Run Seeksoultools

Run tests

Example 1: Basic usage

mkdir -p /demo/myproject/
cd /demo/myproject/
seeksoultools rna run \ 
--fq1 /demo/data/demo3k_R1_001.fastq.gz \ 
--fq2 /demo/data/demo3k_R2_001.fastq.gz \ 
--samplename demo3k \ 
--outdir /demo/myproject/ \ 
--genomeDir /demo/refdata/GRCh38-3.0.0/star \ 
--gtf /path/demo/refdata/GRCh38-3.0.0/genes/genes.gtf \ 
--chemistry MM \ 
--core 4

Example 2: Specify a different version of STAR for analysis. Make sure that the STAR version is compatible with the –genomeDir.

mkdir /demo/myproject/
cd /demo/myproject/
seeksoultools rna run \
--fq1 /demo/data/demo3k_R1_001.fastq.gz \
--fq2 /demo/data/demo3k_R2_001.fastq.gz \
--samplename demo3k \
--outdir /demo/myproject/ \
--genomeDir /demo/refdata/GRCh38/star \
--gtf /path/demo/refdata/GRCh38/genes/genes.gtf \
--chemistry MM \
--core 4 \
--star_path /path/to/cellranger-5.0.0/lib/bin/STAR

Example 3: A sample has multiple sets of fastq files

mkdir /demo/myproject/
cd /demo/myproject/
seeksoultools rna run \
--fq1 /demo/data/demo_S1_L001_R1_001.fastq.gz \
--fq1 /demo/data/demo_S1_L002_R1_001.fastq.gz \
--fq2 /demo/data/demo_S1_L001_R2_001.fastq.gz \
--fq2 /demo/data/demo_S1_L002_R2_001.fastq.gz \
--samplename demo \
--outdir /demo/myproject/ \
--genomeDir /demo/refdata/GRCh38/star \
--gtf /demo/refdata/GRCh38/genes/genes.gtf \
--chemistry MM \
--core 4

Example 4: Customize the structure of R1

seeksoultools rna run \
--fq1 /demo/data/demo3k_R1_001.fastq.gz \
--fq2 /demo/data/demo3k_R2_001.fastq.gz \
--samplename demo \
--outdir /demo/myproject/ \
--genomeDir /demo/refdata/GRCh38/star \
--gtf /demo/refdata/GRCh38/genes/genes.gtf \
--barcode /demo/utils/CLS1.txt \
--barcode /demo/utils/CLS2.txt \
--barcode /demo/utils/CLS3.txt \
--linker /demo/utils/Linker1.txt \
--linker /demo/utils/Linker2.txt \
--structure B9L12B9L13B9U8 \
--core 4

The structure of read1 is represented by B9L12B9L13B9U8, which means it consists of three sections of cell barcode, each with 9 bases, and a UMI section with 8 bases. The linker section between the cell barcode and UMI consists of two parts, with the first part being 12 bases and the second part being 13 bases
Use --barcode to specify the three sections of barcodes sequentially, and use --linker to specify the two sections of linkers sequentially.

Parameter descriptions

Parameters	Descriptions
–fq1	Paths to R1 fastq files.
–fq2	Paths to R2 fastq files.
–samplename	Sample name. A directory will be created named after the sample name in the outdir directory. 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 seeksoultools.
–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: DDV1, DDV2, DD5V1, MM, MM-D, DD-Q. DDV1 corresponds to the 3’ transcriptome-seq kit V1 reagent for the DD platform. DDV2 corresponds to the 3’ transcriptome-seq kit V2 reagent for the DD platform. DD5V1 corresponds to the 5’ transcriptome-seq kit V1 reagent for the DD platform. MM corresponds to the 3’ transcriptome-seq kit reagent for the MM platform. MM-D corresponds to the large-well transcriptome-seq kit for the MM platform. DD-Q corresponds to the full-length rna sequence transcriptome-seq kit for the DD platform.
–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.
–expectNum	Estimated number of captured cells.
–forceCell	When number of cells obtained from analysis is abnormal, add this parameter with expected value N. Seeksoultools will select the top N cells based on UMI from high to low.
–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.