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RNA-Seq pipeline paired-end
The original [BioWardrobe's](https://biowardrobe.com) [PubMed ID:26248465](https://www.ncbi.nlm.nih.gov/pubmed/26248465) **RNA-Seq** basic analysis for a **paired-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 paired-end RNA-Seq data. It performs the following steps: 1. Use STAR to align reads from input FASTQ files according to the predefined reference indices; generate unsorted BAM file and alignment statistics file 2. Use fastx_quality_stats to analyze input FASTQ files and generate quality statistics files 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) 4. Generate BigWig file on the base of sorted BAM file 5. Map input FASTQ files to predefined rRNA reference indices using Bowtie to define the level of rRNA contamination; export resulted statistics to file 6. Calculate isoform expression level for the sorted BAM file and GTF/TAB annotation file using GEEP reads-counting utility; export results to file |
Path: workflows/rnaseq-pe.cwl Branch/Commit ID: b1a5dabeeeb9079b30b2871edd9c9034a1e00c1c |
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advanced-header.cwl
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Path: metadata/advanced-header.cwl Branch/Commit ID: b5e16e359007150647b14dc6e038f4eb8dccda79 |
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THOR - differential peak calling of ChIP-seq signals with replicates
What is THOR? -------------- THOR is an HMM-based approach to detect and analyze differential peaks in two sets of ChIP-seq data from distinct biological conditions with replicates. THOR performs genomic signal processing, peak calling and p-value calculation in an integrated framework. For more information please refer to: ------------------------------------- Allhoff, M., Sere K., Freitas, J., Zenke, M., Costa, I.G. (2016), Differential Peak Calling of ChIP-seq Signals with Replicates with THOR, Nucleic Acids Research, epub gkw680. |
Path: workflows/rgt-thor.cwl Branch/Commit ID: b1a5dabeeeb9079b30b2871edd9c9034a1e00c1c |
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Motif Finding with HOMER with custom background regions
Motif Finding with HOMER with custom background regions --------------------------------------------------- HOMER contains a novel motif discovery algorithm that was designed for regulatory element analysis in genomics applications (DNA only, no protein). It is a differential motif discovery algorithm, which means that it takes two sets of sequences and tries to identify the regulatory elements that are specifically enriched in on set relative to the other. It uses ZOOPS scoring (zero or one occurrence per sequence) coupled with the hypergeometric enrichment calculations (or binomial) to determine motif enrichment. HOMER also tries its best to account for sequenced bias in the dataset. It was designed with ChIP-Seq and promoter analysis in mind, but can be applied to pretty much any nucleic acids motif finding problem. For more information please refer to: ------------------------------------- [Official documentation](http://homer.ucsd.edu/homer/motif/) |
Path: workflows/homer-motif-analysis-bg.cwl Branch/Commit ID: b1a5dabeeeb9079b30b2871edd9c9034a1e00c1c |
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ChIP-Seq pipeline paired-end
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. |
Path: workflows/chipseq-pe.cwl Branch/Commit ID: b1a5dabeeeb9079b30b2871edd9c9034a1e00c1c |
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GSEApy - Gene Set Enrichment Analysis in Python
GSEAPY: Gene Set Enrichment Analysis in Python ============================================== Gene Set Enrichment Analysis is a computational method that determines whether an a priori defined set of genes shows statistically significant, concordant differences between two biological states (e.g. phenotypes). GSEA requires as input an expression dataset, which contains expression profiles for multiple samples. While the software supports multiple input file formats for these datasets, the tab-delimited GCT format is the most common. The first column of the GCT file contains feature identifiers (gene ids or symbols in the case of data derived from RNA-Seq experiments). The second column contains a description of the feature; this column is ignored by GSEA and may be filled with “NA”s. Subsequent columns contain the expression values for each feature, with one sample's expression value per column. It is important to note that there are no hard and fast rules regarding how a GCT file's expression values are derived. The important point is that they are comparable to one another across features within a sample and comparable to one another across samples. Tools such as DESeq2 can be made to produce properly normalized data (normalized counts) which are compatible with GSEA. |
Path: workflows/gseapy.cwl Branch/Commit ID: b1a5dabeeeb9079b30b2871edd9c9034a1e00c1c |
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DESeq - differential gene expression analysis
Differential gene expression analysis ===================================== Differential gene expression analysis based on the negative binomial distribution Estimate variance-mean dependence in count data from high-throughput sequencing assays and test for differential expression based on a model using the negative binomial distribution. 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://bioconductor.org/packages/release/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. |
Path: workflows/deseq.cwl Branch/Commit ID: b1a5dabeeeb9079b30b2871edd9c9034a1e00c1c |
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Cell Ranger Build Reference Indices
Cell Ranger Build Reference Indices =================================== |
Path: workflows/cellranger-mkref.cwl Branch/Commit ID: b1a5dabeeeb9079b30b2871edd9c9034a1e00c1c |
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Build Bismark indices
Copy fasta_file file to the folder and run run bismark_genome_preparation script to prepare indices for Bismark Methylation Analysis. Bowtie2 aligner is used by default. The name of the output indices folder is equal to the genome input. |
Path: workflows/bismark-index.cwl Branch/Commit ID: b1a5dabeeeb9079b30b2871edd9c9034a1e00c1c |
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Trim Galore SMARTer RNA-Seq pipeline paired-end strand specific
https://chipster.csc.fi/manual/library-type-summary.html Modified 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 |
Path: workflows/trim-rnaseq-pe-smarter-dutp.cwl Branch/Commit ID: a1f6ca50fcb0881781b3ba0306dd61ebf555eaba |
