TITLE: Setting a sequencing run with the Rapid Barcoding Sequencing gDNA kit (SQK-RBK004)
AUTHORS: Narjol Gonzalez-Escalona

[DESCRIPTION]
This protocol is to be used after you mastered setting up a MinION sequencing run (dx.doi.org/10.17504/protocols.io.4r3l2oy53v1y/v3) using the rapid sequencing kit from Nanopore (SQK-RAD004). It contains all steps and material need for a successful run. This kit is recommended for rapid preparation time for several different DNA samples that will be run together (equivalent to multiplexing) and limited laboratory equipment. The output is lower than that generated by the ligation sequencing kit. 


preparation time approximately 35 minutes and require a minimum of 400 ng per sample for the sequencing reaction. 



[BEFORE_START]
Before using this protocol:

Make sure that you have extracted the DNA of the bacteria or other organism (if long reads needed, I suggest using a tested DNA extraction kit or in house DNA extraction that renders high quality less sheared DNA).
Quantify the DNA (I suggest using the Qubit Flourometer for determining the DNA concentration).
Check that the PC to be used for the run is compatible with the process. 
Make sure that the MinKnow Software version (software installed in the PC to run the MinION) is up to date (search the community and download the latest version if the version on the PC is not up to date).
Make sure that you have all supplies (see Materials section).

[GUIDELINES]
The Rapid Sequencing Kit contains enough reagents to perform six sequencing runs:

Box 1: Contains all reagents for the sequencing step with barcodes (store at -20C) 
Box 2: Contains all the reagents for flowcell priming (store at -20C) -  Flush Buffer (FB – blue caps) and Flush tether (FLT - purple cap)
 


[STEPS]
SECTION: Preparation of the DNA Library:
1. 
Thaw all reagents in box 1 and 2 of the RBK-004 kit.



SECTION: Preparation of the DNA Library:
2. Take a flow cell from 4-8 °C  and leave it at room temperature (RT).

SECTION: Preparation of the DNA Library:
3. Prepare the DNA to a concentration of 400 ng total in 7.5 µL, or a concentration of 54 ng/ul in a 0.2 mL sterile thin-walled PCR tube. Adjust the volume using nuclease-free water. Mix by gently flicking the tube 5 times. DO NOT vortex. Spin down briefly in a micro-centrifuge. 

SECTION: Preparation of the DNA Library:
4. Prepare individual tubes for each sample. Add 2.5 µL of Fragmentation Mix RB01-12 (one per sample) to each individual tube containing the prepared DNA from the previous step. Mix by gently flicking the tube as above and spin down. 

SECTION: Preparation of the DNA Library:
5. Incubate all tubes in Thermocycler at 30 °C for 1 min, then at 80 °C  for 1 min. Let it cool at RT for 2 min. 

SECTION: Preparation of the DNA Library:
7. Add 1 µL of RAP to the pool tube. Mix gently by flicking 5 times and spin down. Incubate for 5 min. After this step your library DNA is ready for mixing with the sequencing reagents and loading  to the flow cell. While this step is running you can start setting up the priming of the flow cell.

SECTION: Flow cell priming:
8. 
Flow cell priming:

Mix FB and FLT (independently) by vortexing and spin down. Then add 30 µL  of FLT to 1 tube of FB and mix by pipetting thoroughly.   This mixture will be use to prime the flow cell. 

SECTION: Flow cell priming:
9. On the PC attached to the MinION, launch the Minknow Software. Place the RT flow cell in the MinION device and proceed with the flow cell QC by clicking on flow cell check (in the Minknow software) and wait till the program reports back how many live pores are in the flow cell. If live pores below 800, do not continue sequencing (if very important samples) . Contact the vendor if the flow cell is still under warranty for a replacement.
SECTION: Flow cell priming:
10. If the flow cell passed QC then open the priming port by sliding the priming port cover clockwise. 

  Flow cell in the 
SECTION: Flow cell priming:
10.1. By inserting a pipette tip on the priming port, remove approximately 30 µL avoiding introducing bubbles.   

 

SECTION: Flow cell priming:
10.2. Add 800 µL of the FB+FLT solution prepared on step 7 to the priming port, avoiding introducing bubbles, by slowly dialing down the pipette. Wait 5 min.  

SECTION: Flow cell priming:
10.3. After the 5 min, gently open the flow cell SpotON cover and expose the loading port. Add 200 µL of FB+FLT to the priming port  (NOT the SpotON port). Make sure that the loading port does not have any bubble. 

SECTION: Preparation of the sequencing Library:
11. To the tube containing the DNA library add the following:

- 34 µL of the sequencing buffer (SQB) - red 
- 25.5 µL of loading beads (LB) - pink (Thoroughly mixed by vortexing before use)
- 4.5 µL  of nuclease-free water


Mix thoroughly by pipetting and make sure that the solution looks homogenous. This is the sequencing reaction ready to be loaded to the flow cell. 
SECTION: Loading the flow cell
12. Add the 75 µL of the sequencing library to the SpotON port in a dropwise fashion. Make sure that every drop falls into the port and that it goes in, before adding the next drop. 

Replace the cover over the SpotON and close the priming lid port, and close the MinION device lid 

SECTION: Sequencing
13. On the Minknow software select the flowcell and hit Start, then Start sequencing and follow the instructions for setting the sequencing run.  Experiment name (I use the same as the flow cell serial number), then select the kit (RBK004), The run options I leave it mostly default except that I change the time to 2880 min, the basecalling to fast (or high accuracy, your choice), Leave the output default C:/Data (or you can point it to wherever you want the run to be save), then just click start at the end of the sequencing setup and your sequencing run will start.   By default the output format is fast5 and fastq files, with the fastq file containing 4,000 reads. You can modify the number of reads per fastq if needed. You can also demultiplex it or not. I usually leave the barcoding option on. 

Check that the flow cell passes the QC again and look for sequencing performance:

Example below. Passed reads closer to the total reads, meaning most reads passed the QC filter, and there has been few failed reads.

  



  
 



SECTION: Preparation of the DNA Library:
6. Pool barcoded DNA into a single tube to 10 µL total. For example: if 5 samples, then pool 2 µL per sample into a single 0.2 mL sterile thin-walled PCR tube.