I believe you have multiread fast5 files, as generated by recent versions of MinKNOW. A [conversion script][1] is available to convert these to the older, one-read one-file format. AFAIK Albacore is being deprecated and no version will be made supporting the multiread fast5 files, as Albacore is being replaced by Guppy. [1]: https://github.com/nanoporetech/ont_fast5_api/blob/master/README.rst The command line to convert from multi read fast5s to single read fast5s is as follows: $multi_to_single_fast5 -i /path/to/fast5/ -s /path/to/save/output/