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QuantStudio™ 3D Digital PCR System Data Troubleshooting
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Objectives
• Troubleshoot chip images when yellow flags appeared
• Troubleshoot analysis result when yellow flags appeared
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Troubleshooting chip images using the Chip View
• The following slides summarize some of the more
common problems that can effect imaging of Digital PCR
20K Chips depending on which flags appear in the
Instrument touch screen.
• Note: In the examples shown, the conditions triggered a
data flag in the Digital PCR 20K Chip and the conditions
were diagnosed using the Chip View of the
AnalysisSuite™ Software.
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Flag Definitions
• Touch Screen displays Red, Yellow, Green Flags for
each Dye
Re-scan or repeat the experiment, visually inspect chip, not worth
gringing in AnalysisSuiteTM software
Inspect chip in AnalysisSuiteTM software
Chip passed all screening criteria
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Succesful Load
• This a good example of a
succesful load
Manufacturing QC
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Scenario 1: Debris on chip
Possible Cause
• Dust or other debris are
present on the Chip
Sealant during imaging.
Action
• No action required.
• The AnalysisSuite™
Software can compensate
for small quantities of dust
and debris on the Digital
PCR 20K Chip.
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Cutoff chip & debris
Reprocessed
•Ensure customer has latest version of the QS3D software, version 1.1
•Reread chip
Missing chip image
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Scenario 2: Bubble in the Sample Loading Blade
Action
• If possible, use the AnalysisSuite™
Software to filter the low quality data
points, or discard the chip and run the
sample again.
• When loading the Sample Loading
Blades:
• If you are using a manual pipette,
pipette to the first stop.
• If you are using an electronic pipette,
decrease your pipetting speed.
• If a bubble does form in the Sample
Loading Blade, gently tap it to
remove bubble before loading.
Possible Cause
A bubble was present in the Sample Loading Blade when it was used to apply the PCR reaction to the Chip.
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Bubbles in Reaction Mix
To prevent this from happening
•Pipette to the first stop when using a manual pipette
•Pipette very carefully
•Decrease pipetting speed
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Ensure that there are no bubbles in the loaded mix
Bubbles in the blade will
show up as bubbles on the
chip – wells will not be
loaded
Note: The mix may not be evenly distributed in
the blade just after loading. This should be fine,
as it will spread across itself
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Scenario 3: Bridging
Possible Cause
• Excess PCR reaction was present on the Digital PCR 20K Chip after loading it with the Sample Loading Blade.
• The Sample Loading Blade was drawn across the chip too quickly or at an angle shallower than 70-80°.
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Bridging – Manual Load
To minimize the occurrence of bridging:
1. Ensure the loading block is set to 40C
+/- 1C
2. Wait 20 sec after loading before
adding immersion fluid
3. Thermal cycle chips on a tilted
(11deg) flat block
4. Make sure loading blade is at a 70 –
80 deg angle when applying sample
5. Take at least 10 seconds to apply
sample across chip
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Scenario 3: Bridging
Action
• If possible, use the AnalysisSuite™ Software to filter the low quality
data points, or discard the chip and run the sample again.
• To prevent leaving excess PCR reaction on the Digital PCR 20K
Chips between partition (connecting them together, Bridging):
• Confirm that the heated block used to load Digital PCR 20K
Chips is set to 40±1°C for manual loading.
• Make sure to wait 20 seconds after loading a Digital PCR 20K
Chip before pre-wetting it with Immersion Fluid.
• Make sure to install the Tilt Base to the GeneAmp® 9700 system.
You must thermal cycle the Digital PCR 20K Chips at a 11°
angle.
• Make sure to draw the Sample Loading Blade across the chip
slowly (>10 seconds) and at a 70-80° angle.
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Scenario 4: Major Leakage
Possible Cause
• The Digital PCR 20K Chip leaked during thermal cycling or imaging.
• A large bubble was present in the chip (insufficient Immersion Fluid).
• Immersion Fluid was not applied to the chip immediately
after loading (evaporation of the PCR reaction).
• Excess Immersion Fluid is present on the Chip Case Lid.
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Scenario 4 : Major Leakage
Action
• If present, remove excess Immersion Fluid from the chip lid and run
the chip again.
• If possible, use the AnalysisSuite™ Software to filter the low quality
data points.
• Make sure to apply Immersion Fluid to each chip immediately after
loading it with PCR reaction.
• To minimize leakage, when sealing each Digital PCR 20K Chip:
• Wear correctly fitted gloves to prevent the glove material from
snagging during lid application.
• Make sure that the Chip Case Lid is correctly aligned to the Chip
Case.
• Firmly press all four corners when applying the Chip Case Lid.
• Cure the Chip Sealant under ultraviolet light for at least 30
seconds.
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Leaking – 3 different chips
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Leaking - 4 different chips
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To minimize the occurrence of leaking:
1. Leave a small bubble when filling chip with immersion fluid
2. Be careful when covering chip with immersion not to get immersion
fluid on sides of chip base
3. Create a dome of sealant over filling port
When Manually Loading chips:
• Ensure lid is properly aligned to the case
bottom
• Firmly press all 4 corners when applying
the lid
• Firmly hold sides of chips & wiggle back &
forth to ensure seal
• Cure UV glue for a minimum of 30
seconds
When using Chip Loader:
• Ensure that you are using at least
20lbs of pressure when applying lid
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Applying UV-Cured Sealant
Apply sealant to the outer rim of
the chip lid to ensure complete
seal.
Put on enough sealant so that you
create a dome.
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Apply 20 lbs of pressure to seal chip, 15 seconds
•There is no such thing
as too much pressure.
•If you think you are not
applying enough
pressure, you may
increase the hold time
– longer than 15 sec.
•Let off of the pressure
gently, & don’t press
down again. This can
create a vacuum
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Scenario 5 : Evaporation around edges
• Chip was not completely
covered with immersion
fluid (while still in chip nest
on chip loader)
• When using the chip
loader, as soon as the
loading blade is off of the
chip, start to add immersion
fluid
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Apply immersion fluid immediately after loading
Ensure that entire chip is covered
with immersion fluid, even the edges
to prevent evaporation •It’s OK for the oil to overflow into the
chip base, but use caution to make
sure it does not get on the sides on
the chip or that there is too much oil
Yellow around the edges is a
strong indication of evaporation
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Evaporation
Immersion fluid was not
added to chip before
applying chip lid, but was
added after lid was applied.
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Filling the chip chamber with immersion fluid
Leave a small bubble when filling
chip with immersion fluid.
This is to ensure that you do not
get immersion fluid on the outer
rim of the lid.
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Scenario 6 : Condensation
Allow chips to warm to room
temperature after being held at 10
deg C on 9700.
Note: It may appear that there is no
condensation on the chip, but it is
there….
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Effects of Condensation on Chip
This is the same chip, read
directly out of the thermal
cycler, and then allowed to
warm to room temp.
Warming up the chips
increases the:
•data quality
•flag
•separation of the amplified
vs non-amplified peaks.
Note: Condensation was not
visible on the chip
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Scenario 7 : Pooling
Too much sample left in loading blade.
Ensure customer is loading only 14.5 ml of reaction mix
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Loading blade not flush; stuck at beginning of load
Make sure loading blade is flush /
contacting metal surface
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Placing Loading blade onto Chip Loader
Make sure loading blade is
flush / contacting metal surface
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No Sample
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Too much template
Check the copies per reaction in the Analysis software and dilute the
sample so that it is in the digital range
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Dropped Chip
Don’t drop your chip
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• Software demo
• Troubleshooting files
• Practice Files Analysis
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Data Quality Is Flagged
Data meets all quality thresholds
• Instrument cannot clearly identify the population of unamplified wells
• The distribution of unamplified wells on the chip is not uniform
• The concentration is outside of the confidence interval (200-2000
copies/ul) defined by Life Technologies quality metrics
1. Review histogram & adjust threshold if needed. If not bimodal, or a
high concentration or low concentration that is mostly monomodal,
reject chip. If double peak, you can view in relative quant app to look
if clusters are separated in 2-dye space
2. Look to see that unamplified wells are randomly distributed. If not
randomly distributed, reject chip.
3. You can try to raise the QV threshold to see if the histogram gets
more bimodal and the positive/negative distribution gets more uniform
Re-image. If still red, reject the chip. Look for specific failure modes
that are mostly visible on the consumable (leak, large bubble, liquid on
lid if dropped, cracked chip)
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Troubleshooting analysis result
• The following examples summarize some of the more
common problems that can effect the analysis results for a
project as viewed in the AnalysisSuite™ Software. In some of
the examples shown below, the conditions triggered a data
quality flag.
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Scenario 1 : Mono modal peak
• Possible cause:
• PCR reaction does not contain
template (is a negative control).
• PCR reaction is missing template.
• Concentration of sample is too high.
• Action :
• No further action required
• Discard the chip and run the sample
again
• Discard the chip and run the sample
again at a higher dilution.
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Scenario 2 : Poor peak separation
• Possible cause
• Acceptable performance for the
selected assay.
• Excess PCR reaction present on
the chip after loading.
• Low efficiency assay.
• Action
• No further action required
• Discard the chip and run the sample
again.
• Adjust thermal cycling conditions or
use a different assay.
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Scenario 3 : Multi modal peaks
• Possible cause :
• Low efficiency assay.
• Action :
• Increase the quality threshold to
create a more bi-modal distribution, if
possible. Make sure to balance the
number of data points included in the
analysis against the desired data
quality and call separation. If this is
not possible, omit the chip from the
results.
• Adjust thermal cycling conditions or
use a different assay.
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Questions
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The QuantStudio™ 3D Digital PCR System is For Research
Use Only. Not for use in diagnostic procedures.
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