Input structure to run .nd2 files as FOVs

[Boehmin]

I've been trying to run the stitching+registration on my data: .nd2 files, FOVs saved individually: In total 8 rounds a 47 FOVs a 5 channels each

Going through some of the issues on github I saw that some others had similar file structures (img_cycle_FOV.filename), however reading through the ashlar documentation I read that only one image file should be processed per cycle?

How do I go about supplying the right information in params.yml for the ashlar module in mcmicro to recognise that I have multiple FOVs? Is this possible? Attached are myparams.yml and markers.csv the way I ran them currently.
markers.csv
myparams.txt

That's the error I receive (full nextflow log attached
nextflowlog.txt
):

Command exit status:
  1

Command output:
  
          merging tile 1/1
  Cycle 194:
      Channel 0:
  
          merging tile 1/1
      Channel 1:
  
          merging tile 1/1
      Channel 2:
  
          merging tile 1/1
      Channel 3:
  
          merging tile 1/1
      Channel 4:
  
          merging tile 1/1
  Cycle 195:
      Channel 0:
  
          merging tile 1/1
      Channel 1:
  
          merging tile 1/1
      Channel 2:
  
          merging tile 1/1
      Channel 3:
  
          merging tile 1/1
      Channel 4:
  
          merging tile 1/1
  Cycle 196:
      Channel 0:
  
          merging tile 1/1
      Channel 1:
  
          merging tile 1/1
      Channel 2:
  
          merging tile 1/1
      Channel 3:
  
          merging tile 1/1
      Channel 4:
  
          merging tile 1/1

Command error:
  Cycle 196:
      Channel 0:
  
          merging tile 1/1
      Channel 1:
  
          merging tile 1/1
      Channel 2:
  
          merging tile 1/1
      Channel 3:
  
          merging tile 1/1
      Channel 4:
  
          merging tile 1/1Traceback (most recent call last):
    File "/usr/local/bin/ashlar", line 8, in <module>
      sys.exit(main())
    File "/usr/local/lib/python3.10/dist-packages/ashlar/scripts/ashlar.py", line 212, in main
      return process_single(
    File "/usr/local/lib/python3.10/dist-packages/ashlar/scripts/ashlar.py", line 277, in process_single
      writer.run()
    File "/usr/local/lib/python3.10/dist-packages/ashlar/reg.py", line 1228, in run
      tiff.write(
    File "/usr/local/lib/python3.10/dist-packages/tifffile/tifffile.py", line 3049, in write
      iteritem, dataiter = peek_iterator(dataiter)
    File "/usr/local/lib/python3.10/dist-packages/tifffile/tifffile.py", line 21409, in peek_iterator
      first = next(iterator)
    File "/usr/local/lib/python3.10/dist-packages/tifffile/tifffile.py", line 21413, in newiter
      yield from iterator
    File "/usr/local/lib/python3.10/dist-packages/tifffile/tifffile.py", line 21413, in newiter
      yield from iterator
    File "/usr/local/lib/python3.10/dist-packages/tifffile/tifffile.py", line 21413, in newiter
      yield from iterator
    [Previous line repeated 981 more times]
    File "/usr/local/lib/python3.10/dist-packages/ashlar/reg.py", line 1180, in base_tiles
      img = mosaic.assemble_channel(channel)
    File "/usr/local/lib/python3.10/dist-packages/ashlar/reg.py", line 1088, in assemble_channel
      img = self.aligner.reader.read(c=channel, series=si)
    File "/usr/local/lib/python3.10/dist-packages/ashlar/reg.py", line 405, in read
      dtype = self.metadata.pixel_dtype
    File "/usr/local/lib/python3.10/dist-packages/ashlar/reg.py", line 304, in pixel_dtype
      return self._pixel_dtypes[self._metadata.getPixelsType(0).value]
    File "jnius/jnius_export_class.pxi", line 885, in jnius.JavaMethod.__call__
    File "jnius/jnius_export_class.pxi", line 967, in jnius.JavaMethod.call_method
    File "jnius/jnius_conversion.pxi", line 240, in jnius.convert_jobject_to_python
    File "/usr/local/lib/python3.10/dist-packages/jnius/reflect.py", line 203, in autoclass
      cls = MetaJavaClass.get_javaclass(jniname, classparams=(include_protected, include_private))
    File "jnius/jnius_export_class.pxi", line 164, in jnius.MetaJavaClass.get_javaclass
  RecursionError: maximum recursion depth exceeded in comparison

Work dir:
  /scicore/home/rueegg/boehm0002/miniconda3/envs/nextflow/work/9b/3ff3a47792c4b7d4cc97a922918444

Tip: you can replicate the issue by changing to the process work dir and entering the command `bash .command.run`

 -- Check '.nextflow.log' file for details

[ArtemSokolov]

@jmuhlich, @Yu-AnChen Should this be handled using the fileseries interface?

[Boehmin]

@ArtemSokolov are you referring to the Galaxy workflow? This I haven`t tried yet.

[ArtemSokolov]

No, ASHLAR has an undocumented feature called fileseries, which lets you explicitly map parts of the filename to tile number and cycle number.

Some discussions about it can be found in:

• https://forum.image.sc/t/ashlar-stitching-questions-and-developments/67418/11
• https://forum.image.sc/t/ashlar-how-to-pass-multiple-images-to-be-stitched/49864/3

MCMICRO doesn't wrap this feature, so you would need to run ASHLAR "by hand" (using instructions at https://github.com/labsyspharm/ashlar). However, once you have a registered image, you can place it into the registration/ subfolder of your project and add the following to your params.yml to run the rest of the pipeline:

workflow:
  start-at: segmentation

[Boehmin]

Thank you for the advice, I will try this! I do not acquire images in a conventional grid so I`m waiting for some support from the ASHLAR team.