Clean Up Ligation

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This step uses IPB (Illumina Purification Beads) to purify the cfDNA fragments and remove unwanted products, such as unligated adapters. The cfDNA bound beads are washed twice with fresh 80% ethanol. The cfDNA is then eluted with the addition of a PCR mix and UD indexes.

• EPM4 (Enhanced PCR Mix)
• IPB (Illumina Purification Beads)
• RSB (Resuspension Buffer)
• IDT for Illumina DNA/RNA UD Indexes (Set A or B)
• Absolute ethanol (EtOH)
• Nuclease-free water
• Microseal 'B' adhesive seals

This set of reagents contains potentially hazardous chemicals. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Ventilation should be appropriate for handling of hazardous materials in reagents. Wear protective equipment, including eye protection, gloves, and laboratory coat appropriate for risk of exposure. Handle used reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and regulations. For additional environmental, health, and safety information, refer to the SDS at support.illumina.com/sds.html.

• IPB:
– Use IPB at room temperature.
– Aspirate and dispense slowly due to the viscosity of the solution.
• IDT for Illumina DNA/RNA UD Indexes:
– Each well of the UD indexes plate is for single use only.
– When instructed, pierce the foil seal on the plate for only the number of samples being processed.
– When returning the plate to storage, cover the pierced wells with Microseal 'B' to prevent cross-contamination.
1. Prepare the following consumables:
• IPB—Vortex for one minute to disperse the beads.
• RSB—Vortex to mix.
• EPM4—Vortex to mix, and then centrifuge briefly.
2. Combine the following volumes to prepare the PCR Mix. Multiply each volume by the number of samples being processed.
• RSB (22 μl)
• EPM4 (22 μl)

These volumes produce 44 μl PCR Mix per sample, which includes extra volume for accurate pipetting.

3. Vortex PCR Mix to mix, and then place on ice.
4. Combine the following volumes to prepare 80% EtOH. Multiply each volume by the number of samples being processed.
• EtOH (400 μl)
• Nuclease-free water (100 μl)

These volumes produce 500 μl 80% EtOH per sample, which includes extra volume for accurate pipetting.

5. Vortex 80% EtOH to mix.
6. Assign one UDPXXXX index primer per library (XXXX = index primer number).

When sequencing multiple libraries on a single flow cell, assign a different indexing primer to each sample library. Record sample layout orientation and the indexes for each sample library.

7. Prepare the IDT for Illumina DNA/RNA UD Indexes plate.

Make sure to use the IDT for Illumina DNA/RNA UD Indexes plate and not the UMI DNA Index Anchor plate for this procedure.

a. Centrifuge the UD index plate briefly to settle liquid away from the seal.
b. Pierce the seal using a new pipette tip for each well. Pierce only the number of samples being processed.

Bind

1. Briefly vortex IPB to resuspend.
2. Add 80 μl IPB to each well of the LP plate.
3. Adjust pipette to 150 μl, and then pipette 10 times to mix.

Visually inspect each well. If a bead pellet is still observed, pipette up and down to release the pellet.

For optimal results, it is important to mix completely for efficient bead binding.

4. Incubate at room temperature for 5 minutes.

Wash

1. Place the LP plate on a bar magnetic stand for 10 minutes.
2. Without disturbing the beads, remove and discard all supernatant from each well.
3. Wash the beads as follows.
a. Keep on the magnetic stand and add 150 µl 80% EtOH to each well.
b. Wait 30 seconds.
c. Without disturbing the beads, remove and discard all supernatant from each well.
4. Repeat step 3 to wash the beads a second time.
5. Use a 20 μl pipette to remove residual supernatant from each well.

Elute

1. Remove the LP plate from the magnetic stand.
2. Vortex PCR Mix briefly.
3. Add 40 µl PCR Mix to each well.
4. Pipette UD indexes 5 times to mix.
5. Add 10 μl assigned UD indexes to corresponding wells of the LP plate.
6. Seal and shake at 2200 rpm for 1 minute.