When live basecalling is enabled during sequencing in MinKNOW™, read data is initially written to the queued_reads folder before being basecalled. Once basecalling is complete, temporary files are being held in the tmp folder until reaching the output frequency specified during the run setup. Final output files are moved to the appropriate pod5, fastq_pass, fastq_fail, bam_pass and bam_fail folders within the output directory specified at the start of the sequencing run. The default location of all sequencing data generated by MinKNOW is explained in this FAQ.

pod5_skip folder

A pod5_skip folder is created when .pod5 files remain unbasecalled. This can occur:

• During live basecalling, if certain reads are skipped,
• After sequencing, during catch-up basecalling, if the process is manually stopped via the Stop basecalling button.

In both cases, unbasecalled .pod5 files are moved to the pod5_skip folder, located in the same directory as the pod5 and pass/fail folders. These files can still be basecalled later using post-run analysis in MinKNOW or Dorado.

Note: The "Stop basecalling" step may take time to complete. It is important not to interrupt this process to avoid potential data loss. A progress bar in the MinKNOW represents reads being transferred to the pod5_skip folder.

queued_reads folder

If sequencing is unexpectedly interrupted — for example, due to a crash or power outage — MinKNOW may not finish processing read data correctly. In such cases, raw read files may remain in the queued_reads folder, typically found within the /data/ directory. From MinKNOW version 24.11 onwards, most of these reads should be in the .pod5 file format and can be used directly. Some files may be in a .pod5.tmp format and, if using an earlier version of MinKNOW, in .raw format

You can manually convert .pod5.tmp or .raw files to .pod5 using the instructions from this FAQ for Linux or this FAQ for Windows. Once converted, the .pod5 files can then be basecalled using post-run analysis in MinKNOW or Dorado.

tmp folder

The tmp folder, located within /data/reads/, is used by MinKNOW to store temporary working files during sequencing and basecalling. Normally, this folder is managed automatically and does not require user intervention. However, if sequencing or basecalling fails or crashes, some temporary data may be left behind in this folder, including the temporary version of the sequencing_summary.txt file (more details in this FAQ). Any other files in that folder can usually be ignored unless specifically needed for troubleshooting.