snakemake workflow for bulk RNA-seq workflow using STAR-edgeR

public 1yr ago Version: v1.0.0 0 bookmarks

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Bulk RNAseq Workflow
- Usage
  - Step 1: Configure the workflow
  - Step 2: Test and run the workflow

Usage

NOTE this workflow is optimized for the HPC @ Van Andel Institute.

Step 1: Configure the workflow

Move your sequencing reads to raw_data/
Modify the config and samplesheet:
- config/samplesheet/units.tsv; To make a template based in the files in raw_data/ , run ./make_units_template.sh .
  - sample - ID of biological sample; Must be unique.
  - group - Experimental group
  - fq1 - name of read1 fastq
  - fq2 * - name of read2 fastq
- config/config.yaml

Step 2: Test and run the workflow

Test your configuration by performing a dry-run via

snakemake -npr

Execute from within your project directory as a SLURM job.

sbatch bin/run_snake.sh

Code Snippets

shell:
    """
    {params.cat_or_symlink} {output}
    """

SnakeMake From line 25 of rules/qc.smk

shell:
    """
    fastq_screen --threads {threads} --outdir results/fastq_screen/ {input} 
    """

SnakeMake From line 44 of rules/qc.smk

shell:
    """
    fastqc --outdir {params.outdir} {input}
    """

SnakeMake FastQC From line 68 of rules/qc.smk

shell:
    """
    seqtk sample -s 100 {input} {params.num_subsamp} | gzip -c > {output}
    """

SnakeMake seqtk From line 87 of rules/qc.smk

shell:
    """
    sortmerna --threads {threads} {params.fastqs} --workdir {output}  \
    --idx-dir {params.idx_dir}  \
    --ref {params.rfam5s}  \
    --ref {params.rfam5_8s}  \
    --ref {params.silva_arc_16s}  \
    --ref {params.silva_arc_23s}  \
    --ref {params.silva_bac_16s}  \
    --ref {params.silva_bac_23s}  \
    --ref {params.silva_euk_18s}  \
    --ref {params.silva_euk_28s}
    """

SnakeMake From line 124 of rules/qc.smk

shell:
    """
    multiqc -f {params} \
    -o results/multiqc \
    --ignore '*._STARpass1/*' \
    -n multiqc_report.html \
    --cl-config 'max_table_rows: 999999' 
    """

SnakeMake MultiQC From line 180 of rules/qc.smk

shell:
    """
    ln -sr {input} {output} 
    """

SnakeMake From line 26 of rules/RNAseq.smk

shell:
    """
    trim_galore \
    --paired \
    {input} \
    --output_dir {params.outdir} \
    --cores {threads} \
    -q 20 \
    --fastqc
    """

SnakeMake Trim_Galore From line 65 of rules/RNAseq.smk

shell:
    """
    trim_galore \
    {input} \
    --output_dir {params.outdir} \
    --cores {threads} \
    -q 20 \
    --fastqc 
    """

SnakeMake Trim_Galore From line 95 of rules/RNAseq.smk

shell:
    """
    STAR \
    --runThreadN {threads} \
    --genomeDir {params.index} \
    --readFilesIn {params.in_fastqs} \
    --outSAMattrRGline {params.read_groups} \
    --twopassMode Basic \
    --readFilesCommand zcat \
    --outSAMtype BAM Unsorted \
    --outFileNamePrefix {params.outprefix} \
    --quantMode GeneCounts \
    --outStd Log 

    samtools sort -@ {threads} -o {output.bam} {output.unsorted_bam}
    samtools index {output.bam}
    """

SnakeMake SAMtools STAR From line 183 of rules/RNAseq.smk

shell:
    """
    salmon quant \
            -p {threads} \
            -l A \
            -i {input.index} \
            {params.reads} \
            --validateMappings \
            -o {params.outdir}
    """

SnakeMake Quant Salmon From line 219 of rules/RNAseq.smk

shell:
    """
    Rscript --vanilla workflow/scripts/make_sce.R {params.gtf} {params.orgdb} {output.se} {output.sce} {output.sizeFactors}
    """

SnakeMake From line 249 of rules/RNAseq.smk

shell:
    """
    gatk --java-options "-Xms8g -Xmx{resources.mem_gb}g -Djava.io.tmpdir=./tmp" \
        MarkDuplicatesSpark \
        --spark-master local[{threads}] \
        -I {input} \
        -O {output.bam} \
        -M {output.metrics} \
        --conf spark.executor.cores={threads} \
        --conf spark.local.dir=./tmp \
        --conf spark.driver.memory=6g \
        --conf spark.executor.memory=5g

    """

SnakeMake gatk From line 18 of rules/variants.smk

shell:
    """
    gatk --java-options "-Xms8g -Xmx{resources.mem_gb}g -Djava.io.tmpdir=./tmp" \
    SplitNCigarReads \
    -R {params.ref_fasta} \
    -I {input} \
    -O {output} 
    """

SnakeMake gatk From line 48 of rules/variants.smk

shell:
    """
    gatk --java-options "-Xms8g -Xmx{resources.mem_gb}g -Djava.io.tmpdir=./tmp" \
    HaplotypeCaller \
    -R {params.ref_fasta} \
    -I {input.bam} \
    -O {output} \
    -ERC GVCF \
    -dont-use-soft-clipped-bases \
    --native-pair-hmm-threads {threads} \
    --standard-min-confidence-threshold-for-calling 20 \
    {params.dbsnp} \
    {params.contigs}
    """

SnakeMake gatk From line 75 of rules/variants.smk

shell:
    """
    gatk --java-options "-Xms8g -Xmx{resources.mem_gb}g -Djava.io.tmpdir=./tmp" \
    GenomicsDBImport \
    {params.sample_gvcfs} \
    --reader-threads {threads} \
    --genomicsdb-workspace-path {output.genomicsdb} \
    {params.contigs}
    """

SnakeMake gatk From line 108 of rules/variants.smk

shell:
    """
    gatk --java-options "-Xms8g -Xmx{resources.mem_gb}g -Djava.io.tmpdir=./tmp" \
    GenotypeGVCFs \
    -R {params.ref_fasta} \
    -V gendb://{params.genomicsdb} \
    -O {output.vcf}
    """

SnakeMake gatk From line 134 of rules/variants.smk

shell:
    """
    gatk --java-options "-Xms8g -Xmx{resources.mem_gb}g -Djava.io.tmpdir=./tmp" \
    SortVcf \
    -I {input.vcf} \
    -O {output.sorted_vcf} \
    -SD {params.dictionary} 
    """

SnakeMake gatk From line 161 of rules/variants.smk

shell:
    """
    gatk --java-options "-Xms8g -Xmx{resources.mem_gb}g -Djava.io.tmpdir=./tmp" \
    MergeVcfs \
    {params.in_vcfs} \
    --SEQUENCE_DICTIONARY {params.dictionary} \
    --OUTPUT {output.raw} 

    vt peek -r {params.ref_fasta} {output.raw} 2> {output.vt_peek_raw} 
    """

SnakeMake gatk vt From line 193 of rules/variants.smk

shell:
    """
    gatk --java-options "-Xms8g -Xmx{resources.mem_gb}g -Djava.io.tmpdir=./tmp" \
    SelectVariants \
    -R {params.ref_fasta} \
    -V {input} \
    --select-type-to-include {wildcards.var_type} \
    -O {output.raw}

    echo "SelectVariants 1 done." 
    echo "SelectVariants 1 done." 1>&2

    gatk --java-options "-Xms8g -Xmx{resources.mem_gb}g -Djava.io.tmpdir=./tmp" \
    VariantFiltration \
    --R {params.ref_fasta} \
    --V {output.raw} \
    {params.filt_params} \
    -O {output.filt} 

    echo "VariantFiltration done." 
    echo "VariantFiltration done." 1>&2

    gatk --java-options "-Xms8g -Xmx{resources.mem_gb}g -Djava.io.tmpdir=./tmp" \
    SelectVariants \
    -R {params.ref_fasta} \
    -V {output.filt} \
    --exclude-filtered \
    -O {output.pass_only} 

    echo "SelectVariants 2 done." 
    echo "SelectVariants 2 done." 1>&2

    vt peek -r {params.ref_fasta} {output.pass_only} 2> {output.vt_peek_pass} 
    """

SnakeMake gatk vt From line 246 of rules/variants.smk

shell:
    """
    gatk --java-options "-Xms8g -Xmx{resources.mem_gb}g -Djava.io.tmpdir=./tmp" \
    BaseRecalibrator \
    -R {params.ref_fasta} \
    -I {input.bam} \
    --known-sites {input.known_snps_vcf} \
    --known-sites {input.known_indels_vcf} \
    -O {output.recal_table} 

    echo "BaseRecalibrator done." 
    echo "BaseRecalibrator done." 1>&2

    gatk --java-options "-Xms8g -Xmx{resources.mem_gb}g -Djava.io.tmpdir=./tmp" \
    ApplyBQSR \
    -R {params.ref_fasta} \
    -I {input.bam} \
    -bqsr {output.recal_table} \
    -O {output.recal_bam}

    echo "ApplyBQSR done." 
    echo "ApplyBQSR done." 1>&2

    """

SnakeMake gatk From line 302 of rules/variants.smk

shell:
    """
    java -Xms8g -Xmx{resources.mem_gb}g -Djava.io.tmpdir=./tmp -jar $SNPEFF/snpEff.jar eff \
    -v \
    -canon \
    -onlyProtein \
    -stats {output.html_canon} \
    {params.db_id} \
    {input} | \
    bgzip > {output.vcf_canon}

    tabix {output.vcf_canon}


    java -Xms8g -Xmx{resources.mem_gb}g -Djava.io.tmpdir=./tmp -jar $SNPEFF/snpEff.jar eff \
    -v \
    -onlyProtein \
    -stats {output.html} \
    {params.db_id} \
    {input} | \
    bgzip > {output.vcf}

    tabix {output.vcf} 
    """

SnakeMake tabix From line 348 of rules/variants.smk

shell:
    """
    ln -sr {input.vcf} {output.symlink_vcf}
    ln -sr {params.rmd} {output.symlink_rmd}

    cd {params.wd}

    Rscript --vanilla -e "rmarkdown::render('snprelate.Rmd', params = list(in_vcf = '{params.in_vcf}', outdir = '{params.outdir}'))"
    """

SnakeMake From line 394 of rules/variants.smk

shell:
    """
    bamCoverage -b {input.bam} -o {output.bw}  --minMappingQuality 30  \
            --normalizeUsing "None" -p {threads} --binSize 10 \
            --scaleFactor {params.scale_factor} {params.strand}
    """

SnakeMake DeepTools From line 32 of rules/visualisation.smk

shell:
    """
    bigwigAverage -b {input} --binSize 10 -p {threads} -o {output.bw} -of "bigwig"
    """

SnakeMake From line 54 of rules/visualisation.smk

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Created: 1yr ago

Updated: 1yr ago

Maitainers: public

URL: https://github.com/vari-bbc/rnaseq_workflow

Name: rnaseq_workflow

Version: v1.0.0

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License: GNU General Public License v3.0

Keywords:

gatk tabix DeepTools FastQC MultiQC Quant Salmon SAMtools seqtk Snakemake SortMeRNA STAR vt Trim_Galore

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