Light-weight Snakemake workflow for preprocessing and statistical analysis of RNA-seq data

public 1yr ago Version: v1.0 0 bookmarks

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ARMOR ( A utomated R eproducible MO dular R NA-seq) is a Snakemake workflow , aimed at performing a typical RNA-seq workflow in a reproducible, automated, and partially contained manner. It is implemented such that alternative or similar analysis can be added or removed.

ARMOR consists of a Snakefile , a conda environment file ( envs/environment.yaml ) a configuration file ( config.yaml ) and a set of R scripts, to perform quality control, preprocessing and differential expression analysis of RNA-seq data. The output can be combined with the iSEE R package to generate a shiny application for browsing and sharing the results.

By default, the pipeline performs all the steps shown in the diagram below. However, you can turn off any combination of the light-colored steps (e.g STAR alignment or DRIMSeq analysis) in the config.yaml file.

Advanced use : If you prefer other software to run one of the outlined steps (e.g. DESeq2 over edgeR , or kallisto over Salmon ), you can use the software of your preference provided you have your own script(s), and change some lines within the Snakefile . If you think your "custom rule" might be of use to a broader audience, let us know by opening an issue.

Using the ARMOR workflow

Assuming that snakemake and conda are installed (and your system has the necessary libraries to compile R packages), you can use the following commands on a test dataset:

git clone https://github.com/csoneson/ARMOR.git
cd ARMOR && snakemake --use-conda

To use the ARMOR workflow on your own data, follow the steps outlined in the wiki .

Workflow graph

DAG
Blue circles are rules run in R , orange circles from software called as shell commands. Dashed lines and light-colored circles are optional rules, controlled in config.yaml

Contributors

Current contributors include:

Code Snippets

shell:
	'''{params.Rbin} CMD BATCH --no-restore --no-save "--args outtxt='{output}' ncores='{params.ncores}' annotation='{params.flag}' organism='{params.organism}'" {input.script} {log}'''

SnakeMake From line 97 of master/Snakefile

shell:
	'''{params.Rbin} CMD BATCH --no-restore --no-save "--args Routdir='{params.Routdir}' outtxt='{params.outtxt}'" {params.script} {log}'''

SnakeMake From line 136 of master/Snakefile

shell:
	"echo -n 'ARMOR version ' && cat version; "
	"salmon --version; trim_galore --version; "
	"echo -n 'cutadapt ' && cutadapt --version; "
	"fastqc --version; STAR --version; samtools --version; multiqc --version; "
	"bedtools --version"

SnakeMake SAMtools FastQC MultiQC BEDTools STAR Cutadapt Trim_Galore Salmon From line 147 of master/Snakefile

	shell:
	  """
	  if [ {params.anno} == "Gencode" ]; then
      echo 'Salmon version:\n' > {log}; salmon --version >> {log};
  	  salmon index -t {input.txome} -i {params.salmonoutdir} --gencode {params.salmonextraparams}

    else
  	  echo 'Salmon version:\n' > {log}; salmon --version >> {log};
      salmon index -t {input.txome} -i {params.salmonoutdir} {params.salmonextraparams}
    fi
    """

SnakeMake Salmon From line 173 of master/Snakefile

shell:
	'''{params.Rbin} CMD BATCH --no-restore --no-save "--args transcriptfasta='{input.txome}' salmonidx='{input.salmonidx}' gtf='{input.gtf}' annotation='{params.flag}' organism='{params.organism}' release='{params.release}' build='{params.build}' output='{output}'" {input.script} {log}'''

SnakeMake From line 207 of master/Snakefile

shell:
	"echo 'STAR version:\n' > {log}; STAR --version >> {log}; "
	"STAR --runMode genomeGenerate --runThreadN {threads} --genomeDir {params.STARindex} "
	"--genomeFastaFiles {input.genome} --sjdbGTFfile {input.gtf} --sjdbOverhang {params.readlength} "
	"{params.starextraparams}"

SnakeMake STAR From line 230 of master/Snakefile

shell:
	"echo 'FastQC version:\n' > {log}; fastqc --version >> {log}; "
	"fastqc -o {params.FastQC} -t {threads} {input.fastq}"

SnakeMake FastQC From line 255 of master/Snakefile

shell:
	"echo 'FastQC version:\n' > {log}; fastqc --version >> {log}; "
	"fastqc -o {params.FastQC} -t {threads} {input.fastq}"

SnakeMake FastQC From line 275 of master/Snakefile

shell:
	"echo 'MultiQC version:\n' > {log}; multiqc --version >> {log}; "
	"multiqc {params.inputdirs} -f -o {params.MultiQCdir}"

SnakeMake MultiQC From line 324 of master/Snakefile

shell:
	"echo 'TrimGalore! version:\n' > {log}; trim_galore --version >> {log}; "
	"trim_galore -q 20 --phred33 --length 20 -o {params.FASTQtrimmeddir} --path_to_cutadapt cutadapt {input.fastq}"

SnakeMake Cutadapt Trim_Galore From line 346 of master/Snakefile

shell:
	"echo 'TrimGalore! version:\n' > {log}; trim_galore --version >> {log}; "
	"trim_galore -q 20 --phred33 --length 20 -o {params.FASTQtrimmeddir} --path_to_cutadapt cutadapt "
	"--paired {input.fastq1} {input.fastq2}"

SnakeMake Cutadapt Trim_Galore From line 365 of master/Snakefile

shell:
	"echo 'Salmon version:\n' > {log}; salmon --version >> {log}; "
	"salmon quant -i {params.salmonindex} -l A -r {input.fastq} "
	"-o {params.salmondir}/{wildcards.sample} -p {threads} {params.salmonextraparams}"

SnakeMake Quant Salmon From line 392 of master/Snakefile

shell:
	"echo 'Salmon version:\n' > {log}; salmon --version >> {log}; "
	"salmon quant -i {params.salmonindex} -l A -1 {input.fastq1} -2 {input.fastq2} "
	"-o {params.salmondir}/{wildcards.sample} -p {threads} {params.salmonextraparams}"

SnakeMake Quant Salmon From line 416 of master/Snakefile

shell:
	"echo 'STAR version:\n' > {log}; STAR --version >> {log}; "
	"STAR --genomeDir {params.STARindex} --readFilesIn {input.fastq} "
	"--runThreadN {threads} --outFileNamePrefix {params.STARdir}/{wildcards.sample}/{wildcards.sample}_ "
	"--outSAMtype BAM SortedByCoordinate --readFilesCommand gunzip -c "
	"{params.starextraparams}"

SnakeMake STAR From line 443 of master/Snakefile

shell:
	"echo 'STAR version:\n' > {log}; STAR --version >> {log}; "
	"STAR --genomeDir {params.STARindex} --readFilesIn {input.fastq1} {input.fastq2} "
	"--runThreadN {threads} --outFileNamePrefix {params.STARdir}/{wildcards.sample}/{wildcards.sample}_ "
	"--outSAMtype BAM SortedByCoordinate --readFilesCommand gunzip -c "
	"{params.starextraparams}"

SnakeMake STAR From line 469 of master/Snakefile

shell:
	"echo 'samtools version:\n' > {log}; samtools --version >> {log}; "
	"samtools index {input.bam}"

SnakeMake SAMtools From line 488 of master/Snakefile

shell:
	"echo 'bedtools version:\n' > {log}; bedtools --version >> {log}; "
	"bedtools genomecov -split -ibam {input.bam} -bg | LC_COLLATE=C sort -k1,1 -k2,2n > "
	"{params.STARbigwigdir}/{wildcards.sample}_Aligned.sortedByCoord.out.bedGraph; "
	"bedGraphToBigWig {params.STARbigwigdir}/{wildcards.sample}_Aligned.sortedByCoord.out.bedGraph "
	"{input.chrl} {output}; rm -f {params.STARbigwigdir}/{wildcards.sample}_Aligned.sortedByCoord.out.bedGraph"

SnakeMake BEDTools bedGraphToBigWig From line 507 of master/Snakefile

shell:
	'''{params.Rbin} CMD BATCH --no-restore --no-save "--args salmondir='{params.salmondir}' json='{input.json}' metafile='{input.metatxt}' outrds='{output}' annotation='{params.flag}' organism='{params.organism}'" {input.script} {log}'''

SnakeMake From line 539 of master/Snakefile

shell:
    '''{params.Rbin} CMD BATCH --no-restore --no-save "--args metafile='{params.metatxt}' design='{params.design}' contrast='{params.contrast}' outFile='{output}' gtf='{params.gtf}' genome='{params.genome}' fastqdir='{params.fastqdir}' fqsuffix='{params.fqsuffix}' fqext1='{params.fqext1}' fqext2='{params.fqext2}' txome='{params.txome}' run_camera='{params.run_camera}' organism='{params.organism}' {params.genesets} annotation='{params.annotation}'" {input.script} {log};
    cat {output}
    '''

SnakeMake From line 582 of master/Snakefile

shell:
	'''{params.Rbin} CMD BATCH --no-restore --no-save "--args se='{input.rds}' organism='{params.organism}' design='{params.design}' contrast='{params.contrast}' {params.genesets} rmdtemplate='{input.template}' outputdir='{params.directory}' outputfile='edgeR_dge.html'" {input.script} {log}'''

SnakeMake From line 613 of master/Snakefile

shell:
	'''{params.Rbin} CMD BATCH --no-restore --no-save "--args se='{input.rds}' design='{params.design}' contrast='{params.contrast}' ncores='{params.ncores}' rmdtemplate='{input.template}' outputdir='{params.directory}' outputfile='DRIMSeq_dtu.html'" {input.script} {log}'''

SnakeMake From line 644 of master/Snakefile

shell:
	'''{params.Rbin} CMD BATCH --no-restore --no-save "--args se='{input.rds}' gtffile='{input.gtf}' rmdtemplate='{input.template}' outputfile='prepare_shiny.html' {params.p}" {input.script} {log}'''