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Rule smatch

Purpose

The smatch rule examines that for a pair of 2nd-seq FASTQ files, if spatial barcode sequence (HDMI) in read 1 are found in the spatial barcodes map for this chip section. The smatch functions on a per-pair basis for 2nd-seq FASTQ files. This means that for a given chip of interest, which is associated with multiple pairs of 2nd-seq FASTQ files, NovaScope executes smatch for each pair in parallel.

Input Files

  • Per-Chip Spatial Barcode Maps & Manifest File Required input files include the spatial barcode map and manifest file for the chip of interest, which are created by the sbcd2chip rule.

  • The 2nd-seq FASTQ files Required input files also include the read 1 file for a pair of 2nd-seq FASTQ files.

Output Files

The following files are generated for each pair of 2nd-seq FASTQ files in the specified directory path below:

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<output_directory>/match/<flowcell_id>/<chip_id>/<seq2nd_id>

(1) A Matched Spatial Barcode File

Description: A compressed, tab-delimited file containing spatial barcodes matched to the 2nd-seq reads.

File Naming Convention: <seq2st_pair_id>.R1.match.sorted.uniq.tsv.gz

File Format:

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AAAAAAAATAGTTCTGCTAGCTGGTAAGCTA  1  1  7124822  2910007  1  6
AAAAAAAGTGATCAGAGGTGATATTATGCTT  1  1  7382402  2721048  1  6
AAAAAAAGTTCGCACTATACGAACAGGGATC  1  1  8634969  2843056  1  1
  • Column 1: Spatial barcode sequence
  • Column 2: Lane ID, which is defined as 1.
  • Column 3: Tile ID, which is defined as 1.
  • Column 4: X-coordinate within the chip (global X-coordinate).
  • Column 5: Y-coordinate within the chip (global Y-coordinate).
  • Column 6: Number of bases that do not match the expected pattern defined by the format (0 is a perfect match).
  • Column 7: Number of occurrences in the 2nd-seq FASTQ read 1 file.

(2) A "smatch" Image

Description: An image depicting the spatial coordinate distribution of the matched barcodes.

File Naming Convention: <seq2st_pair_id>.R1.match.png

File Visualization:

smatch_image

(3) An Overall Summary of Matching Results

Description: A summary of the count and fraction of 2nd-seq reads based on the matching results.

File Naming Convention: <seq2st_pair_id>.R1.summary.tsv

File Format:

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Type        Reads      Fraction
Total       163383382  1.00000
Miss        80020087   0.48977
Match       83363295   0.51023
Unique      17641021   0.10797
Dup(Exact)  65722274   0.40226
  • Type : The type of statistics, including the following values:
    • Total : All reads in the 2nd-seq FASTQ file.
    • Miss : Reads that do not contain matching spatial barcodes.
    • Match : Reads that match with a spatial barcode.
    • Unique : Unique spatial barcodes that has matches.
    • Dup(Exact) : Duplicate barcodes calculated as Match - Unique.
  • Reads : The number of reads or barcodes that match the type.
  • Fraction : The fraction of the reads (among all reads) that match the type.

(4) A Summary of Matched and Unique Barcodes

Description: A tab-delimited file containing the number of matched and unique spatial barcodes.

File Naming Convention: <seq2st_pair_id>.R1.counts.tsv

File Format:

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id   filepath                 barcodes   matches   unique
1_1  1_1.sbcds.sorted.tsv.gz  175135683  83363295  17641021
  • id: The id is composed of <lane_id>_<tile_id>. Given only one spatial barcode map is created for a chip, the ID is designed as 1_1.
  • filepath: The file name is the corresponding spatial barcode map.
  • barcodes: The number of spatial barcodes in the chip.
  • matches: The number of barcodes match to the expected pattern.
  • unique: The number of unique barcodes match to the expected pattern.

Output Guidelines

Suggested review steps:

  1. Examine summary files to verify that the matched barcode rate isn't low rate, such as < 5%. A low matching rate might indicate a possible sample swap.
  2. Inspect the "smatch" image for an even distribution of matched barcodes across the tissue area. An unexpected pattern may suggest issues with experimental procedures, like unsuccessful tissue permeabilization.

Parameters

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

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upstream:
  smatch:                  
    skip_sbcd: 1            
    match_len: 27           
  visualization:
    drawxy:
      coord_per_pixel: 1000
      intensity_per_obs: 50
      icol_x: 3
      icol_y:

  • The smatch Parameters

    Parameters for smatch, used to pass values to the match-sbcds function in spatula. Below, for each parameter, the corresponding parameter in spatula, description, and the default value in NovaScope are provided.

    Parameter spatula parameter Description Default Value
    skip_sbcd --skip-sbcd The number of initial bases to omit from the read.* 1
    match_len --match-len The length of the spatial barcode to be considered as a perfectmatch. 27
    • skip_sbcd: This is useful if the 1st-seq spatial barcode lacks sufficient bases. When it is absent, NovaScope determines skip_sbcd following the format of fastq2sbcd: 1 for DraI31 and 0 for DraI32.

  • The visualization Parameters

    Parameters for the visualization step, provided to the draw-xy function in spatula.

    Parameter spatula parameter Description Default Value
    coord_per_pixel --coord-per-pixel Coordinates per pixel, as a divisor of input coordinate. 1000
    intensity_per_obs --intensity-per-obs Intensity of points per pixel, max 255. 50
    icol_x --icol-x (0-based) index of X coordinate in input TSV. 3
    icol_y --icol-y (0-based) index of Y coordinate in input TSV. 4

Dependencies

The sbcd2chip requires the successful execution of sbcd2chip to operate as intended. An overview of the rule dependencies are provided in the Workflow Structure.

Code Snippet

The code for this rule is provided in a03_smatch.smk