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Rule sbcd_layout

Purpose

The sbcd_layout rule helps determine the correct spatial map layout for the input flowcell using tile pairs. Specifically, it applies all layouts types on tile pairs and visualizes the corresponding spatial maps, allowing the user to review and choose the correct one based on the alignment of fiducial marks across tiles in a straight line.

Spatial Barcode Map Layout

There are two layout types:

  • 'Top oddshift and bottom evenshift (tobe)': the top layer applies an oddshift (horizontally shifting tiles in odd rows) while the bottom layer uses an evenshift (horizontally shifting tiles in even rows).
  • 'Top evenshift and bottom oddshift (tebo)': the top layer applies an evenshift while the bottom applies an oddshift.

Tile Pairs

Users can define tile pairs for layout examination. Each pair should consist of two tiles from the same layer, but different rows, and the same column. Ideally, use two pairs—one from the top layer and one from the bottom. By default, NovaScope uses the tile pairs "1644, 1544" and "2644, 2544."

Ensure that the tile pairs for spatial map examination are present in the input data by verifying they are listed in the flow cell manifest or by checking for the corresponding per-tile spatial barcode map.

Interpret layer and row information from Tile ID

The Tile ID is a four-digit number with the following structure:

  • The first digit represents the layer: '1' for the top layer and '2' for the bottom.
  • The second digit indicates the row: if the digit is odd, the tile is in an odd row.
  • The last two digits represent the column information.

Input Files

  • Per-Tile Spatial Barcode Maps & Flow Cell Manifest Required inputs are the spatial barcode maps for each tile pair and the manifest file for the corresponding flow cell. Both inputs are generated by the fastq2sbcd rule.

Output Files

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

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<output_directory>/seq1st/<flowcell_id>/image/<seq1st_id>

Oddshift and Evenshift Spatial Barcode Maps

Description: For each tile pair, two images will be provided to display the alignment of the tiles using rowshift and oddshift.

File Naming Convention: 

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<flowcell>.<seq1st_id>.<layer>.<tile1>_<tile2>.<shifttype>.nbcds.png
  • <layer>: "top" or "bottom". NovaScope automatically add layer information in the file name for a better interpretation.
  • <shifttype>: "oddshift" or "evenshift".

File Visualization:

sbcd_layout_image

Output Guidelines

Recommended review steps:

  1. Review the oddshift and evenshift spatial barcode maps for each tile pair. Use the alignment of fiducial marks across the tiles to determine the correct spatial barcode layout. Below are the examples of aligned and misaligned tiles.
sbcd_layout_examples
  1. If the oddshift and evenshift spatial barcode maps indicate the 'tebo' layout, the user should revise the job configuration file by setting layout_shift in seq1st under the input section to 'tebo'. If not, no changes are needed, as the default layout_shift is 'tobe'.

Parameters

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

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upstream:
  sbcd_layout:                                 
    tiles:                                     
     - "1644,1544"
     - "2644,2544"
    colshift: 0.1715
  • The sbcd_layout Parameters
  • Use tile to define tile pairs. One pair should be defined as "<tile1>,<tile2>". NovaScope supports more than one tile pair to be used for layout examination.
  • The colshift defines the distance of horizontal shift. By default, use 0.1715.

Dependencies

The execution of Rule sbcd_layout depends on whether Rule fastq2sbcd functions correctly. An overview of the rule dependencies are provided in the Workflow Structure.

Code Snippet

The code for this rule is provided in b03_sbcd_layout.smk