RNA-Seq analysis

[mgalland]

RNA-Seq analysis

Description

A one-hour demo on how to perform RNA-Seq analysis.

Time and Place

Where: Room D1.113, Science Park 904 When: Tuesday, January 23rd, 2018 at 17:00 PM

Authors

• Fred White (Master student, Molecular Neuroscience@SILS)
• Max van Hooren (PhD student, Plant Cell Biology@SILS)

Level

Novice & intermediate.

Prerequisites

• Being able to open the Anaconda navigator

Tech needed

If you are on a Mac or a linux computer the anaconda navigator is a easy tool to install other packages.

• The Anaconda distribution for data science. Find it here distributions. The Kallisto software and the Sleuth R package can be downloaded using Anaconda.
• Rstudio: A tool to easily run R, you can find it here

  If you are on a windows computer you will have to install kallisto and sleuth manually.

• Kallisto: a software to align RNA-Seq reads to a transcriptome reference. Download and document can be found here.
• Sleuth: a software to perform the statistical analysis of the Kallisto alignment results. Download and documentation can be found here.

Materials needed

The lesson can be found here. The raw files are here. Download the file and unzip it. Inside the zipped folder, you will find the jupyter notebook (with the .ipynb extension) and the sequencing files information. If you choose the zip file with "all results", it will also contain all the intermediate files. On a mac or linux computer you will be able to run the lesson from jupyter, if you are on a windows computer it is easier to copy paste the the commands from the lesson to your terminal and execute from them for there.

Lesson outline

Time to Complete

One hour: half presentation, half hands-on. Going through the complete procces of raw data to differential gene expression is to much to go though in this hour, however the lesson contains enough information on each step to complete on your own.

Lesson outline:

Fred (presentation) Short presentation about mRNA-Seq analysis and workflow.

Max (hands-on)

1. Generation of the transcriptome reference index with Kallisto
2. Alignment of clean RNA-Seq reads with Kallisto
3. Exploration of the results with Sleuth

Expected results

1. A list of differentially expressed genes.
2. Multiple exploratory plots (PCA, Clustering, etc.)
3. New hypothesis
4. UCSC coverage tracks

Glossary

list any new, potentially unfamiliar or confusing terms or jargon and define them here.

Additional Resources & further exploration

• The Kallisto paper in Nature link
• The Sleuth paper in bioXRiv link
• Lesson on read quality check with FastQC link

[mgalland]

@mvhoore1 : 1 - do you want students to install Kallisto and Sleuth by themselves or via Anaconda? Please, specify in the "Tech needed" section. 2 - This sentence is not clear: "The sequence information, and depending on what file you choose also the files for later steps if you want to start later in the lesson" 3 - This sentence is not clear: "The complete lesson you can do on your own." 4 - I would specify a mean and standard deviation common to all your reads. You will spend too much time on this (important parameter but not the main thing for an introduction). Maybe you can say briefly something about read length during the alignment step.