TITLE: Rapid sequencing gDNA Barcoding RBK2004
AUTHORS: Carlos Goller, Carly Sjogren

[DESCRIPTION]
This protocol has been adapted from Oxford Nanopore Technologies. 

You have isolated, purified, and quantified high-molecular weight DNA from your bacterial isolate. You have also worked with your bacterium and grown in the presence of different carbon sources and pH levels to characterize its properties using Biolog GEN3 plates. Now, we have the opportunity to sequence the DNA of your isolate using long-read sequencing and Oxford Nanopore Technologies (ONT) MinION. With the genetic sequences we obtain, we will attempt to assemble the genome of this organism and explore its genomic potential!

 
The MinION sequencer from Oxford Nanopore Technologies (ONT) will allow us to sequence the genomic DNA using a flow cell with hundreds of nanopores. The DNA will be passed through the pores, and changes in current will be interpreted as bases (“base calling”). After performing quality control to remove low quality sequences, we will assemble the sequences to create contigs and scaffolds with the goal of exploring the genome of your isolates. Created with BioRender.com.

After completing this lab you will gain the following lab skills:

Lab safety and proper personal protective equipment (PPE)
Pipetting with micropipettes
Preparing sequencing libraries for the ONT MinION

[BEFORE_START]
Review the “How to use a micropipette” lab we completed at the beginning of the semester. Before coming to the lab watch this 5-minute video, How to Micropipette, that demonstrates 3 key pipetting skills: 
How to hold a micropipette
How to set a micropipette to a desired volume
How to use the plunger and ejector 

In this lab session, we will pipette small volumes of DNA and enzymes to prepare your genomic DNA for sequencing by adding adaptors for the Nanopore sequencing. We will also combine all our samples once we have added adapters to bioinformatically distinguish one sample from another.

 
The Oxford Nanopore Technologies (ONT) Rapid Barcoding Kit will allow us to sequence the genomic DNA from several different genomic DNA samples in one flow cell by using an enzyme to cleave and add barcoded adapter sequences to the ends of the genomic DNA (Step 1). Next, sequencing adapters are added to the pooled  sample (Step 2) before loading and sequencing (Step 3). Created with BioRender.com.


[GUIDELINES]
For any issues with the protocol, ready the attached document thoroughly. It contains the most common issues, and their suggested solutions.
Possible Issues with Rapid Sequencing gNA RBK004

[STEPS]
SECTION: Library Preparation
1. Thaw kit components at Room temperature , spin down briefly using a microfuge and mix by pipetting as indicated by the table below: 

Fragmentation Mix RB01-12: not frozen, briefly spin down, mix well by pipetting 
Rapid Adapter (RAP): not frozen, briefly spin down, mix well by pipetting 
Sequencing Buffer (SQB): thaw at RT, briefly spin down, mix well by pipetting* 
Loading Beads (LB): thaw at RT, briefly spin down, mix by pipetting or vortexing immediately before use 
Flush Buffer (FLB) - 1 tube: thaw at RT, briefly spin down, mix well by pipetting* 
Flush Tether (FLT): thaw at RT, briefly spin down, mix well by pipetting
SECTION: Library Preparation
2. Prepare the DNA in Nuclease-free water.
SECTION: Library Preparation
2.1. Transfer ~400 ng  genomic DNA into a DNA LoBind tube

SECTION: Library Preparation
2.2. Adjust the volume to 7.5 µL  with Nuclease-free water

SECTION: Library Preparation
2.3. Mix by flicking the tube to avoid unwanted shearing
SECTION: Library Preparation
2.4. Spin down briefly in a microfuge
SECTION: Library Preparation
3. In a 0.2 ml thin-walled PCR tube, mix the following:
7.5 µL  400 ng template DNA 
2.5 µL  Fragmentation Mix RB01-12 (one for each sample)

SECTION: Library Preparation
4. Mix gently by flicking the tube, and spin down.
SECTION: Library Preparation
5. Incubate the tube at 30 °C  for 1 min and then at 80 °C for 1 min . Briefly put the tube   on ice  to cool it down

SECTION: Library Preparation
6. Pool the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube.

 

SECTION: Library Preparation
7. Resuspend the AMPure XP beads by vortexing.
SECTION: Library Preparation
8. To the entire pooled barcoded sample from Step 6, add an equal volume of resuspended AMPure XP beads, and mix by flicking the tube.
SECTION: Library Preparation
9. Incubate on a Hula mixer (rotator mixer) for 5 min at Room temperature 

SECTION: Library Preparation
10. Prepare 500 µL  of fresh 70% ethanol in Nuclease-free water.

SECTION: Library Preparation
11. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant
SECTION: Library Preparation
12. Keep the tube on the magnet and wash the beads with 200 µL of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

SECTION: Library Preparation
13. Repeat the previous step.
SECTION: Library Preparation
14. Spin down and place the tube back on the magnet. Pipette off any residual 70% ethanol. Briefly allow to dry.
SECTION: Library Preparation
15. Remove the tube from the magnetic rack and resuspend pellet in 10 µL  of 10 mM Tris-HCl pH 7.5-8.0 with 50 mM NaCl. Incubate for 2 min at Room temperature 

SECTION: Library Preparation
16. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 min 

SECTION: Library Preparation
17. Remove and retain 10 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

SECTION: Library Preparation
18. Add 1 µL  of RAP to 10 µL  of barcoded DNA.

SECTION: Library Preparation
19. Mix gently by flicking the tube, and spin down.
SECTION: Library Preparation
20. Incubate the reaction for 5 min at Room temperature 

 

SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
25. 
 

Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at Room temperature before mixing the reagents by vortexing, and spin down at Room temperature .

SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
26. To prepare the flow cell priming mix, add 30 µL of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at Room temperature 

SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
27. Open the MinION device lid and slide the flow cell under the clip.
SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
28. Slide the priming port cover clockwise to open the priming port.

 

SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
29. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few µl):
SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
29.1. Set a P1000 pipette to 200 µl
SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
29.2. Insert the tip into the priming port
SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
29.3. Turn the wheel until the dial shows 220-230 µL , to draw back 20-30 µL , or until you can see a small volume of buffer entering the pipette tip

 


SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
30. Load 800 µL of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for 5 min . During this time, prepare the library for loading by following the steps below.

SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
31. Thoroughly mix the contents of the Loading Beads (LB) tubes by vortexing.
SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
32. In a new tube, prepare the library for loading as follows:

34 µL Sequencing Buffer (SQB) 
25.5 µL  Loading Beads (LB), mixed immediately before use 
4.5 µL  Nuclease-free water 
11 µL  DNA library
 


SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
33. Complete the flow cell priming:
SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
33.1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
33.2. Load 200 µL of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
34. Mix the prepared library gently by pipetting up and down just prior to loading.
SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
35. 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.

SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
36. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the MinION device lid.
SECTION: Ending the experiment
37. 
SECTION: Ending the experiment
38. 
 

SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
21. Remove a flow cell from the refrigerator and allow it to equilibrate to room temperature.
SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
22. Connect the MinION Mk1B to the computer and carefully insert the flow cell.
SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
23. Open MinKNOW software and run Flow Cell Check.
SECTION: Instrument Setup Priming and loading the SpotON Flow Cell
24. Confirm the number of active pores. A MinION flow cell should have a minimum of 800 active pores as covered by the warranty.