Pipelines to do MinION sequencing of Zika virus

MinION library preparation

Normalize amplicon concentrations

turn on 65 degree Celsius dry bath on now

  1. Divide 1500 ng by the number of non-negative control samples you will run. This gives you the desired amount of DNA for each amplicon pool. Generally this will be:
  • 150 ng of DNA per amplicon pool if running 5 genomes (10 pools) and negative controls.
  • 125 ng of DNA per amplicon pool if running 6 genomes.

note that the PCR control should always be sequenced

  1. Determine how many uL of cleaned-up amplicons you need to add to reach the target amount of DNA. Bring this volume up to 30 uL with nuclease-free water if the full amount of cleaned-up amplicons is not used.

example where target amount is 150 ng per pool

samplepool concentrationvolume ampliconsvolume water
samp1_pool141.8 ng/uL3.6 uL26.4 uL
samp1_pool255.4 ng/uL2.7 uL23.7 uL

End repair and clean-up

  1. Add the following to each sample:
  • 4.2 uL Ultra II End-Prep buffer
  • 1.8 uL Ultra II End-Prep enzyme mix
  1. Mix by inversion and spin down. Total reaction volume is 36 uL.
  2. Incubate at 20 degrees Celsius for 5 minutes. this incubation can also be at room temperature
  3. Incubate at 65 degrees Celsius for 5 minutes.
  4. Perform 1:1 bead clean-up with AMPure beads (36 uL of beads to 36 uL of end-prepped DNA) as described above (5 minute incubations mixing throughout, two 200 uL 80% EtOH washes).

this time resuspend pellet in 15 uL nuclease-free water

Barcoding and pooled clean-up

  1. Thaw a native barcode for each sample. for 6 genomes you'll have 12 barcodes since each pool has it's own barcode
  2. Add 5 uL of one of the barcodes NB01-NB12 to the appropriate sample. Mix by inversion.
  3. Add 20 uL of Blunt/TA Ligase Master Mix to each tube. Mix by inversion. Spin down.
  4. Incubate at room temperature for 10 minutes.
  5. Incubate at 65 degrees Celsius for 10 minutes to kill ligase. Ensure that the temperature is correct. Samples will be pooled after this step so you need to prevent any further ligation that may cause sample-barcode mixtures.
  6. Pool all of the samples together into a single Lo-Bind tube. If you have 12 samples the total volume will be 480 uL.
  7. Add 480 uL AMPure XP beads to the pooled samples (1:1 clean-up). if fewer than 12 samples, add the same volume of beads as pooled samples
  8. Incubate at room temperature on a hula mixer for 5 minutes.
  9. Place tube in magnetic rack and wait until solution fully clears.
  10. Pipette off and discard the supernatant. Be careful to not disturb the pellet.
  11. Perform 2 washes with 80% EtOH as described above. Use sufficient EtOH to cover the pellet (this might be more than 200 uL).
  12. Allow pellet to air dry to the point that the pellet looks matte but not cracked. because of the size of the pellet this can take a while. You can speed this up a bit by incubating for short periods at 65 degrees Celsius. Ensure you keep checking the pellet periodically though so you don't over dry.
  13. Elute the pooled reaction in 39 uL of nuclease-free water, resuspend pellet and incubate at room temperature on a hula mixer for 5 minutes. Remove the supernatant to a clean Lo-Bind tube.

Pooled library quantification

  1. Prepare Qubit standards and sample reactions as described above.
  2. Target is >1000 ng DNA in the remaining 38 uL of pooled library.

MinION adaptor ligation

  1. Add 10 uL BAM to cleaned-up library. Mix by inversion.
  2. Then add 2 uL BHP. Mix by inversion.
  3. Then add 50 uL Blunt/TA Ligase Master Mix. Mix by inversion and spin down.
  4. Incubate at room temperature for 10 minutes. start making the cleaned MyOne C1 beads during this incubation, see directions below
  5. Add 1 uL HPT. Mix by inversion and spin down.
  6. Incubate at room temperature for 10 minutes.

MyONE C1 bead preparation

  1. Vortex MyOne C1 beads until homogenous.

  2. Pipette 50 uL of beads and transfer to new Lo-Bind tube.

  3. Pellet beads on magnetic rack and discard supernatant.

  4. Wash beads twice with 100 uL BBB pipetting to resuspend. Ensure to scrape beads off of tube sides when resuspending pellets. Discard supernatant. Do not let the beads dry after the second wash.

  5. Resuspend cleaned beads in 100 uL BBB. These cleaned beads will be used for adaptor ligation clean-up.

Purify the adapted,tethered library

  1. Add 100 uL washed MyOne C1 beads to the adapted,tethered library.
  2. Incubate at room temperature on a hula mixer for 5 minutes.
  3. Place on magnet. Once solution clears discard the supernatant.
  4. Wash pellet twice with 150 uL of BBB pipetting to resuspend the pellet after each wash.
  5. Spin down, replace tube on magnet and pipette off any residual BBB. Close lid, do not let pellet dry.
  6. Resuspend the beads in 25 uL of ELB. you'll need to scrape the beads off of the tube walls to ensure you fully resuspend the beads. This can take a while, but it's important to do it well to get sufficient library. The tube sides should not show any smear of beads.
  7. Incubate at 37 degrees Celsius for 10 minutes.
  8. Pellet beads on magnetic rack and transfer eluate (tethered,adapted library in ELB) to new Lo-Bind tube.

Adapted, tethered library quantification and normalization

  1. Make up Qubit Master Mix and standards as before.
  2. Quantify adapted, tethered library using 1 uL of library to 199 uL Qubit Master Mix.
  3. Determine the total ng of library, target is >100 ng. if you have >200 ng of library you have enough adapted, tethered library for a second run. You can save this leftover library by keeping it at -20 degrees Celsius.
  4. Normalize the library to have 100 ng of library loaded on the flowcell. Bring library up to 37.5 uL volume with nuclease-free water. multiply the library concentration by 24 uL (amount of library left post quantification) to determine total ng of library.
Libraryconcentrationtotal_amt_libraryvolume_library_neededvolume_nfw
library19.9 ng/uL237.6 ng10.1 uL27.4 uL
  1. Add 37.5 uL of RBF1 at room temperature to the library/nuclease-free water solution. Total library+RBF1 volume will be 75 uL. this volume is specific to R9.4 spot on flow cells. If you're using an older non-spot on flowcell follow the protocol for your flowcell version.
  2. Mix by inversion and spin down.