Explore Workflows

# ChIP-Seq basic analysis workflow for single-read data Reads are aligned to the reference genome with [Bowtie](http://bowtie-bio.sourceforge.net/index.shtml). Results are saved as coordinate sorted [BAM](http://samtools.github.io/hts-specs/SAMv1.pdf) alignment and index BAI files. Optionally, PCR duplicates can be removed. To obtain coverage in [bigWig](https://genome.ucsc.edu/goldenpath/help/bigWig.html) format, average fragment length is calculated by [MACS2](https://github.com/taoliu/MACS), and individual reads are extended to this length in the 3’ direction. Areas of enrichment identified by MACS2 are saved in ENCODE [narrow peak](http://genome.ucsc.edu/FAQ/FAQformat.html#format12) or [broad peak](https://genome.ucsc.edu/FAQ/FAQformat.html#format13) formats. Called peaks together with the nearest genes are saved in TSV format. In addition to basic statistics (number of total/mapped/multi-mapped/unmapped/duplicate reads), pipeline generates several quality control measures. Base frequency plots are used to estimate adapter contamination, a frequent occurrence in low-input ChIP-Seq experiments. Expected distinct reads count from [Preseq](http://smithlabresearch.org/software/preseq/) can be used to estimate read redundancy for a given sequencing depth. Average tag density profiles can be used to estimate ChIP enrichment for promoter proximal histone modifications. Use of different parameters for different antibodies (calling broad or narrow peaks) is possible. Additionally, users can elect to use BAM file from another experiment as control for MACS2 peak calling. ## Cite as *Kartashov AV, Barski A. BioWardrobe: an integrated platform for analysis of epigenomics and transcriptomics data. Genome Biol. 2015;16(1):158. Published 2015 Aug 7. [doi:10.1186/s13059-015-0720-3](https://www.ncbi.nlm.nih.gov/pubmed/26248465)* ## Software versions - Bowtie 1.2.0 - Samtools 1.4 - Preseq 2.0 - MACS2 2.1.1.20160309 - Bedtools 2.26.0 - UCSC userApps v358 ## Inputs | ID | Label | Description | Required | Default | Upstream analyses | | ------------------------- | ---------------------------------------------- | ---------------------------------------------------------------------------------------------------------------------------------------------------------------- | :------: | ------- | ------------------------------- | | **fastq\_file** | FASTQ file | Single-read sequencing data in FASTQ format (fastq, fq, bzip2, gzip, zip) | + | | | | **indices\_folder** | Genome indices | Directory with the genome indices generated by Bowtie | + | | genome\_indices/bowtie\_indices | | **annotation\_file** | Genome annotation file | Genome annotation file in TSV format | + | | genome\_indices/annotation | | **genome\_size** | Effective genome size | The length of the mappable genome (hs, mm, ce, dm or number, for example 2.7e9) | + | | genome\_indices/genome\_size | | **chrom\_length** | Chromosome lengths file | Chromosome lengths file in TSV format | + | | genome\_indices/chrom\_length | | **broad\_peak** | Call broad peaks | Make MACS2 call broad peaks by linking nearby highly enriched regions | + | | | | **control\_file** | Control ChIP-Seq single-read experiment | Indexed BAM file from the ChIP-Seq single-read experiment to be used as a control for MACS2 peak calling | | Null | control\_file/bambai\_pair | | **exp\_fragment\_size** | Expected fragment size | Expected fragment size for read extenstion towards 3' end if *force\_fragment\_size* was set to True or if calculated by MACS2 fragment size was less that 80 bp | | 150 | | | **force\_fragment\_size** | Force peak calling with expected fragment size | Make MACS2 don't build the shifting model and use expected fragment size for read extenstion towards 3' end | | False | | | **clip\_3p\_end** | Clip from 3' end | Number of base pairs to clip from 3' end | | 0 | | | **clip\_5p\_end** | Clip from 5' end | Number of base pairs to clip from 5' end | | 0 | | | **remove\_duplicates** | Remove PCR duplicates | Remove PCR duplicates from sorted BAM file | | False | | | **threads** | Number of threads | Number of threads for those steps that support multithreading | | 2 | | ## Outputs | ID | Label | Description | Required | Visualization | | ------------------------ | ---------------------------------- | ------------------------------------------------------------------------------------ | :------: | ------------------------------------------------------------------ | | **fastx\_statistics** | FASTQ quality statistics | FASTQ quality statistics in TSV format | + | *Base Frequency* and *Quality Control* plots in *QC Plots* tab | | **bambai\_pair** | Aligned reads | Coordinate sorted BAM alignment and index BAI files | + | *Nucleotide Sequence Alignments* track in *IGV Genome Browser* tab | | **bigwig** | Genome coverage | Genome coverage in bigWig format | + | *Genome Coverage* track in *IGV Genome Browser* tab | | **iaintersect\_result** | Gene annotated peaks | MACS2 peak file annotated with nearby genes | + | *Peak Coordinates* table in *Peak Calling* tab | | **atdp\_result** | Average Tag Density Plot | Average Tag Density Plot file in TSV format | + | *Average Tag Density Plot* in *QC Plots* tab | | **macs2\_called\_peaks** | Called peaks | Called peaks file with 1-based coordinates in XLS format | + | | | **macs2\_narrow\_peaks** | Narrow peaks | Called peaks file in ENCODE narrow peak format | | *Narrow peaks* track in *IGV Genome Browser* tab | | **macs2\_broad\_peaks** | Broad peaks | Called peaks file in ENCODE broad peak format | | *Broad peaks* track in *IGV Genome Browser* tab | | **preseq\_estimates** | Expected Distinct Reads Count Plot | Expected distinct reads count file from Preseq in TSV format | | *Expected Distinct Reads Count Plot* in *QC Plots* tab | | **workflow\_statistics** | Workflow execution statistics | Overall workflow execution statistics from bowtie\_aligner and samtools\_rmdup steps | + | *Overview* tab and experiment's preview | | **bowtie\_log** | Read alignment log | Read alignment log file from Bowtie | + | |

https://github.com/datirium/workflows.git

Path: workflows/chipseq-se.cwl

Branch/Commit ID: 91bb63948c0a264334b9007ef85f936768d90d11

https://github.com/joshuagad/cwl-varcall.git

Path: cwl/varcall-standard.cwl

Branch/Commit ID: 2f53e3b76fa2d943bc2fd9733b29bea8db207493

CNV GRIDSS calling

https://gitlab.bsc.es/lrodrig1/structuralvariants_poc.git

Path: structuralvariants/subworkflows/cnv_gridss.cwl

Branch/Commit ID: 70eec658fd1b92c4d0e3b24146820010b5983d41

The original [BioWardrobe's](https://biowardrobe.com) [PubMed ID:26248465](https://www.ncbi.nlm.nih.gov/pubmed/26248465) **ChIP-Seq** basic analysis workflow for a **paired-end** experiment. A [FASTQ](http://maq.sourceforge.net/fastq.shtml) input file has to be provided. The pipeline produces a sorted BAM file alongside with index BAI file, quality statistics of the input FASTQ file, coverage by estimated fragments as a BigWig file, peaks calling data in a form of narrowPeak or broadPeak files, islands with the assigned nearest genes and region type, data for average tag density plot. Workflow starts with step *fastx\_quality\_stats* from FASTX-Toolkit to calculate quality statistics for input FASTQ file. At the same time `bowtie` is used to align reads from input FASTQ file to reference genome *bowtie\_aligner*. The output of this step is an unsorted SAM file which is being sorted and indexed by `samtools sort` and `samtools index` *samtools\_sort\_index*. Depending on workflow’s input parameters indexed and sorted BAM file can be processed by `samtools rmdup` *samtools\_rmdup* to get rid of duplicated reads. If removing duplicates is not required the original BAM and BAI files are returned. Otherwise step *samtools\_sort\_index\_after\_rmdup* repeat `samtools sort` and `samtools index` with BAM and BAI files without duplicates. Next `macs2 callpeak` performs peak calling *macs2\_callpeak* and the next step reports *macs2\_island\_count* the number of islands and estimated fragment size. If the latter is less that 80bp (hardcoded in the workflow) `macs2 callpeak` is rerun again with forced fixed fragment size value (*macs2\_callpeak\_forced*). It is also possible to force MACS2 to use pre set fragment size in the first place. Next step (*macs2\_stat*) is used to define which of the islands and estimated fragment size should be used in workflow output: either from *macs2\_island\_count* step or from *macs2\_island\_count\_forced* step. If input trigger of this step is set to True it means that *macs2\_callpeak\_forced* step was run and it returned different from *macs2\_callpeak* step results, so *macs2\_stat* step should return [fragments\_new, fragments\_old, islands\_new], if trigger is False the step returns [fragments\_old, fragments\_old, islands\_old], where sufix \"old\" defines results obtained from *macs2\_island\_count* step and sufix \"new\" - from *macs2\_island\_count\_forced* step. The following two steps (*bamtools\_stats* and *bam\_to\_bigwig*) are used to calculate coverage from BAM file and save it in BigWig format. For that purpose bamtools stats returns the number of mapped reads which is then used as scaling factor by bedtools genomecov when it performs coverage calculation and saves it as a BEDgraph file whichis then sorted and converted to BigWig format by bedGraphToBigWig tool from UCSC utilities. Step *get\_stat* is used to return a text file with statistics in a form of [TOTAL, ALIGNED, SUPRESSED, USED] reads count. Step *island\_intersect* assigns nearest genes and regions to the islands obtained from *macs2\_callpeak\_forced*. Step *average\_tag\_density* is used to calculate data for average tag density plot from the BAM file.

https://github.com/datirium/workflows.git

Path: workflows/chipseq-pe.cwl

Branch/Commit ID: d6ec0dee61ef65a110e10141bde1a79332a64ab0

https://github.com/common-workflow-language/cwltool.git

Path: cwltool/schemas/v1.0/v1.0/scatter-wf3.cwl

Branch/Commit ID: 03af16c9df2ee77485d4ab092cd64ae096d2e71c

Packed ID: main

https://github.com/arvados/bh20-seq-resource.git

Path: workflows/pangenome-generate/pangenome-generate.cwl

Branch/Commit ID: 80cfaba31a99d0c34722312c1b1a69a139477510

https://github.com/genome/analysis-workflows.git

Path: definitions/subworkflows/single_sample_sv_callers.cwl

Branch/Commit ID: e649fcb1092905c539be026a3f23c82d5b0871d2

https://github.com/common-workflow-language/common-workflow-language.git

Path: v1.0/v1.0/scatter-wf3.cwl

Branch/Commit ID: 1f501e38ff692a408e16b246ac7d64d32f0822c2

Packed ID: main

Workflow runs group-isoforms.cwl tool using scatter for isoforms_file input. genes_filename and common_tss_filename inputs are ignored.

https://github.com/datirium/workflows.git

Path: tools/group-isoforms-batch.cwl

Branch/Commit ID: 7ae3b75bbe614e59cdeaba06047234a6c40c0fe9

https://github.com/genome/analysis-workflows.git

Path: definitions/subworkflows/strelka_and_post_processing.cwl

Branch/Commit ID: 295e7b7f51727c0f2d6cc86ce817449b2e8dba3c