Run
Run Examples
Example 1: ARC Multi-omics Analysis
ARC multi-omics analysis takes both RNA and ATAC library FASTQ files as input and performs RNA quantification, ATAC Peak calling, joint cell calling, matrix generation, and report generation.
seekarctools arc run \
--rnafq1 /path/to/demo/demo_GE_S1_L001_R1_001.fastq.gz \
--rnafq2 /path/to/demo/demo_GE_S1_L001_R2_001.fastq.gz \
--atacfq1 /path/to/demo/demo_arc_S2_L002_R1_001.fastq.gz \
--atacfq2 /path/to/demo/demo_arc_S2_L002_R2_001.fastq.gz \
--samplename demo \
--outdir /path/to/outdir \
--refpath /path/to/reference/GRCh38 \
--chemistry DD_AG \
--include-introns \
--core 16Example 2: RNA-only Library Analysis
RNA-only library analysis is intended for datasets containing only RNA/GEX libraries. The workflow performs barcode processing, STAR alignment, UMI quantification, cell calling, expression matrix generation, Seurat RDS generation, and GEX quality-control summary generation.
seekarctools rna run \
--rnafq1 /path/to/demo/demo_GE_S1_L001_R1_001.fastq.gz \
--rnafq2 /path/to/demo/demo_GE_S1_L001_R2_001.fastq.gz \
--samplename demo \
--outdir /path/to/outdir \
--refpath /path/to/reference/GRCh38 \
--chemistry DD_AG_RNA \
--include-introns \
--core 16Example 3: Rerun Peak Calling or Cell Calling with Adjusted Thresholds
If the Peaks or cells identified with the default parameters do not meet the analysis requirements, use arc retry to skip previous steps such as alignment and rerun the downstream workflow using existing intermediate files. This saves time while allowing the results to be optimized.
seekarctools arc retry \
--samplename demo \
--outdir /path/to/outdir \
--refpath /path/to/reference/GRCh38 \
--core 16 \
--qvalue 0.01 \
--snapshift 0 \
--extsize 200 \
--min_len 200 \
--min_atac_count 1000 \
--min_gex_count 500NOTE
Ensure that all files generated by the previous run remain complete and have not been deleted or moved. The --outdir parameter must point to the output directory used for the initial seekarctools arc run. --min_atac_count and --min_gex_count must be used together; specifying only one of them will not take effect.This command is only available for ARC multi-omics analysis. RNA-only library analysis currently does not provide a retry command.
Parameter Description
| Parameter | Description |
|---|---|
--rnafq1 | Path to the RNA library R1 FASTQ file |
--rnafq2 | Path to the RNA library R2 FASTQ file |
--atacfq1 | Path to the ATAC library R1 FASTQ file |
--atacfq2 | Path to the ATAC library R2 FASTQ file |
--samplename | Sample name |
--chemistry | Library chemistry. Use DD_AG or DD5_AG for ARC multi-omics analysis, and DD_AG_RNA for RNA-only library analysis |
--outdir | Output directory. Default: ./ |
--skip_misB | When enabled, base mismatches are not allowed in the Barcode. By default, one mismatch is allowed |
--skip_misL | When enabled, base mismatches are not allowed in the Linker. By default, one mismatch is allowed |
--skip_multi | When enabled, reads that can be corrected to multiple whitelist Barcodes are discarded. By default, they are corrected to the most frequent Barcode |
--skip_len | When enabled, short-read filtering after adapter trimming is skipped, and short reads are retained for downstream analysis |
--core | Number of threads used for analysis |
--memory | Memory limit in GB used to generate the Seurat RDS for RNA-only library analysis. Default: 32 |
--include-introns | When enabled, intronic reads are included in gene expression quantification. By default, only exonic reads are used |
--refpath | Path to the reference genome directory |
--star_path | Path to an external STAR executable. Specify this path if the reference genome index was built with a particular STAR version |
--scoremin | STAR relaxed-alignment parameter corresponding to --outFilterScoreMinOverLread |
--matchnmin | STAR relaxed-alignment parameter corresponding to --outFilterMatchNminOverLread |
--pairendAlignment | Enables STAR paired-end alignment for RNA-only library analysis. By default, processed R2 reads are aligned in single-end mode |
--pairendMinR1Len | Minimum retained R1 length for RNA paired-end alignment. Default: 20 |
--expect_cells | Expected number of recovered cells for RNA-only library analysis. Default: 3000 |
--pvalue | p-value threshold for the ambient RNA test during RNA cell calling. Default: 0.01 |
--force_cells | Forces the top N Barcodes ranked by UMI count to be called as cells in RNA-only library analysis. Default: 0, which enables automatic cell calling |
--rscript_path | Path to Rscript used for RNA-only library analysis. Default: Rscript |
--qvalue | Minimum FDR (q-value) threshold for Peak calling. Default: 0.001 |
--nolambda | When enabled, MACS3 uses the background lambda as the local lambda and does not account for local bias around candidate Peak regions |
--snapshift | MACS3 Peak-calling shift size. Default: 0 |
--extsize | MACS3 Peak-calling extension size. Default: 400 |
--min_len | Minimum Peak length. Default: 400 |
--broad | Enables broad Peak calling and generates results in UCSC gappedPeak format |
--broad_cutoff | Threshold for broad Peak calling. Default: 0.1 |
--min_atac_count | Cell-calling override that defines the minimum number of ATAC transposition events within Peak regions for a cell Barcode |
--min_gex_count | Cell-calling override that defines the minimum number of gene expression UMI counts for a cell Barcode |
--keep_mito | When enabled, mitochondrial reads are retained. By default, mitochondrial reads are filtered |
--strict_cell_calling | When enabled, an ambient RNA background test is added to K-Means-based cell calling for more stringent cell identification |
--max_cells | Limits the maximum number of automatically identified cells. No limit is applied if this parameter is not specified |
-h, --help | Displays command-line help |