RNA-seq data analysis workshop

Lecturers: Dr. Seyed Amir Malekpour and Dr. Najmeh Salehi, form School of Biological Sciences at Institute for Research in Fundamental Sciences (IPM).  

In this workshop we focus on running RNA-seq pipelines to discover differentially expressed genes across two or more conditions. As a case study, we use a real RNA-seq dataset of 7 paired Prostate cancer samples, before and after treatment. We show how to download data from Sequence Read Archive (SRA) and process them to call differentially expressed genes (DEGs) and pathways that are enriched with such genes.

 This will be an 8-hour tutorial, given in two days, and covering following topics:

• A brief introduction to basic shell commands in Linux
• High-throughput technologies to study mRNAs
• Gene Expression Omnibus (GEO) and Sequence Read Archive (SRA) database
• SRA Toolkit to download short reads from SRA
• RNA-seq library preparation
• Raw fast files, e.g. fastq
• Quality control of raw files with fastqc
• Mapping/Aligning short reads to the reference genome with hisat2
• Standard formats for alignment files, e.g. SAM (Sequence Alignment Map) or BAM
• Processing SAM files with samtools
• Post-alignment quality control with RSeQC
• Deriving statistics on ribosomal contaminations and novel splicing events in RNA samples
• Gene transfer format (GTF) file usage
• Differential gene expression analysis across two or more conditions with DESeq2
• PCA analysis for differentially expressed genes
• Gene Set Enrichment Analysis with DAVID and GSEA packages

Target Audience: Graduate students, postdoctoral scholars, and principal investigators currently working with RNA-seq data, or about to embark on projects that require such data analysis.

Computer requirements: You will need to have your own laptop/desktop computer and you will have access to the IPM server to run the codes in Linux environment online. Downstream analyses, e.g. in DEG calling and gene set enrichment analysis, are performed in windows operating system.