MNase-seq analysis pipeline using BWA and DANPOS2.

public 1yr ago Version: 1.0.0 0 bookmarks

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Introduction

nfcore/mnaseseq is a bioinformatics analysis pipeline used for DNA sequencing data obtained via micrococcal nuclease digestion.

The pipeline is built using Nextflow , a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It comes with docker containers making installation trivial and results highly reproducible.

Pipeline summary

Raw read QC ( FastQC )
Adapter trimming ( Trim Galore! )
Alignment ( BWA )
Mark duplicates ( picard )
Merge alignments from multiple libraries of the same sample ( picard )
1. Re-mark duplicates ( picard )
2. Filtering to remove:
  - reads mapping to blacklisted regions ( SAMtools , BEDTools )
  - reads that are marked as duplicates ( SAMtools )
  - reads that arent marked as primary alignments ( SAMtools )
  - reads that are unmapped ( SAMtools )
  - reads that map to multiple locations ( SAMtools )
  - reads containing > 4 mismatches ( BAMTools )
  - reads that are soft-clipped ( BAMTools )
  - reads that have an insert size within specified range ( BAMTools ; paired-end only )
  - reads that map to different chromosomes ( Pysam ; paired-end only )
  - reads that arent in FR orientation ( Pysam ; paired-end only )
  - reads where only one read of the pair fails the above criteria ( Pysam ; paired-end only )
3. Alignment-level QC and estimation of library complexity ( picard , Preseq )
4. Create normalised bigWig files scaled to 1 million mapped reads ( BEDTools , bedGraphToBigWig )
5. Calculate genome-wide coverage assessment ( deepTools )
6. Call nucleosome positions and generate smoothed, normalised coverage bigWig files that can be used to generate occupancy profile plots between samples across features of interest ( DANPOS2 )
7. Generate gene-body meta-profile from DANPOS2 smoothed bigWig files ( deepTools )
Merge filtered alignments across replicates ( picard )
1. Re-mark duplicates ( picard )
2. Remove duplicate reads ( SAMtools )
3. Create normalised bigWig files scaled to 1 million mapped reads ( BEDTools , wigToBigWig )
4. Call nucleosome positions and generate smoothed, normalised coverage bigWig files that can be used to generate occupancy profile plots between samples across features of interest ( DANPOS2 )
5. Generate gene-body meta-profile from DANPOS2 smoothed bigWig files ( deepTools )
Create IGV session file containing bigWig tracks for data visualisation ( IGV ).
Present QC for raw read and alignment results ( MultiQC )

Quick Start

i. Install nextflow

ii. Install either Docker or Singularity for full pipeline reproducibility (please only use Conda as a last resort; see docs )

iii. Download the pipeline and test it on a minimal dataset with a single command

nextflow run nf-core/mnaseseq -profile test,<docker/singularity/conda/institute>

Please check nf-core/configs to see if a custom config file to run nf-core pipelines already exists for your Institute. If so, you can simply use -profile <institute> in your command. This will enable either docker or singularity and set the appropriate execution settings for your local compute environment.

iv. Start running your own analysis!

nextflow run nf-core/mnaseseq -profile <docker/singularity/conda/institute> --input design.csv --genome GRCh37

See usage docs for all of the available options when running the pipeline.

Documentation

The nf-core/mnaseseq pipeline comes with documentation about the pipeline, found in the docs/ directory:

Credits

The pipeline was originally written by The Bioinformatics & Biostatistics Group for use at The Francis Crick Institute , London.

The pipeline was developed by Harshil Patel .

Many thanks to others who have helped out along the way too, including (but not limited to): @crickbabs .

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines .

For further information or help, don't hesitate to get in touch on Slack (you can join with this invite ).

Citation

If you use nf-core/mnaseseq for your analysis, please cite it using the following doi: 10.5281/zenodo.6581372 .

You can cite the nf-core publication as follows:

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x .
ReadCube: Full Access Link

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

Code Snippets

"""
check_design.py $design design_reads.csv
"""

NextFlow From line 288 of master/main.nf

"""
bwa index -a bwtsw $fasta
mkdir BWAIndex && mv ${fasta}* BWAIndex
"""

NextFlow BWA From line 344 of master/main.nf

"""
gtf2bed $gtf > ${gtf.baseName}.bed
"""

NextFlow GFFutils From line 367 of master/main.nf

"""
cat $bed | awk -v FS='\t' -v OFS='\t' '{ if(\$6=="+") \$3=\$2+1; else \$2=\$3-1; print \$1, \$2, \$3, \$4, \$5, \$6;}' > ${bed.baseName}.tss.bed
"""

NextFlow From line 388 of master/main.nf

"""
samtools faidx $fasta
cut -f 1,2 ${fasta}.fai > ${fasta}.sizes
$blacklist_filter > ${fasta}.include_regions.bed
"""

NextFlow SAMtools From line 418 of master/main.nf

"""
[ ! -f  ${name}.fastq.gz ] && ln -s $reads ${name}.fastq.gz
fastqc -q -t $task.cpus ${name}.fastq.gz
"""

NextFlow FastQC From line 456 of master/main.nf

"""
[ ! -f  ${name}_1.fastq.gz ] && ln -s ${reads[0]} ${name}_1.fastq.gz
[ ! -f  ${name}_2.fastq.gz ] && ln -s ${reads[1]} ${name}_2.fastq.gz
fastqc -q -t $task.cpus ${name}_1.fastq.gz
fastqc -q -t $task.cpus ${name}_2.fastq.gz
"""

NextFlow FastQC From line 461 of master/main.nf

"""
[ ! -f  ${name}.fastq.gz ] && ln -s $reads ${name}.fastq.gz
trim_galore --cores $cores --fastqc --gzip $c_r1 $tpc_r1 $nextseq ${name}.fastq.gz
"""

NextFlow Trim_Galore From line 524 of master/main.nf

"""
[ ! -f  ${name}_1.fastq.gz ] && ln -s ${reads[0]} ${name}_1.fastq.gz
[ ! -f  ${name}_2.fastq.gz ] && ln -s ${reads[1]} ${name}_2.fastq.gz
trim_galore --cores $cores --paired --fastqc --gzip $c_r1 $c_r2 $tpc_r1 $tpc_r2 $nextseq ${name}_1.fastq.gz ${name}_2.fastq.gz
"""

NextFlow Trim_Galore From line 529 of master/main.nf

"""
bwa mem \\
    -t $task.cpus \\
    -M \\
    -R $rg \\
    ${index}/${bwa_base} \\
    $reads \\
    | samtools view -@ $task.cpus -b -h -F 0x0100 -O BAM -o ${prefix}.bam -
"""

NextFlow SAMtools BWA From line 566 of master/main.nf

"""
samtools sort -@ $task.cpus -o ${prefix}.sorted.bam -T $name $bam
samtools index ${prefix}.sorted.bam
samtools flagstat ${prefix}.sorted.bam > ${prefix}.sorted.bam.flagstat
samtools idxstats ${prefix}.sorted.bam > ${prefix}.sorted.bam.idxstats
samtools stats ${prefix}.sorted.bam > ${prefix}.sorted.bam.stats
"""

NextFlow SAMtools From line 602 of master/main.nf

"""
picard -Xmx${avail_mem}g MergeSamFiles \\
    ${'INPUT='+bam_files.join(' INPUT=')} \\
    OUTPUT=${name}.sorted.bam \\
    SORT_ORDER=coordinate \\
    VALIDATION_STRINGENCY=LENIENT \\
    TMP_DIR=tmp
samtools index ${name}.sorted.bam

picard -Xmx${avail_mem}g MarkDuplicates \\
    INPUT=${name}.sorted.bam \\
    OUTPUT=${prefix}.sorted.bam \\
    ASSUME_SORTED=true \\
    REMOVE_DUPLICATES=false \\
    METRICS_FILE=${prefix}.MarkDuplicates.metrics.txt \\
    VALIDATION_STRINGENCY=LENIENT \\
    TMP_DIR=tmp

samtools index ${prefix}.sorted.bam
samtools idxstats ${prefix}.sorted.bam > ${prefix}.sorted.bam.idxstats
samtools flagstat ${prefix}.sorted.bam > ${prefix}.sorted.bam.flagstat
samtools stats ${prefix}.sorted.bam > ${prefix}.sorted.bam.stats
"""

NextFlow SAMtools Picard From line 660 of master/main.nf

"""
picard -Xmx${avail_mem}g MarkDuplicates \\
    INPUT=${bam_files[0]} \\
    OUTPUT=${prefix}.sorted.bam \\
    ASSUME_SORTED=true \\
    REMOVE_DUPLICATES=false \\
    METRICS_FILE=${prefix}.MarkDuplicates.metrics.txt \\
    VALIDATION_STRINGENCY=LENIENT \\
    TMP_DIR=tmp

samtools index ${prefix}.sorted.bam
samtools idxstats ${prefix}.sorted.bam > ${prefix}.sorted.bam.idxstats
samtools flagstat ${prefix}.sorted.bam > ${prefix}.sorted.bam.flagstat
samtools stats ${prefix}.sorted.bam > ${prefix}.sorted.bam.stats
"""

NextFlow SAMtools Picard From line 684 of master/main.nf

"""
sed 's/MIN_INSERT_SIZE/${params.min_insert}/g' <$bamtools_filter_config >bamtools_filter.json
sed -i -e 's/MAX_INSERT_SIZE/${params.max_insert}/g' bamtools_filter.json
sed -i -e 's/MAX_MISMATCH/${params.max_mismatch}/g' bamtools_filter.json

samtools view \\
    $filter_params \\
    $dup_params \\
    $multimap_params \\
    $blacklist_params \\
    -b ${bam[0]} \\
    | bamtools filter \\
        -out ${prefix}.sorted.bam \\
        -script bamtools_filter.json

samtools index ${prefix}.sorted.bam
samtools flagstat ${prefix}.sorted.bam > ${prefix}.sorted.bam.flagstat
samtools idxstats ${prefix}.sorted.bam > ${prefix}.sorted.bam.idxstats
samtools stats ${prefix}.sorted.bam > ${prefix}.sorted.bam.stats

$name_sort_bam
"""

NextFlow SAMtools BamTools From line 737 of master/main.nf

"""
bampe_rm_orphan.py ${bam[0]} ${prefix}.bam --only_fr_pairs

samtools sort -@ $task.cpus -o ${prefix}.sorted.bam -T $prefix ${prefix}.bam
samtools index ${prefix}.sorted.bam
samtools flagstat ${prefix}.sorted.bam > ${prefix}.sorted.bam.flagstat
samtools idxstats ${prefix}.sorted.bam > ${prefix}.sorted.bam.idxstats
samtools stats ${prefix}.sorted.bam > ${prefix}.sorted.bam.stats
"""

NextFlow SAMtools From line 807 of master/main.nf

"""
preseq lc_extrap -v -output ${prefix}.ccurve.txt -bam ${bam[0]}
"""

NextFlow preseq From line 846 of master/main.nf

"""
picard -Xmx${avail_mem}g CollectMultipleMetrics \\
    INPUT=${bam[0]} \\
    OUTPUT=${prefix}.CollectMultipleMetrics \\
    REFERENCE_SEQUENCE=$fasta \\
    VALIDATION_STRINGENCY=LENIENT \\
    TMP_DIR=tmp
"""

NextFlow Picard From line 883 of master/main.nf

"""
picard -Xmx${avail_mem}g CollectMultipleMetrics \\
    INPUT=${bam[0]} \\
    OUTPUT=${prefix}.CollectMultipleMetrics \\
    REFERENCE_SEQUENCE=$fasta \\
    VALIDATION_STRINGENCY=LENIENT \\
    TMP_DIR=tmp
"""

NextFlow Picard From line 925 of master/main.nf

"""
SCALE_FACTOR=\$(grep 'mapped (' $flagstat | awk '{print 1000000/\$1}')
echo \$SCALE_FACTOR > ${prefix}.scale_factor.txt
genomeCoverageBed -ibam ${bam[0]} -bg -scale \$SCALE_FACTOR $pe_fragment $extend | sort -T '.' -k1,1 -k2,2n >  ${prefix}.bedGraph

bedGraphToBigWig ${prefix}.bedGraph $sizes ${prefix}.bigWig

find * -type f -name "*.bigWig" -exec echo -e "bwa/mergedLibrary/bigwig/"{}"\\t0,0,178" \\; > ${prefix}.bigWig.igv.txt
"""

NextFlow bedGraphToBigWig From line 961 of master/main.nf

"""
plotFingerprint \\
    --bamfiles ${bam[0]} \\
    --plotFile ${prefix}.plotFingerprint.pdf \\
    $extend \\
    --labels $prefix \\
    --outRawCounts ${prefix}.plotFingerprint.raw.txt \\
    --outQualityMetrics ${prefix}.plotFingerprint.qcmetrics.txt \\
    --skipZeros \\
    --numberOfProcessors $task.cpus \\
    --numberOfSamples $params.fingerprint_bins
"""

NextFlow DeepTools From line 993 of master/main.nf

"""
bamToBed -i $ibam > ${prefix}.bed
"""

NextFlow From line 1034 of master/main.nf

"""
danpos.py dpos \\
    $bed \\
    --span 1 \\
    --smooth_width 20 \\
    --width 40 \\
    --count 1000000 \\
    --out ./result/ \\
    $pe_params
mv ./result/*/* .

wigToBigWig -clip ${prefix}.Fnor.smooth.wig $sizes ${prefix}.Fnor.smooth.bigWig

awk -v FS='\t' -v OFS='\t' 'FNR > 1 { print \$1, \$2-1, \$3, "Interval_"NR-1, \$6, "+" }' ${prefix}.Fnor.smooth.positions.xls > ${prefix}.Fnor.smooth.positions.bed
awk -v FS='\t' -v OFS='\t' 'FNR > 1 { print \$1, \$4-1, \$4, "Interval_"NR-1, \$6, "+" }' ${prefix}.Fnor.smooth.positions.xls > ${prefix}.Fnor.smooth.positions.summit.bed

find * -type f -name "*.bigWig" -exec echo -e "bwa/mergedLibrary/danpos/"{}"\\t0,0,178" \\; > ${prefix}.danpos.bigWig.igv.txt
find * -type f -name "*.bed" -exec echo -e "bwa/mergedLibrary/danpos/"{}"\\t0,0,178" \\; > ${prefix}.danpos.bed.igv.txt
"""

NextFlow wigToBigWig From line 1068 of master/main.nf

"""
computeMatrix scale-regions \\
    --regionsFileName $bed \\
    --scoreFileName $bigwig \\
    --outFileName ${prefix}.computeMatrix.mat.gz \\
    --outFileNameMatrix ${prefix}.computeMatrix.vals.mat.gz \\
    --regionBodyLength 1000 \\
    --beforeRegionStartLength 3000 \\
    --afterRegionStartLength 3000 \\
    --skipZeros \\
    --samplesLabel $name \\
    --numberOfProcessors $task.cpus

plotProfile --matrixFile ${prefix}.computeMatrix.mat.gz \\
    --outFileName ${prefix}.plotProfile.pdf \\
    --outFileNameData ${prefix}.plotProfile.tab
"""

NextFlow DeepTools From line 1110 of master/main.nf

"""
picard -Xmx${avail_mem}g MergeSamFiles \\
    ${'INPUT='+bam_files.join(' INPUT=')} \\
    OUTPUT=${name}.sorted.bam \\
    SORT_ORDER=coordinate \\
    VALIDATION_STRINGENCY=LENIENT \\
    TMP_DIR=tmp
samtools index ${name}.sorted.bam

picard -Xmx${avail_mem}g MarkDuplicates \\
    INPUT=${name}.sorted.bam \\
    OUTPUT=${prefix}.sorted.bam \\
    ASSUME_SORTED=true \\
    REMOVE_DUPLICATES=true \\
    METRICS_FILE=${prefix}.MarkDuplicates.metrics.txt \\
    VALIDATION_STRINGENCY=LENIENT \\
    TMP_DIR=tmp

samtools index ${prefix}.sorted.bam
samtools flagstat ${prefix}.sorted.bam > ${prefix}.sorted.bam.flagstat
samtools idxstats ${prefix}.sorted.bam > ${prefix}.sorted.bam.idxstats
samtools stats ${prefix}.sorted.bam > ${prefix}.sorted.bam.stats
"""

NextFlow SAMtools Picard From line 1182 of master/main.nf

"""
ln -s ${bams[0]} ${prefix}.sorted.bam
ln -s ${bams[1]} ${prefix}.sorted.bam.bai
touch ${prefix}.MarkDuplicates.metrics.txt
samtools flagstat ${prefix}.sorted.bam > ${prefix}.sorted.bam.flagstat
samtools idxstats ${prefix}.sorted.bam > ${prefix}.sorted.bam.idxstats
samtools stats ${prefix}.sorted.bam > ${prefix}.sorted.bam.stats
"""

NextFlow SAMtools From line 1206 of master/main.nf

"""
samtools sort -n -@ $task.cpus -o ${prefix}.bam -T $prefix ${bam[0]}
"""

NextFlow SAMtools From line 1238 of master/main.nf

"""
SCALE_FACTOR=\$(grep 'mapped (' $flagstat | awk '{print 1000000/\$1}')
echo \$SCALE_FACTOR > ${prefix}.scale_factor.txt
genomeCoverageBed -ibam ${bam[0]} -bg -scale \$SCALE_FACTOR $pe_fragment $extend | sort -T '.' -k1,1 -k2,2n >  ${prefix}.bedGraph

bedGraphToBigWig ${prefix}.bedGraph $sizes ${prefix}.bigWig

find * -type f -name "*.bigWig" -exec echo -e "bwa/mergedReplicate/bigwig/"{}"\\t0,0,178" \\; > ${prefix}.bigWig.igv.txt
"""

NextFlow bedGraphToBigWig From line 1281 of master/main.nf

"""
bamToBed -i $ibam > ${prefix}.bed
"""

NextFlow From line 1319 of master/main.nf

"""
danpos.py dpos \\
    $bed \\
    --span 1 \\
    --smooth_width 20 \\
    --width 40 \\
    --count 1000000 \\
    --out ./result/ \\
    $pe_params
mv ./result/*/* .

wigToBigWig -clip ${prefix}.Fnor.smooth.wig $sizes ${prefix}.Fnor.smooth.bigWig

awk -v FS='\t' -v OFS='\t' 'FNR > 1 { print \$1, \$2-1, \$3, "Interval_"NR-1, \$6, "+" }' ${prefix}.Fnor.smooth.positions.xls > ${prefix}.Fnor.smooth.positions.bed
awk -v FS='\t' -v OFS='\t' 'FNR > 1 { print \$1, \$4-1, \$4, "Interval_"NR-1, \$6, "+" }' ${prefix}.Fnor.smooth.positions.xls > ${prefix}.Fnor.smooth.positions.summit.bed

find * -type f -name "*.bigWig" -exec echo -e "bwa/mergedReplicate/danpos/"{}"\\t0,0,178" \\; > ${prefix}.danpos.bigWig.igv.txt
find * -type f -name "*.bed" -exec echo -e "bwa/mergedReplicate/danpos/"{}"\\t0,0,178" \\; > ${prefix}.danpos.bed.igv.txt
"""

NextFlow wigToBigWig From line 1353 of master/main.nf

"""
computeMatrix scale-regions \\
    --regionsFileName $bed \\
    --scoreFileName $bigwig \\
    --outFileName ${prefix}.computeMatrix.mat.gz \\
    --outFileNameMatrix ${prefix}.computeMatrix.vals.mat.gz \\
    --regionBodyLength 1000 \\
    --beforeRegionStartLength 3000 \\
    --afterRegionStartLength 3000 \\
    --skipZeros \\
    --samplesLabel $name \\
    --numberOfProcessors $task.cpus

plotProfile --matrixFile ${prefix}.computeMatrix.mat.gz \\
    --outFileName ${prefix}.plotProfile.pdf \\
    --outFileNameData ${prefix}.plotProfile.tab
"""

NextFlow DeepTools From line 1395 of master/main.nf

"""
cat *.txt > igv_files.txt
igv_files_to_session.py igv_session.xml igv_files.txt ../reference_genome/${fasta.getName()} --path_prefix '../'
"""

NextFlow From line 1444 of master/main.nf

"""
echo $workflow.manifest.version > v_pipeline.txt
echo $workflow.nextflow.version > v_nextflow.txt
fastqc --version > v_fastqc.txt
trim_galore --version > v_trim_galore.txt
echo \$(bwa 2>&1) > v_bwa.txt
samtools --version > v_samtools.txt
bedtools --version > v_bedtools.txt
echo \$(bamtools --version 2>&1) > v_bamtools.txt
echo \$(plotFingerprint --version 2>&1) > v_deeptools.txt || true
picard MarkDuplicates --version &> v_picard.txt  || true
echo \$(R --version 2>&1) > v_R.txt
python -c "import pysam; print(pysam.__version__)" > v_pysam.txt
preseq &> v_preseq.txt
danpos.py --version > v_danpos.txt
multiqc --version > v_multiqc.txt
scrape_software_versions.py &> software_versions_mqc.yaml
"""

NextFlow SAMtools FastQC MultiQC BEDTools Picard Trim_Galore preseq From line 1473 of master/main.nf

"""
multiqc . -f $rtitle $rfilename $custom_config_file \\
    -m custom_content -m fastqc -m cutadapt -m samtools -m picard -m preseq -m deeptools
"""

NextFlow SAMtools FastQC MultiQC Picard Cutadapt DeepTools preseq From line 1555 of master/main.nf

"""
markdown_to_html.py $output_docs -o results_description.html
"""

NextFlow From line 1582 of master/main.nf

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Created: 1yr ago

Updated: 1yr ago

Maitainers: public

URL: https://nf-co.re/mnaseseq

Name: mnaseseq

Version: 1.0.0

Badge:

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License: None

Keywords:

bigWig preseq BamTools bedGraphToBigWig BEDTools BWA Cutadapt DeepTools FastQC GFFutils MultiQC Picard SAMtools Trim_Galore wigToBigWig Sequence analysis

Refs:

https://nf-co.re/mnaseseq

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