When live basecalling is on during sequencing in MinKNOW™, read data is initially written to the "queued_reads" folder before they are basecalled. Once basecalled, the pod5 data is moved to the corresponding pass and fail folders in the experiment output location selected when setting up the sequencing run in MinKNOW™. A "pod5_skip" folder is generated when there are pod5 files that remain unbasecalled either during live basecalling or catch-up basecalling after sequencing has completed and catch-up basecalling has been manually cancelled. The "pod5_skip" folder is located in the same folder where the "fastq_pass", "fastq_fail", "pod5_pass" and "pod5_fail" folders are found.

MinKNOW will automatically start the catch-up basecalling mode to continue basecalling after sequencing has finished. Catch-up basecalling may take several extra hours to complete depending on the yield. If the process is terminated by clicking the "StopBasecalling" button, MinKNOW™ will put unbasecalled data in the "pod5_skip" folder in .pod5 format to be basecalled later. The "Stop basecalling" step can take a long time to complete, it is important that you let this finish to avoid losing any data. You can monitor the process in the progress bar at the top of the MinKNOW™ interface, where it will count the number of reads that have been moved to the "pod5_skip" folder.)

If MinKNOW™ ends a run without clearing up correctly, e.g. due to MinKNOW crash or unexpected computer shutdown, raw read data can be left in the "queued_reads" folder, located in /data/queued_reads, without the data being converted to pod5 files. These raw data files (ending in .raw) can be converted to .pod5 files as per the instructions, for Windows or Linux, Mac, and Mk1C. Once converted, the resulting .pod5 files can be basecalled using the post-run analysis function in MinKNOW or the command-line .