I started watching videos from the Nanopore Learning Lesson Library. The Knowledge Exchange video “Getting the most out of a flow cell” caught my attention. Connie O’Donnell was the host and was joined by Anne-Marie Wadsworth who discussed tips to maximize flow cell use. Wadsworth explained that all flow cells have a maximum run time determined by several factors. They noted that there is also the option to “Run Until” a certain value is obtained. The ability to run multiple samples in a single flow cell can be achieved using barcoded kits. It is also possible to run multiple samples one after the next. Wadsworth recommended barcoding even when washing and reusing a flow cell. In a second case, a user desires the maximum output. Wadsworth noted that if DNA is high purity and reasonable length, MinKNOW software will attempt to maximize sequencing. The MinKNOW graphs help identify pore time and pore occupancy. Wadsworth defined pore occupancy as the “proportion of time spent in “sequencing” compared to “pore.” High pore occupancy can be visualized within 30 min or an hour. Wadsworth noted that low pore occupancy can be identified when pores are available and not sequencing. Essentially, actively sequencing pores (light green) are desired. Pore state graphs can identify contamination in samples (black channels throughout a run). The translocation speed is controlled by the motor that requires fuel from the buffer. As fuel is used, the speed drops. If there is severe drop off, there may be too many adapter molecules. Speed can be recovered with a refuel flush. The Wash Kit can be used to recover unavailable channels during a run. However, the wash kit won’t recover unrecoverabled pores. When washed, a flow cell will need an adjusted voltage bias. There is a table with these values in the Nanopore Community. Bubbles should be avoided. Refuelling is the replenishment of motor fuel by addition of components from the Flow Cell Priming Kit. The translocation speed and Q score can help identify when refueling is needed. For refuelling, the run is paused, the air bubble is removed, and buffer is introduced. Disk space can be monitored, and a run can be paused if necessary to move files and increase usable space. I found the refuelling information very useful in this video. I will likely share this video as an assignment in the Portable Genome Sequencing course.
