RNAseq - Alignment and differential expression analysis

1. Input data
2. Trimming and quality control
3. Alignment to transcriptome
4. Genome coverage
5. Expression analysis - quantification
6. Differential expression - protein-coding genes
7. Differential expression - all genes
8. Differential expression - isoform level
9. Differential expression - combined files
10. Alternative splicing analysis
11. MultiQC report
12. UCSC hub
13. Methods summary

UCSC Genome Browser: use the previous link to display the data tracks automatically, or copy the the URL https://bw:bw@lichtlab.cancer.ufl.edu/reports/NSD//GE7114-NSD2deg/hub/hub.txt and paste it into the "My Hubs" form in this page.

WashU EpiGenome Browser: use the previous link to display the data tracks automatically, or copy the following URL into the "Datahub by URL Link" field: https://bw:bw@lichtlab.cancer.ufl.edu/reports/NSD//GE7114-NSD2deg/hub/hub.json.

Short reads were trimmed using trimmomatic (v 0.36) [1], and QC on the original and trimmed reads was performed using FastQC (v 0.11.4) [2] and MultiQC [3].

The reads were aligned to the transcriptome using STAR version 2.7.9a [4].

Transcript abundance was quantified using RSEM (RSEM v1.3.1) [5].

Differential expression analysis was performed using DESeq2 [6], with an FDR-corrected P-value threshold of 0.05. The output files were further filtered to extract transcripts showing a 2.0-fold change in either direction. Results were reported for protein-coding genes only, and for all transcript types.

Alternative splicing analysis was performed using rMATS version v4.1.0 [7].

1. Bolger, A. M., Lohse, M., and Usadel, B. (2014). Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics, btu170.
2. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
3. Philip Ewels, Mans Magnusson, Sverker Lundin and Max Kaller (2016). MultiQC: Summarize analysis results for multiple tools and samples in a single report. Bioinformatics | doi: 10.1093/bioinformatics/btw354 | PubMed: 27312411
4. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 29(1):15-21 | doi: 10.1093/bioinformatics/bts635
5. Li B and Dewey CN (2011). RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12:323 | doi: 10.1186/1471-2105-12-323
6. Love MI, huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15,550 (2014). | doi: 10.1186/s13059-014-0550-8
7. Shen S., Park JW., Lu ZX., Lin L., Henry MD., Wu YN., Zhou Q., Xing Y. rMATS: Robust and Flexible Detection of Differential Alternative Splicing from Replicate RNA-Seq Data. PNAS, 111(51):E5593-601 | doi: 10.1073/pnas.1419161111