Beer DNA sequencing

Requirements

The most important: 7.5µl DNA extracted as described in the DNA extraction protocol

Before starting Prepare DNA

Starting beer DNA sequencing

Library preparation

Needed consumables
Needed material

Do

  1. Prepare tubes with 7.5 µl extracted DNA and 2.5 µl FRA
  2. Mix gently by flicking the tube, and spin down
  3. Put the tubes into a thermocycler: 1 min at 30℃, 1 min at 80℃
  4. Keep on ice until next step
  5. Attach adapter by adding 1 µl of RAP into the tube
  6. Mix gently by flicking the tube, and spin down
  7. Incubate the reaction for 5 min at room temperature
  8. Store the library on ice until ready to load

Priming and loading the SpotON Flow Cell

Needed consumables
Needed material

Prepare loading port

  1. Mix the Sequencing Buffer (SQB) and Flush Buffer (FB) tubes by vortexing, spin down and return to ice
  2. Spin down the Flush Tether (FLT) tube, mix by pipetting, and return to ice
  3. Open priming port to check for small bubbles
  4. If there are bubbles remove them by taking some liquid from the port
  5. To remove use the P1000 pipette set to 200 µl
  6. Remove the liquid by turning the weel but only until 220-230 µl
  7. Removing more than 30 µl will damage the pores in the array because they need to be covered by the buffer at all times

Prepare flowcell priming mix

  1. Add 30 µl of Flush Tether (FLT) directly to the Flush Buffer (FB) Eppi tube and mix by pipetting up and down
  2. Load 800 μl of the priming mix into the flow cell via the priming port, avoiding introduction of air bubbles
  3. Wait for 5 minutes

Prepare library for loading

  1. Use a new tube
  2. Mix the following reagents together: 34 μl Sequencing Buffer (SQB), 25.5 μl Loading Beads (LB) (mix before adding), 4.5 μl nuclease-free water and 11 μl DNA library

Load sample

  1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible
  2. Load 200 μl of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding introduction of air bubbles
  3. Mix the prepared library gently by pipetting up and down prior to loading
  4. Add 75 μl of sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next
  5. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and the MinION lid

Protocol: Software setup

MinKNOW

MinKNOW is the program needed to connect your computer with MinION. It has a graphic user interface for configuring and running the sequencing. Furthermore, it has integrated base-caller software to convert the raw nanopore signals to the strings of nucleotides, in fastq format.

Installation:

Starting the software:

Requirements:

Either you find the “MinION Software” botton directly or you open it via the terminal using the path “/opt/ui/MinKNOW”

Launching MinKNOW and running the sequencing

After having the software installed, the major steps would be connect, initialize and configure the MinION within the MinKNOW software. Afterwards the device would be ready for loading and sequencing the data.