Pipelines to do MinION sequencing of Zika virus
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Two-step RT-PCR for amplicon generation

Perform the following in a clean pre-PCR hood or in a pre-PCR designated area. The hood and all pipettes/tips entering the hood should be cleaned with 10% bleach and 70% ethanol prior to use.

Reverse Transcription

  1. For each sample, mix the following in a PCR tube (preferably individually-hinged strip tubes):
  • 7uL RNA
  • 1 uL random hexamers (50 ng/uL)
  1. Mix by inversion then spin down.
  2. Heat to 70 degrees Celsius for 7 minutes, then place on ice to prevent secondary structure from re-forming.
  3. Add the following to each tube:
  • 10 uL ProtoScript II Reaction Mix
  • 2 uL ProtoScript II Enzyme Mix

Note: you can also make the this as a mastermix, and pipette once per sample.

  1. Place on the thermocycler and run the following program:
  • 25 degrees Celsius for 5 minutes **note: this step can be swapped for a 5 minute hold at room temperature.
  • 48 degrees Celsius for 15 minutes
  • 80 degrees Celsius for 5 minutes
  • Hold at 10 degrees Celsius

Amplification PCR

  1. Make the following mastermix in a 1.5 mL tube. You will need both a Pool 1 mastermix and a Pool 2 mastermix.
  • 8 uL Q5 Reaction Buffer
  • 0.8 uL 10 mM dNTPs
  • 0.4 uL Q5 DNA polymerase
  • 24.4 uL Nuclease-free water
  • 2.4 uL of primer pool (either pool 1 OR pool 2)
  1. Label 2 0.2mL PCR tubes for each sample.
  2. Pipette 36 uL of mastermix into the corresponding tubes.
  3. Add 4 uL of cDNA into the corresponding tubes.
  4. Mix by inversion and spin down.
  5. Place on the thermocycler and run the following program:
  • Step 1: 98 degrees Celsius for 30 seconds (initial denaturation)
  • Step 2: perform 40 cycles of
    • 98 degrees Celsius for 15 seconds
    • 65 degrees Celsius for 5 minutes
  • Step 3: Hold at 4 degrees Celsius.

**note: If generating amplicons for Miseq run rather than MinION run, you should do only 35 cycles of amplification, since you will have additional cycles of amplification during library prep.