Pipelines to do MinION sequencing of Zika virus

Viral RNA extraction

We use the QiaAMP Viral RNA mini kit, and the protocol below is based off of the manufacturer's instructions. The protocol below is written out assuming a clinical sample volume of 200 uL. Note that you need 4x the amount of Buffer AVL as serum, so set Buffer AVL and carrier RNA amounts based off of your available sample volume. The volume of carrier RNA that should be used is 1/100th the volume of buffer AVL added.

We've also run this protocol on the QIAcube. To make the protocol compatible we use 140 uL of serum/urine and elution volume of 50 uL

Other equivalent extraction methods can also be used (e.g. Trizol, Omega etc.)

In addition to the samples, remember to have an extraction negative control (add nuclease-free water instead of serum/urine).

  1. Remove serum samples from freezer and allow to come up to room temperature.
  2. Prepare stock solution of Buffer AVL + carrier RNA. Per sample add:
  • 800 uL Buffer AVL
  • 8 uL Carrier RNA (previously resuspended in Buffer AVE)
  1. Pipette 800 uL of Buffer AVL containing carrier RNA into 2.0 ml tube.
  2. Add 200 uL of serum or urine to the tube with Buffer AVL.
  3. Mix sample and buffer AVL by pulse-vortexing for 15 seconds.
  4. Incubate mixture at room temperature for 10 minutes. Spin down.
  5. Add 800 uL of 100% Ethanol to the sample + Buffer AVL mixture.
  6. Mix by pulse vortexing for 15 seconds.
  7. Spin down fluid from tube walls.
  8. Take 2ml collection tubes and put the columns in the tubes.
  9. Perform the following 3 times in order to have run all 1800 uL of ethanol+sample+Buffer AVL mixture through the filter column.
  • Pipette 600 uL ethanol+sample+Buffer AVL mixture into the column directly onto the filter. do not touch tip to filter.
  • Centrifuge at room temperature at 8000 rpm for 1 minute. Take column out and place in a new 2 mL collection tube.
  1. Add 500 uL of Buffer AW1 to the column. this volume does not need to be increased even if the sample volume was greater than 200 uL.
  2. Spin sample at 8000 rpm for 1 minute. Transfer column to a new 2 mL collection tube.
  3. Add 500 uL of Buffer AW2.
  4. Spin at full speed (14,000 RPM) for 3 minutes. Place column in a new collection tube and spin at full speed for 1 minute.
  5. Place column in clean, labelled 1.5 mL tube. Add 50 uL of Buffer AVE at room temperature to column.
  6. Incubate at room temp for 1 minute.
  7. Centrifuge at room temp at 8000 rpm for 1 minute.
  8. Place extracted RNA in eluate on ice if proceeding directly to PCR or store in freezer.