RNA-Seq workflow
RNA-Seq workflow

Methods

Adapters will be trimmed with CutAdapt (Martin) to remove adapters and low-quality bases and overall quality will assessed with FastQC (FastQC), FastQCScreen (Wingett and Andrews, 2018), and MultiQC (Ewels et al., 2016). Reads will be aligned and quantified using RSEM/STAR (Li and Dewey, 2011; Dobin et al., 2013). Differential expression modeling will use the DESeq2 (Love et al., 2014). Intra and inter group variance will be assessed with Principal Component Analysis. Candidate DEGs are visualized with volcano plots filtered to the subset of DEGs where Benjamini-Hochberg adjusted p-value ≤ 0.05 (Benjamini and Hochberg, 1995). Functional analysis using Advaita iPathwayGuide (Draghici et al., 2007; iPathwayGuide) will assess enrichment of KEGG pathways and Gene Ontology (GO) concepts.

References

Benjamini,Y. and Hochberg,Y. (1995) Controlling the false discovery rate: A practical and powerful approach to multiple testing. Journal of the Royal Statistical Society. Series B (Methodological), 57, 289–300.
Dobin,A. et al. (2013) STAR: Ultrafast universal RNA-seq aligner. Bioinformatics (Oxford, England), 29, 15–21.
Draghici,S. et al. (2007) A systems biology approach for pathway level analysis. Genome Research, 17, 1537–1545.
Ewels,P. et al. (2016) MultiQC: Summarize analysis results for multiple tools and samples in a single report. Bioinformatics, 32, 3047–3048.
FastQC https://github.com/s-andrews/FastQC.
iPathwayGuide.
Li,B. and Dewey,C.N. (2011) RSEM: Accurate transcript quantification from RNA-seq data with or without a reference genome. BMC bioinformatics, 12, 323.
Love,M.I. et al. (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15, 550.
Martin,M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal, 17, 3.
Wingett,S.W. and Andrews,S. (2018) FastQ screen: A tool for multi-genome mapping and quality control. F1000Research, 7, 1338.