Rapid Sequencing with Oxford Nanopore Sequencing

1 – Introduction

 

• In the Rapid Library Preparation, the motor protein is attached to the DNA using a transposase which simultaneously cleaves the DNA and adds an index and sequencing adapter.
  

Materials checklist

Reagents

• Rapid Barcoding Kit (SQK-RBK114.24)

• PCR Product
• Freshly prepared 70% ethanol in nuclease-free water (500 µL per PCR reaction to be cleaned up)
• MinION Mk1B 
• Flow cell (R10.4.1), Flongle (R10.4.1) and Flongle adapter
• Flongle Expansion Kit (EXP-FLP002)

 

Shared Supplies and Equipment

• Container with cracked or crushed ice
• Micropipettes and tips (2–100 µL)
• Microcentrifuge
• Microcentrifuge tube rack
• PCR tubes
• 1.5ml Microcentrifuge tubes
• Qubit (™) tubes (optional)
• Qubit™ Fluorometer (Invitrogen™), Q32856), Nanodrop™, QIAxpert® or other for DNA quantification  (optional)

 Rapid Index Attachment to Amplicon (Everyone)

1. In a PCR tube mix:

Reagent	Volume Per Sample
DNA/PCR	10ul
Rapid Barcodes	1.5ul
Total	11.5ul

1. Using a thermocycler, incubate the tubes at 30°C for 2 minutes and then 80°C for 2 minutes. 
2. Briefly spin down tubes.
3. Pool all samples into a 1.5ml tube.

Clean Up 

1. Resuspend Ampure beads by vortexing.
2. Add equal volume Ampure beads to 1.5ml tube with pooled samples (Volume of beads = 11.5 X number of samples).
3. Incubate for 10 minutes at room temperature, flicking occasionally. 
4. Spin down for 15 seconds.
5. Place the tube on a magnetic rack and allow Ampure beads to pellet. 
6. Pipette off supernatant and discard.
7. Add 1 ml of 80% ethanol then pipette and discard supernatant without disturbing pellet
8. Repeat the previous step. 
9. Briefly spin down, remove any residual ethanol. Allow to dry for 30 seconds.
10. Add 15ul of water.
11. Incubate for 10 minutes at room temperature.
12. Add tube to magnetic rack and allow beads to pellet.
13. Transfer 11ul of supernatant to a new 1.5ml tube
14.  For PCRs: Transfering a maximum of 800 ng of the DNA library.
    If necessary, take forward only the necessary volume for 800 ng of DNA library and make up the rest of the volume to 11 μl using Elution Buffer (EB). 

(For genomic DNA load 3-20fmol)

https://www.bioline.com/media/calculator/01_07.html

Library Preparation (Instructor)

1. In a 1.5ml tube dilute Rapid Adapter (RA) as follows:

 

Reagent	Volume
Rapid Adapter (RA)	1.5ul
Adapter Buffer (ADB)	3.5ul
Total	5ul

1. Add 1μl of diluted Rapid Adapter to indexed DNA.
2. Incubate for 10 min at room temperature.

 

Flow cell Priming (Instructor)

 

1. Mix 117ul FCF (Flow Cell Flush, from the Flongle expansion kit)  and 3ul of FCT (Flow Cell tether, from the Rapid Barcoding kit). 
2. Peel back the seal from the Flongle flow cell
3. Slowly expel liquid from the pipette by turning the dial clockwise until a bead of liquid forms at the tip.
4. Place the pipette tip in the sample port, keeping the pipette vertical.
5. Turn the dial clockwise to load the flush buffer and primer into the flow cell, stopping right before all liquid is entirely expelled to avoid pipetting air into the flow cell.

 

 

Flow cell Loading (Instructor)

 

1. Make sequencing mix:

 

Reagents	Volume
Sequencing Buffer (SB)	15ul
Loading Beads (LIB)	10ul
DNA Library	5ul
Total	30ul

 

1. Pipette up sequencing mix, slowly expel liquid from the pipette by turning the dial clockwise until a bead of liquid forms at the tip.
2. Place the pipette tip in the sample port, keeping the pipette vertical.
3. Turn the dial clockwise to load to the flush buffer and primer the flow cell port slowly. Stop rotation right before all liquid is entirely expelled to avoid pipetting air into the flow cell.
4. Seal flow cell port with sticker and run sequencing.