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Day7: RNA-seq and Differential Expression

Welcome to Day 7 of the Short-Read Sequencing Analysis Workshop.The videos for day 7 will focus on analyzing RNA-seq data. Considerations for library and sequencing format and how these may affect your analysis will be discussed in addition to going through an example RNA-seq mapping using Tophat2 and differential expression analysis using DESeq.

Day 7 Videos

Video1: RNA-Seq overview The first video gives a general overview of some of the choices that will affect your RNA-seq analysis such as what protocol was used for library construction (polyA selection vs. ribosomal subtraction, stranded vs. non-stranded, and single-end vs. paired-end).

Video2: How to process RNA-Seq data  This video will go through some of the mapping considerations that need to be accounted for with different types of library and sequencing preps. At the end you will be walked through an example data set consisting of a 2x100 paired-end strand specific library from mouse with poly-A mRNA. Tophat2 will be used for mapping the example data set to show you how to work with spliced data and what the typical output from Tophat2 looks like.

Video3: Differential Expression using DESeq The last video will be reviewing some of the concepts of differential expression with RNA-Seq data, starting with how programs like DESeq estimate dispersion of natural variability instead of using fold-change to call differentially expressed transcripts. You will be introduced to the R programming package in order to work through a DESeq example.

Day 7 Files

Day7 In-class slides for RNA-Seq (available by the beginning of class)

Day7 In-class slides for DESeq (available by the beginning of class)

Day7 Homework (available by the beginning of class)

Day7 Homework solutions (available the day after class)

Day7 Additional information and links

Tophat2 Paper 

Tophat2 Manual To use Tophat2 you must also have Bowtie2 and Samtools installed and in your path, see Day 5 for links and manuals for those programs

DESeq Documentation 

The R Project This is the website for the R Project, you can read about using R, get the manual, and download R from this site

Seyednasrollah, et al (2015) This paper compares 8 different differential expression software packages using real data sets