Sequencing And Fragment Applications - Thermo Scientific appliedbiosystems SeqStudio Flex Series User Manual

Observation
"Calibration from manual" is
reported for spectral calibration
status and pull-up peaks are
observed

Sequencing and fragment applications

Observation
Spikes are present in raw
and/or analyzed fluorescence
data
Poor resolution, low size
quality, or a low-quality
sequencing result is observed
Details: Results are color-
coded based on quality. For
more information, see "Sample
QC and quality alerts" on
page 172.
SeqStudio ™ Flex Series Genetic Analyzer with Instrument Software v1.0 User Guide
Possible cause
Auto-spectral calibration was
not used because the sample
possibly contains overlapping
peaks that are labelled with
different dyes. For more
information, see "Auto-spectral
calibration during an injection"
on page 150.
IMPORTANT!
Spectral
calibration status is reported
for informational and tracking
purposes only. This calibration
status does not reflect the
quality of the sample or affect
the results. However, use of
the manual spectral calibration
instead of the auto-spectral
calibration can increase the
likelihood of pull-up peaks.
Possible cause
Trace impurities passed the
detector.
The samples degraded over
time while on the instrument.
Appendix A Troubleshooting
Sample and data troubleshooting
Recommended action
Perform a manual spectral calibration, then
repeat the run. See "Run a spectral
calibration" on page 310.
IMPORTANT!
When running samples that
contain overlapping peaks that are labelled
with different dyes, manual spectral calibration
is required after changing the capillary array or
moving the instrument.
Recommended action
For fragment analysis, increasing the
Minimum peak half width setting in the
analysis settings (under Common settings)
can reduce the identification of spikes as
peaks.
Note: Secondary sequencing and fragment
analysis software can recognize and ignore
spikes.
Repeat the injection if necessary.
Thermal breakdown of samples is normal.
Samples are stable for 16–24 hours on the
instrument.
Use Hi‑Di ™ Formamide to prepare the
samples. Sample stability is optimal in Hi‑Di
Formamide.
Prepare the sample according to the protocol
provided with the kits for sample preparation.
See "Sample preparation guidelines"
page 73.
IMPORTANT!
Do not resuspend samples in
water.
A
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on
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