I'm looking for some advice from anyone who has experience of using simulated playback or specifically Readfish for selective sequencing (a.k.a. adaptive sampling) on ONT. I am currently trying to test the simulated playback feature (using a simulated Minion device as I don't have permanent access to an actual device currently).
The issue I am having currently is with getting the simulated playback to work. Does anyone know of documentation/have a protocol for how to do this? I am following the steps on the Readfish readme, and cannot find any ONT documentation on this feature of Minknow, but currently receiving a very unhelpful error (MinION Mk1B · MS00000 20 October, 10:18:43 Unable to open Playback file '' as a source for playback. -- this is despite selecting a bulk FAST5 file downloaded from the Readfish GitHub page provided expressly for this purpose).
Which version of MinKNOW are you using? Have you seen the requirement of dorado and MinKNOW combinations noted on readfish site.
Adaptive sampling can be a tricky thing. Some people report having good success where as for others it is an exercise in frustration. You may not know until you actually try it out with your own libraries.
The error message with the empty quotes around the playback file path suggests that MinKNOW isn't picking up the selected FAST5 file correctly, even if you've browsed to it in the Run Options under Simulated Playback. This is a common hiccup in the UI—double-check that the full path appears in the selection box after browsing (it should show something like /path/to/PLSP57501_20170308_FNFAF14035_MN16458_sequencing_run_NOTT_Hum_wh1rs2_60428.fast5 for the R9.4.1 example file from the Readfish repo).
If it's still blank, try restarting MinKNOW entirely (stop the service if on Linux, or quit/relaunch on Mac) to clear any cached settings, then add your simulated MinION position again via python -m minknow_api.examples.manage_simulated_devices --add MS00000 before starting the run. Ensure you're on MinKNOW >=5.7 for the current method, and confirm no prior runs are still processing in the background, as that can interfere.
The Readfish README has the full protocol under the Simulated Playback section—it's solid for this setup. If the issue persists after verifying the file selection, it might be worth checking the MinKNOW logs for more details on why the path is resolving empty.
Which version of MinKNOW are you using? Have you seen the requirement of
doradoandMinKNOWcombinations noted onreadfishsite.Adaptive sampling can be a tricky thing. Some people report having good success where as for others it is an exercise in frustration. You may not know until you actually try it out with your own libraries.