build_traces¶

Invoke¶

Inside the folder with your input data, run:

pyhim -C build_traces

build_traces

Inputs¶

Name shape	Quantity	Mandatory	Description
parameters.json	1	Yes	Parameter file.

Outputs¶

Name shape	Quantity	Description

Relevant options¶

Parameters to run this script will be read from the buildsPWDmatrix field of parameters.json.

"tracing_method": ["masking","clustering"], # list of methods it will use
"mask_expansion": 8,# number of pixels masks will be expanded to assign localizations
"masks2process":{"nuclei":"DAPI","mask1":"mask0"}, # masks identities to process
"KDtree_distance_threshold_mum": 1, # threshold distance used for KDtree clustering
"barcode_coordinates_BEDfile": "", # optional BED file used to annotate barcodes with genomic coordinates

Optional BED-driven barcode annotation¶

If barcode_coordinates_BEDfile is set to a valid BED file path, build_traces will load that file and use it to enrich each saved trace entry with genomic metadata.

Example in parameters.json (buildsPWDmatrix section):

"buildsPWDmatrix": {
  "tracing_method": ["masking", "clustering"],
  "mask_expansion": 8,
  "masks2process": {"nuclei": "DAPI"},
  "KDtree_distance_threshold_mum": 1,
  "barcode_coordinates_BEDfile": "resources/barcode_coordinates.bed"
}

When a BED file is provided:

Barcode identities are validated against the BED mapping.
If a 5th BED column is present, barcode IDs can be remapped before writing the trace table.
Chrom, Chrom_Start, and Chrom_End columns in the output trace table are populated from BED coordinates.

BED file format expected by `build_traces`¶

Use a tab-separated BED-like file (no header), with at least 4 columns:

chrom (e.g. chr2L, chrX)
start (genomic start, basepairs)
end (genomic end, basepairs)
barcode (barcode ID expected in localizations)
(optional) barcode_remap (barcode ID written to output table after remapping)

Example:

chr2L	150000	151000	1
chr2L	220000	221000	2	102
chr2L	300000	301000	3

In this example, barcode 2 is remapped to 102 when traces are saved, while all entries receive genomic coordinates from columns 1 to 3.

Output images:

_XYZ_ROI*.png

	full image	zoomed images
3D mask
3D mask
3D KDtree