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beer-dna-sequencing.md

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layout default
title Beer DNA sequencing
image /images/protocols/beer-dna-sequencing.jpg

Requirements 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:

  • Please search for "MinION Software" in the Nanopore downloads web page https://community.nanoporetech.com/downloads.
  • Select your host operating system and follow the link to the installation instructions.
  • Since the supported systems and the installation procedure is constantly updated, please always refer to the community website for details. (You need to have an account to access the community website)

Setting up the software:

Requirements:

  • You have to have a account at nanopor MinION to start the software

Do:

  • Attech the MinION divice to your computer (connected to through the USB 3.0 port)
  • Open the divice; remove the addapter and add a flowcell instead
  • To start the software you either find the "MinION Software" botton directly or you can open it via the terminal using the path "/opt/ui/MinKNOW"
  • After starting the software, first select the flow cell type. You should find the type disctiption on the flowcell package
  • Click on the botton 'Check flow cell' and 'start test'
  • On the right side under massanges you will see when the check is compleated and how may nanopores are avalabel for your sequencing run.

Requirements DNA

The most important: 7.5µl DNA extracted as described in the [DNA extraction protocol]({% link _protocols/beer-dna-extraction.md %})

Before starting Prepare DNA

  • DNA ~ 400 ng should be in 7.5 µl nuclease-free water
  • DNA should be mixed by flicking the tube and spin down briefly in a centrifuge
  • All kit reagents should be:
  1. Thaw to room temperature
  2. Briefly spin down
  3. Mix well by pipetting (up and down)
  • Once thawed, keep all the kit components on ice
  • Always repeat the mixing step before using each reagent

Starting beer DNA sequencing

Library preparation

Needed consumables
  • ~400 ng high molecular weight genomic DNA
  • Fragmentation Mix (FRA)
  • Rapid Adapter (RAP)
  • Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
  • 0.2 ml PCR tubes
  • Ice
Needed material
  • Thermal cycler at 30° C and 80° C
  • P2 pipette and tips
  • P10 pipette and tips
  • centrifuge

Do

  1. Prepare a tube 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
  • Flush Tether (FLT)
  • Sequencing Buffer (SQB)
  • Flush Buffer (FB)
  • Loading Beads (LB)
  • Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
  • 1.5 ml Eppendorf DNA
  • MinIon diveice including Flow cell
  • Ice
Needed material
  • MinION
  • P100 pipette and tips
  • P1000 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • Vortex
  • centrifuge

Prepare substance of the priming mix

  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

Prepare loading port

  1. Open priming port to check for small bubbles
  2. To remove bubbles take some liquid from the port by use the P1000 pipette set to 200 µl
  3. Remove the liquid by turning the weel but only until 220-230 µl
  4. Note: 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
  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

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.