Explore Workflows

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

Path: definitions/subworkflows/bam_to_trimmed_fastq_and_biscuit_alignments.cwl

Branch/Commit ID: 31602b94b34ff55876147c7299e1bec47e8d1a31

https://github.com/ncbi/pgap.git

Path: task_types/tt_bact_get_kmer_reference.cwl

Branch/Commit ID: 6d8d29a2156b93a75f1d1c6952738bd63f6bd98e

A basic analysis workflow for paired-read CUT&RUN and CUT&TAG sequencing experiments. These sequencing library prep methods are ultra-sensitive chromatin mapping technologies compared to the ChIP-Seq methodology. Its primary benefits include 1) length filtering, 2) a higher signal-to-noise ratio, and 3) built-in normalization for between sample comparisons. This workflow utilizes the tool MACS2 which calls enriched regions in the target sequence data by identifying the top regions by area under a poisson distribution (of the alignment pileup). This workflow is loosely based on the [CUT-RUNTools-2.0 pipeline](https://github.com/fl-yu/CUT-RUNTools-2.0) pipeline, and the ChIP-Seq pipeline from [BioWardrobe](https://biowardrobe.com) [PubMed ID:26248465](https://www.ncbi.nlm.nih.gov/pubmed/26248465) was used as a CWL template. ### __Inputs__ *General Info (required\*):* - Experiment short name/Alias* - a unique name for the sample (e.g. what was used on tubes while processing it) - Cells* - sample cell type or organism name - Conditions* - experimental condition name - Catalog # - catalog number for cells from vender/supplier - Primary [genome index](https://scidap.com/tutorials/basic/genome-indices) for peak calling* - preprocessed genome index of sample organism for primary alignment and peak calling - Secondary [genome index](https://scidap.com/tutorials/basic/genome-indices) for spike-in normalization* - preprocessed genome index of spike-in organism for secondary alignment (of unaligned reads from primary alignment) and spike-in normalization, default should be E. coli K-12 - FASTQ file for R1* - read 1 file of a pair-end library - FASTQ file for R2* - read 2 file of a pair-end library *Advanced:* - - Number of bases to clip from the 3p end - used by bowtie aligner to trim <int> bases from 3' (right) end of reads - Number of bases to clip from the 5p end - used by bowtie aligner to trim <int> bases from 5' (left) end of reads - Call samtools rmdup to remove duplicates from sorted BAM file? - toggle on/off to remove duplicate reads from analysis - Fragment Length Filter will retain fragments between set base pair (bp) ranges for peak analysis - drop down menu - `default_below_1000` retains fragments <1000 bp - `histones_130_to_300` retains fragments between 130-300 bp - `TF_below_130` retains fragments <130 bp - Max distance (bp) from gene TSS (in both directions) overlapping which the peak will be assigned to the promoter region - default set to `1000` - Max distance (bp) from the promoter (only in upstream directions) overlapping which the peak will be assigned to the upstream region - default set to `20000` - Number of threads for steps that support multithreading - default set to `2` ### __Outputs__ Intermediate and final downloadable outputs include: - IGV with gene, BigWig (raw and normalized), and stringent peak tracks - quality statistics and visualizations for both R1/R2 input FASTQ files - coordinate sorted BAM file with associated BAI file for primary alignment - read pileup/coverage in BigWig format (raw and normalized) - cleaned bed files (containing fragment coordinates), and spike-in normalized peak-called BED files (also includes \"narrow\" and \"broad\" peaks). - stringent peak call bed file with nearest gene annotations per peak ### __Data Analysis Steps__ 1. Trimming the adapters with TrimGalore. - This step is particularly important when the reads are long and the fragments are short - resulting in sequencing adapters at the ends of reads. If adapter is not removed the read will not map. TrimGalore can recognize standard adapters, such as Illumina or Nextera/Tn5 adapters. 2. Generate quality control statistics of trimmed, unmapped sequence data 3. (Optional) Clipping of 5' and/or 3' end by the specified number of bases. 4. Mapping reads to primary genome index with Bowtie. - Only uniquely mapped reads with less than 3 mismatches are used in the downstream analysis. Results are then sorted and indexed. Final outputs are in bam/bai format, which are also used to extrapolate effects of additional sequencing based on library complexity. 5. (Optional) Removal of duplicates (reads/pairs of reads mapping to exactly the same location). - This step is used to remove reads overamplified during amplification of the library. Unfortunately, it may also remove \"good\" reads. We usually do not remove duplicates unless the library is heavily duplicated. 6. Mapping unaligned reads from primary alignment to secondary genome index with Bowtie. - This step is used to obtain the number of reads for normalization, used to scale the pileups from the primary alignment. After normalization, sample pileups/peak may then be appropriately compared to one another assuming an equal use of spike-in material during library preparation. Note the default genome index for this step should be *E. coli* K-12 if no spike-in material was called out in the library protocol. Refer to [Step 16](https://www.protocols.io/view/cut-amp-tag-data-processing-and-analysis-tutorial-e6nvw93x7gmk/v1?step=16#step-4A3D8C70DC3011EABA5FF3676F0827C5) of the \"CUT&Tag Data Processing and Analysis Tutorial\" by Zheng Y et al (2020). Protocol.io. 7. Formatting alignment file to account for fragments based on paired-end BAM. - Generates a filtered and normalized bed file to be used as input for peak calling. 8. Call enriched regions using MACS2. - This step called peaks (broad and narrow) using the MACS2 tool with default parameters and no normalization to a control sample. 9. Generation and formatting of output files. - This step collects read, alignment, and peak statistics, as well asgenerates BigWig coverage/pileup files for display on the browser using IGV. The coverage shows the number of fragments that cover each base in the genome both normalized and unnormalized to the calculated spike-in scaling factor. ### __References__ - Meers MP, Tenenbaum D, Henikoff S. (2019). Peak calling by Sparse Enrichment Analysis for CUT&RUN chromatin profiling. Epigenetics and Chromatin 12(1):42. - Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25.

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

Path: workflows/cutandrun-macs2-pe.cwl

Branch/Commit ID: 36fd18f11e939d3908b1eca8d2939402f7a99b0f

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

Path: definitions/subworkflows/single_sample_sv_callers.cwl

Branch/Commit ID: 4bc0a4577d626b65a4b44683e5a1ab2f7d7faf4c

Some workflow engines won't stage files in our nested structure, so parse it out here

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

Path: definitions/subworkflows/sequence_align_and_tag_adapter.cwl

Branch/Commit ID: 60edaf6f57eaaf02cda1a3d8cb9a825aa64a43e2

https://github.com/ncbi/pgap.git

Path: bacterial_annot/wf_bacterial_annot_pass3.cwl

Branch/Commit ID: ce433f771ebf5677c9f40858e2ae91b1a7e75d30

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

Path: tests/wf/sec-wf.cwl

Branch/Commit ID: 280a852e74aec08cf79687e8004e17b1ab464534

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

Path: definitions/subworkflows/duplex_alignment.cwl

Branch/Commit ID: 31602b94b34ff55876147c7299e1bec47e8d1a31

https://github.com/reanahub/reana-demo-root6-roofit.git

Path: workflow/cwl/workflow-htcondorcern.cwl

Branch/Commit ID: 1fbe52138b98a85a15fde58d385b21b637c18be8

https://github.com/cancerit/workflow-seq-import.git

Path: cwls/chksum_seqval_wf_paired_fq.cwl

Branch/Commit ID: edef7383acae1215a34ddfa6388224570c582c9f