Hybridize Single-Stranded Probes

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This step binds single-stranded enrichment probe oligonucleotides to targeted regions of the cfDNA libraries.

Follow this procedure when using single-stranded probes in 1-plex or 4-plex enrichment.

• EHB2 (Enrich Hyb Buffer 2)
• NHB2 (Hyb Buffer 2 + IDT NXT Blockers)
• Enrichment Probe Panel, single stranded
• 1.7 ml microcentrifuge tube
• 96-well PCR plate, semiskirted
• 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.

• NHB2:
– Precipitates and separates during storage.
– Must be at room temperature before use.
• Enrichment probe panel:
– Enrichment probe panel refers to the chosen enrichment oligonucleotide panel from Illumina or a third-party vendor to run with this workflow.
– For information on the required specifications for enrichment probe panels, refer to Enrichment Probe Panel Requirements.
– Enrichment probes must be single-stranded.
1. Prepare the following consumables:
• EHB2:
a. Vortex for 30 seconds.
b. If crystals and cloudiness are observed, repeat vortex, or pipette up and down to mix until the solution is clear.
• NHB2:
a. Vortex three times for 10 seconds on the maximum setting.
b. Centrifuge briefly.
c. Pipette up and down from the bottom of the tube.
d. If crystals and cloudiness are observed, repeat vortex, or pipette up and down to mix until the solution is clear.

Warm tube in hand and repeat vortex if needed.

• Enrichment Probe Panel—Vortex to mix, and then centrifuge briefly.
2. Prepare the ALS plate:
a. Pipette to mix, and then centrifuge briefly.
b. Place on a peg magnetic stand for at least one minute before starting the procedure.
3. Label a new 96-well semiskirted PCR plate HYB.
4. Save the following HYB program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Set the reaction volume to 25 µl
• 98°C for 5 minutes
• 19 cycles for 1 minute each
• From 96°C, decrease by 2°C per cycle until reaching 58°C
• 58°C for 30 minutes
• Hold at 58°C
1. [4-plex enrichment] Avoiding bead carryover to the new plate, transfer supernatant as follows to create a 4-plex enrichment pool.
a. Leave the ALS plate on the magnetic stand. Transfer 2 µl supernatant from each of the four libraries with nonoverlapping indexes from the ALS plate to a single well of the HYB plate.
b. Repeat step a until each of the 4-plex enrichment pools have been added to new wells of the HYB plate.
c. Adjust pipette to 5 µl, and then pipette each enrichment pool 5 times to mix.
2. [1-plex enrichment] Avoiding bead carryover to the new plate, transfer supernatant as follows to prepare 1-plex enrichment libraries.
a. Leave the ALS plate on the magnetic stand. Transfer 7.5 µl supernatant from each well of the ALS plate to the corresponding well of the HYB plate.
3. Seal the ALS plate with Microseal 'B' and store at -25°C to -15°C.
4. In the order listed, combine the following volumes to prepare the HYB Master Mix. Multiply each volume by the number of enrichment reactions.
• NHB2 (14.4 μl)
• Enrichment Panel (2.9 μl)
• EHB2 (2.9 μl)

These volumes produce 20.2 μl HYB Master Mix per sample, which includes extra volume for accurate pipetting.

Add reagents in the order listed. After EHB2 is added, the master mix might become cloudy over time.

5. Vortex HYB Master Mix to mix, and then centrifuge briefly.

If the master mix forms a phase separation appearance over time, repeat the vortex.

6. Add 17.5 μl HYB Master Mix to each well of the HYB plate, and then pipette 10 times to mix.
7. Seal the plate, and then centrifuge briefly.
8. Place on the preprogrammed thermal cycler and run the HYB program.
9. When the program reaches the 58°C hold, immediately proceed to Capture Hybridized Probes.