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RNASeq Analysis Worklow

This workflow can be run in with the whole genome, or with a specific list of genes of interest.

Initiate Nextflow Workflows

Required Tools

This pipeline uses Nextflow, a bioinformatics workflow tool and Singularity, a containerization tool.

Make sure both tools rae installed before running this pipeline. If running on a HPC cluster then load required modules.

module load nextflow/20.01.0 singularity/3.5.3

RNA Design File

The design file must named design.txt and be in tab seperated format for the workflows. All RNA workflows can be run usin the same design file format. You can run in single-end mode with blank cells in the FqR2 column.

SampleID SampleGroup SubjectID SampleName FqR1 FqR2
SRR1551069 monocytes 53 53_Monocytes SRR1551069_1.fastq.gz SRR1551069_2.fastq.gz
SRR1551068 neutrophils 53 53_Neutrophils SRR1551068_1.fastq.gz SRR1551068_2.fastq.gz
SRR1551055 monocytes 21 21_Monocytes SRR1551055_1.fastq.gz SRR1551055_2.fastq.gz
SRR1551054 neutrophils 21 21_Neutrophils SRR1551054_1.fastq.gz SRR1551054_2.fastq.gz
SRR1551048 monocytes 20 20_Monocytes SRR1551048_1.fastq.gz SRR1551048_2.fastq.gz
SRR1551047 neutrophils 20 20_Neutrophils SRR1551047_1.fastq.gz SRR1551047_2.fastq.gz
SRR1550987 monocytes 44 44_Monocytes SRR1550987_1.fastq.gz SRR1550987_2.fastq.gz
SRR1550986 neutrophils 44 44_Neutrophils SRR1550986_1.fastq.gz SRR1550986_2.fastq.gz

RNA Parameters

  • --input
    • directory containing the design file and fastq files
    • default is set to '${basedir}/fastq'
    • eg: --input '/project/shared/bicf_workflow_ref/workflow_testdata/rnaseq/fastq'
  • --output
    • directory for the analysis output
    • default is set to '${basedir}/analysis'
    • eg: --output '${basedir}/output'
  • --genome
    • directory containing all reference files for the various tools. This includes the genome.fa, gencode.gtf, genenames.txt, ect.
    • default is set for use on UTSW BioHPC.
    • eg: --genome '/project/shared/bicf_workflow_ref/human/grch38_cloud/rnaref'
  • --stranded
    • option for -s flag in featurecount used in geneabundance calculations
    • default is set to '0'
    • eg: --stranded '0'
  • --pairs
    • select either 'pe' (paired-end) or 'se' (single-end) based on read inputs. Select 'pe' when both R1 and R2 are present. If only R1, then select 'se'.
    • default is set to 'pe'
    • eg: --pairs 'pe'
  • --align
    • select the algorithm/tool for alignment from 'hisat' or 'star'
    • default is set to 'hisat'
    • eg: --align 'hisat'
  • --markdups
    • select either picard (Mark Duplicates) or null (do not Mark Duplicates)
    • default is set to 'picard'
    • eg: --align 'picard'

RNA Run Workflow Testing

Human PE

module load nextflow/20.01.0 singularity/3.5.3
base=$repoClonedDirectory
datadir='/project/shared/bicf_workflow_ref/workflow_testdata/rnaseq'

nextflow -C ${base}/workflow/configs/nextflow.config run ${base}/workflow/main.nf --design  ${datadir}/design.rnaseq.txt --input ${datadir} --output analysis

Mouse SE

module load nextflow/20.01.0 singularity/3.5.3
base=$repoClonedDirectory
datadir='/project/shared/bicf_workflow_ref/workflow_testdata/rnaseq'

nextflow -C ${base}/workflow/configs/nextflow.config run -with-dag flowchart.png -with-timeline mouse_timeline.html -with-report mouse_report.html ${base}/workflow/main.nf --design  ${datadir}/mouse_se.design.txt --input ${datadir} --pairs se --output analysis