QuantStudio™ 3D Digital PCR System Data Troubleshooting · •The following slides summarize some...

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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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