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Day 8: RNA-Seq I, Introduction to RNA-Seq

Day 8: RNA-Seq I, Introduction to RNA-Seq

Welcome to Day 8 of the Short-Read Sequencing Analysis Workshop. This year we have split up the RNA-Seq analysis portion into 2 days. This first day we will focus on the different kinds or RNA-Seq experiments, considerations for library prep, and the theory behind mapping spliced RNA-seq reads and determining expression levels. The in-class portion will be spent performing read mapping for a human RNA-Seq data set, visualization of the data, and creating a counts table for expression levels over each gene/transcript. The second day will be spent focusing on differential expression analysis.


Videos for Day 8

Video 1: Introduction to RNA-Seq Analysis (5:35)  The first video serves as a basic introduction to RNA-Seq, what kinds of experiments and questions can be asked, considerations for library prep depending on the type of experiment/question, and the difference between steady state and nascent RNA-Seq.

Video 2: Theory of Mapping Spliced RNA-Seq Reads (8:01)  The second video discusses choices for mapping RNA-Seq data to a reference, and introduces the splice-aware aligner program Tophat2.

Video 3: How to run Tophat2 (6:56)  This video outlines considerations for running Tophat2, how to run the program, and the different output files that will be created.

Video 4: TBA  This video introduces different methods used to quantify gene expression and uses HTSeq-Counts to create a count table that can be used for one type of differential expression analysis.


Files for Day 8

Day 8 Slides

Day 8 Homework

Day 8 Homework Answers


Additional Resources

Description and Manual for HTSeq-count

Tophat2 Paper

Tophat2 Manual

HISAT2 program - this is a newly-released program that is a replacement for Tophat2. 

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