Open Laolga opened 4 months ago
Hello,
Thanks for the custom pipelines and for this nice protocol!
On a similar note, I'm having trouble understanding the exact input I should use to run the DNA-based pipeline.
Would it be possible to give me a specific example?
Please provide if possible an example samplesheet.csv file and also a specific value for the "--directory" parameter assuming that I already have my FASTQ files ready, so I don't need to run cellranger to produce them from the BCL files.
Thanks in advance, Maria
Hello Maria,
Here is an example of a few runs and how to set up the software. It assumes the fastq files are in the directory that bcl2fastq would create. So you need to make that path and put your fastq's in it if you don't have that path already.
Create conda environment
conda create -n cutadapt -c bioconda -c conda-forge cutadapt python=3.9 bwa pysam samtools numpy3
Download dna10x pipeline from github
Create sample sheet in directory with the pipeline
Download reference or use 10x cellranger references
Make Directories Within the dna10x directory with all the associated .py files create the following path mkdir PTO035/outs/fastq_path/PTO035/PTO035/
Run pipeline <With exisitng fastqs, assumed to be located in dna10x/PTO035/outs/fastq_path/PTO035/PTO035/> nohup python dna10x.py --samplesheet ss.csv -d PTO035 -b /opt/cellranger-atac-2.0.0/lib/python/atac/barcodes/737K-arc-v1.txt -t 16 -r /opt/refdata-cellranger-arc-GRCh38-2020-A-2.0.0/fasta/genome.fa -i 1000 -m 0.9 -c -sf -p 1 -rc -ad CTGTCTCTTATACACATCT &
<With BCL to fastq, may need to install bcl2fastq> nohup python dna10x.py --bcl ~/230407_NB551203_0654_AH5LM2BGXT --samplesheet ss.csv -d PTO035 -b /opt/cellranger-atac-2.0.0/lib/python/atac/barcodes/737K-arc-v1.txt -t 16 -r /opt/refdata-cellranger-arc-GRCh38-2020-A-2.0.0/fasta/genome.fa -i 1000 -m 0.9 -p 1 -rc -ad CTGTCTCTTATACACATCT -c &
Tim
Dear authors, Please provide information needed to execute your pipeline: 1) what is the format of the samplesheet 2) how can one know barcodes before running any analysis? 3) What is BARCODE_START_CYCLE? 4) What is rc? 5) What is ad?