Rule `historef`¶

Purpose¶

The goal of historef rule is to match the histology image with the spatial coordinates of the spatial digital gene expression (SGE) matrix. This is achieved by aligning fiducial markers observable in both the histology image and the composite image of "sbcd", "smatch", and "sge" images. The current pipeline allows multiple input histology files.

Input Files¶

A histology image Th histology image with fiducial markers is required. The historef identifies fiducial marks by detecting the brightness in the input histology image, so it is crucial that fiducial markers are the brightest area in the histology image.
The composite image The composite image, which shows "sbcd", "smatch", and "sge" images side-by-side, will also be applied to historef.

Output Files¶

The rule generates the following output in the specified directory path:

1	`<output_directory>/histology/<flowcell_id>/<chip_id>/aligned/<run_id>`

(1) A referenced Histology File¶

Description: The referenced histology file, which is in GeoTIFFformat, allows the coordinate transformation between the SGE matrix and the input histology image.

File Naming Convention:

1	`<magnification><flowcell_abbreviation>-<chip_id>-<species>-<figtype>.tif"`

The magnification and figtype are derived from the magnification and figtype fields within the histology in the input section of the job configuration file.
The flowcell_abbreviation is derived by splitting the flowcell_id, which is sourced from the flowcell field in input section of the job configuration file, by "-" and taking the first part.

File Visualization:

The image displayed above only serves an initial glimpse into the results but has been substantially reduced in size and is presented in PNG format.

For an in-depth examination, access the full-size referenced histology file in the example datasets for NovaScope-exemplary-downstream-analysis (NEDA).

(2) A Re-sized Referenced Histology File¶

Description: A TIFF file shares the identical dimensions with both the "smatch" image and the "sge" image, acilitating the comparison of consistency between the histology file and these images.

File Naming Convention:

1	`<magnification><flowcell_abbreviation>-<chip_id>-<species>-<figtype>-fit.tif"`

The magnification and figtype are derived from the magnification and figtype fields within the histology section of the job configuration file.
The flowcell_abbreviation is derived by splitting the flowcell_id, which is sourced from the flowcell field in input section of the job configuration file, by "-" and taking the first part.

File Visualization:

An example re-sized referenced histology TIFF is provided in the access the full-size referenced histology file in the example datasets for NovaScope-exemplary-downstream-analysis (NEDA).

Output Guidelines¶

To verify the accuracy of the alignment, it is recommended to compare the re-sized referenced histology file against the "smatch" image and the "sge" image, ensuring a precise match with the histology images. A clear visibility of fiducial marks in both images indicates an accurate match with submicrometer resolution upon overlay. If the fiducial marks are insufficiently visible or aligned incorrectly, manual adjustment of the histology images is required.

Parameters¶

The following parameter in the job configuration file will be applied in this rule.

histology:
    min_buffer_size: 1000   
    max_buffer_size: 2000
    step_buffer_size: 100
    raster_channel: 1      

The histology Parameters
- min_buffer_size, max_buffer_size and step_buffer_size will create a list of buffer size help historef to do the alignment. For example, the default min_buffer_size, max_buffer_size and step_buffer_size are 1000, 2000, and 100, respectively, and this will return a buffer size list of 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000.
- raster_channel specifies which channel from the "sge" image will used for historef alignment

Dependencies¶

Rule historef commences only after Rule dge2sdge has successfully executed. An overview of the rule dependencies are provided in the Workflow Structure.

Code Snippet¶

The code for this rule is provided in b02_historef.smk

Rule historef¶