drneavin
commented
1 year ago Hi @Tan-GuangHui,
I think there might be a couple issues here. First, you are indicating —ignore true which might be the reason that you aren’t getting an error output but you also don’t get an output.
Second, the file you’re using for the barcode file isn’t formatted correctly. It should have a single barcode per line, not comma separated with multiple fields. I’m guessing there is a single barcode for each spot that can be specified but without seeing more of the bam file I can’t be sure how that would be formatted. Further, the barcode might be specified with a different tag in the bam than is used for single cell droplet data. Can you send the output of one bam read or head of the bam file? I might be able to work out what your barcode tag would need to be.
Cheers, Drew
Jylab-Genetics
I now have a spatial transcriptome sequencing bam file with a file size of 58g. VCF files for individual variations in fetuses and mothers, Souforce is now used to distinguish cells from maternal and fetal sources. My script is as follows： singularity exec Demuxafy.sif souporcell_pipeline.py \ -i sorted.bam\ -b BeadLocationsForR.csv \ -f ARS-UCD1.2_Btau5.0.1Y.fa \ -t 40\ -o /work/home/luo_funong/Tan_Guang-hui/mutai_qufen/out \ -k 2 \ --known_genotypes /work/home/luo_funong/Tan_Guang-hui/mutai_qufen/mothers.QC.vcf /work/home/luo_funong/Tan_Guang-hui/mutai_qufen/fetal.QC.vcf \ --known_genotypes_sample_names mothers fetal --ignore true
The location information in the bam file is shown in the following figure：The CX and cy columns correspond to the BeadLocationsForR coordinates below
BeadLocationsForR.csv file
The running error is as follows： usage: souporcell_pipeline.py [-h] -i BAM -b BARCODES -f FASTA -t THREADS -o OUT_DIR -k CLUSTERS [-p PLOIDY] [--min_alt MIN_ALT] [--min_ref MIN_REF] [--max_loci MAX_LOCI] [--restarts RESTARTS] [--common_variants COMMON_VARIANTS] [--known_genotypes KNOWN_GENOTYPES] [--known_genotypes_sample_names KNOWN_GENOTYPES_SAMPLE_NAMES [KNOWN_GENOTYPES_SAMPLE_NAMES ...]] [--skip_remap SKIP_REMAP] [--no_umi NO_UMI] [--umi_tag UMI_TAG] [--cell_tag CELL_TAG] [--ignore IGNORE] [--aligner ALIGNER] souporcell_pipeline.py: error: unrecognized arguments: /work/home/luo_funong/Tan_Guang-hui/mutai_qufen/fetal.QC.vcf
When I use an individual SNP file, there is no error reported, but there is no result file output, even after running for 10 hours