Hybridize Probes

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This step adds capture probes to pooled libraries to target regions of interest. The procedure uses enrichment reagents and oligos from an enrichment panel.

• EHB2 (Enrichment Hyb Buffer 2)
• NHB2 (Hyb Buffer 2 + IDT NXT Blockers)
• Illumina Viral Surveillance Panel v2 panel
• 96-well PCR plate, semiskirted
• Microseal 'B' adhesive film
• NHB2
– Precipitates and separates during storage.
– Follow the preparation instructions before first use.
1. Preheat the microheating system to 50°C.
2. Prepare the following consumables:
• EHB2—Vortex to mix. If crystals and cloudiness are observed, repeat vortex, or pipette up and down to mix well until the solution is clear.
• NHB2—Vortex to mix. Lay in the preheated microheating system and incubate for 5 minutes.
• Illumina Viral Surveillance Panel v2—Vortex to mix.
• SMB4—If you are proceeding to the next procedure immediately after the 90 minute hold in the NF-HYB program, bring to room temperature. If you are extending the hold time, bring to room temperature at least 30 minutes before the NF-HYB program ends.
3. Mix preheated NHB2 as follows.
a. Vortex for 30 seconds.
b. Pipette to resuspend fully. Keep warm until use to prevent the reformation of precipitates.
4. If EHB2 or NHB2 appears crystallized or cloudy, vortex or pipette until clear.
5. 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
• 95°C for 5 minutes
• 18 cycles of 1 minute each:
• 94°C for the first cycle
• Decrease 2°C per subsequent cycle
• 58°C for 90 minutes
• Hold at 58°C for ≤ 24 hours

The minimum hybridization time at 58°C is 90 minutes. Extending the hold to up to 24 hours is optional for overnight hybridization. Without the extended hold, total program time is ~2 hours.

1. Add the following reagents to each well of a new PCR plate in the order listed.
a. Three-plex pre-enriched library pool (7.5 µl)
b. NHB2 (12.5 µl)
c. Illumina Viral Surveillance Panel v2 (2.5 µl)
d. EHB2 (2.5 µl)
2. Pipette 10 times to mix, and then seal.
3. Centrifuge at 280 × g for 3 seconds.

EHB2 can make the reaction appear cloudy, which is normal.

4. Place on the preprogrammed thermal cycler and run the HYB program.
5. Allow to incubate at 58°C for 90 minutes to 24 hours.

Each well contains a volume of 25 µl.

If the reaction falls below room temperature precipitates can form, impacting the run results.