# Galaxy RNA-Seq Step-by-Step Tutorial/Protocol

## Background

### File Types

• FASTQ - text file; like a FASTA but with extra lines for read quality scores for each base. This is what you usually get from the sequencing center.
• BAM - Binary compresssed sequences Alignment/Map format - Contains a copy of the reference genome, and every short read that was mapped, including the short reads location and alignment. It can also contain the "left-over" reads from the source FASTQ's that did NOT align to the reference genome!
• BED - text file; a list of locations, in genomic coordinates.
• GTF Gene Transfer Format - text file; contains gene/transcript annotations for a sequence/genome, also in genomic coordinates.
• FASTA text file; contains a list of named sequences. Can be used to hold the sequence of a reference genome to which RNAseq reads will be mapped.

For this tutorial, we will have 2 mouse samples, sequenced with paired-end reads on an Illumina machine. That gives us 4 FASTQ files to upload (forward and reverse sequences for each sample).

FTP site for compressed fastq.gz files

Or they can be found inside Galaxy at Shared Data / Data Libraries / Tutorial Data Sets / RNAseq Tutorial

### Set filetype and genome

To start, you must move the data (FASTQ) from the sequencing center into the Galaxy instance, be sure to specify the filetype (fastqsanger for UAB and HudsonAlpha) and the organism that was sequenced ("Genome database"), in this tutorial, "mm9" for mouse.

### Check quality of data

At this step, we check the quality of sequencing. This says nothing about the quality of the sample, or whether it was the right sample. We'll check that later.

[NGS: QC and manipulation > FASTQ QC > Fastqc] or []

## Align using TopHat: inputs

For the moment, TopHat is the only NGS aligner for transcript data - it's the only one that handles splicing. In addition, it can be set to detect indels relative to the reference genome.

We'll run TopHat once for each sample (twice, in this case).

### Menu: [NGS: RNA Analysis > Tophat for Illumina ]

FASTQs As we're doing "paired" reads, we will need to provide 2 FASTQ files: the forward and reverse reads. In order to have a place to specify the 2nd FASTQ file, we must set the "Is this library mate-paired?" pulldown to "Paired-end", then a second pulldown will appear to specify the 2nd FASTQ.
Mean Inner Distance between Mate Pairs is a value you must get from your sequencing center. This is the mean fragment length of the molecules sequenced, minus the part sequenced. For many RNAseq experiments, it is around 150-175.
Genome (Built-in or FASTA from history)
We must provide it with the reference genome to align to. In our case, this is Mouse, and we'll use the already installed "mm9" genome build. If you have an genome that is not on the list, you can either have us add it, or you can upload a FASTA file of the genome into your history, and point TopHat at that.
TopHat settings to use
Defaults
This is the easy thing to do, but, as the manual said, "There is no such thing (yet) as an automated gearshift in splice junction identification. It is all like stick-shift driving in San Francisco. In other words, running this tool with default parameters will probably not give you meaningful results."
Full parameter list
The most common thing to change
• Allow indel search (from NO to YES)
• Minimum isoform fraction (to 0 if looking for rare isoforms)
• Maximum/minimum intron length (defaults are for Mammals; other critters will do better with stricter settings)

## TopHat output

accepted_hits
Two binary files: .BAM (data) and .BAI (index)
These are the actual paired reads mapped to their position on the genome, and split across exon junctions. This can be visualized in IGV or IGB, but you must download both .BAM and .BAI files to the same directory.
splice_junctions
BED file (list of genomic locations, no sequence) listing all the places TopHat had to split a read into two pieces to span an exon junction. This can be visualized at UCSC or in IGV, etc.
deletions (if indel search is on)
insertions (if indel search is on)

## QC alignment

* %mapped
* visualize in IGV or IGB


* Denovo vs existing annotation
* UAB Modified Reference annotations