Explore Workflows

Runs RNA-Seq BioWardrobe basic analysis with pair-end data file.

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

Path: workflows/rnaseq-pe.cwl

Branch/Commit ID: 89082ba8d398033dee84c37a84c6547466c1f2d2

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

Path: definitions/subworkflows/qc_exome_no_verify_bam.cwl

Branch/Commit ID: da335d9963418f7bedd84cb2791a0df1b3165ffe

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

Path: cwltool/schemas/v1.0/v1.0/count-lines6-wf.cwl

Branch/Commit ID: 5ae5798f1c0c8d2178986b77cfd74edff510877a

https://github.com/common-workflow-language/cwl-v1.2.git

Path: tests/count-lines4-wf.cwl

Branch/Commit ID: a5073143db4155e05df8d2e7eb59d9e62acd65a5

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

Path: definitions/subworkflows/qc_exome.cwl

Branch/Commit ID: b7d9ace34664d3cedb16f2512c8a6dc6debfc8ca

Note: should be updated The original [BioWardrobe's](https://biowardrobe.com) [PubMed ID:26248465](https://www.ncbi.nlm.nih.gov/pubmed/26248465) **RNA-Seq** basic analysis for **strand specific single-read** 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-read RNA-Seq data. It performs the following steps: 1. Use STAR to align reads from input FASTQ file according to the predefined reference indices; generate unsorted BAM file and alignment statistics file 2. Use fastx_quality_stats to analyze input FASTQ file and generate quality statistics file 3. 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 file 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/rnaseq-se-dutp.cwl

Branch/Commit ID: 3fc68366adb179927af5528c27b153abaf94494d

https://github.com/common-workflow-language/cwl-v1.1.git

Path: tests/count-lines4-wf.cwl

Branch/Commit ID: 664835e83eb5e57eee18a04ce7b05fb9d70d77b7

Experimental pipeline for Cut-n-Run analysis. Uses mapping results from the following experiment types: - `chipseq-pe.cwl` - `trim-chipseq-pe.cwl` - `trim-atacseq-pe.cwl` Note, the upstream analyses should not have duplicates removed

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

Path: workflows/trim-chipseq-pe-cut-n-run.cwl

Branch/Commit ID: 730b40bc403263b724399a952c0f3e2d28f13519

# Differential gene expression analysis This differential gene expression (DGE) analysis takes as input samples from two experimental conditions that have been processed with an RNA-Seq workflow (see list of \"Upstream workflows\" below). DESeq estimates variance-mean dependence in count data from high-throughput sequencing assays, then tests for DGE based on a model which assumes a negative binomial distribution of gene expression (aligned read count per gene). ### Experimental Setup and Results Interpretation The workflow design uses as its fold change (FC) calculation: condition 1 (c1, e.g. treatment) over condition 2 (c2, e.g. control). In other words: `FC == (c1/c2)` Therefore: - if FC<1 the log2(FC) is <0 (negative), meaning expression in condition1<condition2 (gene is downregulated in c1) - if FC>1 the log2(FC) is >0 (positive), meaning expression in condition1>condition2 (gene is upregulated in c1) In other words, if you have input TREATMENT samples as condition 1, and CONTROL samples as condition 2, a positive L2FC for a gene indicates that expression of the gene in TREATMENT is greater (or upregulated) compared to CONTROL. Next, threshold the p-adjusted values with your FDR (false discovery rate) cutoff to determine if the change may be considered significant or not. It is important to note when DESeq1 or DESeq2 is used in our DGE analysis workflow. If a user inputs only a single sample per condition DESeq1 is used for calculating DGE. In this experimental setup, there are no repeated measurements per gene per condition, therefore biological variability in each condition cannot be captured so the output p-values are assumed to be purely \"technical\". On the other hand, if >1 sample(s) are input per condition DESeq2 is used. In this case, biological variability per gene within each condition is available to be incorporated into the model, and resulting p-values are assumed to be \"biological\". Additionally, DESeq2 fold change is \"shrunk\" to account for sample variability, and as Michael Love (DESeq maintainer) puts it, \"it looks at the largest fold changes that are not due to low counts and uses these to inform a prior distribution. So the large fold changes from genes with lots of statistical information are not shrunk, while the imprecise fold changes are shrunk. This allows you to compare all estimated LFC across experiments, for example, which is not really feasible without the use of a prior\". In either case, the null hypothesis (H0) tested is that there are no significantly differentially expressed genes between conditions, therefore a smaller p-value indicates a lower probability of the H0 occurring by random chance and therefore, below a certain threshold (traditionally <0.05), H0 should be rejected. Additionally, due to the many thousands of independent hypotheses being tested (each gene representing an independent test), the p-values attained by the Wald test are adjusted using the Benjamini and Hochberg method by default. These \"padj\" values should be used for determination of significance (a reasonable value here would be <0.10, i.e. below a 10% FDR). Further Analysis: Output from the DESeq workflow may be used as input to the GSEA (Gene Set Enrichment Analysis) workflow for identifying enriched marker gene sets between conditions. ### DESeq1 High-throughput sequencing assays such as RNA-Seq, ChIP-Seq or barcode counting provide quantitative readouts in the form of count data. To infer differential signal in such data correctly and with good statistical power, estimation of data variability throughout the dynamic range and a suitable error model are required. Simon Anders and Wolfgang Huber propose a method based on the negative binomial distribution, with variance and mean linked by local regression and present an implementation, [DESeq](http://www.bioconductor.org/packages/3.8/bioc/html/DESeq.html), as an R/Bioconductor package. ### DESeq2 In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. [DESeq2](http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html), a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. ### __References__ - Anders S, Huber W (2010). “Differential expression analysis for sequence count data.” Genome Biology, 11, R106. doi: 10.1186/gb-2010-11-10-r106, http://genomebiology.com/2010/11/10/R106/. - Love MI, Huber W, Anders S (2014). “Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.” Genome Biology, 15, 550. doi: 10.1186/s13059-014-0550-8.

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

Path: workflows/deseq.cwl

Branch/Commit ID: bf80c9339d81a78aefb8de661bff998ed86e836e

Wrapped in a workflow SoupX tool for easy access to Cell Ranger pipeline compressed outputs.

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

Path: tools/soupx-subworkflow.cwl

Branch/Commit ID: ebbf23764ede324cabc064bd50647c1f643726fa