Setting up a portable RNA-Seq pipeline for CBIO

# Wiki

Pertinent points for setup of NGI-RNAseq pipeline on UCT hex

*Main pipeline source code is at https://github.com/SciLifeLab/NGI-RNAseq

*Currently used pipeline source code however is at https://github.com/ewels/nf-core-RNAseq (this was kindly customized for us by the authors for easy configuration on hex and includes a config file 'uct_hex.config') so that this 'profile' can be called as a flag on the command line (further customization may be required following testing).

*Additional overview on NGI-RNAseq pipeline at https://scilifelab.github.io/courses/rnaseq/1711/slides/pipeline.pdf 

*Software requirements will be met using Singularity - the image has been downloaded and stored here /scratch/DB/bio/singularity-containers/ngi-rnaseq.img using the command: singularity pull --name ngi-rnaseq.img docker://scilifelab/ngi-rnaseq 

Note that the singularity image path has been specified in the aforementioned uct_hex.config file so no need to specify on job submission.

* First test: nextflow run SciLifeLab/NGI-RNAseq --help | ewels/nf-core-RNAseq

* Reference genomes and annotation files should be placed in /scratch/DB/bio/rna-seq (iGenomes GRCh37 has been pulled to /scratch/DB/bio/rna-seq/references/ from https://ewels.github.io/AWS-iGenomes/) and this location is referenced in our custom uct_hex.config file under the parameter igenomes_base = '/scratch/DB/bio/rna-seq/references'

In order to download /scratch/DB/bio/rna-seq/references/ from https://ewels.github.io/AWS-iGenomes/ Andrew had to install aws tools on hex, which should be loaded as follows:

module load python/anaconda-python-2.7

aws configure

You may then be prompted for a key and a security key (you need to register an aws account to get this, which is free but you still need to specify credit card details – see https://console.aws.amazon.com)

For reproducibility please specify the pipeline version used when running the pipeline using the -r flag (e.g. –r 1.3.1)

The basic run will look something like this:

nextflow run ewels/nf-core-RNAseq --reads '/researchdata/fhgfs/katie/NGI-RNAseq-test/*_R{1,2}.fastq.gz' --genome GRCh37 --outdir /researchdata/fhgfs/katie/NGI-RNAseq-test/nextflow-output -profile uct_hex --email katie.viljoen@uct.ac.za

Human RNAseq test data to be used: http://h3data.cbio.uct.ac.za/assessments/RNASeq/practice/ (downloaded to /researchdata/fhgfs/katie/NGI-RNAseq-test)

First test run:

qsub -I -q UCTlong -d pwd

nextflow run ewels/nf-core-RNAseq --reads '/researchdata/fhgfs/katie/NGI-RNAseq-test/*_R{1,2}.fastq.gz' --genome GRCh37 --outdir /researchdata/fhgfs/katie/NGI-RNAseq-test/nextflow-output -profile uct_hex --email katie.viljoen@uct.ac.za