TITLE: Multiplexing microbial gDNA with SQK-RBK004 rapid kit
AUTHORS: Ian Boostrom, Maria João Carvalho, Refath Farzana, Edward AR Portal, Kirsty Sands

[DESCRIPTION]
Purpose

This protocol is intended to guide users in creating genomic libraries for MinION & GridION sequencing, using Oxford Nanopore’s SQK-RBK004 rapid kit with high molecular weight microbial gDNA.

[BEFORE_START]
QC of the MinION flow cell to quantify active nanopores:
     ·Let the flow cell reach room temperature (~30 minutes)
     ·Open MinKnow & select ‘Flow Cell Check’
     ·Click Start on the bottom right of the screen
     ·Upon completion, record number of active pores

Have gDNA ready.

UV a MIDI plate (can re-use plate as long as clean wells are used).
Warm SPRI beads (AMPure or Mag-Bind) slightly.
Thaw Barcodes and RAP.
Prepare Ethanol (80%).
Get ice.


[GUIDELINES]
This is a modified protocol intended to reduce DNA shearing. An unmodified SQK-RBK004 protocol from the Oxford Nanopore Community can be found here. (Note: An Oxford Nanopore Community account is required.)

[STEPS]
SECTION: SPRI bead concentration
3. In the MIDI plate, aliquot gDNA to SPRI beads at a 1:1 ratio. Gently pipette-mix 10 times, then leave the sample at room temperature for 2 minutes. 
SECTION: Initial DNA Quantification
1. Use Qubit fluorometer (or equivalent) to determine concentration of gDNA in samples.

For all samples with values less than 40 ng/µL, perform SPRI bead concentration as indicated in the following section.
SECTION: Priming the Flow Cell
33. Load 800μl of the flow cell priming mix into the flow cell via the priming port.


Wait for 5 minutes. In this 5 minute step prepare the DNA with loading beads mixture.
SECTION: SPRI bead concentration
4. Place MIDI plate with samples on the magnetic plate for 2 minutes, then remove the supernatant ensuring the SPRI+DNA pellet is not disturbed.
SECTION: SPRI bead concentration
2. Use a pipette to estimate the total volume of gDNA. Record this value
SECTION: SPRI bead concentration
5. Perform 2x ethanol washes as with Illumina prep (on the magnet add 200 µL of 80% ethanol, immediately remove & repeat).

Once the second ethanol wash has been removed, incubate the plate for a maximum of 5 minutes to air dry. 

***DO NOT OVER DRY.***
SECTION: SPRI bead concentration
6. Take the MIDI plate off the magnet and add between 15-30 µL of molecular grade water. 

Gently pipette mix 10 times.
SECTION: SPRI bead concentration
8. Adjust concentrations to 53.3 ng/µL (acceptable limits between 40-60 ng/µL) and begin rapid barcoding.
SECTION: SPRI bead concentration
7. Place MIDI plate back on the magnet.

With the plate on the magnet, remove 2µL for Qubit measurement using the BR kit.
SECTION: Rapid Barcoding
9. In 8-strip PCR tubes, aliquot 7.5 μL of gDNA at 53.3ng/μL.
SECTION: Rapid Barcoding
11. Mix gently by flicking the tube, and spin down.
SECTION: Rapid Barcoding
13. Briefly put the tube on ice to cool it down.
SECTION: Rapid Barcoding
14. In a 500 µL reaction tube, pool all barcoded samples in your desired ratios. 

This will yield a total volume of 120μL when preparing 12 isolates.
SECTION: Rapid Barcoding
15. Aliquot a 1:1 ratio of SPRI beads to the pool of barcoded samples. 

This will be 120µL of SPRI beads if preparing 12 isolates.
SECTION: Rapid Barcoding
16. Gently pipette-mix 10 times, then leave the sample at room temperature for 2 minutes.
SECTION: Rapid Barcoding
17. Rotate the reaction tube containing the barcoded pool for 5 minutes.
SECTION: Rapid Barcoding
18. Place the reaction tube containing barcoded pool against the magnet for 5 minutes

Alternatively, an additional clean well in the MIDI plate can be used for this step.
SECTION: Rapid Barcoding
10. To each aliquot, add 2.5 μL of fragmentation mix barcode. Use a different barcode for each sample.
SECTION: Rapid Barcoding
12. In a suitable thermal cycler or heat-blocks, incubate the tube at 30 °C for 1 minute and then at 80 °C for 1 minute.
SECTION: Rapid Barcoding
19. If the mix is not clear at 5 minutes, wait until it is clear to move on to the next step.
SECTION: Rapid Barcoding
22. Off the magnet, add 15 μL of molecular grade water, gently pipette mix 10 times, and incubate at RT for 2 minutes.
SECTION: Rapid Barcoding
21. Repeat the 2x ethanol wash steps as in step 5.
SECTION: Rapid Barcoding
20. Remove the supernatant – and place into a new 500µL reaction tube. Store this until protocol is complete.
SECTION: Rapid Barcoding
26. Prepare reagents for the next section by thawing:

·Sequencing Buffer (SQB)
·Loading beads (LB)
·Flush tether (FLT)
·One tube of flush buffer (FB)

Place these on ice once thawed.
SECTION: Rapid Barcoding
25. Incubate the reaction for up to 30 minutes at room temperature, and then place on ice until you are ready to prepare the final library mixture and load into the flow cell.
SECTION: Rapid Barcoding
24. Transfer 10 μL to a 500 μL reaction tube and add 1 μL of RAP. 

Mix gently by flicking the tube and spin down.
SECTION: Rapid Barcoding
23. Qubit 2 μL, which should leave 10 μL comfortably. Record the value.

Qubit results should be between 50ng/μL to 150ng/μL – as this means the total load volume is between 500ng and 1500 μL on the flow cell.
SECTION: Preparing Reagents for Priming &amp; Loading of the Pooled Library
27. Immediately before priming the flow cell:

·Mix SQB and FLB by vortexing, spin down and place back on ice.

·Mix FLT via pipetting, spin down and place back on ice.

·Set a P1000 pipette to 200µL
SECTION: Priming the Flow Cell
28. A useful video for beginners can be found here: https://www.youtube.com/watch?v=CC11Jlydqrc

Take care to avoid introducing any air during pipetting, as the array of pores must never be exposed to air.
SECTION: Priming the Flow Cell
29. Flip back the MinION lid and slide the priming port cover clockwise so that the priming port is visible. 
SECTION: Priming the Flow Cell
31. Visually confirm there is continuous buffer from the priming port across the sensor array.
SECTION: Priming the Flow Cell
32. Prepare the flow cell priming mix: 

·Add 30μL of thawed FLT directly to the thawed and mixed FLB.

·Mix by pipetting up and down carefully.
SECTION: Priming the Flow Cell
30. Attach a 1250 µL tip to the P1000 pipette, and insert the tip into the flow cell's priming port.

Turn the pipette's volume-adjustment wheel until you can see a small volume of buffer entering the pipette tip, or the dial shows 220-230 µL. Removing more than 20-30 µL of buffer risks damaging the pores in the array.
SECTION: Loading the Pooled Library
38. Close all ports.

Close the lid of the MinION device.
SECTION: Loading the Pooled Library
37. Mix the final 75µL DNA library mixture by pipetting & load to the flow cell via the SpotON sample port in a dropwise fashion.
SECTION: Loading the Pooled Library
36. Avoiding the introduction of air bubbles, load an additional 200 µL of priming mix into the flow cell via the priming port (not the SpotON sample port). 

***You load this 200µL in the same place as you did when adding 800µL of priming mix, however this time with the SpotON port open (you will see some fluid moving up & down by the SpotON port).***
SECTION: Loading the Pooled Library
35. Gently lift the SpotON sample port cover to make the SpotON port accessible.
SECTION: Loading the Pooled Library
34. Prepare the DNA with loading beads mixture:

     34 µL SQB

     25.5 μL LB –pipette-mix the loading beads before removing

     4.5 μL – molecular grade water

     11 μL DNA library

75μl total
SECTION: MinKNOW
39. To open the MinKNOW GUI, double–click the MinKNOW icon located on the Windows Desktop or Ubuntu Launcher.

Wait for the MinKNOW GUI to load, select the local MinION, and click Connect.

Follow on-screen instructions, selecting SQK-RBK004 as the kit. Confirm your other settings and select Start.
SECTION: MinKNOW
40. Upon sequencing completion, upload fast5 data to a GPU partition for basecalling with the most recent version of ONT's Guppy basecalling software.