Day 1 - RNA-Seq Analysis

Keywords

Alignment, BAM, FASTA, FASTQ, QC

Authors

• Jenny Drnevich @jenny
• Radhika Khetani @radhika
• Jessica Kirkpatrick [email protected]

Type

• Both

Description

Day 1 starts at the very beginning of a typical RNA-Seq workflow, explaining the sequencing technology and considerations for experimental design, then starts with hands-on application of working with sequencing data fresh off the sequencer.

Aims

This day covers an overview of Illumina sequencing technology and proper experimental design, a review of Unix/Linux, common file formats of sequence data, how to do sequencing QC and quality trimming, and an introduction to alignment.

Prerequisites

• Basic UNIX and how to submit jobs to a computing cluster

Target audience

Graduates students/post docs/beginning faculty

Learning objectives

• Be able to describe the rough basics of Illumina sequencing and how to design an experiment
• Be able to describe the first steps in a typical RNA-Seq workflow.
• Be able to follow and modify UNIX scripts for QC and trimming

Materials

• Lecture on Illumina sequencing and experimental design
• Lecture on file formats, FastQC and trimming
• Lecture on alignment theory
• Review of Linux
• Linux/R comparison sheet
• Script templates
• Example sequencing report
• Explanation of strandedness

Data

• tarball of yeast genome, gene models, STAR index files and 12 fastq files
• list of files in tarball
• md5sum for the tarball

Timing

6 hours contact time; practicals intersperced with lectures; easily can fit into 1 day + lunch and breaks

Content stability

Should be stable

Technical requirements

• UNIX server with FastQC >= 0.11.2, Trimmomatic >= 0.30,

Literature references