Dear Yingying,
Thank you for your detailed message.
The issue you’re encountering with Release 226 is due to changes in GTDB-Tk itself. Since GTDB-Tk v2.4.0, FastANI has been replaced by skani, and as a result, older versions of the software (like 2.3.2) cannot find the FastANI directory, which no longer exists in the latest database.
We strongly recommend updating GTDB-Tk to the latest version when using the most recent database releases, such as R226.
Cheers,
Pierre