Видео

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!

Hi Maria, I would suggest looking on bioinformatics forums such as StackOverflow or Biostars, because other people are likely to have come across the same problem as you. Alternatively, you could post your problem on the Nanopore Community website and someone with specific ONT knowledge will respond.

make sure you activate that conda environment and run conda list to confirm medaka is actually there

@@pandora-id-netconsortium1846 thank you for the suggestion.

@@thomasmatthew7759 thanks

Hi Shubham, Have you connected more than one MinION to your computer? On the flow cell check screen, if you have multiple MinIONs, you need to tell MinKNOW which one the flow cell you want to QC is in (even if you don't have a flow cell in the other). Usually (depending on the version of MinKNOW), you have to click on one of the white squares (representing the MinIONs) in the top left corner of the QC screen. Thanks, Linzy

Hi Alejandro, I'd recommend signing up for the Nanopore Community site, they have lots of really interesting information about how ONT technology can be applied: nanoporetech.com/community.

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.

@@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.

@@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

The best cure for bubbles is to carefully avoid adding them in the first place. But if there is a bubble before the ASIC panel, you could test to see if it is stuck in it's position, or whether it will move. If it is stuck in place, you might be able to add your buffer without disturbing it, if you are slow and careful. If it does move, you could try and suck it back up through the priming port, but be careful to make sure not to suck up so much that the pores are exposed to air from the other end of the channel.

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

these are commercial buffers that come with flow cell wash kit

Hi Annie, when you next run samples, you will make up flush buffer and load that into the buffer port. This will flush all of the storage buffer into the waste channel.

Hi Luis. The flow cells will just click into place (of course you need to make sure they are the correct way round, to ensure the electronics on the bottom fit), but we recommend being gentle with it! Once MinKNOW has displayed the number of active pores, you can then remove the flow cell (it's easiest to do so by gently lifting the waste port end up and then slowly wiggling the flow cell out - you can see Linzy do this in the video). There are no locking mechanisms in place on either the flow cell or MinION (it just has small magnets to hold the lid down)

@@pandora-id-netconsortium1846did QC 1432 pores on the flowcel. How to unplug safely the minION? I tried to eject ....but windows response is "device running" so i am closing software and then turning off the computer. Then unplug. As i get ready for my first run. Flushing is done while placed on the the minION ? Looking forward

Hi Maurine, it doesn't really matter, all that will happen if you add more buffer and the waste reaches the port, is that it will just bubble out of it. Equally, removing the waste shouldn't affect the ASIC panel section of the flow cell, so you can remove it if you wish

Hi Juliana, For each sample, you need to input 400 ng DNA, which is ~40 ng/ul once you account for the barcode you added. You then pool these and take 10 ul from that, which will still be 40 ng/ul. More than getting an exact concentration though, it's more important to get equal amounts of DNA across all the samples. This is why we've found that normalising it to fmol rather than ng and kb works best (as you are unlikely to get all of your extractions exactly the same size and this means you will have more or less DNA when you input).

@@pandora-id-netconsortium1846 This is very useful! thanks! another question: The protocol says that this kit is for gDNA with >30kb fragments. My gDNA is between 10 to 20kb. Can I still using it?

@@julianasoto5152 Hello thank you for the video. I have 3000kb fragment the HIV pol region can I still use it? is there a special primer adaptors I need to add on my nested step?

Hi Luis, As you can tell from the video, I had a terrible cold when I recorded this video and the audio at home wasn't great! As there's about 4 hours' worth of recordings, I don't plan to re-record it (I don't really have time), but you should find all the information you need on the TGHN hub pages and in the accompanying PDF. You're more than welcome to ask any questions here too and I'll try to answer them for you. pandora.tghn.org/sequencing/sequencing-tutorials/#ref6

@@pandora-id-netconsortium1846 information provided was excellent. Looking for PCR sequencing video. That applies the 4 primer protocol of ONT.

Glad it helped!

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!

Hi Dr Osama, you will find all the accompanying information on our TGHN pages: pandora.tghn.org/covid-19-diagnostic-tools/molecular-diagnostics/ and a link directly to the protocol from the video here: media.tghn.org/medialibrary/2022/02/Outbreak_diagnostics_SARS-CoV-2_-_Protocol.pdf

There is no chance of contaminating the run if it is a new flow cell, because samples need barcodes, adaptors etc. to be sequenced and recognised. When washing a flow cell for re-use, you should flush the wash buffer through with the SpotON port closed. That should ensure that any DNA from previous samples will be broken down. The longer you leave it, the less the chance of cross-contamination, but that is of course not completely guaranteed. If reusing a flow cell, it's recommended the samples are very different (e.g. tuberculosis and then SARS-CoV-2), so that if contamination has occurred, the contaminating reads can be removed post-sequencing. I hope that's helpful.

Hi Melanie, what we mean when we (and others) refer to the 'priming' or 'sequencing' buffer, is the combination of Flush Buffer (FB) and Flush Tether (FLT) that you then apply to the priming port, prior to loading your sample into the SpotON port. With most sequencing kits, you will also receive the Flow Cell Priming Kit (EXP-FLP002), which is made up of these components. The preparing and addition of the priming/sequencing buffer is shown in another of our tutorial videos: ruclips.net/video/P5y4lfpCUtw/видео.html which you might find useful. I hope that helps!

@@pandora-id-netconsortium1846 Many thanks for the information and additional video!

You're welcome!

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/

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: ruclips.net/channel/UCrQEBjyA199n3FYtiGWSm9Aplaylists

Nope, no air bubbles here! We've found that doing each of the steps (sucking up a bit first, then creating a small blob before you add buffer in) really eliminates air bubble risks. Also worth keeping an eye on the channel between the priming port and the sensor array (where the pores are), so you can check for any potential air movement and stop before you let air hit the pores (if there's air in the channel, you'll see that it's pale grey in colour, rather than black)

Hi Kevin, that depends how long you run it for and what you are doing. If you are sequencing a small part of the genome, or only need to know species level (e.g. metagenomics), then you can run for a short period (~ a few hours) and get the info you need, then wash the flow cell and reuse. However, if you need detail down to SNP level for WGS, and/or you're running multiple samples, to get the detail you need you'll probably need to run it for the full 48-72 hours, so there'll be no point washing it because there won't be many pores left. Once you wash the flow cell, you should QC it again immediately and it will tell you how many active pores you've got left to work with. In theory one flow cell can be used multiple times, but you need to consider data quality and application!