Run SeekSpace® Tools

Run tests

Example 1: Basic usage

Set up the necessary configuration files for the analysis, including paths to the sample data, chemistry versions, genome index, gene annotation file, etc. Run the SeekSpace® Tools using the following command:

seekspacetools run \
--fq1 /path/to/demo/demo_expression_S8_L007_R1_001.fastq.gz \
--fq2 /path/to/demo/demo_expression_S8_L007_R2_001.fastq.gz \
--spatialfq1 /path/to/demo/demo_spatial_S7_L007_R1_001.fastq.gz \
--spatialfq2 /path/to/demo/demo_spatial_S7_L007_R2_001.fastq.gz \
--hdmifq /path/to/demo/2P231224030A4.fq.gz \
--samplename demo \
--outdir /path/to/outdir \
--genomeDir /path/to/GRCh38/star \
--gtf /path/to/GRCh38/genes/genes.gtf \
--chemistry DDVS \
--core 4 \
--include-introns \
--forceCell 80000 \
--min_umi 200 \
--chip_id 2P231224030A4 \
--DAPI /path/to/demo/2P231224030A4.tif

• Note：--HE is optional. If an H&E image is provided, the path to the H&E image must be specified.

Example 2: Skip read processing steps and start from image processing:

seekspacetools run \
--fq1 /path/to/demo/demo_expression_S8_L007_R1_001.fastq.gz \
--fq2 /path/to/demo/demo_expression_S8_L007_R2_001.fastq.gz \
--spatialfq1 /path/to/demo/demo_spatial_S7_L007_R1_001.fastq.gz \
--spatialfq2 /path/to/demo/demo_spatial_S7_L007_R2_001.fastq.gz \
--hdmifq /path/to/demo/2P231224030A4.fq.gz \
--samplename demo \
--outdir /path/to/outdir \
--genomeDir /path/to/GRCh38/star \
--gtf /path/to/GRCh38/genes/genes.gtf \
--chemistry DDVS \
--core 4 \
--include-introns \
--forceCell 80000 \
--min_umi 200 \
--chip_id 2P231224030A4 \
--DAPI /path/to/demo/2P231224030A4.tif \
--alignment_file /path/to/demo/parameters.json \
--skip

• Note：--outdir specifies the path of directory that has outputs from the first run of seekspacetools.

Parameter descriptions

Parameters	Descriptions
–fq1	Paths to R1 fastq files of RNA library
–fq2	Paths to R2 fastq files of RNA library
–spatialfq1	Paths to R1 fastq files of spatial library
–spatialfq2	Paths to R2 fastq files of spatial library
–hdmifq	Path to HDMI fastq file of HDMI library
–samplename	Sample name. Only digits, letters, and underscores are supported.
–outdir	output directory. Default: ./
–genomeDir	The path of the reference genome generated by STAR. The version needs to be consistent with the STAR used by SeekSpace® Tools.
–gtf	Path to the GTF file for the corresponding species.
–core	Number of threads used for the analysis.
–chemistry	Reagent type, with each type corresponding to a combination of --shift, --pattern, --structure, --barcode, and --sc5p. Available options: DDVS. DDVS corresponds to the SeekSpace® Single Cell Spatial Transcriptomic Kit.
–skip_misB	If enabled, no base mismatch is allowed for barcode. Default is 1.
–skip_misL	If enabled, no base mismatch is allowed for linker. Default is 1.
–skip_multi	If enabled, discard reads that can be corrected to multiple white-listed barcodes. Barcodes are corrected to the barcode with the highest frequency by default.
–forceCell	Add this parameter with expected value N, SeekSpace® Tools will select the top N cells based on UMI from high to low. Default is 80000.
–min_umi	Minimum number of UMI for a cell. Cells with fewer UMI than this value will be discarded. Default is 200.
–include-introns	When disabled, only exon reads are used for quantification. When enabled, intron reads are also used for quantification.
–star_path	Path to another version of STAR for alignment. The version must be compatible with the --genomeDir version. The default --star_path is the STAR in the environment.
–chip_id	Chip ID
–DAPI	Slide image with DAPI staining in TIFF format.
–HE	Slide image with H&E staining in TIFF format.
–alignment_file	alignment file of image alignment parameters.
–skip	Skip read processing steps and continue from image processing steps.