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Rule fastq2sbcd

Purpose

The fastq2sbcd rule aims to generate a spatial barcode map per-tile basis for an input 1st-seq FASTQ file.

Input Files

The only input file required by fastq2sbcd is the 1st-seq FASTQ file. Ensure the raw FASTQ files are correctly formatted and listed in the job configuration file.

Output Files

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

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<output_directory>/seq1st/<flowcell_id>/sbcds/<seq1st_id>

(1) Per-Tile Spatial Barcode Maps

Description: The spatial barcode map for each tile is stored in a zipped, tab-separated file. These files map the barcodes to their coordinates within the tile, facilitating easier matching with 2nd-Seq sequences through reverse-complemented barcodes.

File Naming Convention: <lane_id>_<tile_id>.sbcds.sorted.tsv.gz

File Format: The format of the spatial barcode map is outlined below with an example:

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AAAAAAAAAAAAGCGACCGGGTAATATATGT 3   2456    1036    35446   1
AAAAAAGGTACCCGCAGTGCGGACAAACGAA 3   2456    23448   29731   1
AAAAAGACGAGTAAAAGTGACTGTTAATTAC 3   2456    29794   1799    1
  • Column 1: Spatial barcode sequence (HDMI, typically 32 base pairs).
  • Column 2: Lane ID.
  • Column 3: Tile ID.
  • Column 4: X-coordinate within the tile (local X-coordinate).
  • Column 5: Y-coordinate within the tile (local Y-coordinate).
  • Column 6: Number of mismatching bases from expected pattern

(2) Flow Cell Manifest

Description: The manifest file provides summary statistics for each tile within the input FASTQ file, with each tile's statistics presented in a separate row.

File Naming Convention: manifest.tsv

File Format:

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id      filepath                        barcodes    matches mismatches  xmin    xmax    ymin    ymax
3_2456  3_2456.sbcds.sorted.tsv.gz      3460541     3377518 83023       1027    32949   1000    37059
3_2556  3_2556.sbcds.sorted.tsv.gz      3416413     3334054 82359       1036    32958   1000    37059
  • id: The id is composed of <lane_id>_<tile_id>.
  • filepath: The file name is the corresponding spatial barcode map.
  • barcodes: The number of barcodes in the tile.
  • matches: The number of barcodes match to the expected pattern.
  • mismatches: The number of barcodes don't match to the expected pattern.
  • xmin: The minimum X-coordinate across all barcodes within the tile (i.e., mimimum local X-coordinate).
  • xmax: The maximum X-coordinate across all barcodes within the tile (i.e., maximum local X-coordinate).
  • ymin: The minimum Y-coordinate across all barcodes within the tile (i.e., mimimum local Y-coordinate).
  • ymax: The maximum Y-coordinate across all barcodes within the tile (i.e., maximum local Y-coordinate).

Output Guidelines

For accuracy purposes, it's recommended to examine the manifest.tsv file to verify:

  1. A full lane typically comprises 936 tiles, with each tile having 3 million or more reads;
  2. The majority of reads are expected to align with the anticipated HDMI patterns.

Parameters

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

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upstream:
  fastq2sbcd:
    format: DraI32
  • The format Parameter This parameter specifies the HDMI-oligo seed library used. The default setting is DraI32, which corresponds to HDMI32-DraI. Please see details for the seed HDMI-oligo library in the original publication of SeqScope.

Dependencies

Rule fastq2sbcd operates independently without dependencies on preceding rules. An overview of the rule dependencies are provided in the Workflow Structure.

Code Snippet

The code for this rule is provided in a01_fastq2sbcd.smk.