake the sample. Ensure that you are
using:
– Enough sequencing template
– Enough primer and/or a sufficient
concentration of primer
Examine the analyzed trace. A het indel
typically has single peaks at the 5' end, then
part-way through the trace, two peaks appear
in almost every position to the end of the
trace. This pattern occurs when one copy of
the gene has an insertion or deletion relative to
the other copy of the gene.
When aligning your sequence to a reference
sequence, a series of bases may have been
inserted or deleted in an allele. These indels
can be encountered in any number of bases
after the gene-specific priming region. To
confirm that the het indel is present in
both directions of your target, check the
sequencing in the opposite direction.
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.