Explore Workflows

This ATAC pipeline is based on original [BioWardrobe's](https://biowardrobe.com) [PubMed ID:26248465](https://www.ncbi.nlm.nih.gov/pubmed/26248465) **ChIP-Seq** basic analysis workflow for a **single-read** experiment with Trim Galore. The pipeline was adapted for ATAC-Seq single-read data analysis by updating genome coverage step. ### Data Analysis Steps SciDAP starts from the .fastq files which most DNA cores and commercial NGS companies return. Starting from raw data allows us to ensure that all experiments have been processed in the same way and simplifies the deposition of data to GEO upon publication. The data can be uploaded from users computer, downloaded directly from an ftp server of the core facility by providing a URL or from GEO by providing SRA accession number. Our current pipelines include the following steps: 1. Trimming the adapters with TrimGalore. This step is particularly important when the reads are long and the fragments are short as in ATAC -resulting in sequencing adapters at the end of read. If adapter is not removed the read will not map. TrimGalore can recognize standard adapters, such as Nexterra/Tn5 adapters. 2. QC 3. (Optional) trimming adapters on 5' or 3' end by the specified number of bases. 4. Mapping reads with BowTie. Only uniquely mapped reads with less than 3 mismatches are used in the downstream analysis. Results are saved as a .bam file. 5. Reads mapping to chromosome M are removed. Since there are many copies of chromosome M in the cell and it is not protected by histones, some ATAC libraries have up to 50% of reads mapping to chrM. We recommend using OMNI-ATAC protocol that reduces chrM reads and provides better specificity. 6. (Optional) Removal of duplicates (reads/pairs of reads mapping to exactly same location). This step is used to remove reads overamplified in PCR. Unfortunately, it may also remove \"good\" reads. We usually do not remove duplicates unless the library is heavily duplicated. Please note that MACS2 will remove 'excessive' duplicates during peak calling ina smart way (those not supported by other nearby reads). 7. Peakcalling by MACS2. (Optionally), it is possible to specify read extension length for MACS2 to use if the length determined automatically is wrong. 8. Generation of BigWig coverage files for display on the browser. Since the cuts by the Tn5 transposome are 9bp apart, we show coverage by 9bp reads rather than fragments as in ChIP-Seq. The coverage shows the number of fragments at each base in the genome normalized to the number of millions of mapped reads. This way the peak of coverage will be located at the most accessible site. ### Details _Trim Galore_ is a wrapper around [Cutadapt](https://github.com/marcelm/cutadapt) and [FastQC](http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to consistently apply adapter and quality trimming to FastQ files, with extra functionality for RRBS data. In outputs it returns coordinate sorted BAM file alongside with index BAI file, quality statistics of the input FASTQ file, reads coverage in a form of 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 (on the base of BAM file). 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 unsorted SAM file which is being sorted and indexed by `samtools sort` and `samtools index` *samtools\_sort\_index*. Based 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 input BAM and BAI files return. Otherwise step *samtools\_sort\_index\_after\_rmdup* repeat `samtools sort` and `samtools index` with BAM and BAI files. Right after that `macs2 callpeak` performs peak calling *macs2\_callpeak*. On the base of returned outputs the next step *macs2\_island\_count* calculates the number of islands and estimated fragment size. If the last one is less that 80bp (hardcoded in the workflow) `macs2 callpeak` is rerun again with forced fixed fragment size value (*macs2\_callpeak\_forced*). If at the very beginning it was set in workflow input parameters to force run peak calling with fixed fragment size, this step is skipped and the original peak calling results are saved. In the next step workflow again calculates the number of islands and estimates fragment size (*macs2\_island\_count\_forced*) for the data obtained from *macs2\_callpeak\_forced* step. If the last one was skipped the results from *macs2\_island\_count\_forced* step are equal to the ones obtained from *macs2\_island\_count* step. 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 on the base of input BAM file and save it in BigWig format. For that purpose bamtools stats returns the number of mapped reads number which is then used as scaling factor by bedtools genomecov when it performs coverage calculation and saves it in BED format. The last one is then being sorted and converted to BigWig format by bedGraphToBigWig tool from UCSC utilities. To adapt the pipeline for ATAC-Seq data analysis we calculate genome coverage using only the first 9 bp from every read. 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 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 on the base of BAM file.

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

Path: workflows/trim-atacseq-se.cwl

Branch/Commit ID: 2c486543c335bb99b245dfe7e2f033f535efb9cf

https://github.com/cr-ste-justine/chujs-alignment-workflow.git

Path: workflows/kfdrc_alignment_CramOnly_wf.cwl

Branch/Commit ID: 682ec407000059b7f397e5faaeff1317af1d9402

https://github.com/CERIT-SC/fireprot.git

Path: stability.cwl

Branch/Commit ID: a2b0c18f117dcf2b0d7e33c59c0f180b6a9bf709

### Devel version of QuantSeq 3' FWD, FWD-UMI or REV for single-read mRNA-Seq data

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

Path: workflows/trim-quantseq-mrnaseq-se-strand-specific.cwl

Branch/Commit ID: 2c486543c335bb99b245dfe7e2f033f535efb9cf

Runs ChIP-Seq BioWardrobe basic analysis with paired-end input data files.

https://github.com/Barski-lab/workflows.git

Path: workflows/trim-chipseq-pe.cwl

Branch/Commit ID: 12edfc2207507e53c6b5bb21e50decb5535a12f7

1. Convert input SRA file into pair of upsrtream and downstream FASTQ files (run fastq-dump) 2. Analyze quality of FASTQ files (run fastqc with each of the FASTQ files) 3. If any of the following fields in fastqc generated report is marked as failed for at least one of input FASTQ files: \"Per base sequence quality\", \"Per sequence quality scores\", \"Overrepresented sequences\", \"Adapter Content\", - trim adapters (run trimmomatic) 4. Align original or trimmed FASTQ files to reference genome, calculate genes and isoforms expression (run RSEM) 5. Count mapped reads number in sorted BAM file (run bamtools stats) 6. Generate genome coverage BED file (run bedtools genomecov) 7. Sort genearted BED file (run sort) 8. Generate genome coverage bigWig file from BED file (run bedGraphToBigWig)

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

Path: workflows/xenbase-rnaseq-se.cwl

Branch/Commit ID: 7518b100d8cbc80c8be32e9e939dfbb27d6b4361

This is a subworkflow of the main oxog_varbam_annotat_wf workflow - this is not meant to be run as a stand-alone workflow!

https://github.com/svonworl/OxoG-Dockstore-Tools.git

Path: annotator_sub_wf.cwl

Branch/Commit ID: b38a8a4785746b8267913ea5389e21ae6dc921a3

This workflow will run OxoG, variantbam, and annotate. Run this as `dockstore --script --debug workflow launch --descriptor cwl --local-entry --entry ./oxog_varbam_annotate_wf.cwl --json oxog_varbam_annotat_wf.input.json `

https://github.com/svonworl/OxoG-Dockstore-Tools.git

Path: oxog_varbam_annotate_wf.cwl

Branch/Commit ID: b38a8a4785746b8267913ea5389e21ae6dc921a3

The original [BioWardrobe's](https://biowardrobe.com) [PubMed ID:26248465](https://www.ncbi.nlm.nih.gov/pubmed/26248465) **RNA-Seq** basic analysis for a **pair-end** experiment. A corresponded input [FASTQ](http://maq.sourceforge.net/fastq.shtml) file has to be provided. Current workflow should be used only with the single-end RNA-Seq data. It performs the following steps: 1. Trim adapters from input FASTQ files 2. Use STAR to align reads from input FASTQ files according to the predefined reference indices; generate unsorted BAM file and alignment statistics file 3. Use fastx_quality_stats to analyze input FASTQ files and generate quality statistics files 4. Use samtools sort to generate coordinate sorted BAM(+BAI) file pair from the unsorted BAM file obtained on the step 1 (after running STAR) 5. Generate BigWig file on the base of sorted BAM file 6. Map input FASTQ files to predefined rRNA reference indices using Bowtie to define the level of rRNA contamination; export resulted statistics to file 7. Calculate isoform expression level for the sorted BAM file and GTF/TAB annotation file using GEEP reads-counting utility; export results to file

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

Path: workflows/trim-rnaseq-pe.cwl

Branch/Commit ID: 7518b100d8cbc80c8be32e9e939dfbb27d6b4361

This is a subworkflow of the main oxog_varbam_annotat_wf workflow - this is not meant to be run as a stand-alone workflow!

https://github.com/svonworl/OxoG-Dockstore-Tools.git

Path: oxog_sub_wf.cwl

Branch/Commit ID: b38a8a4785746b8267913ea5389e21ae6dc921a3