Error: No such file or directory: '/outputpath/samplename/step3/filtered_feature_bc_matrix/barcodes.tsv.gz'

• First step: Check the output directory. If raw_feature_bc_matrix exists and the files under this directory are not abnormally small, run the cell identification script for test:

Rscript /path/seeksoultools/lib/python3.XX/site-packages/seeksoultools/utils/cell_identify.R -i outputpath/step3/raw_feature_bc_matrix -e 3000

• If this script fails (e.g., due to a missing R package), then it’s probably environment issue. So run the source activate seeksoultools, or export PATH=/path/seeksoultools/bin:$PATH in order to activate environment, followed by rerunning the script.
• Tip: When installing seeksoultools, avoid creating soft links to another disk. Install it directly, ensure the environment name is unique, and use absolute paths when running scripts.
• If the files in the raw_feature_bc_matrix directory are abnormally small (e.g., only a few bytes), the GTF file may be problematic. For GTF format requirements, refer to the "Q&A 23: How to Build a Reference Genome?" section.