Explore Workflows

Devel version of Single-Cell Cell Ranger Aggregate ================================================== Workflow calls \"cellranger aggr\" command to combine output files from \"cellranger count\" (the molecule_info.h5 file from each run) into a single feature-barcode matrix containing all the data. When combining multiple GEM wells, the barcode sequences for each channel are distinguished by a GEM well suffix appended to the barcode sequence. Each GEM well is a physically distinct set of GEM partitions, but draws barcode sequences randomly from the pool of valid barcodes, known as the barcode whitelist. To keep the barcodes unique when aggregating multiple libraries, we append a small integer identifying the GEM well to the barcode nucleotide sequence, and use that nucleotide sequence plus ID as the unique identifier in the feature-barcode matrix. For example, AGACCATTGAGACTTA-1 and AGACCATTGAGACTTA-2 are distinct cell barcodes from different GEM wells, despite having the same barcode nucleotide sequence. This number, which tells us which GEM well this barcode sequence came from, is called the GEM well suffix. The numbering of the GEM wells will reflect the order that the GEM wells were provided in the \"molecule_info_h5\" and \"gem_well_labels\" inputs. When combining data from multiple GEM wells, the \"cellranger aggr\" pipeline automatically equalizes the average read depth per cell between groups before merging. This approach avoids artifacts that may be introduced due to differences in sequencing depth. It is possible to turn off normalization or change the way normalization is done through the \"normalization_mode\" input. The \"none\" value may be appropriate if you want to maximize sensitivity and plan to deal with depth normalization in a downstream step.

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

Path: workflows/cellranger-aggr.cwl

Branch/Commit ID: 4ab9399a4777610a579ea2c259b9356f27641dcc

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

Path: definitions/subworkflows/filter_sv_vcf.cwl

Branch/Commit ID: ae75b938e6e8ae777a55686bbacad824b3c6788c

### Pipeline for Lexogen's QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina [Lexogen original documentation](https://www.lexogen.com/quantseq-3mrna-sequencing/) * Cost-saving and streamlined globin mRNA depletion during QuantSeq library preparation * Genome-wide analysis of gene expression * Cost-efficient alternative to microarrays and standard RNA-Seq * Down to 100 pg total RNA input * Applicable for low quality and FFPE samples * Single-read sequencing of up to 9,216 samples/lane * Dual indexing and Unique Molecular Identifiers (UMIs) are available ### QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina The QuantSeq FWD Kit is a library preparation protocol designed to generate Illumina compatible libraries of sequences close to the 3’ end of polyadenylated RNA. QuantSeq FWD contains the Illumina Read 1 linker sequence in the second strand synthesis primer, hence NGS reads are generated towards the poly(A) tail, directly reflecting the mRNA sequence (see workflow). This version is the recommended standard for gene expression analysis. Lexogen furthermore provides a high-throughput version with optional dual indexing (i5 and i7 indices) allowing up to 9,216 samples to be multiplexed in one lane. #### Analysis of Low Input and Low Quality Samples The required input amount of total RNA is as low as 100 pg. QuantSeq is suitable to reproducibly generate libraries from low quality RNA, including FFPE samples. See Fig.1 and 2 for a comparison of two different RNA qualities (FFPE and fresh frozen cryo-block) of the same sample.  Figure 1 | Correlation of gene counts of FFPE and cryo samples.  Figure 2 | Venn diagrams of genes detected by QuantSeq at a uniform read depth of 2.5 M reads in FFPE and cryo samples with 1, 5, and 10 reads/gene thresholds. #### Mapping of Transcript End Sites By using longer reads QuantSeq FWD allows to exactly pinpoint the 3’ end of poly(A) RNA (see Fig. 3) and therefore obtain accurate information about the 3’ UTR.  Figure 3 | QuantSeq read coverage versus normalized transcript length of NGS libraries derived from FFPE-RNA (blue) and cryo-preserved RNA (red). ### Current workflow should be used only with the single-end RNA-Seq data. It performs the following steps: 1. Separates UMIes and trims adapters from input FASTQ file 2. Uses ```STAR``` to align reads from input FASTQ file according to the predefined reference indices; generates unsorted BAM file and alignment statistics file 3. Uses ```fastx_quality_stats``` to analyze input FASTQ file and generates quality statistics file 4. Uses ```samtools sort``` and generates coordinate sorted BAM(+BAI) file pair from the unsorted BAM file obtained on the step 2 (after running STAR) 5. Uses ```umi_tools dedup``` and generates final filtered sorted BAM(+BAI) file pair 6. Generates BigWig file on the base of sorted BAM file 7. Maps input FASTQ file to predefined rRNA reference indices using ```bowtie``` to define the level of rRNA contamination; exports resulted statistics to file 8. Calculates isoform expression level for the sorted BAM file and GTF/TAB annotation file using GEEP reads-counting utility; exports results to file

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

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

Branch/Commit ID: 4ab9399a4777610a579ea2c259b9356f27641dcc

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/)

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

Path: workflows/homer-motif-analysis-bg.cwl

Branch/Commit ID: 2005c6b7f1bff6247d015ff6c116bd9ec97158bb

The \"main\" workflow. Takes fastq files generated using the seCLIP protocol (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5991800/) and outputs candidate RBP binding regions (peaks). runs: wf_get_peaks_se.cwl through scatter across multiple samples.

https://github.com/yeolab/eclip.git

Path: cwl/wf_get_peaks_scatter_se.cwl

Branch/Commit ID: 49a9bcda10de8f55fab2481f424eb9cdf2e5b256

Workflow converts input BAM file into bigWig and bedGraph files. Input BAM file should be sorted by coordinates (required by `bam_to_bedgraph` step). If `split` input is not provided use true by default. Default logic is implemented in `valueFrom` field of `split` input inside `bam_to_bedgraph` step to avoid possible bug in cwltool with setting default values for workflow inputs. `scale` has higher priority over the `mapped_reads_number`. The last one is used to calculate `-scale` parameter for `bedtools genomecov` (step `bam_to_bedgraph`) only in a case when input `scale` is not provided. All logic is implemented inside `bedtools-genomecov.cwl`. `bigwig_filename` defines the output name only for generated bigWig file. `bedgraph_filename` defines the output name for generated bedGraph file and can influence on generated bigWig filename in case when `bigwig_filename` is not provided. All workflow inputs and outputs don't have `format` field to avoid format incompatibility errors when workflow is used as subworkflow.

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

Path: tools/bam-bedgraph-bigwig.cwl

Branch/Commit ID: 9d5cbdd3ea0bb417518115d8092584254598a440

Workflow maps FASTQ files from `fastq_files` input into reference genome `reference_star_indices_folder` and insilico generated `insilico_star_indices_folder` genome (concatenated genome for both `strain1` and `strain2` strains). For both genomes STAR is run with `outFilterMultimapNmax` parameter set to 1 to discard all of the multimapped reads. For insilico genome SAM file is generated. Then it's splitted into two SAM files based on strain names and then sorted by coordinates into the BAM format. For reference genome output BAM file from STAR slignment is also coordinate sorted.

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

Path: subworkflows/allele-vcf-alignreads-se-pe.cwl

Branch/Commit ID: 99695ff7739364d02a1cb33cd6407c8693a7aaab

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

Path: cwltool/schemas/v1.0/v1.0/scatter-valuefrom-wf5.cwl

Branch/Commit ID: 1e3f5404b7d5af02e3dec0faea31352111ad7cd8

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

Path: task_types/tt_taxonomy_check_16S.cwl

Branch/Commit ID: 4def84df33963fc9ac9d5c5f804b911d01a0d9ad

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.

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

Path: workflows/gseapy.cwl

Branch/Commit ID: 1131f82a53315cca217a6c84b3bd272aa62e4bca