Calling spots

Once registered, the spots can be called. This is done with the iss call command. The process is then different depending on the type of acquisition. See details for barcode, genes, and hybridisation acquisitions.

Overview

The command is run as follows:

iss call --path relative/path/to/data --genes --barcodes --hyridisation

By default, the script will perform only missing steps. There is a --force-redo flag that will force the script to re-run all steps, even if the output files already exist.

        flowchart TD
start[Start] --> setup_hyb_spot_calling;


setup_hyb_spot_calling --> extract_hyb_spots_all(((extract_hyb_spots_all)))

style extract_hyb_spots_all fill:#E1BEE7,stroke:#424242
style setup_hyb_spot_calling stroke:#000000,fill:#E1BEE7

start[Start] --> setup_channel_correction;


setup_channel_correction --> setup_barcode_calling;

setup_barcode_calling --> basecall_tile(((basecall_tile)))

style basecall_tile fill:#E1BEE7,stroke:#424242
style setup_channel_correction stroke:#000000,fill:#E1BEE7
style setup_barcode_calling stroke:#000000,fill:#E1BEE7


setup_channel_correction --> setup_omp;
subgraph setup_omp
    get_reference_spots --> get_cluster_means --> make_gene_templates
end

subgraph diagnostics
    check_omp_setup(check_omp_setup);
    check_omp_thresholds(check_omp_thresholds);
    check_omp_alpha_thresholds(check_omp_alpha_thresholds);
end

setup_omp --> diagnostics;
setup_omp --> batch_call(((extract_tile)))
style batch_call fill:#E1BEE7,stroke:#424242

style setup_omp stroke:#000000,fill:#E1BEE7
style setup_channel_correction stroke:#000000,fill:#E1BEE7


style check_omp_setup fill:#BBDEFB,stroke:#616161,color:#000000
style check_omp_thresholds fill:#BBDEFB,stroke:#616161,color:#000000
style check_omp_alpha_thresholds fill:#BBDEFB,stroke:#616161,color:#000000

style get_cluster_means fill:#C8E6C9,stroke:#000000
style get_reference_spots fill:#C8E6C9,stroke:#000000
style make_gene_templates fill:#C8E6C9,stroke:#000000

style setup_omp fill:#AAAAAA, stroke:#424242
style diagnostics fill:#AAAAAA, stroke:#424242
    

Calling genes

        flowchart TD
start[Start] --> setup_channel_correction;


setup_channel_correction --> setup_omp;
subgraph setup_omp
    get_reference_spots --> get_cluster_means --> make_gene_templates
end

subgraph diagnostics
    check_omp_setup(check_omp_setup);
    check_omp_thresholds(check_omp_thresholds);
    check_omp_alpha_thresholds(check_omp_alpha_thresholds);
end

setup_omp --> diagnostics;
setup_omp --> batch_call(((extract_tile)))
style batch_call fill:#E1BEE7,stroke:#424242

style setup_omp stroke:#000000,fill:#E1BEE7
style setup_channel_correction stroke:#000000,fill:#E1BEE7


style check_omp_setup fill:#BBDEFB,stroke:#616161,color:#000000
style check_omp_thresholds fill:#BBDEFB,stroke:#616161,color:#000000
style check_omp_alpha_thresholds fill:#BBDEFB,stroke:#616161,color:#000000

style get_cluster_means fill:#C8E6C9,stroke:#000000
style get_reference_spots fill:#C8E6C9,stroke:#000000
style make_gene_templates fill:#C8E6C9,stroke:#000000

style setup_omp fill:#AAAAAA, stroke:#424242
style diagnostics fill:#AAAAAA, stroke:#424242
    

Calling barcodes

        flowchart TD
start[Start] --> setup_channel_correction;


setup_channel_correction --> setup_barcode_calling;

setup_barcode_calling --> batch_call(((basecall_tile)))

style batch_call fill:#E1BEE7,stroke:#424242
style setup_channel_correction stroke:#000000,fill:#E1BEE7
style setup_barcode_calling stroke:#000000,fill:#E1BEE7
    

Calling hybridisation spots

        flowchart TD
start[Start] --> setup_hyb_spot_calling;


setup_hyb_spot_calling --> batch_call(((extract_hyb_spots_all)))

style batch_call fill:#E1BEE7,stroke:#424242
style setup_hyb_spot_calling stroke:#000000,fill:#E1BEE7