rna-seq_wf

RNA-seq pipeline documentation

• Make sure to have installed:

  • Docker

    apt-get install ca-certificates curl
    install -m 0755 -d /etc/apt/keyrings
    curl -fsSL https://download.docker.com/linux/ubuntu/gpg -o /etc/apt/keyrings/docker.asc
    chmod a+r /etc/apt/keyrings/docker.asc
    echo "deb [arch=$(dpkg --print-architecture) signed-by=/etc/apt/keyrings/docker.asc] https://download.docker.com/linux/ubuntu $(. /etc/os-release && echo "$VERSION_CODENAME") stable" | tee /etc/apt/sources.list.d/docker.list > /dev/null
    apt-get update
    apt-get install docker-ce docker-ce-cli containerd.io docker-buildx-plugin docker-compose-plugin
    docker ps

  • Conda

    wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh
    bash Miniconda3-latest-Linux-x86_64.sh
    ...
    exec bash

  • Add conda-forge as priority

    conda config --add channels conda-forge

• Requirements

  • Install nextflow
  • Install docker
  • Make sure your system has at least 36 nodes/CPUs

• Clone the repo

  git clone https://github.com/uclanelsonlab/nl-rna-seq_wf.git

• Run the pipeline for your sample, it expects the FASTQ files to be at s3://ucla-rare-diseases/UCLA-UDN/rnaseq/fastq to download

  cd nl-rna-seq_wf/
  chmod u+x -R modules/
  nextflow run main.nf --sample_name SH1311-P-muscle --proband SH1311-P --library SN_7RNA_S-24-0479_XA044

• Check if you have your outputs on S3:

  aws s3 ls s3://ucla-rare-diseases/UCLA-UDN/Analysis/UDN_cases/ --recursive | grep SH1311-P-muscle

• The expected outputs:

  • flagstat_rrna: Ribosomal contamination stats for human_rRNA_strict.fasta (*.rrna.flagstat.txt)
  • flagstat_globinrna: Ribosomal contamination stats for human_globinRNA.fa (*.globinrna.flagstat.txt)
  • reads_gene: STAR's tab output (*.ReadsPerGene.out.tab.gz)
  • reads_gene_log: STAR's log output (*.ReadsPerGene.log.out)
  • final_log: STAR's final log (*.Log.final.out)
  • sj_tab: STAR's junctions output (*.SJ.out.tab.gz)
  • sj_tab_gz: Home made script output of junctions recreated from STAR's output (*.bam2SJ.out.tab.gz)
  • all_rare_junctions: Home made script output of all junctions in TSV (*_rare_junctions_all.tsv)
  • rare_junctions: Home made script output of junctions in spreadsheet (*_rare_junctions_filtered.xlsx)
  • gene_counts: Subreads featureCounts output (*.gene_id.exon.ct)
  • gene_counts_short: Subreads featureCounts output (*.gene_id.exon.ct.short.txt)
  • gene_counts_summary: Subreads featureCounts output (*.gene_id.exon.ct.summary)
  • rna_cram: CRAM aligned file output from STAR (*.hg19_rna.normal.cram)
  • rna_crai: CRAM's index aligned file output from STAR (*.hg19_rna.normal.cram.crai)