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PANDORA-ID-NET Consortium
Добавлен 6 фев 2020
This channel showcases the work undertaken by the Pandora-ID-Net consortium, the Pan-African network for rapid research, response and preparedness for infectious disease epidemics. For more information and up to date news, follow us on Twitter @PandoraIDNet or visit our website www.pandora-id.net
Overview of the Oxford Nanopore tuberculosis sequencing pipeline
This presentation by Dr Linzy Elton from the Centre for Clinical Microbiology, UCL, UK, gives an overview of the recently published Oxford Nanopore Technologies tuberculosis drug resistance and lineage diagnostics pipeline. This video covers each of the steps needed to optimise sequencing of M. tuberculosis:
- Culture methods
- DNA extraction
- DNA quantification (and the importance of good DNA)
- Library preparation kits
- Basecalling algorithms
- Analysis programmes
- Logistical considerations for LMICs
For more information about the tuberculosis sequencing pipeline, including more detailed tutorials, links to the paper and laboratory manual, please visit PANDORA-ID-NET's TGHN hub page: pandora...
- Culture methods
- DNA extraction
- DNA quantification (and the importance of good DNA)
- Library preparation kits
- Basecalling algorithms
- Analysis programmes
- Logistical considerations for LMICs
For more information about the tuberculosis sequencing pipeline, including more detailed tutorials, links to the paper and laboratory manual, please visit PANDORA-ID-NET's TGHN hub page: pandora...
Просмотров: 176
Видео
CPHIA 23 - Lizeka Tandwa "An African-centred ethical framework for research during epidemics"
Просмотров 4711 месяцев назад
Welcome to our series of talks recorded from our CHPIA 2023 side event. In this video, Lizeka Tandwa discusses an African-centred ethical framework for research during epidemics and emergencies in Africa. Lizeka Tandwa is a bioethicist and holds an MSc (Med) in Bioethics and Health Law and Bachelor of Health Sciences (BHSc) in Biomedical Sciences (Hons) qualifications from the University of the...
CPHIA 23 - Faiza Osman "detecting and evaluating arbovirus surveillance in Sudan"
Просмотров 2511 месяцев назад
Welcome to our series of talks recorded from our CHPIA 2023 side event. In this video Dr Faiza Osman discusses the application of integrated assessment plans and methodology to detect and evaluate arbovirus surveillance in Sudan; problems and constrains: Lessons from PANDORA-ID-NET in Sudan. Dr Faiza Mohamed Osman is head of the Department of Epidemiology, Nutrition and Refugees Health/Institut...
CPHIA 23 - Danny Asogun "PANDORA-ID-NET in Nigeria: Achievements and next steps"
Просмотров 3111 месяцев назад
Welcome to our series of talks recorded from our CHPIA 2023 side event. In this video Professor Danny Asogun discusses PANDORA-ID-NET's achievements in Nigeria since 2018. Professor Danny Asogun is a public health Physician with interest in highly infectious disease epidemiology and Social medicine at Ambrose Alli University, Ekpoma and Irrua Specialist Teaching Hospital (ISTH), Nigeria. Pionee...
CPHIA 23 - Najmul Haider "Modifiable Risk factors for Lassa fever infection in Sierra Leone"
Просмотров 3011 месяцев назад
Welcome to our series of talks recorded from our CHPIA 2023 side event. In this video Dr Najmul Haider discusses the modifiable Risk factors for Lassa fever virus infection in Sierra Leone, 2018-2020. Dr Najmul Haider is an epidemiologist and public health researcher, working as a Lecturer of Epidemiology at Keele University, UK. Dr Haider obtained his doctoral degree from the Technical Univers...
CPHIA 23 - Intro & Isobella Honeyborne "A new tool for cross-species surveillance of Lassa Fever"
Просмотров 4711 месяцев назад
Welcome to our series of talks recorded from our CHPIA 2023 side event. This video includes a brief introduction to PANDORA-ID-NET by Professor Francine Ntoumi, then Dr Isobella Honeyborne talks about a new tool for cross-species surveillance of Lassa Fever. Dr Isobella Honeyborne BSc (Hons), DPhil, from University College London, UK. She is interested in infectious diseases that disproportiona...
Basic bioinformatics for Oxford Nanopore sequencing data analysis
Просмотров 5 тыс.Год назад
This presentation, led by Dr John Tembo from HerpeZ, Zambia demonstrates how to basecall using Guppy (processing raw fast5 files into fastq files) on Mac and Windows computers, how to concatenate (combine) these files into a single fastq for downstream processing, and how to use FastQC to analyse the quality of your fastq files. For more information about this workshop, please visit the worksho...
Introduction to sequencing and Oxford Nanopore Technologies
Просмотров 1,6 тыс.Год назад
This presentation by Dr John Tembo of HerpeZ, Zambia explains the basics and brief history of sequencing and describes and compares how Sanger, Illumina and Oxford Nanopore sequencing works. He then discusses in further detail Oxford Nanopore's range of devices and kits, as well as the basic sequencing workflow (which is then described in greater detail across the rest of the presentations). Fo...
DNA quality analysis
Просмотров 452Год назад
This presentation by Dr John Tembo looks at ways of quantifying and identifying the quality of extracted DNA. He explains why quality and quantity of DNA is so important for long read sequencing and which methods provide the most accurate results. For more information about this workshop, please visit the workshop page: pandora.tghn.org/training/tuberculosis-sequencing-using-oxford-nanopore-uni...
Use of genomics for tuberculosis research, surveillance and diagnosis in Zambia
Просмотров 299Год назад
This presentation by Dr Kabengele Keith Siame of the Tropical Disease Research Centre in Ndola, Zambia looks at the current diagnosis tools for tuberculosis in Zambia and why they are inadequate for tracking drug resistance and transmission patterns. Dr Siame discusses how sequencing, especially whole genome sequencing, can be used to fill these information gaps and improve diagnostics and surv...
The TB sequencing pipeline (setting up a sequencing workshop and issues with Mycobacteria)
Просмотров 284Год назад
This presentation by Dr Linzy Elton from the Centre for Clinical Microbiology, UCL (UK) firstly describes the infrastructure and logistics that must be considered before setting up a sequencing laboratory (with emphasis on resource poor settings) and then discusses some of the difficulties of extracting DNA from, and then sequencing the Mycobacteria using Oxford Nanopore Technologies. For more ...
Library preparation with the rapid barcoding 96 kit (SQK-RBK110.96)
Просмотров 1,5 тыс.Год назад
In this presentation, Dr Linzy Elton from the Centre for Clinical Microbiology, UCL (UK) explains how to prepare a DNA library for the Rapid Barcoding 96 Kit. Note that the RBK110.96 is an update of the original RBK004 kit, which we have previously used (this kit still uses kit 10 chemistry). For more information about this workshop, please visit the workshop page: pandora.tghn.org/training/tub...
Using TB-Profiler to analyse tuberculosis sequencing data
Просмотров 742Год назад
This presentation, led by Dr Linzy Elton from the Centre for Clinical Microbiology at UCL in the UK, looks at some common processed file types you might come across (e.g. .sam, .bam, .fasta and .vcf) as well as how to use the server-based version of the data analysis programme TB-Profiler to identify drug resistance SNPs and lineage information. For more information about this workshop, please ...
Optimising CTAB DNA extraction for Oxford Nanopore sequencing of tuberculosis
Просмотров 399Год назад
In this presentation Caren Kabanda from HerpeZ, Zambia gives an overview of the CTAB DNA extraction method commonly used for extracting DNA from M. tuberculosis and how to optimise it for whole genome sequencing using Oxford Nanopore Technologies. For more information about this workshop, please visit the workshop page: pandora.tghn.org/training/tuberculosis-sequencing-using-oxford-nanopore-uni...
Centre for Clinical Microbiology at UCL: Our antimicrobial resistance projects
Просмотров 3452 года назад
To celebrate World Antimicrobial Awareness Week (18-24th November), we've put together a video of our researchers talking about their antimicrobial resistance projects and how we're tackling the growing global issue of AMR! You can read more about the projects mentioned in the video on our CCM website: www.ucl.ac.uk/infection-immunity/research/research-department-infection/lab-research-groups/c...
Bioinformatics part 4: Downstream analysis programmes
Просмотров 4,5 тыс.3 года назад
Bioinformatics part 4: Downstream analysis programmes
Bioinformatics part 3: Assembling/aligning sequencing data using command line interfaces (CLIs)
Просмотров 6 тыс.3 года назад
Bioinformatics part 3: Assembling/aligning sequencing data using command line interfaces (CLIs)
Bioinformatics part 2: Processing fastq files for downstream applications
Просмотров 10 тыс.3 года назад
Bioinformatics part 2: Processing fastq files for downstream applications
ONT bioinformatics part 1: introduction
Просмотров 12 тыс.3 года назад
ONT bioinformatics part 1: introduction
Starting an Oxford Nanopore sequencing run using MinKNOW software
Просмотров 17 тыс.3 года назад
Starting an Oxford Nanopore sequencing run using MinKNOW software
Oxford Nanopore Technologies Rapid Barcode Kit - Library preparation protocol (SQK-RBK004)
Просмотров 15 тыс.3 года назад
Oxford Nanopore Technologies Rapid Barcode Kit - Library preparation protocol (SQK-RBK004)
How to wash a Flow Cell (and how to empty the waste channel)
Просмотров 15 тыс.3 года назад
How to wash a Flow Cell (and how to empty the waste channel)
Oxford Nanopore flow cell priming and loading tutorial
Просмотров 20 тыс.3 года назад
Oxford Nanopore flow cell priming and loading tutorial
Oxford Nanopore flow cell QC tutorial
Просмотров 12 тыс.3 года назад
Oxford Nanopore flow cell QC tutorial
PANDORA-ID-NET postdoc discusses plague on ABC news
Просмотров 573 года назад
PANDORA-ID-NET postdoc discusses plague on ABC news
COVID-19 FAQ series: Why is COVID-19 changing and what does it mean for us?
Просмотров 1063 года назад
COVID-19 FAQ series: Why is COVID-19 changing and what does it mean for us?
COVID-19 FAQ series: Which under lying conditions make you most vulnerable to COVID-19?
Просмотров 693 года назад
COVID-19 FAQ series: Which under lying conditions make you most vulnerable to COVID-19?
Dr Chiara Montaldo - Question and Answer session
Просмотров 1254 года назад
Dr Chiara Montaldo - Question and Answer session
Presentation 5: Introduction to the International Health Regulations (IHR)
Просмотров 6 тыс.4 года назад
Presentation 5: Introduction to the International Health Regulations (IHR)
Presentation 2: Clinical management of ill traveller: Points of entry and at the hospital level
Просмотров 844 года назад
Presentation 2: Clinical management of ill traveller: Points of entry and at the hospital level
Thanks.
Could you please spot more light on the tether?
Many thaks for sharing this! How can i get the prtocol from A-Z? Thnanks
Could you please help me to understand the tether story, what is it, why do i add it? is it added to the sample in the library preparation step? does it bind to the flow cell or DNA or both? Many thanks in advance!
The audio is poor, please can you look into that, I find the lecture very important to me as I have recently generated some raw sequence data using nanopore MinIon.
Hi there, Should we remove storage buffer before priming the flow cell for next run?
🎯 Key Takeaways for quick navigation: 00:35 🧬 *Sequencing is about determining the genetic makeup of an organism, and methods include Sanger sequencing, Next Generation sequencing, and Oxford Nanopore technology.* 02:29 🧬 *Genetic sequencing helps determine species, relatedness of organisms, identify mutations, and aids in surveillance by analyzing transmission patterns.* 05:47 🧬 *Sanger sequencing involves terminating DNA fragments of different lengths, with fluorescence detection during capillary electrophoresis to determine the sequence.* 11:48 🧬 *Illumina sequencing uses fluorescently tagged nucleotides, Bridge amplification, and cycles to generate sequences with short reads (35-600 base pairs).* 18:34 🧬 *Oxford Nanopore sequencing involves amplifying DNA, passing it through a pore, and decoding the electrical signals produced by different nucleotides.* 20:19 💡 *Oxford Nanopore Technologies offers portable sequencing devices like the mk1c, suitable for remote regions, with lower cost compared to larger platforms.* 26:44 💻 *Different sequencing platforms include the MinION, GridION, PromethION, each suited for varying sample sizes and lab requirements.* 29:42 💰 *Sequencing platforms vary in cost and capabilities, with starter packs available for different platforms like MinION and GridION.* 31:54 🔍 *Depth in sequencing refers to the number of reads at a specific position, influencing confidence in identifying bases; minimum depth requirements differ for Illumina and Oxford Nanopore.* 32:23 🧬 *Sequencing depth is crucial for accurate representation of the whole genome. Higher depth is better, and a minimum coverage of about 40 is generally considered across the entire genome.* 33:06 🧩 *Coverage in sequencing refers to how much of the reference sequence is covered by the aligned fragments. Gaps in the sequence indicate positions with insufficient information.* 33:52 🧬 *Large deletion events in the genome may impact sequencing results, leading to the loss of information, especially in organisms with complex genomes.* 36:09 🧬 *Workflow of loading a flow cell includes priming ports, addition of priming solution, and waste ports for removing unwanted materials, crucial for successful sequencing.* 37:38 📊 *Oxford Nanopore sequencing allows real-time reading of barcodes, providing insights into the frequency of specific DNA sequences during the sequencing process.* 38:32 💻 *The number of barcodes used in sequencing varies based on the complexity of the organism. For whole genomes, around 12 barcodes are common, but the choice depends on the organism.* 39:15 💽 *Flow cells can be reused, especially for organisms with small genomes like viruses. Considerations for the number of reads, organism size, and desired depth impact the choice of barcodes.* 40:39 🔍 *Before starting sequencing, calculate the number of reads needed based on the genome size, desired depth, and the organism being sequenced to optimize cost and data quality.* 43:10 🧬 *Bioinformatics plays a crucial role in cleaning up and assembling sequenced fragments into a whole genome. Downstream processing involves tasks like creating phylogenetic trees.* 45:32 💻 *Minimum computer specs for bioinformatics work include Windows 10, Linux (Ubuntu), or macOS (Catalina), 16 GB RAM, and a powerful CPU and GPU. Invest in the best possible computer for future-proofing.* 46:15 📂 *External drives for storage should have a minimum capacity of 1 TB, with additional storage as needed. Focus on processing power with a robust GPU, CPU, and ample RAM for efficient bioinformatics work.* 48:45 🌐 *Reliable internet is crucial for nanopore sequencing initiation, although permanent internet may not be necessary. Data should be saved directly onto the internal drive to avoid potential loss.* 49:42 📊 *Storage location is essential due to the large amount of data generated. Consideration should be given to research storage drives, and savings should be organized to manage data efficiently.* Made with HARPA AI
This will be really helpful for my undergrad thesis
hola me pasas el ppt?
i would like to listen to these but the audio keeps glitching out
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?
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!
test the storage buffer within the device ;)
Hi, Very helpful vedio. I am a PhD student at McGill University. I am working with Nanopore sequencing and having issue with medaka. I also installed it in conda environment but its not working. Is there any chance you could help in this regard, please. Once again, the vedio was so much informative and helping for me. Thanks.
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
While flow cell check, I received an error “no position selected”. Please guide what can I do
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
Hello! I am in Mexico, the video was very helpful, what applications are useful with Oxford Nanopore specifically in the human diagnostic setting or in any other area? Thanks! Thumbed up the video :D
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.
helpful
Brilliant video
Quite helpful and informative. Thank you
Quite helpful and informative.
When more than 50% of my pores die. Should i buy a new unit or can i do maintenance or replace anything?
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
Great job.. keep going
Thanks a lot for the detailed explanation!
While you prime the flow cell or before you load your sample, if you notice a bubble in the flow cell channel, but hasn't reached to the sensor array yet, how would you try getting rid of the bubble? What if there is a big bubble already before priming, how do you get rid of it, we are not meant to pipette out more than 20-30 ul, what if the bubble is not completely tackled by taking 30 ul out? Thanks.
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.
Please upload a video on data analysis using minknow after sequencing
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
You made mention of being able to make the A and B buffers, do you where I can find information on how to make them?
these are commercial buffers that come with flow cell wash kit
I'm doing it right now, but the voltage option doesn't appear
How to remove storage buffer before next use?
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.
Need to understand all the hidden T&C. This involves WHO.. and we do not want they controlling and powering the power we had now in the country.
few Q need a proper how to turn on an off the equipment. say you have done your calibration check. now what just pull the tests cell out?
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
Err errr errr errr errr errr errr errr errrr
Thanks for sharing this!
thank you!
Excellent video, thank you so much!
How much waste should be removed from the waste port before washing?
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
What is the ideal concentration of the pooled library?
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?
Information super useful. Is there one with improved audio?
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.
Thanks it was helpful!
Glad it helped!
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 ?
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 iam dr osama can you send me prosedure step by step
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
When you place the spot-on cap back on (especially when reusing flow cells, is there a risk of introducing contaminants from previous experiments?
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.
Thank you! A very useful video. Every video I have watched refers to a priming or sequencing buffer, but I can’t find what this buffer is. Can I make this buffer, or is there a reagent I can buy?
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!
really helpful. THANK YOU VERY MUCH
You're welcome!
We are planning to buy this... kindly suggest it's fissiblity
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/
Do you know how to analyse the obtained results.....???
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
That's very informative, thank you. However, it seems to me that you did create an air bubble there, no?
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)
How many times can I use one cell flow?
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!
why not see step 72oC. taq polymerase DNA the best at 72oC. and take image at 72
ThankS to DR Rorpopor Herbal on RUclips who helps me cured my PCR God bless you DR Rorpopor Herbal his herbal medication is very infective and active❣️❣️❣️
If a cat were to walk through covid spit on sidewalk and then cat walks all over your outdoor property including vehicles,tables, benches etc. is it possible that a human could contract the virus? Please comment. Please
Thanks for sharing this video