Question: Why is my Space Ranger run failing with manual alignment? I'm getting this error message:
[error] Loupe manual alignment file seems to be created with different image(s) than was passed to the pipeline
Answer: The error message indicates that the image used in Loupe Browser for the manual alignment was different than the image that was used as input for the Space Ranger pipeline (--image flag). The solution is to use the same image for both manual alignment in Loupe Browser and tissue selection in the Space Ranger count pipeline.
There is a temptation to use a lower resolution image in Loupe Browser manual alignment and tissue selection because the smaller files are easier to transfer. This doesn't work because the alignment information output by Loupe Browser in the JSON file relies on pixel coordinates. If the resolution of the image changes, so do the pixel coordinates thereby invalidating the alignment.
The alignment JSON file contains a checksum generated by Loupe Browser from the image. When Space Ranger runs it generates a new checksum from the image it was given. It compares that checksum to the one in the JSON file and if they are different the pipeline aborts with the error message above.
Please note that images cannot be modified in any way (e.g. color modification, greyscale, size modification, etc.) after manual alignment and before running Space Ranger. Any changes require the generation of a new manual alignment (JSON file).
In some instances Space Ranger can be run with automatic alignment; however, if automatic alignment fails, a new Space Ranger run is required. Failed Space Ranger runs cannot be restarted from intermediate points with a new image.