/snRNAseq-NextFlow

Primary LanguagePython

NextFlow pipeline for 10X snRNA-seq data

Dependencies

Singularity (v. 3) and NextFlow (>= v. 20.10.0). Containers with the software for each step are pulled from the Sylabs cloud library (https://cloud.sylabs.io/library).

Configuration

Paths to reference files must be included in the nextflow.config file -- check that file and change paths accordingly. These include:

  1. STAR indices (compatible with STAR v. 2.7.9a)
  2. GTF files
  3. Barcode whitelist (for Chromium v3, that is the 3M-february-2018.txt file; for v2, that is the 737K-august-2016.txt file; for multiome, that is 737K-arc-v1.txt)

You'll also need to set the params.results variable -- either in the nextflow.config file itself, or on the command line when you run the pipeline ('--results /path/to/results') as well as the 10X Chromium chemistry version ('V2', 'V3', or 'multiome')

Lastly, you'll need to include information about each RNA-seq library, including the genome to which it should be mapped, and the paths to the fastq files for each readgroup. Organize this information in a JSON file, as in library-config.json. For each readgroup, the '1' fastq file corresponds to the sequencing read including the UMI and the nucleus index; the '2' fastq file refers to the sequencing read representing the actual transcript. Also, note that the 'genome' attribute is given as a list (because I will be adding the ability to map to multiple genomes, in the case that nuclei from multiple species are mixed together).

Running

Once you have all of the above information, you can run the pipeline as follows (in this case, indicating the path to the results on the command line):

nextflow run -resume -params-file library-config.json --chemistry multiome --results /path/to/results /path/to/main.nf

You may wish to run under nohup so that the pipeline continues to run in the background and does not terminate upon logging out of the server (nohup nextflow run ... &)

Output

  • dropkick/*: Results of running dropkick per-library (QC plots, as well as per-barcode dropkick score that can be used for selecting quality barcodes for downstream analysis)
  • multiqc/fastq/*: multiqc summaries of fastqc results
  • multiqc/star/*: multiqc summaries of STAR logs
  • prune/*: filtered bam files (duplicates NOT removed)
  • qc/*: Per-barcode QC metrics and QC metric plots
  • starsolo/*: starsolo output. Count matrices derived using a variety of counting methods (see STAR manual) are in starsolo/{library}/{library}.Solo.out/*