Amplicon Indexing

This step amplifies the tagmented amplicons using a PCR program and adds unique dual indexes (10 base pair Index 1 (i7) adapters, and 10 base pair Index 2 (i5) adapters), and additional adapter sequences required for sequencing cluster generation.

• EPM (Enhanced PCR Mix)
• Index adapters
• Nuclease-free water
• [1-24 samples] 1.7 ml tube
• [24-48 samples] 15 ml tube
• 96-well PCR plate
• Index adapter plates
– Do not add samples to the index plate wells.
– Index plate wells are considered single use and should not be reused.
1. Prepare the following consumables:
• EPM—Invert to mix, and then keep on ice.
• Index adapters—Vortex to mix, and then centrifuge at 1000 × g for 1 minute.
2. Open each prepared index adapter plate seal as follows.
• 48 samples: Align a new 96-well PCR plate above the index adapter plate, and then press down to puncture the foil seal. Discard the PCR plate.
• < 48 samples: Pierce the foil seal on the index adapter plate with a new pipette tip for each well. Pierce wells for only the number of samples to be processed.
3. Save the following IMAP-Flu TAG PCR program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Set the reaction volume to 50 µl
• 72°C for 3 minutes
• 98°C for 3 minutes
• 7 cycles of:
• 98°C for 20 seconds
• 60°C for 30 seconds
• 72°C for 1 minute
• 72°C for 3 minutes
• Hold at 10°C
1. In the appropriately sized tube, combine the following volumes to prepare PCR Master Mix. Multiply each volume by the number of samples. Reagent overage is included to account for small pipetting errors.
• EPM (24 µl)
• Nuclease-free water (24 µl)
2. Vortex PCR Master Mix to mix.
3. Keep the TAG1 plate on the magnetic stand and remove TWB remaining from the Post Tagmentation Clean Up procedure.
4. Use a 20 µl pipette to remove any remaining TWB.
5. Remove the TAG1 plate from the magnetic stand.
6. Add 40 µl PCR Master Mix to each well.
7. Add 10 µl index adapters to each well of the PCR plate.
8. Seal and shake at 1600 rpm for 1 minute.
9. If liquid is visible on the seal, centrifuge at 500 x g for 1 minute.
10. Inspect to make sure beads are resuspended. To resuspend, set pipette to 35 µl with the plunger down, and then slowly pipette to mix.
11. Place on the preprogrammed thermal cycler and run the IMAP-Flu TAG PCR program.