NF-RNASEQ Pipeline Methods

Please note that the methods below are for projects delivered after April 7th, 2025.

Methods for projects delivered before April 7th, 2025 can be found here: https://soccin.github.io/pwg-docs/methods/rnaSeq.html

Sequences are aligned with STAR[1] using –twopassMode basic command line option. Aligned reads are sorted and indexed with samtools[2] , and the duplicates are marked but not removed using picard [3]. Several QC programs are then run. RSeQC[4] and qualimap[5] provide various rnaseq qc metrics. dupRadar[6] provides QC of duplication in samples. A script runs DESeq2 [7] and provides a number of output files to look at samples in the dataset.

Methods for nf-differentialabundance pipeline:

DESeq2[7] is run to generate differential expression results between each comparison, as well as normalized counts and variance stabilized counts. MSKCC developed R scripts are run to generate various plots to visualize the samples and the differential expression. In addition scripts from shinyngs[8] are run to generate additional plots.

For full calls, please inquire with BIC (full commands should be seen in pipeline_info files).

References:

  1. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013 Jan 1;29(1):15-21. doi: 10.1093/bioinformatics/bts635. Epub 2012 Oct 25. PMID: 23104886; PMCID: PMC3530905.
  2. Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, Whitwham A, Keane T, McCarthy SA, Davies RM, Li H. Twelve years of SAMtools and BCFtools. Gigascience. 2021 Feb 16;10(2):giab008. doi: 10.1093/gigascience/giab008. PMID: 33590861; PMCID: PMC7931819.
  3. “Picard Toolkit.” 2019. Broad Institute, GitHub Repository.  https://broadinstitute.github.io/picard/; Broad Institute
  4. Wang L, Wang S, Li W. RSeQC: quality control of RNA-seq experiments. Bioinformatics. 2012 Aug 15;28(16):2184-5. doi: 10.1093/bioinformatics/bts356. Epub 2012 Jun 27. PMID: 22743226.
  5. García-Alcalde F, Okonechnikov K, Carbonell J, Cruz LM, Götz S, Tarazona S, Dopazo J, Meyer TF, Conesa A. Qualimap: evaluating next-generation sequencing alignment data. Bioinformatics. 2012 Oct 15;28(20):2678-9. doi: 10.1093/bioinformatics/bts503. Epub 2012 Aug 22. PMID: 22914218.
  6. Sayols S, Scherzinger D, Klein H. dupRadar: a Bioconductor package for the assessment of PCR artifacts in RNA-Seq data. BMC Bioinformatics. 2016 Oct 21;17(1):428. doi: 10.1186/s12859-016-1276-2. PMID: 27769170; PMCID: PMC5073875.
  7. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. doi: 10.1186/s13059-014-0550-8. PMID: 25516281; PMCID: PMC4302049.
  8. Jonathan R Manning (2022). Shiny apps for NGS etc based on reusable components created using Shiny modules. Computer software. Vers. 1.5.3. Jonathan Manning, Dec. 2022. Web.

 

Reference files:

Human:

  • Genome: UCSC hg38
  • GTF: gencode v46

Mouse

  • Genome: mm39
  • gtf: Gencode vM36
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