IV. Differential analysis at the gene level and transcript level

Airway example

The data used in this workflow comes from an RNA-seq experiment where airway smooth muscle cells were treated with dexamethasone, a synthetic glucocorticoid steroid with anti-inflammatory effects (Himes et al. 2014). Glucocorticoids are used, for example, by people with asthma to reduce inflammation of the airways. In the experiment, four human airway smooth muscle cell lines were treated with 1 micromolar dexamethasone for 18 hours. For each of the four cell lines, we have a treated and an untreated sample. For more description of the experiment see the article, PubMed entry PMID: 24926665, and for raw data see the GEO entry GSE52778.

We will conduct an analysis at the gene and transcript level by starting from quantification using the fast alignment-free transcript mapper Salmon.

This can be done as follows:

• Building index with salmon

salmon index --gencode -t gencode.v32.transcripts.fa -i gencode.v32_salmon_index

• Mapping one sample with salmon:

salmon quant -i gencode.v32_salmon_index -l A --gcBias -1 SRR1039508_subset_1.fastq -2 SRR1039508_subset_2.fastq --validateMappings -o quant/SRR1039508_subset_quant

More details can be found in Intro to salmon. The mapped output from Salmon can be found at: data.

1. Import the data with tximeta package: see tximeta vignette on bioconductor
2. Run a DE analysis on the gene-level counts with quasi-likelihood workflow of edgeR
3. Run a DE analysis on the transcript level counts with quasi-likelihood workflow of edgeR
4. Run a transcript usage analysis

Tip: I had to use the argument importer=read.delim in the tximeta function because the default function to read the files returned an error on my laptop.