Workshop 3: Intro to RNAseq analysis

Fursham Hamid

Overview

Duration: 4 hours (over 2 days)

Objectives

1. Describe key reasons for learning shell.
2. Navigate your file system using the command line.
3. View, search within, copy, move, and rename files. Create new directories.
4. Retrieve RNAseq files using shell
5. Employ the grep command to search for information within files.
6. Print the results of a command to a file.
7. Explain how a FASTQ file encodes per-base quality scores.
8. Interpret a FastQC plot summarizing per-base quality across all reads.
9. Clean FASTQ reads using Trimmomatic.
10. Align and quantify reads using Kallisto
    

Before starting the lesson

In this workshop, we will be using UNIX Shell to navigate around our computer filesystem and to process publicly-available RNAseq data. Please refer to this setup instructions to check if your operating system comes pre-installed with Shell.

We will also be using several command-line programs to process reads from RNAseq experiments. These programs will be required only for day 2 of the workshop, and we will briefly go through the installation process during day 1. For instructions on installing these software, please refer to this setup instructions.

During the lesson, we will be using etherpad, an online collaborative platform for our discussions and activities. Below are the links to the lesson pads:

1. Lesson 1
2. Lesson 2

You may bookmark this page before the lesson commence!

Lesson schedule

Day 1 [11th May 2021]

10:00	1. Introducing the Shell	What is a command shell and why would I use one? How can I move around on my computer? How can I see what files and directories I have? How can I specify the location of a file or directory on my computer?
10:20	2. Navigating files and directories	How can I perform operations on files outside of my working directory? What are some navigational shortcuts I can use to make my work more efficient?
10:40	3. Working with files and directories	How can I view and search file contents? How can I create, copy and delete files and directories?
10:50	Break
11:00	4. Retrieving and inspecting RNAseq files	How do I download files from the internet? How do I compress/decompress large files? How do I preview large files in Shell?
11:30	5. Overview of RNAseq pipeline and installing softwares	What is the general outline of an RNAseq data analysis? How do I install command-line software?
12:00	Finish

Day 2 [25th May 2021]

10:00	1. Check-in and brief lesson overview
10:15	2. Recap of commands learnt so far
10:20	3. Overview of RNAseq pipeline	What is the general workflow of a typical RNAseq pipeline? What tools are available to process RNAseq reads based on my desired research outcome?
10:30	4. Quality control of RNAseq reads	How do I check the quality of my sequencing reads to make an informed decision on the degree of trimming needed? How do I write a script to perform repititive task?
10:50	Break
11:00	5. Read trimming	How do I trim my reads to obtain high-quality sequences for subsequent steps of the workflow? How do I determine the adaptor sequence to remove from my reads?
11:30	6. Mapping and quantifying reads to the transcriptome	What are indices? How do I determine the strandness of my reads? How do I align my reads to the transcriptome?
11:50	Feedback and questions