Starting an Oxford Nanopore sequencing run using MinKNOW software

This video is a recording from a training session for one of our studies funded by BSAC (Twitter @AmrCovid) - link below:
The tutorial shows you how to prepare a DNA library using the Rapid Barcoding kit (SQK-RBK004). Please note that this is a practice session and (as mentioned in the video), we are practicing on a USED flow cell for training purposes, but the steps are the same.
All the documentation and links mentioned in the video can be found on our The Global Health Network's hub page pandora.tghn.org/sequencing/s...

[@gitikarajbongshi687]

Please upload a video on data analysis using minknow after sequencing

[@pandora-id-netconsortium1846]

Hi Gitika, MinKNOW isn't designed to analyse your data, it's designed to run the sequencing and basecall the reads to generate fastq files. If you are interested in analysing your data, have a look at our bioinformatics videos and resources: pandora.tghn.org/sequencing/sequencing-tutorials/#ref6

[@kevinsanders6057]

I'm doing it right now, but the voltage option doesn't appear

[@lamiouss]

Hello, I am wondering if it possible to have the commande Line paramétrer used for each analyzing step. Exemple : for guppy I need to know the commande Line also for minimap2. Do you know from where can I have the python code used ?

[@pandora-id-netconsortium1846]

Hi Iamia,
Have a look at our series of 4 bioinformatics videos (they're in the same playlist), you will see that the commands are all on there. There is also an accompanying PDF that you can use. The majority of the programmes used e.g. Guppy and MiniMap2 run on Linux/Mac (some also run on Windows), so you will need to be familiar with them. We also have provided a beginners guide to bioinformatics, so if you don't yet know how to use them, you can look through the resources: pandora.tghn.org/sequencing/bioinformatics/
Good luck!

[@anushkar55]

Hello, I see that the SQK-RBK004 kit is recommended if you need a PCR-free method of multiplexing but we still select "PCR" in the kit selection?

[@pandora-id-netconsortium1846]

Hi Anushkar,
The options at the top are just to help narrow down the kits to make it easier to select, but if you know the one you have used, then you can find it in the list.
You're correct that if you're using the RBK004 kit, you would choose 'PCR-free', but in the video, we're making the point that you can use the buttons to narrow your choice.
The most important thing is making sure you get the kit you used correct, as each kit has different barcodes, and choosing the wrong one will confuse Guppy/Dorado!

[@TheScientificGurus]

Do you know how to analyse the obtained results.....???

[@pandora-id-netconsortium1846]

Again, it really depends on what information you want to obtain from the sequencing data. But have a look at the rest of the videos in this playlist, as we've made step by step tutorials on the basic steps including basecalling, QC, aligning and assembling and downstream analysis options: th-cam.com/channels/rQEBjyA199n3FYtiGWSm9A.htmlplaylists

[@TheScientificGurus]

We are planning to buy this... kindly suggest it's fissiblity

[@pandora-id-netconsortium1846]

It depends what you mean by feasibility. If you're talking in terms of would this suit your lab then we suggest you look at our sequencing information on our TGHN hub pages: pandora.tghn.org/sequencing/

[@hikaroto2791]

When more than 50% of my pores die. Should i buy a new unit or can i do maintenance or replace anything?

[@pandora-id-netconsortium1846]

Hi Hikaroto. When you say 50% of your pores have died, do you mean when you QC the flow cell before using it? Or during the run?
If you mean when you QC, ONT have a MinION flow cell warranty of 800, less than that and you can request a new one (see pandora.tghn.org/sequencing/sequencing-tutorials/#ref1 for more info)
If you mean during a run, this is normal, you will lose pores over time (how many depends on what you are sequencing, how long for etc.) You can wash and reuse a flow cell (pandora.tghn.org/sequencing/sequencing-tutorials/#ref3) but you won't regain pores that have become clogged etc.
I hope that's helpful.

[@hikaroto2791]

@@pandora-id-netconsortium1846 thank you for answering, I mean before using, during the initial checks after receiving the product and the checks, the QC before the run. When do I know for the flow cells it's time to replace it, or time to buy a new unit? Flow cells costs less than purchasing the whole device i assume.

[@pandora-id-netconsortium1846]

@@hikaroto2791 If you QC the flow cells and they are below the 800 pore warranty, Nanopore will send you a new one. Have a look at our TGHN page on flow cell QC for more details: pandora.tghn.org/sequencing/sequencing-tutorials/#ref1