Thermo Scientific Applied Biosystems SeqStudio Genetic Analyzer User Manual page 178

A
Appendix A Troubleshooting
Sample and data troubleshooting
Observation
Dye blobs are seen in the
sequencing data
Extra peaks are present in the
sequencing traces
178
Possible cause
Impurities remained in the
sample after the sample
purification. The impurities
cause dye blobs to appear in
the sequencing data.
There was renaturation of the
sample.
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.
SeqStudio
Recommended action
Improve the sample purification method. See
"Sample preparation guidelines" on page 37
for guidelines.
Heat-denature the samples prepared with
fresh Hi‑Di Formamide, then immediately
™
place the samples on ice.
Check the raw data, the raw data signal
intensity, and average raw signal‑to‑noise ratio,
then:
• Increase the injection time and reinject.
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.
Genetic Analyzer Instrument and Software User Guide
™