bulk RNAseq

Fast RNAseq pipeline

Here we show how to run a NextFlow pipeline for RNAseq from species with well annotated genes, like human or mouse.

Raw data

This is an example using a public dataset.

We got the following links from sra-explore, a web-based app that help you to find public datasets and provides a script to download the data.

We use the following files:

#!/usr/bin/env bash
curl -L ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR307/SRR307897/SRR307897_1.fastq.gz -o SRR307897_GSM758559_CshlLong_RnaSeq_GM12878_cell_longPolyA_1.fastq.gz
curl -L ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR307/SRR307897/SRR307897_2.fastq.gz -o SRR307897_GSM758559_CshlLong_RnaSeq_GM12878_cell_longPolyA_2.fastq.gz
curl -L ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR307/SRR307912/SRR307912_1.fastq.gz -o SRR307912_GSM758566_CshlLong_RnaSeq_H1-hESC_cell_longPolyA_1.fastq.gz
curl -L ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR307/SRR307912/SRR307912_2.fastq.gz -o SRR307912_GSM758566_CshlLong_RnaSeq_H1-hESC_cell_longPolyA_2.fastq.gz
  • create a folder raw
  • type cd raw
  • copy that into a file called download.sh
  • type bash download.sh.

Wait until is finished, then come back to parent folder cd ...

Annotation

We need the transcriptome fasta file as well:

wget ftp://ftp.ensembl.org/pub/release-96/fasta/homo_sapiens/cdna/Homo_sapiens.GRCh38.cdna.all.fa.gz

Environment

If you are in the PILM-cluster, follow this instructions: [PRIVATE PILM]

If you want to try from scratch, follow these instructions to install miniconda, and then type:

  • conda -n create -y nf-fastrnaseq nextflow salmon multiqc fastqc
  • conda activate nf-fastrnaseq

Get the pipeline

If you are in the PILM-cluster, follow this instructions: PRIVATE PILM

If you want to try from scratch, download the pipeline from GitHub: PUBLIC PILM.

Now that you know where the file paired.nf is, you can run the following command to get the pipeline working:

nextflow run paired.nf -w working -name tiny -with-report all-report -with-timeline all-time --outdir results -qs 6 --reads "raw/*{1,2}.fastq.gz" --transcriptopme Homo_sapiens.GRCh38.cdna.all.fa.gz

Updated on 29 Dec 2018