Troubleshooting

Refer to the following solutions and workarounds if the Infinium Assay assay operates in an unexpected manner. If the issue cannot be resolved using this guide, contact Illumina Technical Support.

Pre-Hybridization

Symptom

Probable Cause

Resolution / Comment

No blue pellet observed in wells after 20 minute centrifugation.

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Original DNA sample degraded.

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Precipitation reaction solution was not mixed thoroughly before centrifugation.

Invert plate several times and centrifuge again.

If pellets appear, the solutions were not mixed before centrifuging. If pellets do not appear, the original DNA samples may be degraded.

Note: If samples were degraded, repeat the Amplify DNA (Make) step of the protocol you are performing.

Inspect wells for complete mixing before the 20 minute centrifugation.

Either PM1 or 2-propanol was not added.

Add missing reagent to wells.

Invert plate several times and centrifuge plate again.

Inspect wells for complete mixing before 20 minute centrifugation.

Blue color observed on absorbent pad after precipitation supernatant was decanted.

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Precipitation reaction solution was not thoroughly mixed before centrifugation.

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The plate was centrifuged at less than 3000 × g, or for less than the recommended time.

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Supernatant was not removed immediately after centrifugation.

The samples are lost. Repeat the Amplify DNA (Make) step of the protocol you are performing.

Check centrifuge program to make sure that correct speed was selected.

Blue pellet did not dissolve back into solution after vortexing.

An air bubble formed at the bottom of the well that prevented the pellet from mixing with RA1 or IHX.

Pulse centrifuge plate to 280 × g to remove air bubble, then revortex plate at 1800 rpm for 1 minute.

Vortex speed is not fast enough.

Check the vortexer speed setting, as the speed setting may drift over time. Recalibrate if necessary.

Revortex plate at 1800 rpm for 1 minute.

The reaction plate did not incubate for long enough.

Incubate the plate for an additional 30 minutes.

Make sure that the cover is properly seated to prevent evaporation.

Excessive drying.

Do not dry the pellet for longer than 40 minutes.

Hybridization to XStain

#	Symptom	Probable Cause	Resolution / Comment
1	There was not enough reagent to dispense to all BeadChips.	Make sure to centrifuge the reagent tubes at 280 × g after thawing.	Check pipette calibration. A gravimetric test using water is a quick and easy way to check for accurate pipette dispensing volumes. Recalibrate pipettes yearly.
		Programmable pipettor may be set incorrectly.	Check to see if the programmable pipettors have an overasp volume that is discarded.
		Excessive evaporation after heat denaturing.	Foil heat sealer should have been used. Ensure proper seating.
2	Cap mat deformed or melted while heat-denaturing DNA samples, non-EX assays only.	Foil heat sealer should have been used.	Samples have been ruined. Repeat experiment. Use the foil heat sealer for all temperatures ≥ 45°C.
3	A small amount of precipitate was present in the hybridization solution.	A small amount of precipitate is normal, and does not affect data quality.	Continue with experiment.
4	Large precipitate cannot be resuspended.	Excessive evaporation after heat denaturing.	Foil heat sealer should have been used. Ensure proper sealing.
5	BeadChips are still wet on the underside after 55 minutes in the vacuum desiccator.	The BeadChips must dry for a longer period. XC4 may be old and must be replaced with fresh XC4. An old bottle of ethanol may have absorbed atmospheric water. Replace with a fresh bottle of ethanol.	Dry BeadChips longer under vacuum desiccator. Lab temperature and humidity affect drying time.
6	After coating the BeadChips with XC4, some uncoated areas remain.	During the coating process, a bubble formed between the BeadChips and prevented the XC4 solution from reaching the surface.	Briefly place the staining rack with BeadChips back into the wash dish containing XC4. Gently move BeadChips back and forth while moving up and down, breaking the surface of the solution. The back-and-forth movement is especially important when processing 16 or 24 BeadChips.
7	During XStain, the liquid in the Flow-Through Chamber dropped below the bottom edge of the glass back plate reservoir.	Glass back plates may not have been completely clean, causing capillary gap failure. Make sure that the correct spacer was used to assemble the Flow Through Chamber.	Before reassembling the Flow-Through Chambers, clean the glass back plates.
7		The Flow-Through Chambers may not be securely assembled.	Attach the metal clamps to the Flow-Through Chambers as described in the Reference Guide for the assay.

Imaging

Symptom

Probable Cause

Resolution / Comment

The iScan was unable to find all the fiducials during scanning.

XC4 coating was not properly removed from BeadChip edges.

Rewipe the edges of BeadChips with ProStat EtOH wipes and rescan.

BeadChips were not seated correctly in BeadChip carrier.

Reseat BeadChips in BeadChip carrier.

The scanning process did not generate any intensity signal.

The absence of an intensity image may be due to failures in any experimental steps upstream of scanning.

Repeat the experiment.

Check the staining controls. If the controls do not generate a signal either, then the staining reagents may be compromised.

The scanning process generated a low assay signal. The Hyb controls display expected intensities.

A low assay accompanied by normal performance from the sample-independent controls signal indicates a sample-dependent failure. The error may have occurred in any experimental steps between amplification and hybridization.

Repeat experiment.

Make sure that there is a DNA pellet after precipitation.

During resuspension, make sure that the DNA pellet dissolves properly. The blue color in the pellet should disappear.

The scanning process generated a low assay signal. The Hyb controls display low intensities.

A low assay signal accompanied by low sample-independent controls indicates a sample-independent failure related to assay processing. The error probably occurred after hybridization.

To eliminate the scanner as the problem, make sure that other chips that are scanned at the same time or in the same batch are yielding normal assay signals. If other chips show the same low signal, contact Illumina Technical Support to analyze the data in GenomeStudio to determine possible root causes.

Some spots on the BeadChip have low to 0 intensity.

Bubbles in the reagents can cause this phenomenon by preventing reagents from reaching the BeadChip surface.

Centrifuge all reagent tubes before using to prevent bubbles.

Always perform a system flush before running the experiment.

(Low to 0 intensity areas on the BeadChip may not have a negative effect on array data due to randomness and oversampling of BeadChips.)

The scanner returns red stripes indicating a failed scan.

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Assay process failure.

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Sample failure.

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Consumable failure.

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Instrument failure.

Move the chip to another position in the carrier and rescan. If this fails, contact Illumina Technical Support.

Miscellaneous

#	Symptom	Probable Cause	Resolution / Comment
1	Genotyping results do not correlate with samples.	The samples may have been mixed up.	Check the sample sheet and lab tracking form to determine the location of DNA samples during amplification.
			Check to make sure that the correct sample was loaded onto the correct BeadChip or part of the BeadChip.
			Repeat the experiment. After resuspending the DNA pellet, make sure to consolidate the samples back to the original amplification position, if necessary.
2	Solution foams excessively when dispensed.	Pipetting may have been too vigorous.	Pipette gently without forming bubbles. Try centrifuging the plate to 280 × g to remove bubbles.
3	Heat block cooled to less than 37°C when it was set to 37°C after the 95°C incubation.	Heat block overshot set temperature.	Wait for heat block to reequilibrate to 37°C. Many heat blocks will overshoot the bottom set temperature when cooling from a previously set higher temperature.