Explore Workflows

### 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: a8eaf61c809d76f55780b14f2febeb363cf6373f

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

Path: definitions/subworkflows/pindel_region.cwl

Branch/Commit ID: 5be54bf09092c53e6c7797a875f64a360d511d7f

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

Path: task_types/tt_kmer_top_n.cwl

Branch/Commit ID: e4d6182d5f7a6a880e5f2b21273cf40d25e187df

https://github.com/EBI-Metagenomics/pipeline-v5.git

Path: workflows/subworkflows/func_summaries.cwl

Branch/Commit ID: 4b98d8bf882bc96d924b5d2d4e6d9c188fa7b273

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

Path: subworkflows/xenbase-sra-to-fastq-pe.cwl

Branch/Commit ID: 3b2e0de49d9ee6fd9a8c9580b6a02d0f7e4c8f7c

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

Path: tests/wf/mut2.cwl

Branch/Commit ID: 596aab620489cd2611f4bc1d9a4fc914ddf34514

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

Path: definitions/subworkflows/strelka_process_vcf.cwl

Branch/Commit ID: 76a35e7d885790f30559beb31f3b58770e343afd

https://github.com/tmooney/cancer-genomics-workflow.git

Path: definitions/subworkflows/molecular_alignment.cwl

Branch/Commit ID: 0db1a5f1ceedd4416ac550787c27b99c87dbe985

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

Path: definitions/subworkflows/varscan_germline.cwl

Branch/Commit ID: efbbe5ed51f6ac583e87a348785c72818a33f56e

https://github.com/d3b-center/canine-dev.git

Path: subworkflows/canine_annotation_module.cwl

Branch/Commit ID: 462aaebbd442e84ea101b45b716df0174b88512e