 Genomic DNA sequencing by Oxford Nanopore Minayon. The learning goals in this video are Know the principle of long-read sequencing Know the basic procedure of genomic DNA sequencing by Minayon Know the interaction of dry and wet lab operations during a sequencing project In this video, you will go through the whole sequencing process of a bacterial genome by using Minayon The genomic DNA selecting for long-read protocol contains 10 steps In this video, we will start from step 2 1. Overview 2. Library preparation 3. Preparing for an experiment 4. Library preparation 5. Preparing to load a library 6. Loading a library 7. Starting a mino run 8. Data exchange using the metric core agent 9. Topping up the flow cell with library 10. Completing the experiment Minayon is a portable real-time device for DNA and RNA sequencing Each consumable flow cell can now generate 5 to 10 GB of DNA sequencing data Ultra-long-read lengths are possible Hundreds of kilobases, as you can choose your fragment length The Minayon strings data in real-time You can watch this video about the background of nanopore sequencing Library preparation In the experiment in this video, these equipment are used Nanopore sequencing kit Spot-on flow cells API to ME workflows Mino script Library preparation kit Please note that the equipment might change due to the type of sequencing Preparing for an experiment Mino software runs on the host computer to which the Minayon is connected Mino carries out several core tasks Data acquisition Real-time analysis and feedback of experimental progression Data streaming while providing device control including selecting the round parameters And ensuring that the platform chemistry is performing correctly to run the samples Specific parameters need to be filled into the Mino interface This will decide the type of sequencing experiment Assemble the Minayon spot-on flow cell And connect the Minayon to the host computer After assembling the Minayon, go back to the Mino interface and start the experiment Flow cells are shipped with a QC DNA molecule present in the buffer This molecule processes a distinctive nanopore signal The Mino software uses this signal to validate the integrity of the nanopore array Before use and provides the user with an estimation of the number of Mutaneously available channels for the experiment Active pores are reported in four groups Each of which may be used in charn when running long experiments Example a 48-hour sequencing round Shorter experiments will use fewer than four groups Library preparation To perform genomic DNA sequencing, a DNA library must be made Here you see a flow chart of a library preparation It generally contains three core steps Fragmenting or sizing the target sequences to a desired length Converting targets to double-stranded DNA Attaching oligonucleotide adapters to the end of target fragments The DNA repair step is performed to repair nicks in the DNA In order to maximize the rate length This step is performed with the use of NAPNAC's FFPE repair mix And this mixture is incubated at 20 minutes at 20 degrees DNA is purified with ampoule beads The DNA will bind to the beads on a hula mixer for five minutes And subsequent, the beads are washed two times with ethanol The beads are allowed to dry to the air A nucleus-free water is added Gently agitate the tube and incubate for 10 minutes at room temperature To harvest the DNA, the tube is placed back in the magnetic rack And the DNA is removed from the tube The end repair step is performed to repair the DNA at the end And the detailing of double-stranded DNA fragments This step is performed with the use of NAPNAC's end repair The detailing module by PCR The incubation steps are 25 minutes at 20 degrees And 25 minutes at 65 degrees DNA is purified with ampoule beads The DNA will bind to the beads on a hula mixer for five minutes And subsequent, the beads are washed two times with ethanol The beads are allowed to dry to the air A nucleus-free water is added Gently agitate the tube And incubate for 10 minutes at room temperature To harvest the DNA, the tube is placed back in the magnetic rack And the DNA is removed from the tube Adapter ligation is performed to add the adapters and the tether Taking the end prepped DNA, the following reagents are added Adapter mixed Hairpin adapter NAPNAC's quick ligation reaction buffers Quick T4 DNA ligase And nucleus-free water This mix will be incubated for 10 minutes at room temperature Hereafter, HP tether is added And subsequent incubated for 10 minutes at room temperature The DNA will be purified using my1C1 streptavidin beads To use these beads, the shipping buffer needs to be exchanged by bead binding buffer The adapted and tethered DNA library is now purified with my1C1 streptavidin beads The DNA will bind to the beads in a hula mixer for five minutes And subsequent, the beads are washed two times with bead binding buffer The DNA is eluated by elution buffer The beads with elution buffer are incubated at 37 degrees for 10 minutes The beads are pelleted in the magnet and the DNA is removed from the tube This library is called the pre-sequencing mix Store the pre-sequencing mix in an ice bucket Preparing to load a library Before the library is loaded, the following reagents are added Running buffer with fuel and nucleus-free water Before the library is loaded, the minion needs to be primed with buffer for two times Loading a library Now, the DNA library has been loaded to the minion Starting a mino round Start a sequencing round Starting a mino protocol script Progression of mino protocol script Checking progress at the beginning of the experiment The data is stored in a computer that is connected to minion If necessary, the following two steps can be performed Data is changed using the metric core agents Topping up the flow cell with library Completing the experiment Now you have learned the principle of nanopore sequencing The process of genomic DNA sequencing by using Oxford Nanopore Minion The dry and wet lab interaction when performing a sequencing project What follows is the intensive data analysis using the sequencing output