HRIBO: High-throughput annotation by Ribo-seq workflow for analyzing bacterial Ribo-seq data

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We present HRIBO (High-throughput annotation by Ribo-seq), a workflow to enable reproducible and high-throughput analysis of bacterial Ribo-seq data. The workflow performs all required pre-processing steps and quality control. Importantly, HRIBO outputs annotation-independent ORF predictions based on two complementary prokaryotic-focused tools, and integrates them with additional computed features. This facilitates both the rapid discovery of ORFs and their prioritization for functional characterization.

For a detailed description of this workflow, the installation, usage and examples, please refer to the ReadTheDocs documentation .

HRIBO installs all dependencies via conda . Once you have conda installed simply type: 

 conda create -c bioconda -c conda-forge -n snakemake snakemake source activate snakemake

Basic usage

The retrieval of input files and running the workflow locally and on a server cluster via a queuing system is working as follows. Create a project directory and change into it: 

 mkdir project cd project

Retrieve the HRIBO from GitHub: 

 git clone git@github.com:gelhausr/HRIBO.git

The workflow requires a genome sequence (fasta), an annotation file (gtf) and the sequencing results files (fastq). We recommend retrieving both the genome and the annotation files from Ensembl Genomes . Copy the genome and the annotation file into the project folder, decompress them and name them genome.fa and annotation.gtf.

Create a folder fastq and copy your compressed fastq.gz files into the fastq folder.

Please copy the template of the sample sheet and the config file into the HRIBO folder. 

 cp HRIBO/templates/config.yaml HRIBO/ cp HRIBO/templates/samples.tsv HRIBO/

Customize the config.yaml with the used adapter sequence and optionally with the path to a precomputed STAR genome index. For correct removal of reads mapping to ribosomal genes please specify the taxonomic group of the used organism (Eukarya, Bacteria, Archea). Now edit the sample sheet corresponding to your project, using one line per sequencing result, stating the used method (RIBO for ribosome profiling, RNA for RNA-seq), the applied condition (e.g. A, B, CTRL, TREAT), the replicate (e.g. 1, 2,..) and the filename. Following is an example:

method condition replicate fastqFile
RIBO A 1 "fastq/FP-ctrl-1-2.fastq.gz"
RIBO B 1 "fastq/FP-treat-1-2.fastq.gz"
RNA A 1 "fastq/Total-ctrl-1-2.fastq.gz"
RNA B 1 "fastq/Total-treat-1-2.fastq.gz"

Now you can start your workflow.

Run Snakemake locally: 

 snakemake --use-conda -s HRIBO/Snakefile --configfile HRIBO/config.yaml --directory ${PWD} -j 20 --latency-wait 60

Run Snakemake on the cluster:

Edit cluster.yaml according to your queuing system and cluster hardware. The following example works for Grid Engine: 

 snakemake --use-conda -s HRIBO/Snakefile --configfile HRIBO/config.yaml --directory ${PWD} -j 20 --cluster-config HRIBO/cluster.yaml --cluster "qsub -N {cluster.jobname} -cwd -q {cluster.qname} -pe {cluster.parallelenvironment} -l {cluster.memory} -o {cluster.logoutputdir} -e {cluster.erroroutputdir} -j {cluster.joinlogs} -M <email>" --latency-wait 60

Once the workflow has finished you can request a automatically generated report.html file with the following command: 

 snakemake --report report.html

Code Snippets

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shell:
    "mkdir -p auxiliary; HRIBO/scripts/enrich_annotation.py -a {input.annotation} -o {output}"
SnakeMake From line 21 of rules/auxiliary.smk 
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    """
    mkdir -p auxiliary;
    awk -F'\\t' '/^[^#]/ {{printf "%s\\t%s\\t%s\\t%s\\t%s\\t%s\\t%s\\t%s\\tID=uid%s;\\n", $1, $2, $3, $4, $5, $6, $7, $8, NR-1}}' {input} > {output}
    """
SnakeMake From line 32 of rules/auxiliary.smk 
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    "mkdir -p auxiliary; HRIBO/scripts/samples_to_xlsx.py -i {input} -o {output}"
SnakeMake From line 46 of rules/auxiliary.smk 
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shell:
    "mkdir -p auxiliary; HRIBO/scripts/generate_excel.py -t {input.total} -r {input.reads} -g {input.genome} -o {output}"
SnakeMake From line 59 of rules/auxiliary.smk 
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shell:
    "mkdir -p auxiliary; HRIBO/scripts/generate_excel.py -t {input.total} -r {input.reads} -g {input.genome} -o {output}"
SnakeMake From line 72 of rules/auxiliary.smk 
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shell:
    "mkdir -p auxiliary; HRIBO/scripts/generate_excel_reparation.py -t {input.total} -r {input.reads} -g {input.genome} -o {output}"
SnakeMake From line 85 of rules/auxiliary.smk 
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shell:
    "mkdir -p auxiliary; HRIBO/scripts/generate_read_table.py -r {input.reads} -t {input.total} -o {output}"
SnakeMake From line 97 of rules/auxiliary.smk 
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shell:
    "mkdir -p auxiliary; HRIBO/scripts/generate_read_table.py -r {input.reads} -t {input.total} -o {output}"
SnakeMake From line 109 of rules/auxiliary.smk 
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shell:
    """
    mkdir -p auxiliary;
    if [ -z {params.contrasts} ]
    then
        HRIBO/scripts/generate_excel_overview.py -a {input.annotation} -g {input.genome} -t {input.totalreads} --mapped_reads_reparation {input.reparation} -o {output}
    else
        HRIBO/scripts/generate_excel_overview.py -a {input.annotation} -c {params.contrasts} -g {input.genome} -t {input.totalreads} --mapped_reads_reparation {input.reparation} -o {output}
    fi
    """
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shell:
    """
    mkdir -p auxiliary;
    if [ -z {params.contrasts} ]
    then
        HRIBO/scripts/generate_excel_overview.py -a {input.annotation} -g {input.genome} -t {input.totalreads} --mapped_reads_deepribo {input.deepribo} --mapped_reads_reparation {input.reparation} -o {output}
    else
        HRIBO/scripts/generate_excel_overview.py -a {input.annotation} -c {params.contrasts}  -g {input.genome} -t {input.totalreads} --mapped_reads_deepribo {input.deepribo} --mapped_reads_reparation {input.reparation} -o {output}
    fi
    """
SnakeMake From line 150 of rules/auxiliary.smk 
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shell:
    """
    if [ -z {params.contrasts} ]
    then
        mkdir -p auxiliary; HRIBO/scripts/generate_excel_overview.py -a {input.annotation} -g {input.genome} --xtail {input.xtail} --deltate {input.deltate} --riborex {input.riborex} -t {input.totalreads} --mapped_reads_reparation {input.reparation} -o {output}
    else
        mkdir -p auxiliary; HRIBO/scripts/generate_excel_overview.py -c {params.contrasts} -a {input.annotation} -g {input.genome} --xtail {input.xtail} --deltate {input.deltate} --riborex {input.riborex} -t {input.totalreads} --mapped_reads_reparation {input.reparation} -o {output}
    fi
    """
SnakeMake From line 177 of rules/auxiliary.smk 
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shell:
    """
    if [ -z {params.contrasts} ]
    then
        mkdir -p auxiliary; HRIBO/scripts/generate_excel_overview.py -a {input.annotation} -g {input.genome} --xtail {input.xtail} --deltate {input.deltate} --riborex {input.riborex} -t {input.totalreads} --mapped_reads_deepribo {input.deepribo} --mapped_reads_reparation {input.reparation} -o {output}
    else
        mkdir -p auxiliary; HRIBO/scripts/generate_excel_overview.py -c {params.contrasts} -a {input.annotation} -g {input.genome} --xtail {input.xtail} --deltate {input.deltate} --riborex {input.riborex} -t {input.totalreads} --mapped_reads_deepribo {input.deepribo} --mapped_reads_reparation {input.reparation} -o {output}
    fi
    """
SnakeMake From line 204 of rules/auxiliary.smk 
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shell:
    """
    mkdir -p tracks;
    HRIBO/scripts/concatenate_gff.py {input.reparation_orfs} {input.currentAnnotation} -o {output}
    """
SnakeMake From line 10 of rules/conditionals.smk 
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run:
    shell("mkdir -p deepribo; mv {input} deepribo/DeepRibo_model_v1.pt")
SnakeMake From line 18 of rules/deepribo.smk 
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shell:
    "mkdir -p coverage_deepribo; HRIBO/scripts/coverage_deepribo.py --alignment_file {input.bam} --output_file_prefix coverage_deepribo/{wildcards.condition}-{wildcards.replicate}"
SnakeMake From line 32 of rules/deepribo.smk 
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shell:
    """
    mkdir -p coverage_deepribo
    bedtools genomecov -bg -ibam {input.bam} -strand + > {output.covfwd}
    bedtools genomecov -bg -ibam {input.bam} -strand - > {output.covrev}
    """
SnakeMake BEDTools From line 45 of rules/deepribo.smk 
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shell:
    """
    mkdir -p deepribo/{wildcards.condition}-{wildcards.replicate}/0/;
    mkdir -p deepribo/{wildcards.condition}-{wildcards.replicate}/1/;
    DataParser.py {input.covS} {input.covAS} {input.asiteS} {input.asiteAS} {input.genome} deepribo/{wildcards.condition}-{wildcards.replicate} -g {input.annotation}
    """
SnakeMake From line 65 of rules/deepribo.smk 
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shell:
    "mkdir -p deepribo; Rscript HRIBO/scripts/parameter_estimation.R -f {input} -o {output}"
SnakeMake From line 80 of rules/deepribo.smk 
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shell:
    """
    mkdir -p deepribo;
    DeepRibo.py predict deepribo/ --pred_data {wildcards.condition}-{wildcards.replicate}/ -r {params.rpkm} -c {params.cov} --model {input.model} --dest {output} --num_workers {threads}
    """
SnakeMake From line 96 of rules/deepribo.smk 
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shell:
    "mkdir -p tracks; HRIBO/scripts/create_deepribo_gff.py -c {wildcards.condition} -r {wildcards.replicate} -i {input} -o {output}"
SnakeMake From line 110 of rules/deepribo.smk 
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shell:
    "mkdir -p tracks; HRIBO/scripts/concatenate_gff.py {input} -o {output}"
SnakeMake From line 121 of rules/deepribo.smk 
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shell:
    "mkdir -p tracks; HRIBO/scripts/concatenate_gff.py {input.merged_gff} -o {output}"
SnakeMake From line 132 of rules/deepribo.smk 
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shell:
    "mkdir -p tracks; HRIBO/scripts/merge_duplicates_deepribo.py -i {input.ingff} -o {output.merged} -a {input.annotation}"
SnakeMake From line 145 of rules/deepribo.smk 
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shell:
    "mkdir -p auxiliary; HRIBO/scripts/generate_excel_deepribo.py -t {input.total} -r {input.reads} -g {input.genome} -o {output}"
SnakeMake From line 159 of rules/deepribo.smk 
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shell:
    """
    mkdir -p tracks;
    HRIBO/scripts/concatenate_gff.py {input.deepribo_orfs} {input.reparation_orfs} {input.currentAnnotation} -o {output}
    """
SnakeMake From line 172 of rules/deepribo.smk 
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run:
    if not os.path.exists("contrasts"):
        os.makedirs("contrasts")
    for f in CONTRASTS:
        print(f)
        open(f"contrasts/{f}", 'a').close()
SnakeMake From line 4 of rules/diffex_contrast.smk 
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shell:
    """
    mkdir -p diffex_input/riborex/;
    python3 HRIBO/scripts/prepare_diffex_input.py -r {input.rawreads} -c {wildcards.contrast}  -t riborex -o diffex_input/riborex/
    """
SnakeMake From line 22 of rules/diffex_contrast.smk 
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shell:
    """
    mkdir -p diffex_input/xtail/;
    python3 HRIBO/scripts/prepare_diffex_input.py -r {input.rawreads} -c {wildcards.contrast} -t xtail -o diffex_input/xtail/
    """
SnakeMake From line 40 of rules/diffex_contrast.smk 
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shell:
    """
    mkdir -p deltate;
    HRIBO/scripts/prepare_deltate_input.py -c {params.contrast} -r {input.rawreads} -b bam/ -o {params.out_dir}
    """
SnakeMake From line 31 of rules/diffex_deltate.smk 
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shell:
    """
    mkdir -p deltate;
    touch {output.fcribo}
    touch {output.fcrna}
    touch {output.fcte}
    touch deltate/{params.contrast}/Result_figures.pdf
    DTEG.R {input.ribo} {input.rna} {input.samples} 0 deltate/{params.contrast}/ || true
    cp deltate/{params.contrast}/Result_figures.pdf {output.fig}
    """
SnakeMake From line 55 of rules/diffex_deltate.smk 
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shell:
    """
    python3 HRIBO/scripts/generate_excel_deltate.py -a {input.annotation} -g {input.genome} -i {input.deltate_ribo} -r {input.deltate_rna} -t {input.deltate_te} -o {output.xlsx_sorted} --padj_cutoff {params.padj_cutoff} --log2fc_cutoff {params.log2fc_cutoff}
    """
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shell:
    """
    python3 HRIBO/scripts/merge_differential_expression.py {input.deltate} -o {output} -t deltate
    """
SnakeMake From line 94 of rules/diffex_deltate.smk 
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shell:
    """
    mkdir -p riborex;
    HRIBO/scripts/riborex.R -r {input.ribo} -m {input.rna} -c {input.cv} -x {output.table};
    """
SnakeMake From line 12 of rules/diffex_riborex.smk 
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shell:
    """
    python3 HRIBO/scripts/generate_excel_riborex.py -a {input.annotation} -g {input.genome} -i {input.riborex_out} -o {output.xlsx_sorted} --padj_cutoff {params.padj_cutoff} --log2fc_cutoff {params.log2fc_cutoff}
    """
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shell:
    """
    python3 HRIBO/scripts/merge_differential_expression.py {input.riborex} -o {output} -t riborex
    """
SnakeMake From line 44 of rules/diffex_riborex.smk 
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shell:
    """
    mkdir -p xtail;
    HRIBO/scripts/xtail.R -r {input.ribo} -m {input.rna} -c {input.cv} -x {output.table} -f {output.fcplot} -p {output.rplot};
    """
SnakeMake From line 14 of rules/diffex_xtail.smk 
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shell:
    """
    python3 HRIBO/scripts/generate_excel_xtail.py -a {input.annotation} -g {input.genome} -i {input.xtail_out} -o {output.xlsx_sorted} --padj_cutoff {params.padj_cutoff} --log2fc_cutoff {params.log2fc_cutoff}
    """
SnakeMake From line 33 of rules/diffex_xtail.smk 
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shell:
    """
    python3 HRIBO/scripts/merge_differential_expression.py {input.xtail} -o {output} -t xtail
    """
SnakeMake From line 46 of rules/diffex_xtail.smk 
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shell:
    "mkdir -p genomeSegemehlIndex; echo \"Computing Segemehl index\"; segemehl.x --threads {threads} -x {output.index} -d {input.genome} 2> {log}"
SnakeMake From line 11 of rules/mapping.smk 
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shell:
    """
    mkdir -p sammulti; segemehl.x -e -d {input.genome} -i {input.genomeSegemehlIndex} {params.fastq} --threads {threads} -o {output.sammulti} 2> {log}
    """
SnakeMake From line 40 of rules/mapping.smk 
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shell:
    """
    set +e
    mkdir -p sam
    awk '$2 == "4"' {input.sammulti} > {input.sammulti}.unmapped
    gawk -i inplace '$2 != "4"' {input.sammulti}
    samtools view -H <(cat {input.sammulti}) | grep '@HD' > {output.sam}
    samtools view -H <(cat {input.sammulti}) | grep '@SQ' | sort -t$'\t' -k1,1 -k2,2V >> {output.sam}
    samtools view -H <(cat {input.sammulti}) | grep '@RG' >> {output.sam}
    samtools view -H <(cat {input.sammulti}) | grep '@PG' >> {output.sam}
    cat {input.sammulti} |grep -v '^@' | grep -w 'NH:i:1' >> {output.sam}
    exitcode=$?
    if [ $exitcode -eq 1 ]
    then
        exit 1
    else
        exit 0
    fi
    """
SnakeMake SAMtools From line 54 of rules/mapping.smk 
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shell: "if [ \"{params.method}\" == \"NOTSET\" ]; then HRIBO/scripts/sam_strand_inverter.py --sam_in_filepath={input.sam} --sam_out_filepath={output.sam}; else cp {input.sam} {output.sam}; fi"
SnakeMake From line 82 of rules/mapping.smk 
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shell:
    "mkdir -p bammulti; samtools view -@ {threads} -bh {input.sam} | samtools sort -@ {threads} -o {output} -O bam"
SnakeMake SAMtools From line 92 of rules/mapping.smk 
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shell:
    "mkdir -p rRNAbam; samtools view -@ {threads} -bh {input.sam} | samtools sort -@ {threads} -o {output} -O bam"
SnakeMake SAMtools From line 103 of rules/mapping.smk 
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shell:
    "mkdir -p maplink; ln -s {params.inlink} {params.outlink}"
SnakeMake From line 115 of rules/mapping.smk 
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shell:
    "mkdir -p tracks; cat {input.reparation} >> {output}.unsorted; bedtools sort -i {output}.unsorted > {output};"
SnakeMake BEDTools From line 9 of rules/merge.smk 
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shell:
    "mkdir -p tracks; HRIBO/scripts/concatenate_gff.py {input.mergedGff} -o {output}"
SnakeMake From line 20 of rules/merge.smk 
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shell:
    "mkdir -p tracks; HRIBO/scripts/merge_duplicates_reparation.py -i {input} -o {output}"
SnakeMake From line 31 of rules/merge.smk 
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shell:
    "mkdir -p tracks; HRIBO/scripts/reannotate_orfs.py -a {input.annotation} -c {input.reparation} -o {output}"
SnakeMake From line 43 of rules/merge.smk 
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shell:
    "mkdir -p tracks; HRIBO/scripts/annotation_unite.py -a {input} -o {output}"
SnakeMake From line 54 of rules/merge.smk 
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shell:
    """
    mkdir -p metageneprofiling;
    HRIBO/scripts/read_length_statistics.py -a {input.bamfiles} -r {params.readlengths} -o metageneprofiling/ > {log}
    """
SnakeMake From line 17 of rules/metageneprofiling.smk 
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shell:
    """
    mkdir -p metageneprofiling;
    if [ {params.colorList} == nocolor ]; then
        colorList="";
    else
        colorList="--color_list {params.colorList}";
    fi;
    HRIBO/scripts/metagene_profiling.py -b {input.bam} -g {input.genome} -a {input.annotation} -o {output.meta} \
        --read_lengths {params.readlengths} \
        --normalization_methods {params.normalizationMethods} \
        --mapping_methods {params.mappingMethods} \
        --positions_in_ORF {params.positionsInORF} \
        --positions_out_ORF {params.positionsOutORF} \
        --filtering_method {params.filteringMethods} \
        --neighboring_genes_distance {params.neighboringGenesDistance} \
        --rpkm_threshold {params.rpkmThreshold} \
        --length_cutoff {params.lengthCutoff} \
        --output_formats {params.outputFormats} \
        --include_plotly_js {params.includePlotlyJS} \
        ${{colorList}}; > {log}
    """
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shell:
    """
    mkdir -p pca;
    sed -e '1s/-/_/g' {input.rawreads} > {output.rawreads};
    HRIBO/scripts/preparePCAinput.py -s {input.samples} -o {output.meta};
    """
SnakeMake From line 11 of rules/pca.smk 
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shell:
    """
    mkdir -p pca;
    HRIBO/scripts/analyse_variance.R -r {input.rawreads} -m {input.meta} -o pca/;
    """
SnakeMake From line 32 of rules/pca.smk 
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shell:
    """
    mkdir -p pca;
    HRIBO/scripts/plot_PCA.py -r {input.rld} -p {input.pvar} -c {input.cor} -o pca/;
    """
SnakeMake From line 49 of rules/pca.smk 
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shell:
    "mkdir -p genomes; cp {input.genome} genomes/genome.fa"
SnakeMake From line 7 of rules/preprocessing.smk 
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shell:
    "mkdir -p annotation; cp {input.annotation} annotation/annotation.gff"
SnakeMake From line 16 of rules/preprocessing.smk 
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shell:
    "mkdir -p annotation; HRIBO/scripts/gtf2gff3.py -a {input} -o {output}"
SnakeMake From line 25 of rules/preprocessing.smk 
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shell:
    "mkdir -p qc/4unique; fastqc -o qc/4unique -t {threads} -f sam_mapped {input.sam}; mv qc/4unique/{params.prefix}_fastqc.html {output.html}; mv qc/4unique/{params.prefix}_fastqc.zip {output.zip}"
SnakeMake FastQC From line 13 of rules/qcauxiliary.smk 
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shell:
    "mkdir -p qc/3mapped; fastqc -o qc/3mapped -t {threads} -f sam_mapped {input.sam}; mv qc/3mapped/{params.prefix}_fastqc.html {output.html}; mv qc/3mapped/{params.prefix}_fastqc.zip {output.zip}"
SnakeMake FastQC From line 28 of rules/qcauxiliary.smk 
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shell:
    "mkdir -p qc/5removedrRNA; fastqc -o qc/5removedrRNA -t {threads} {input}; mv qc/5removedrRNA/{params.prefix}_fastqc.html {output.html}; mv qc/5removedrRNA/{params.prefix}_fastqc.zip {output.zip}"
SnakeMake FastQC From line 43 of rules/qcauxiliary.smk 
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shell:
    """
    mkdir -p qc/all;
    column3=$(cut -f3 auxiliary/unambigous_annotation.gff | sort | uniq)
    if [[ " ${{column3[@]}} " =~ "gene" ]];
    then
        featureCounts -T {threads} -t gene -g ID -a {input.annotation} -o {output.txt} {input.bam};
    else
        touch {output.txt};
    fi
    """
SnakeMake FeatureCounts From line 55 of rules/qcauxiliary.smk 
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shell:
    """
    mkdir -p qc/trnainall;
    column3=$(cut -f3 auxiliary/unambigous_annotation.gff | sort | uniq)
    if [[ " ${{column3[@]}} " =~ "tRNA" ]];
    then
        featureCounts -T {threads} -t tRNA -g ID -a {input.annotation} -o {output.txt} {input.bam};
    else
        touch {output.txt};
    fi
    """
SnakeMake FeatureCounts From line 76 of rules/qcauxiliary.smk 
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shell:
    """
    mkdir -p qc/rrnainall;
    column3=$(cut -f3 auxiliary/unambigous_annotation.gff | sort | uniq)
    if [[ " ${{column3[@]}} " =~ "rRNA" ]];
    then
        featureCounts -T {threads} -t rRNA -g ID -a {input.annotation} -o {output.txt} {input.bam};
    else
        touch {output.txt};
    fi
    """
SnakeMake FeatureCounts From line 97 of rules/qcauxiliary.smk 
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shell:
    """
    mkdir -p qc/rrnainallaligned;
    column3=$(cut -f3 auxiliary/unambigous_annotation.gff | sort | uniq)
    if [[ " ${{column3[@]}} " =~ "rRNA" ]];
    then
        featureCounts -T {threads} -t rRNA -g ID -a {input.annotation} -o {output.txt} {input.bam};
    else
        touch {output.txt};
    fi
    """
SnakeMake FeatureCounts From line 118 of rules/qcauxiliary.smk 
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shell:
    """
    mkdir -p qc/rrnainuniquelyaligned;
    column3=$(cut -f3 auxiliary/unambigous_annotation.gff | sort | uniq)
    if [[ " ${{column3[@]}} " =~ "rRNA" ]];
    then
        featureCounts -T {threads} -t rRNA -g ID -a {input.annotation} -o {output.txt} {input.bam};
    else
        touch {output.txt};
    fi
    """
SnakeMake FeatureCounts From line 139 of rules/qcauxiliary.smk 
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shell:
    "mkdir -p coverage; bedtools genomecov -ibam {input} -bg > {output}"
SnakeMake BEDTools From line 159 of rules/qcauxiliary.smk 
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shell:
    "mkdir -p qc/1raw; fastqc -o qc/1raw -t {threads} {input.fastq}; mv qc/1raw/{params.prefix}_fastqc.html {output.html}; mv qc/1raw/{params.prefix}_fastqc.zip {output.zip}"
SnakeMake FastQC From line 13 of rules/qc.smk 
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shell:
    "mkdir -p qc/2trimmed; fastqc -o qc/2trimmed -t {threads} {input}; mv qc/2trimmed/{params.prefix}_fastqc.html {output.html}; mv qc/2trimmed/{params.prefix}_fastqc.zip {output.zip}"
SnakeMake FastQC From line 27 of rules/qc.smk 
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shell:
    """
    mkdir -p qc/1raw
    fastqc -o qc/1raw -t {threads} {input.fastq1}; mv qc/1raw/{params.prefix1}_fastqc.html {output.html1}; mv qc/1raw/{params.prefix1}_fastqc.zip {output.zip1}
    fastqc -o qc/1raw -t {threads} {input.fastq2}; mv qc/1raw/{params.prefix2}_fastqc.html {output.html2}; mv qc/1raw/{params.prefix2}_fastqc.zip {output.zip2}
    """
SnakeMake FastQC From line 46 of rules/qc.smk 
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shell:
    """
    mkdir -p qc/2trimmed;
    fastqc -o qc/2trimmed -t {threads} {input}; mv qc/2trimmed/{params.prefix1}_fastqc.html {output.html1}; mv qc/2trimmed/{params.prefix1}_fastqc.zip {output.zip1}
    fastqc -o qc/2trimmed -t {threads} {input}; mv qc/2trimmed/{params.prefix2}_fastqc.html {output.html2}; mv qc/2trimmed/{params.prefix2}_fastqc.zip {output.zip2}
    """
SnakeMake FastQC From line 68 of rules/qc.smk 
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shell:
    "export LC_ALL=en_US.utf8; export LANG=en_US.utf8; multiqc -f -d --exclude picard --exclude gatk -z -o {params.dir} qc/1raw qc/2trimmed qc/3mapped qc/4unique qc/5removedrRNA qc/all qc/trnainall qc/rrnainallaligned qc/rrnainuniquelyaligned qc/rrnainall trimmed  2> {log}"
SnakeMake gatk MultiQC Picard From line 114 of rules/qc.smk 
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shell:
    """
    if [ "{params.features}" == None ]; then
        features="";
    else
        features="--use_features {params.features}";
    fi;
    mkdir -p readcounts
    HRIBO/scripts/call_featurecounts.py -b {input.bam} -s 1 --with_O --for_diff_expr -o {output} -t {threads} -a {input.annotation} ${{features}}
    """
SnakeMake From line 13 of rules/readcounting.smk 
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shell:
    """
    mkdir -p readcounts
    HRIBO/scripts/call_featurecounts.py -b {input.bam} -s 1 --with_O -o {output} -t {threads} -a {input.annotation}
    """
SnakeMake From line 34 of rules/readcounting.smk 
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shell:
    """
    mkdir -p readcounts
    HRIBO/scripts/call_featurecounts.py -b {input.bam} -s 1 --with_O -o {output} -t {threads} -a {input.annotation}
    """
SnakeMake From line 50 of rules/readcounting.smk 
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shell:
    """
    mkdir -p readcounts
    HRIBO/scripts/call_featurecounts.py -b {input.bam} -s 1 --with_O -o {output} -t {threads} -a {input.annotation}
    """
SnakeMake From line 66 of rules/readcounting.smk 
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shell:
    """
    mkdir -p readcounts
    HRIBO/scripts/call_featurecounts.py -b {input.bam} -s 1 --with_O --with_M --fraction -o {output} -t {threads} -a {input.annotation}
    """
SnakeMake From line 82 of rules/readcounting.smk 
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shell:
    """
    mkdir -p auxiliary
    HRIBO/scripts/call_featurecounts.py -b {input.bam} -s 1 --with_O --fraction -o {output} -t {threads} -a {input.annotation}
    """
SnakeMake From line 98 of rules/readcounting.smk 
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shell:
    """
    mkdir -p readcounts; HRIBO/scripts/map_reads_to_annotation.py -i {input.reads} -a {input.annotation} -o {output}
    """
SnakeMake From line 113 of rules/readcounting.smk 
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shell:
    """
    mkdir -p readcounts; HRIBO/scripts/map_reads_to_annotation.py -i {input.reads} -a {input.annotation} -o {output}
    """
SnakeMake From line 127 of rules/readcounting.smk 
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shell:
    """
    mkdir -p readcounts; HRIBO/scripts/map_reads_to_annotation.py -i {input.reads} -a {input.annotation} -o {output}
    """
SnakeMake From line 141 of rules/readcounting.smk 
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shell:
    """
    mkdir -p readcounts; HRIBO/scripts/map_reads_to_annotation.py -i {input.reads} -a {input.annotation} -o {output}
    """
SnakeMake From line 155 of rules/readcounting.smk 
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shell:
    """
    mkdir -p readcounts; HRIBO/scripts/map_reads_to_annotation.py -i {input.reads} -a {input.annotation} -o {output}
    """
SnakeMake From line 169 of rules/readcounting.smk 
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shell:
    "mkdir -p readcounts; HRIBO/scripts/total_mapped_reads.py -b {input.bam} -m {output.mapped} -l {output.length}"
SnakeMake From line 184 of rules/readcounting.smk 
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shell:
    "mkdir -p readcounts; HRIBO/scripts/total_mapped_reads.py -b {input.bam} -m {output.mapped} -l {output.length}"
SnakeMake From line 197 of rules/readcounting.smk 
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shell:
    "mkdir -p readcounts; HRIBO/scripts/total_mapped_reads.py -b {input.bam} -m {output.mapped} -l {output.length}"
SnakeMake From line 210 of rules/readcounting.smk 
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run:
    outputName = os.path.basename(input[0])
    shell("mkdir -p uniprotDB; mv {input} uniprotDB/{outputName}; gunzip uniprotDB/{outputName}")
SnakeMake From line 10 of rules/reparation.smk 
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shell:
    "mkdir -p reparation; if [ uniprotDB/uniprot_sprot.fasta.bak does not exist ]; then cp -p uniprotDB/uniprot_sprot.fasta uniprotDB/uniprot_sprot.fasta.bak; fi; mkdir -p {params.prefix}/tmp; reparation.pl -bam {input.bam} -g {input.genome} -gtf {input.gtf} -db {input.db} -out {params.prefix} -threads {threads}; if [ uniprotDB/uniprot_sprot.fasta does not exist ]; then cp -p uniprotDB/uniprot_sprot.fasta.bak uniprotDB/uniprot_sprot.fasta; fi;"
SnakeMake From line 34 of rules/reparation.smk 
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shell:
    "mkdir -p tracks; HRIBO/scripts/create_reparation_gff.py -c {wildcards.condition} -r {wildcards.replicate} -i {input} -o {output}"
SnakeMake From line 45 of rules/reparation.smk 
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shell:
    "mkdir -p tracks; HRIBO/scripts/concatenate_gff.py {input} -o {output}"
SnakeMake From line 56 of rules/reparation.smk 
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shell:
    """
    mkdir -p annotation; awk -F'\\t' '$3 == "rRNA" || $3 == "tRNA"' {input.annotation} | awk -F'\\t' '{{print $1 FS $4 FS $5 FS "." FS "." FS $7}}' > {output.annotation}
    """
SnakeMake From line 9 of rules/rrnafiltering.smk 
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shell:
    "mkdir -p norRNA; mkdir -p mapuniqnorrna; bedtools intersect -v -a {input.mapuniq} -b {input.annotation} > {output.bam}"
SnakeMake BEDTools From line 23 of rules/rrnafiltering.smk 
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shell:
    "mkdir -p trimlink; ln -s {params.inlink} {params.outlink};"
SnakeMake From line 18 of rules/trimming.smk 
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shell:
    "mkdir -p trimlink; ln -s {params.inlink1} {params.outlink1}; ln -s {params.inlink2} {params.outlink2};"
SnakeMake From line 35 of rules/trimming.smk 
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shell:
    "mkdir -p trimmed; cutadapt -j {threads} {params.adapter3} {params.adapter5} {params.quality} {params.filtering} -o {output.fastq} {input.fastq}"
SnakeMake Cutadapt From line 54 of rules/trimming.smk 
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shell:
    "mkdir -p trimmed; cutadapt -j {threads} {params.adapter3q} {params.adapter5q} {params.adapter3p} {params.adapter5p} {params.quality} {params.filtering} -o {output.fastq1} -p {output.fastq2} {input.fastq1} {input.fastq2}"
SnakeMake Cutadapt From line 74 of rules/trimming.smk 
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shell:
    "samtools faidx {rules.retrieveGenome.output}"
SnakeMake SAMtools From line 11 of rules/visualization.smk 
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shell:
    "mkdir -p genomes; cut -f1,2 {input[0]} > genomes/sizes.genome"
SnakeMake From line 23 of rules/visualization.smk 
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shell:
    "mkdir -p genomes; HRIBO/scripts/reverse_complement.py --input_fasta_filepath genomes/genome.fa --output_fasta_filepath genomes/genome.rev.fa"
SnakeMake From line 34 of rules/visualization.smk 
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shell:
    "mkdir -p tracks; HRIBO/scripts/motif_to_gff.py --input_genome_fasta_filepath {input.fwd} --input_reverse_genome_fasta_filepath {input.rev} --motif_string ATG --output_gff3_filepath {output}"
SnakeMake From line 46 of rules/visualization.smk 
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shell:
    "mkdir -p tracks; HRIBO/scripts/motif_to_gff.py --input_genome_fasta_filepath {input.fwd} --input_reverse_genome_fasta_filepath {input.rev} --motif_string GTG,TTG,CTG --output_gff3_filepath {output}"
SnakeMake From line 58 of rules/visualization.smk 
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shell:
    "mkdir -p tracks; HRIBO/scripts/motif_to_gff.py --input_genome_fasta_filepath {input.fwd} --input_reverse_genome_fasta_filepath {input.rev} --motif_string TAG,TGA,TAA --output_gff3_filepath {output}"
SnakeMake From line 71 of rules/visualization.smk 
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shell:
    "mkdir -p tracks; HRIBO/scripts/motif_to_gff.py --input_genome_fasta_filepath {input.fwd} --input_reverse_genome_fasta_filepath {input.rev} --motif_string AAGG --output_gff3_filepath {output}"
SnakeMake From line 83 of rules/visualization.smk 
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shell:
    "samtools index -@ {threads} maplink/{params.prefix}"
SnakeMake SAMtools From line 98 of rules/visualization.smk 
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shell:
    "samtools index -@ {threads} bammulti/{params.prefix}"
SnakeMake SAMtools From line 112 of rules/visualization.smk 
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shell:
    "samtools index -@ {threads} rRNAbam/{params.prefix}"
SnakeMake SAMtools From line 126 of rules/visualization.smk 
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shell:
    "mkdir -p totalmappedtracks; mkdir -p totalmappedtracks/raw; mkdir -p totalmappedtracks/mil; mkdir -p totalmappedtracks/min; HRIBO/scripts/mapping.py --mapping_style global --bam_path {input.bam} --wiggle_file_path totalmappedtracks/ --no_of_aligned_reads_file_path {input.stats} --library_name {params.prefix};"
SnakeMake From line 148 of rules/visualization.smk 
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shell:
    "mkdir -p uniquemappedtracks; mkdir -p uniquemappedtracks/raw; mkdir -p uniquemappedtracks/mil; mkdir -p uniquemappedtracks/min; HRIBO/scripts/mapping.py --mapping_style global --bam_path {input.bam} --wiggle_file_path uniquemappedtracks/ --no_of_aligned_reads_file_path {input.stats} --library_name {params.prefix};"
SnakeMake From line 170 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 182 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 194 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 206 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 218 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 230 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 242 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 254 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 266 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 278 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 290 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 302 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 314 of rules/visualization.smk 
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shell:
    "mkdir -p globaltracks; mkdir -p globaltracks/raw; mkdir -p globaltracks/mil; mkdir -p globaltracks/min; HRIBO/scripts/mapping.py --mapping_style global --bam_path {input.bam} --wiggle_file_path globaltracks/ --no_of_aligned_reads_file_path {input.stats} --library_name {params.prefix};"
SnakeMake From line 336 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 348 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 360 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 372 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 384 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 396 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 408 of rules/visualization.smk 
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shell:
    "mkdir -p centeredtracks; mkdir -p centeredtracks/raw; mkdir -p centeredtracks/mil; mkdir -p centeredtracks/min; HRIBO/scripts/mapping.py --mapping_style centered --bam_path {input.bam} --wiggle_file_path centeredtracks/ --no_of_aligned_reads_file_path {input.stats} --library_name {params.prefix};"
SnakeMake From line 430 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 441 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 453 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 465 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 477 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 489 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 501 of rules/visualization.smk 
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shell:
    "mkdir -p fiveprimetracks; mkdir -p fiveprimetracks/raw; mkdir -p fiveprimetracks/mil; mkdir -p fiveprimetracks/min; HRIBO/scripts/mapping.py --mapping_style first_base_only --bam_path {input.bam} --wiggle_file_path fiveprimetracks/ --no_of_aligned_reads_file_path {input.stats} --library_name {params.prefix};"
SnakeMake From line 523 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 535 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 547 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 559 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 571 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 583 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 595 of rules/visualization.smk 
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shell:
    "mkdir -p threeprimetracks; mkdir -p threeprimetracks/raw; mkdir -p threeprimetracks/mil; mkdir -p threeprimetracks/min; HRIBO/scripts/mapping.py --mapping_style last_base_only --bam_path {input.bam} --wiggle_file_path threeprimetracks/ --no_of_aligned_reads_file_path {input.stats} --library_name {params.prefix};"
SnakeMake From line 617 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 629 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 641 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 653 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 665 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.fwd} {input.genomeSize} {output.fwd}"
SnakeMake wigToBigWig From line 677 of rules/visualization.smk 
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shell:
    "wigToBigWig {input.rev} {input.genomeSize} {output.rev}"
SnakeMake wigToBigWig From line 689 of rules/visualization.smk 
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shell:
    "mkdir -p tracks; multiBamSummary bins --smartLabels --bamfiles {input.bam} -o {output} -p {threads};"
SnakeMake From line 702 of rules/visualization.smk 
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shell:
    "mkdir -p figures; plotCorrelation -in {input.npz} --corMethod spearman --skipZeros --plotTitle \"Spearman Correlation of Read Counts\" --whatToPlot heatmap --colorMap RdYlBu --plotNumbers -o {output.correlation} --outFileCorMatrix SpearmanCorr_readCounts.tab"
SnakeMake From line 713 of rules/visualization.smk 
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shell:
    "mkdir -p tracks; cat {input[0]} | grep -v '\tgene\t' > tracks/annotation-woGenes.gtf; gtf2bed < tracks/annotation-woGenes.gtf > tracks/annotation.bed"
SnakeMake GFFutils From line 724 of rules/visualization.smk 
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shell:
    "mkdir -p tracks; cut -f1-6 {input[0]} > tracks/annotationNScore.bed6;  awk '{{$5=1 ; print ;}}' tracks/annotation.bed6 > tracks/annotation.bed6; bedToBigBed -type=bed6 -tab tracks/annotation.bed6 {input[1]} tracks/annotation.bb"
SnakeMake From line 736 of rules/visualization.smk 
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shell:
    """
    set +e
    mkdir -p tracks/color
    bigWigToWig {input.infwd} {params.unzippedfwd}
    bigWigToWig {input.inrev} {params.unzippedrev}
    sed -i '2s/^/track type=wiggle_0 visibility=full color=0,0,128 autoscale=on\\n/' {params.unzippedfwd}
    sed -i '2s/^/track type=wiggle_0 visibility=full color=0,130,200 autoscale=on\\n/' {params.unzippedrev}
    gzip -f {params.unzippedfwd}
    gzip -f {params.unzippedrev}
    """
SnakeMake ucsc-bigwigtowig From line 752 of rules/visualization.smk 
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shell:
    """
    set +e
    mkdir -p tracks/color
    cp {input.rbs} ./tracks/color/
    cp {input.start} ./tracks/color/
    cp {input.stop} ./tracks/color/
    sed -i '1s/^/##track type=wiggle_0 visibility=full color=145,30,180 autoscale=on\\n/' {output.outrbs}
    sed -i '1s/^/##track type=wiggle_0 visibility=full color=210,245,60 autoscale=on\\n/' {output.outstart}
    sed -i '1s/^/##track type=wiggle_0 visibility=full color=230,25,75 autoscale=on\\n/' {output.outstop}
    """
SnakeMake From line 774 of rules/visualization.smk
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Free

Created: 1yr ago
Updated: 1yr ago
Maitainers: public
URL: https://github.com/RickGelhausen/HRIBO
Name: hribo
Version: 1.7.1
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Copyright: Public Domain
License: GNU General Public License v3.0
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