TITLE: Nanopore Rapid PCR Barcoding for Genomic Samples
AUTHORS: Carlos Goller, Carly Sjogren

[DESCRIPTION]
This protocol is for Rapid sequencing of DNA using PCR Barcoding (SQK-RPB004) and has been adapted from Oxford Nanopore Technologies.

[BEFORE_START]
Overall time is about 30 to 40 minutes. 

COLLECT THESE:
Equipment:
-Microfuge
-Timer
-Thermal Cycler
-Pipette and tips P2, P10, P20, P100, P200, P1000.

Consumables:
-1.5 ml Eppendorf DNA LoBind tubes
-0.2 ml thin-walled PCR tubes
-Nuclease-free water
-Agencourt AMPure XP Beads
-LongAmp Taq 2X Master Mix
-Fresh 70% ethanol in nuclease-free water
-10 mM Tris-HCl pH 8.0 with 50 mM NaCl

Materials:
-1-5 ng high molecular weight genomic DNA
-Rapid PCR Barcoding Kit
-Flow Cell Priming Kit


[STEPS]
SECTION: Checklist
1. Gather Materials, Consumables and Equipment.
These are listed in the Materials tab also

Equipment:
-Microfuge
-Timer
-Thermal Cycler
-Pipette and tips: P2, P10, P20, P100, P200, P1000

Consumables:
-1.5 ml Eppendorf DNA LoBind Tubes
-0.2 ml thin-walled PCR tubes
-Nuclease-free water
-Agencourt AMPure XP Beads
-LongAmp Taq 2x Master Mix
-Freshly prepped 70% ethanol in Nuclease-free water
-10 mM Tris-HCl pH 8.0 with 50 mM NaCl

Materials:
-1-5 ng high molecular weight Genomic DNA
-Rapid PCR Barcoding Kit
-Flow Cell Priming Kit



SECTION: Library Preparation
2. Thaw and prepare the reagents as follows:
    -Barcodes (RLB 01-12A) at Room Temp
    -Fragmentation Mix (FRM) on ice
    -Rapid Adaptor (RAP) on ice
SECTION: Library Preparation
2.1. Prepare the DNA in Nuclease-free water:
    -Transfer 1-5 ng genomic DNA into a DNA LoBind tube
    -Adjust volume to 3 µL  with nuclease-free water
    -Mix thoroughly by flicking (Avoid unwanted shearing)
    -Spin down briefly in Microfuge

SECTION: Library Preparation
2.2. In a thin 0.2 ml thin-walled PCR tube, Mix the following:
    -3 µL 3 1-5 ng template DNA
    -1 µL 1 Fragmentation Mix (FRM)

Mix Gently by flicking tube and spin down.
SECTION: Library Preparation
2.3. Incubate tube in a thermal cycler at 30C for 1 minute.1 min 
Then for 1 minute at 80C1 min 

Briefly put tube on ice to cool down
SECTION: Library Preparation
3. Amplifying using following cycling conditions:
    -Initial Denaturation 3 min 
    -Denaturation15 s 
    -Annealing 15 s 
    -Extension6 min 
    -Final extension 6 min 
    -HOLD AT 4C


SECTION: Library Preparation
2.4. Set up a PCR reaction as follows in 0.2 ml thin-walled PCR tube:
    -20 µL 2 Nuclease-free water
    - 4 µL Tagmented DNA
    -1 µL  RLB (01,12A, at 10μM)
    -25 µL  LongAmp Taq 2X Master Mix
Mix gently by flicking tube and spin down
SECTION: Library Preparation
3.1. Transfer sample to clean 1.5ml Eppendorf DNA LoBind tube and Resuspend AMPure XP Beads by vortexing
    -Add 30 µL  of resuspended AMPure XP Beads to reaction, mix by Pipetting

SECTION: Library Preparation
3.2. Incubate on Hula Mixer 5 min 

Prepare 500 µL  of fresh 70% ethanol in Nuclease-Free water during incubation

SECTION: Library Preparation
3.3. Take tube from Hula Mixer and put on a magnet. Once solution looks clear pipette off supernatant

While on magnet wash beads with 200 µL  of 70% ethanol.
 
Remove ethanol using a pipette and discard.

SECTION: Library Preparation
3.4. Repeat above washing and removal step.
SECTION: Library Preparation
3.5. Spin down and place tube on magnetic rack. Pipette off any residual ethanol in the tube and keep the top open to allow the pellet to dry for about 30 seconds.
 

SECTION: Library Preparation
3.6. Take tube out of magnetic rack and resuspend pellet in 10 µL of 10 mM Tris-HCl pH 8.0 with 50 mM NaCl.

Incubate 2 min

SECTION: Library Preparation
3.7. Put tube back on magnetic rack and wait until elute is clear and colorless. Once clear, remove and retain 10 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube. Dispose the pelleted beads and previous tube. 
 


SECTION: Library Preparation
4. Quantify 1 µL of eluted sample using a Qubit Fluorometer. 


SECTION: Library Preparation
5. Pool all barcoded libraries in desired ratios to a total of 50-100 fmoles in 10 µL of 10 mM Tris-HCl pH 8.0 with 50 mM NaCl.
 


SECTION: Library Preparation
5.1. Add 1 µL of RAP to barcoded DNA. Mix by gently flicking tube and spin down.

SECTION: Library Preparation
5.2. Incubate reaction for 5 min 

 

SECTION: Priming and Loading the SpotON Flow Cell
6.  
Thaw the following at Room Temperature:
-Sequencing Buffer (SQB)
-Loading Beads (LB)
-Flush Tether (FLT)
-One tube of Flush Tether (FB)

Once thawed, mix reagents by vortexing then spin down at room temperature.
 

SECTION: Priming and Loading the SpotON Flow Cell
6.1. Prepare the flow cell priming mix:
- add 30 µL of thawed and mixed Flush Tether (FLT) directly to tube of thawed and mixed Flush Buffer (FB).
- Mix by vortexing at room temperature. 
SECTION: Priming and Loading the SpotON Flow Cell
6.2. Open the MinION device lid and slide the flow cell under the clip. Slide the priming port cover clockwise to open the priming port.
 

SECTION: Priming and Loading the SpotON Flow Cell
6.3. After opening priming port, check for small air bubbles under the cover. Draw back small volume to remove any bubbles (a few μl):
 -Set a P1000 to 200 µL
- Insert tip into priming port
- Turn wheel on pipette until dial shows 220-230 µL (This allows you to draw back 20-30 µL), or until you can see a SMALL amount of buffer entering pipette tip.

 
- Load 800 µL of the priming mix into the flow cell via the priming port. Avoid introduction of air bubbles. Wait 5 min . During this 5 minutes, proceed to the next sub-step

SECTION: Priming and Loading the SpotON Flow Cell
6.4. Thoroughly mix the contents of the Loading Beads (LB) tubes by vortexing. 

 
 In a new tube, prepare the library for loading as follows:
- 34 µL of Sequencing Buffer (SQB)
- 25.5 µL Loading Beads (LB), mixed IMMEDIATELY before use.
-4.5 µL Nuclease-free water
-11 µLDNA library

SECTION: Priming and Loading the SpotON Flow Cell
6.5. Complete the flow cell priming:
-Gently lift the SpotON sample port cover to make the SpotOn sample port accessible.
-Load 200 µL of priming mix into flow cell via the priming port (NOT the SpotON sample port), avoid introduction of air bubbles

SECTION: Priming and Loading the SpotON Flow Cell
6.6. Mix prepared library gently by pipetting up and down prior to loading

Add 75 µL of sample to flow cell via SpotON sample port in dropwise fashion. Ensure each drop flows into port before adding the next drop

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.