Illumina Infinium HTS Assay Experienced User Card

Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

FOR RESEARCH USE ONLY

ILLUMINA PROPRIETARY

Part # 15045736 Rev. A

October 2013

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Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

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Part # 15045736 Rev. A

Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

Amplify DNA (Pre-Amp)

Estimated Time

Robot time:

• 30 minutes for 48 samples

• 1 hour or 96 samples

Incubation time: ~20–24 hours

Consumables

Item

MA1

Quantity

1 tube

(per 96 samples)

Storage

Room temperature

Supplied By

Illumina

MA2

MSM

0.1N NaOH

96-well 0.8 ml microplate (MIDI)

WG#DNA plate with 48 or 96

DNA samples (50 ng/μl)

1 tube

(per 96 samples)

1 tube

(per 96 samples)

15 ml

(per 96 samples)

1 plate

1 plate

-15°C to -25°C

-15°C to -25°C

2°C to 8°C

-15°C to -25°C

Illumina

Illumina

General lab supplier

General lab supplier

User

Preparation

[_] 1 Preheat the Illumina Hybridization Oven in the post-amp area to 37°C and allow the temperature to equilibrate.

[_] 2 In the Sample Sheet, enter the Sample_Name and Sample_Plate for each Sample_Well.

[_] 3 Apply an MSA3 barcode label to a new MIDI.

[_] 4 Thaw MA1, MA2, and MSM tubes to room temperature.

[_] 5 Thaw DNA samples to room temperature.

Steps to Make the MSA3 Plate

[_] 1 If you do not already have a WG#-DNA plate, add DNA into one of the following:

• MIDI plate: 20 μl to each WG#-DNA plate well

• TCY plate: 10 μl to each WG#-DNA plate well

Apply a barcode label to the new WG#-DNA plate.

[_] 2 At the robot PC, select MSA3 Tasks | Make MSA3.

Part # 15045736 Rev. A

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Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

[_] 3 Select the WG#-DNA plate type (MIDI or TCY).

[_] 4 (Other than Illumina LIMS) Make sure that the Use Barcodes checkbox is cleared. In the

Basic Run Parameters pane, enter the Number of DNA samples (48 or 96) that are in the plate.

NOTE

If you are using Illumina LIMS, you cannot change the number of DNA samples on this screen. However, the Illumina LIMS software processes the correct number of samples.

NOTE

If you are using Illumina LIMS, then you must click Run and select batches before the robot bed map displays the correct layout for the WG#-DNA plates.

[_] 5 Remove caps from MA1, MA2, and MSM tubes, then place the tubes in the robot standoff tube rack according to the bed map.

[_] 6 Vortex the sealed plate at 1600 rpm for 1 minute.

[_] 7 Centrifuge to 280 × g for 1 minute.

[_] 8 Add 15 ml 0.1 N NaOH to the quarter reservoir, then place the reservoir on the robot bed according to the bed map.

[_] 9 Place the WG#-DNA and MSA3 plates on the robot bed according to the bed map.

[_] 10 (Other than Illumina LIMS) At the robot PC, click Run.

[_] 11 (Illumina LIMS) At the robot PC:

[_] a Make sure that the Use Barcodes checkbox is cleared.

[_] b Click Run to start the process. Log in if prompted.

[_] 12 (Illumina LIMS only) Select the batch you want to run, and then click OK.

[_] 13 (Illumina LIMS only) Click OK to confirm the required DNAs.

[_] 14 Observe the robot run to make sure that there are no problems.

After the robot adds the 0.1N NaOH to the DNA in the MSA3 plate, follow the instructions at the prompt.

[_] 15 Seal the plate with a cap mat.

[_] 16 Vortex the sealed MSA3 plate at 1600 rpm for 1 minute.

[_] 17 Centrifuge to 280 × g at 22°C for 1 minute.

NOTE

When you remove a cap mat, set it aside, upside down, in a safe location for use later in the protocol. When you place the cap mat back on the plate, be sure to match it to its original plate and orient it correctly.

[_] 18 Place the MSA3 plate back on the robot bed in its original position, and then click OK.

[_] 19 When the robot finishes, seal the MSA3 plate with a cap mat.

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Part # 15045736 Rev. A

Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

[_] 20 Centrifuge to 280 × g for 1 minute.

[_] 21 Record the location of DNA samples in the lab tracking worksheet.

[_] 22 (Illumina LIMS) In the Illumina LIMS left pane, click Infinium HTS | Incubate MSA3.

[_] 23 Scan the barcode of the MSA3 plate, click Verify, and then click Save.

INCUBATION

Incubate in the Illumina Hybridization Oven for 20–24 hours at 37°C.

[_] 24 Proceed to the next step.

Part # 15045736 Rev. A

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Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

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Part # 15045736 Rev. A

Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

Fragment DNA (Post-Amp)

This process enzymatically fragments the amplified DNA samples. An end-point fragmentation is used to prevent over-fragmentation.

Estimated Time

Robot time:

• 5 minutes for 48 samples

• 10 minutes for 96 samples

Incubation time: 1 hour

Consumables

Item

FMS

Quantity Storage

1 tube (per 96 samples) -15°C to -25°C

Supplied By

Illumina

Preparation

[_] 1 Preheat the heat block with the MIDI plate insert to 37°C.

[_] 2 Thaw FMS tubes to room temperature. Gently invert at least 10 times to mix contents.

[_] 3 Pulse centrifuge to 280 × g.

[_] 4 Remove the MSA3 plate from the Illumina Hybridization Oven.

[_] 5 Remove the cap mat.

[_] 6 If you plan to Resuspend the MSA3 plate today, remove the RA1 from the freezer to thaw.

Steps to Fragment the MSA3 Plate

[_] 1 Centrifuge the MSA3 plate to 50 × g for 1 minute.

[_] 2 At the robot PC, select MSA3 Tasks | Fragment MSA3.

[_] 3 (Other than Illumina LIMS) Make sure that the Use Barcodes checkbox is cleared. In the

Basic Run Parameters pane, change the value for Number of MSA3 plates and Number of

DNA samples per plate to indicate the number of samples being processed.

NOTE

If you are using Illumina LIMS, you cannot change the number of DNA samples on this screen. However, the LIMS software processes the correct number of samples.

[_] 4 Place the MSA3 plate on the robot bed according to the bed map.

[_] 5 Remove the plate seal.

[_] 6 Place FMS tubes in the robot tube rack according to the bed map. Remove the cap.

[_] 7 (Other than Illumina LIMS) At the robot PC, click Run.

[_] 8 (Illumina LIMS) At the robot PC:

[_] a Make sure the Use Barcodes check box is checked.

[_] b Click Run to start the process. Log in if prompted.

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Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

[_] 9 When the robot finishes, click OK in the message box.

[_] 10 Remove the MSA3 plate from the robot bed and seal it with a cap mat.

[_] 11 Vortex at 1600 rpm for 1 minute.

[_] 12 Centrifuge to 50 × g for 1 minute at 22°C.

[_] 13 Place the sealed plate on the 37°C heat block for 1 hour.

[_] 14 Do one of the following:

• Proceed to Precipitate the MSA3 Plate (Post-AMP). Leave plate in 37°C heat block until you have completed the preparatory steps. Do not leave the plate in the 37°C heat block for longer than 2 hours.

• If you do not plan to proceed to the next step immediately, store the sealed MSA3 plate at -15°C to -25°C.

SAFE STOPPING POINT

Now is a good stopping point in the process.

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Part # 15045736 Rev. A

Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

Precipitate DNA (Post-Amp)

PM1 and 2-propanol are added to the MSA3 plate to precipitate the DNA samples.

Estimated Time

Robot time:

• 10 minutes for 48 samples

• 20 minutes for 96 samples

Incubation and dry time: 2 hours

Consumables

Item

PM1

100% 2-propanol

Quantity Storage

1 tube (per 96 samples) 2°C to 8°C

32 ml (per 96 samples) Room temperature

Supplied By

Illumina

General lab supplier

Preparation

[_] 1 Preheat the heat block to 37°C.

[_] 2 If you froze the MSA3 plate, thaw it to room temperature, then pulse centrifuge to 50 × g.

[_] 3 Thaw PM1 to room temperature. Centrifuge to 280 × g for 1 minute.

Steps to Precipitate the MSA3 Plate (Post-AMP)

[_] 1 At the robot PC, select MSA3 Tasks | Precip MSA3.

[_] 2 (Other than Illumina LIMS) Make sure the Use Barcodes check box is cleared. In the Basic

Run Parameters pane, change the value for Number of MSA3 plates and Number of DNA

samples per plate to indicate the number of samples being processed.

NOTE

If you are using Illumina LIMS, you cannot change the number of DNA samples on this screen. However, the LIMS software processes the correct number of samples.

[_] 3 Remove the cap mat and place the MSA3 plate on the robot bed according to the bed map.

[_] 4 Place a half reservoir in the reservoir frame, according to the robot bed map, and add PM1 as follows:

• For 96 samples: 1 tube

[_] 5 Place a full reservoir in the reservoir frame, according to the robot bed map, and add

2-propanol as follows:

• For 96 samples: 32 ml

[_] 6 (Non-Illumina LIMS) At the robot PC, click Run.

[_] 7 (Illumina LIMS) At the robot PC:

[_] a Ensure the Use Barcodes check box is checked.

[_] b Click Run to start the process. Log in if prompted.

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Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

[_] 8 When prompted, remove the MSA3 plate from the robot bed. Do not click OK in the message box yet.

[_] 9 Seal the MSA3 plate with the same cap mat removed earlier.

[_] 10 Vortex the sealed plate at 1600 rpm for 1 minute.

[_] 11 Incubate at 37°C for 5 minutes.

[_] 12 Centrifuge to 50 × g at room temperature for 1 minute.

NOTE

Set centrifuge to 4°C in preparation for the next centrifuge step.

[_] 13 Remove the cap mat and discard it.

[_] 14 Place the MSA3 plate back on the robot bed according to the bed map.

[_] 15 At the robot PC, click OK.

[_] 16 When prompted, seal the plate with a new, dry cap mat.

[_] 17 Invert the plate at least 10 times to mix contents thoroughly.

[_] 18 Incubate at 4°C for 30 minutes.

[_] 19 (Illumina LIMS) In the Illumina LIMS left sidebar, click Infinium HTS | Spin MSA3. At the robot PC, click Run.

[_] 20 Centrifuge to 3,000 × g at 4°C for 20 minutes. Immediately remove the MSA3 plate from centrifuge.

[_] 21 Remove the cap mat and discard it.

Perform the next step immediately to avoid dislodging the blue pellet. If any delay occurs, repeat the preceding centrifuge step and this one before you proceed to the next step.

[_] 22 Quickly invert the MSA3 plate and drain the liquid onto an absorbent pad to decant the supernatant. Then smack the plate down on a dry area of the pad, avoiding the liquid that was drained onto the pad.

[_] 23 Tap firmly several times for 1 minute or until all wells are devoid of liquid.

[_] 24 Leave the uncovered, inverted plate on the tube rack for 1 hour at room temperature to air dry the pellet.

At this point, blue pellets should be present at the bottoms of the wells.

[_] 25 If you are using Illumina LIMS:

[_] a Scan the barcode of the MSA3 plate and click Verify and then click Save. Illumina LIMS records the centrifugation step and queues the plate for the next step.

[_] 26 Do one of the following:

• Proceed to Resuspend DNA (Post-Amp).

• If you do not plan to proceed to the next step immediately, seal the MSA3 plate with a new cap mat and store at -15°C to -25°C.

SAFE STOPPING POINT

Now is a good stopping point in the process.

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Part # 15045736 Rev. A

Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

Resuspend DNA (Post-Amp)

RA1 is added to the MSA3 plate to resuspend the precipitated DNA samples.

Estimated Time

Robot time:

• 15 minutes for 96 samples

Incubation time: 1 hour

Consumables

Item

RA1

Quantity

7 ml per 96 samples

Storage

-15°C to -25°C

Supplied By

Illumina

Preparation

[_] 1 If you stored the MSA3 plate at -15°C to -25°C, thaw it to room temperature. Remove the cap mat and discard it.

[_] 2 Preheat the Illumina Hybridization Oven to 48°C.

[_] 3 Preheat the heat sealer. Allow 20 minutes.

[_] 4 Thaw RA1 to room temperature. Invert several times to re-dissolve solution.

Steps to Resuspend the MSA3 Plate

[_] 1 At the robot PC, select MSA3 Tasks | Resuspend MSA3.

[_] 2 (Other than Illumina LIMS) Make sure the Use Barcodes check box is cleared. In the Basic

Run Parameters pane, change the value for Number of MSA3 plates and Number of DNA

samples per plate to indicate the number of samples being processed.

NOTE

If you are using Illumina LIMS, you cannot change the number of DNA samples on this screen. However, the LIMS software processes the correct number of samples.

[_] 3 Place the MSA3 plate on the robot bed according to the bed map.

[_] 4 Place a quarter reservoir in the reservoir frame, according to the robot bed map, and add

RA1 as follows:

• 7 ml for 96 samples

[_] 5 (Non-Illumina LIMS) At the robot PC, click Run.

[_] 6 (Illumina LIMS) At the robot PC:

[_] a Ensure the Use Barcodes check box is checked.

[_] b Click Run to start the process. Log in if prompted.

[_] 7 Click OK in the message box. Remove the MSA3 plate from the robot bed.

[_] 8 Apply a foil seal to the MSA3 plate by firmly holding the heat sealer block down for 5 full seconds.

Part # 15045736 Rev. A

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Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

[_] 9 Place the sealed plate in the Illumina Hybridization Oven and incubate for 1 hour at 48°C.

[_] 10 Vortex the plate at 1800 rpm for 1 minute.

[_] 11 Pulse centrifuge to 280 × g.

[_] 12 Do one of the following:

• Proceed to Hybridize to BeadChip (Post-Amp). If you plan to do so immediately, it is safe to leave the RA1 at room temperature.

• If you do not plan to proceed to the next step immediately, store the sealed MSA3 plate at -15°C to -25°C for no more than 24 hours. Store at -80°C if storing for more than 24 hours. Store RA1 at -15°C to -25°C.

SAFE STOPPING POINT

Now is a good stopping point in the process.

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Part # 15045736 Rev. A

Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

Hybridize to BeadChip (Post-Amp)

Dispense the fragmented, resuspended DNA samples onto BeadChips. Incubate the BeadChips in the Illumina Hybridization Oven to hybridize the samples onto the BeadChips.

Estimated Time

Robot time:

• 24x1 HTS BeadChip: ~25 minutes for 4 BeadChips (96 samples)

Incubation time: 16–24 hours

Consumables

Item

PB2

Quantity

(per 96 Samples)

1 tube

Storage

Room temperature

Supplied By

Illumina

BeadChips

Hyb chambers

Hyb chamber gaskets

Hyb chamber inserts

Robot BeadChip alignment fixtures

Robot Tip Alignment Guide-G

(one-piece guide)

4

1

1

4

2

2

Illumina

Illumina

Illumina

Illumina

Illumina

Illumina

1% aqueous Alconox solution As needed User

Preparation

[_] 1 If frozen, thaw MSA3 plate to room temperature, and then pulse centrifuge the MSA3 plate to 280 × g.

[_] 2 Preheat the heat block to 95°C.

[_] 3 Preheat the Illumina Hybridization Oven to 48°C and set the rocker speed to 5.

Prepare the Robot Tip Alignment Guide

[_] 1 Make sure that you have the correct Robot Tip Alignment Guide for the Infinium assay you are running. The barcode says Guide-G.

[_] 2 Wash and dry the entire one-piece Robot Tip Alignment Guide. See Wash Robot Tip

Alignment Guide at the end of the Hybridize Multi-BeadChip steps for washing instructions.

[_] 3 Place the assembled Robot Tip Alignment Guides on the lab bench until it is time to place them on the robot bed.

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Illumina Infinium HTS Assay, Automated Protocol

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Assemble the Hybridization Chambers

[_] 1 Prepare the hyb chambers.

[_] a Place the BeadChip Hyb Chamber gaskets into the BeadChip Hyb Chambers.

[_] b Dispense 400 μl PB2 into the humidifying buffer reservoirs in the Hyb Chambers.

[_] c After you fill the Hyb Chamber reservoirs with PB2, place the lid on the Hyb Chamber right away to prevent evaporation. It is not necessary to lock down the lid.

[_] d Leave the closed Hyb Chambers on the bench at room temperature until the BeadChips are loaded with DNA sample. Load BeadChips into the Hyb Chamber within one hour.

NOTE

You can also prepare the hyb chambers later, during the 30 minute cool down.

[_] 2 Place the hyb chamber inserts into the hyb chambers.

[_] 3 Remove the BeadChips from 2°C to 8°C storage but do not unpackage.

[_] 4 (Illumina LIMS only) In the Illumina LIMS left sidebar, click Infinium HTS | Confirm

BeadChips for Hyb.

[_] 5 Scan the barcode of the MSA3 plate and all the BeadChips you plan to hybridize with the plate. Click Verify.

[_] 6 Place the resuspended MSA3 plate on the heat block to denature the samples at 95°C for 20 minutes.

[_] 7 After the 20 minute incubation, remove the MSA3 plate from the heat block and place it on the benchtop at room temperature for 30 minutes.

[_] 8 After the 30 minute cool down, pulse centrifuge the MSA3 plate to 280 × g.

Load BeadChips

[_] 1 Remove all BeadChips from their ziplock bags and mylar packages.

When handling the BeadChip, avoid contacting the beadstripe area and sample inlets.

[_] 2 Place BeadChips into the Robot BeadChip Alignment Fixtures with the barcode end aligned to the ridges on the fixture.

[_] 3 At the robot PC, select MSA3 Tasks | Hyb Multi-BC2.

[_] 4 Choose the appropriate BeadChip from the BeadChip Selection dialog box.

[_] 5 (Non-Illumina LIMS) Make sure the Use Barcodes checkbox is cleared. In the Basic Run

Parameters pane, change the value for Number of MSA3 plates and Number of DNA

samples per plate to indicate the number of samples being processed.

NOTE

If you are using Illumina LIMS, you cannot change the number of DNA samples on this screen. However, the LIMS software processes the correct number of samples.

[_] 6 Place the Robot BeadChip Alignment Fixtures onto the robot bed according to the bed map.

[_] 7 Place the MSA3 plate onto the robot bed according to the bed map. Remove the foil seal.

[_] 8 (Non-Illumina LIMS) At the robot PC, click Run.

[_] 9 (Illumina LIMS only) At the robot PC:

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Part # 15045736 Rev. A

Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

[_] a Ensure the Use Barcodes check box is checked.

[_] b Click Run to start the process. Log in if prompted.

[_] 10 At the robot PC, click OK to confirm you have placed the Robot Tip Alignment Guide on top of the Robot BeadChip alignment fixture. The robot scans the barcode on the Robot Tip

Alignment Guide to confirm the correct tip guide is being used.

The robot dispenses sample to the BeadChips.

[_] 11 Click OK in the message box.

[_] 12 Carefully remove the Robot BeadChip alignment fixtures from the robot bed and visually inspect all sections of the BeadChips. Make sure DNA sample covers all of the sections of each bead stripe. Record any sections that are not completely covered.

Set up Multi-BeadChip for Hybridization

[_] 1 Make sure the Illumina Hybridization Oven is set to 48°C.

[_] 2 Carefully remove each BeadChip from the Robot BeadChip alignment fixtures when the robot finishes.

CAUTION

For optimal performance, take care to keep the Hyb Chamber inserts containing

BeadChips steady and level when lifting or moving. Avoid shaking and keep parallel to the lab bench at all times. Do not hold by the sides near the sample inlets.

[_] 3 Carefully place each BeadChip in a Hyb Chamber insert, orienting the barcode end so that it matches the barcode symbol on the insert.

[_] 4 Load the Hyb Chamber inserts containing loaded BeadChips inside the Illumina Hyb

Chamber. Position the barcode over the ridges indicated on the Hyb Chamber.

[_] 5 (Illumina LIMS) In the Illumina LIMS left sidebar click Infinium HTS | Infinium Prepare

Hyb Chamber.

[_] 6 Scan the barcodes of the PB2 tubes and scan the BeadChip barcodes. Click Verify, and then click Save.

[_] 7 Position the lid onto the Hyb Chamber by applying the backside of the lid first and then slowly bringing down the front end to avoid dislodging the Hyb Chamber inserts.

[_] 8 Close the clamps on both sides of the Hyb Chamber so that the lid is secure and even on the base (no gaps).

NOTE

Keep the Hyb Chamber steady and level when moving it or transferring it to the

Illumina Hybridization Oven.

[_] 9 Place the Hyb Chamber in the 48°C Illumina Hybridization Oven so that the clamps of the

Hyb Chamber face the left and right side of the oven and the Illumina logo on top of the Hyb

Chamber is facing you.

OVERNIGHT INCUBATION

Incubate at 48°C for at least 16 hours but no more than 24 hours.

[_] 10 Proceed to Wash BeadChip (Post-Amp) after the overnight incubation.

Part # 15045736 Rev. A

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Illumina Infinium HTS Assay, Automated Protocol

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Resuspend XC4 Reagent for XStain BeadChip

[_] 1 Add 330 ml 100% EtOH to the XC4 bottle.

[_] 2 Shake vigorously for 15 seconds.

[_] 3 Leave the bottle upright on the lab bench overnight.

[_] 4 Shake the XC4 bottle vigorously to ensure complete resuspension. If any coating is visible, vortex at 1625 rpm until it is in complete suspension. After it is resuspended, use XC4 at room temperature.

Wash the Robot Tip Alignment Guide

For optimal performance, wash and dry the Robot Tip Alignment Guides after every run.

[_] 1 Soak the tip guide inserts in a 1% aqueous Alconox solution (one part Alconox to 99 parts water) using a 400 ml Pyrex beaker for 5 minutes.

NOTE

Do not use bleach or ethanol to clean the tip guide inserts.

[_] 2 After the 5 minute soak in the 1% Alconox solution, thoroughly rinse the tip guides with

DiH

2

O at least three times to remove any residual detergent.

[_] 3 Dry the Robot Tip Alignment Guide using a Kimwipe or lint-free paper towels. Use a laboratory air gun to dry. Be sure to inspect the tip guide channels, including the top and bottom. Tip guides should be completely dry and free of any residual contaminates before next use.

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Part # 15045736 Rev. A

Illumina Infinium HTS Assay, Automated Protocol

Experienced User Card

Wash BeadChip (Post-Amp)

Prepare the BeadChips for the staining process.

Estimated Time

• 20 minutes for 4 BeadChips

• 30 minutes for 8 BeadChips

Consumables

Item

PB1

Quantity

550 ml for 1 to 8

BeadChips

700 ml for 9 to 16

BeadChips

850 ml for 17 to 24

BeadChips

1 (per 8 BeadChips) Multi-sample BeadChip alignment fixture

Te-Flow LCG flow-through chambers, with black frames,

LCG spacers, LCG glass back plates, and clamps

Wash dish

Wash rack

1 (per BeadChip)

2 (up to 8 BeadChips)

1 (up to 8 BeadChips)

Storage

Room temperature

Supplied By

Illumina

Illumina

Illumina

Illumina

Illumina

Preparation

[_] 1 Remove each Hyb Chamber from the Illumina Hybridization Oven. Let cool on the benchtop for 30 minutes before opening.

[_] 2 While the Hyb Chamber is cooling:

[_] a Fill two wash dishes with PB1 (200 ml per wash dish). Label each dish "PB1".

[_] b Fill the Multi-Sample BeadChip Alignment Fixture with 150 ml PB1.

[_] c Separate the clear plastic spacers from the white backs.

[_] d Clean the glass back plates if necessary.

Steps to Wash BeadChip

[_] 1 Attach the wire handle to the rack and submerge the wash rack in the wash dish containing

200 ml PB1.

[_] 2 Remove the Hyb Chamber inserts from the Hyb Chambers.

[_] 3 Remove BeadChips from the Hyb Chamber inserts one at a time.

[_] 4 Remove the cover seal from each BeadChip.

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Illumina Infinium HTS Assay, Automated Protocol

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NOTE

To make sure that no solution splatters on you, Illumina recommends removing the cover seal over an absorbent cloth or paper towels, preferably in a hood.

[_] a Using powder-free gloved hands, hold the BeadChip securely and by the edges in one hand. Avoid contact with the sample inlets. Make sure that the barcode is facing up and closest to you, and that the top side of the BeadChip is angled slightly away from you.

[_] b Remove the entire seal in a single, continuous motion. Start with a corner on the barcode end and pull with a continuous upward motion away from you and towards the opposite corner on the top side of the BeadChip. Do not touch the exposed arrays.

[_] 5 Immediately and carefully slide each BeadChip into the wash rack, one at a time, making sure that the BeadChip is completely submerged in the PB1.

[_] 6 Repeat steps

4

through

5

until all BeadChips (a maximum of 8) are transferred to the submerged wash rack.

NOTE

You can use the two 200 ml PB1 wash dishes for up to 24 BeadChips. However, use 150 ml of fresh

PB1 for every 8 BeadChips in the Multi-Sample BeadChip Alignment Fixture.

[_] 7 After all BeadChips are in the wash rack, move the wash rack up and down for 1 minute, breaking the surface of the PB1 with gentle, slow agitation.

[_] 8 Move the wash rack to the other wash dish containing clean PB1. Make sure the BeadChips are completely submerged.

[_] 9 Move the wash rack up and down for 1 minute, breaking the surface of the PB1 with gentle, slow agitation.

[_] 10 When you remove the BeadChips from the wash rack, inspect them for remaining residue.

[_] 11 If you are processing more than 8 BeadChips

[_] a Assemble the flow-through chambers for the first 8 BeadChips, as described in the next section, and place them on the lab bench in a horizontal position.

NOTE

Keep the flow-through chambers in a horizontal position on the lab bench until all assembled flow-through chambers are ready to be loaded into the chamber rack. Do not place the flow-through chambers in the chamber rack until all BeadChips are prepared in flow-through chambers.

[_] b Return to this procedure and follow the steps described above to wash the next set of 8

BeadChips.

[_] c Repeat for each remaining set of 8 BeadChips.

Assemble Flow-Through Chambers

NOTE

Confirm that you are using the correct Infinium LCG glass back plates and spacers before assembling the flow-through chambers. Refer to the following image for the correct flow-through chamber components.

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Part # 15045736 Rev. A

Illumina Infinium HTS Assay, Automated Protocol

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Figure 1 Correct LCG Back Plates and Spacers

[_] 1 If you have not done so, fill the Multi-sample BeadChip Alignment Fixture with 150 ml PB1.

If you plan to process more than 4 BeadChips, this 150 ml of PB1 can be reused for an additional set of 4 BeadChips. Use 150 ml of fresh PB1 for every additional set of 8

BeadChips.

[_] 2 For each BeadChip to be processed, place a black frame into the Multi-Sample BeadChip

Alignment Fixture pre-filled with PB1.

[_] 3 Place each BeadChip to be processed into a black frame, aligning its barcode with the ridges stamped onto the Alignment Fixture.

NOTE

Inspect the surface of each BeadChip for residue left by the seal. Use a pipette tip to remove any residue under buffer and be careful not to scratch the bead area.

[_] 4 Place a clear LCG spacer onto the top of each BeadChip. Use the alignment fixture grooves to guide the spacers into proper position.

NOTE

Be sure to use the clear plastic spacers, not the white ones.

[_] 5 Place the alignment bar onto the alignment fixture.

The groove in the alignment bar fits over the tab on the alignment fixture.

[_] 6 Place a clean LCG glass back plate on top of the clear spacer covering each BeadChip. The plate reservoir is at the barcode end of the BeadChip, facing inward to create a reservoir against the BeadChip surface.

[_] 7 Attach the metal clamps to the flow-through chambers as follows:

[_] a Gently push the glass back plate up against the alignment bar with one finger.

[_] b Place the first metal clamp around the flow-through chamber so that the clamp is approximately 5 mm from the top edge.

[_] c Place the second metal clamp around the flow-through chamber at the barcode end, approximately 5 mm from the reagent reservoir.

[_] 8 Using scissors, trim the ends of the clear plastic spacers from the flow-through chamber assembly. Slip scissors up over the barcode to trim the other end.

[_] 9 Immediately wash the Hyb Chamber reservoirs with DiH

2

O and scrub them with a small cleaning brush, ensuring that no PB2 remains in the Hyb Chamber reservoir.

[_] 10 If you are using Illumina LIMS:

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[_] a In the Illumina LIMS left sidebar, click Infinium HTS | Wash BeadChip.

[_] b Scan the reagent barcodes and the BeadChip barcodes. Click Verify and then click Save.

Illumina LIMS records the data and queues the BeadChips for the next step.

[_] 11 Proceed to Extend and Stain (XStain) BeadChip (Post-Amp).

CAUTION

Place all assembled flow-through chambers on the lab bench in a horizontal position while you perform the preparation steps for the XStain BeadChip. Do not place the flow-through chambers in the chamber rack until the preparation is complete.

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Extend and Stain (XStain) BeadChip (Post-Amp)

In this process, you use RA1 reagent to wash away unhybridized and non-specifically hybridized DNA sample. LX1 and LX2 are added to condition the BeadChip surface for the extension reaction. Dispense EML reagent into the flow-through chambers to extend the primers hybridized to DNA on the BeadChip. This reaction incorporates labeled nucleotides into the extended primers. 95% formamide/1 mM EDTA is added to remove the hybridized DNA. After neutralization using the XC3 reagent, the labeled extended primers undergo a multi-layer staining process on the chamber rack. Next, you disassemble the flow-through chambers and wash the BeadChips in the PB1 reagent, coat them with XC4, and then dry them.

Estimated Time

Robot time:

• ~2 hours and 45 minutes for 8 BeadChips

• ~3 hours for 16 BeadChips

• ~3 hours and 10 minutes for 24 BeadChips

Dry time: 55 minutes

Consumables

Item

RA1

Storage

-15°C to -25°C

Supplied By

Illumina

LX1

LX2

EML

XC3

SML (Make sure that all SML tubes indicate the same stain temperature on the label)

ATM

Quantity

10 ml for 1–8

BeadChips

20 ml for 9–16

BeadChips

30 ml for 17–24

BeadChips

2 tubes (per 8

BeadChips)

2 tubes (per 8

BeadChips)

2 tubes (per 8

BeadChips)

50 ml for 1–8

BeadChips

100 ml for 9–16

BeadChips

150 ml for 17–24

BeadChips

2 tubes (per 8

BeadChips)

2 tubes (per 8

BeadChips)

-15°C to -25°C

-15°C to -25°C

-15°C to -25°C

Room temperature

-15°C to -25°C

-15°C to -25°C

Illumina

Illumina

Illumina

Illumina

Illumina

Illumina

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Item

PB1

XC4

Alconox Powder Detergent

EtOH

95% formamide/1 mM EDTA

Quantity

310 ml for 1–8

BeadChips

285 ml for 9–24

BeadChips

310 ml for 1–8

BeadChips

285 ml for 9–24

BeadChips

As needed

As needed

15 ml for 1–8

BeadChips

17 ml for 9–16

BeadChips

25 ml for 17–24

BeadChips

Storage

Room temperature

Room temperature

Room temperature

Room temperature

-15°C to -25°C

Supplied By

Illumina

Illumina

General lab supplier

General lab supplier

General lab supplier

Preparation

[_] 1 Place all reagent tubes in a rack in the order in which they will be used. If frozen, allow them to thaw to room temperature, and then gently invert the reagent tubes at least 10 times to mix contents.

[_] 2 Make sure the water circulator is filled to the appropriate level.

[_] 3 Turn on the water circulator and set it to 44°C using the Circulator Manager in the automation control software.

[_] 4 Remove bubbles trapped in the Chamber Rack.

[_] 5 Test several locations on the Chamber Rack, using the Illumina Temperature Probe. All locations should be at 44°C ± 0.5°C. If the temperature on the probe is not within ± 0.5°C, contact Illumina Technical Support.

Single-Base Extension and Stain

CAUTION

The remaining steps must be performed without interruption.

[_] 1 Slide the chamber rack into column 36 on the robot bed. Make sure that it is seated properly.

[_] 2 At the robot PC, select XStain Tasks | XStain LCG BeadChip.

[_] 3 In the Basic Run Parameters pane, enter the number of BeadChips.

[_] 4 If you plan on imaging the BeadChip immediately after the staining process, turn on the iScan or HiScan now to allow the lasers to stabilize.

[_] 5 Place a quarter reservoir in the reservoir frame, according to the robot bed map, and add

95% formamide/1 mM EDTA as follows:

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• 15 ml to process 8 BeadChips

• 17 ml to process 16 BeadChips

• 25 ml to process 24 BeadChips

[_] 6 Place a half reservoir in the reservoir frame, according to the robot bed map, and add RA1 in the following volumes:

• 10 ml to process 8 BeadChips

• 20 ml to process 16 BeadChips

• 30 ml to process 24 BeadChips

[_] 7 Place a full reservoir in the reservoir frame, according to the robot bed map, and add XC3 in the following volumes:

• 50 ml to process 8 BeadChips

• 100 ml to process 16 BeadChips

• 150 ml to process 24 BeadChips

[_] 8 Place each reagent tube (LX1, LX2, EML, SML, ATM) in the robot tube rack according to the bed map, and remove their caps.

[_] 9 When prompted, enter the stain temperature indicated on the SML tube.

[_] 10 Do not load the BeadChips yet.

[_] 11 When the chamber rack reaches 44°C, quickly place each flow-through chamber assembly into the first row of the chamber rack. Refer to the robot bed map for the correct layout.

[_] 12 At the robot PC, click OK.

[_] 13 When the robot finishes, immediately remove the flow-through chambers from the chamber rack. Place horizontally on the lab bench at room temperature.

Wash and Coat 8 BeadChips

[_] 1 Pour 310 ml PB1 per 8 BeadChips into a wash dish.

[_] 2 Place the staining rack inside the wash dish.

[_] 3 For each BeadChip:

[_] a Use the dismantling tool to remove the two metal clamps from the flow-through chamber.

[_] b Remove the glass back plate, the spacer, and then the BeadChip.

[_] c Immediately place each BeadChip into the staining rack that is in the wash dish with the barcode facing away from you. All chips should be completely submerged.

[_] 4 Slowly move the staining rack up and down 10 times, breaking the surface of the reagent.

[_] 5 Allow the BeadChips to soak for an additional 5 minutes.

[_] 6 Shake the XC4 bottle vigorously to ensure complete resuspension. If necessary, vortex until completely dissolved.

[_] 7 Pour 310 ml XC4 into a wash dish.

CAUTION

Do not let the XC4 sit for longer than 10 minutes.

[_] 8 Move the BeadChip staining rack into the XC4 dish.

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[_] 9 Slowly move the staining rack up and down 10 times, breaking the surface of the reagent.

[_] 10 Allow the BeadChips to soak for an additional 5 minutes.

[_] 11 Lift the staining rack out of the solution and place it on a tube rack with the staining rack and BeadChips horizontal, barcodes facing up.

[_] 12 Remove the BeadChips from the staining rack with locking tweezers, working from top to bottom. Place each BeadChip on a tube rack to dry. Remove the rack handle if it facilitates removal of the BeadChips.

[_] 13 Dry the BeadChips in the vacuum desiccator for 50–55 minutes at 675 mm Hg (0.9 bar).

[_] 14 Make sure that the XC4 coating is dry before continuing to the next step.

[_] 15 Clean the underside of each BeadChip with a ProStat EtOH wipe or Kimwipe soaked in

EtOH.

CAUTION

Do not touch the stripes with the wipe or allow EtOH to drip onto the stripes.

[_] 16 Clean and store the glass back plates and Hyb Chamber components.

[_] 17 If you are using Illumina LIMS:

[_] a In the Illumina LIMS left sidebar, click Infinium HTS | Coat BC2.

[_] b Scan the reagent barcodes and BeadChip barcodes. Click Save. Illumina LIMS records the data and queues the BeadChips for the next step.

[_] 18 Do one of the following:

• Proceed to Image BeadChip (Post-AMP).

• Store the BeadChips in the Illumina BeadChip Slide Storage Box inside a vacuum desiccator at room temperature. Be sure to image the BeadChips within 72 hours.

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Image BeadChip (Post-Amp)

Follow the instructions in the iScan System User Guide or HiScan System User Guide to scan your

BeadChips. Use the Infinium LCG scan setting for your BeadChip.

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Part # 15045736 Rev. A