1. RNA Sequencing Day 7 Wooohoooo!
Aaron Odell
June 17th 2015

2. Outline For The Day Map raw fastq files to reference genome
Convert mapped data to visualization ready files
“Quantify” mapped data over reference genome genes/annotations
Differential Expression

3. Data Set Mouse RNA Seq mm10 Reference Genome (.fasta file)
PolyA Strand Specific Library. Ethanol treatment
2 x 100 Paired End Reads = 2 Fastq Files
mm10 Reference Genome (.fasta file)
Ensemble mm10 gene annotation file (.gtf)
Due to time and compute restraints we will only be working with Chromosome 19
Pull up what a gtf looks like

4. What Mapping Program To Use?...
Need program that is able to handle spliced reads…
Tophat2, GSNAP, STAR, etc …
We will be using Tophat2
Excellent spliced alignment program evaluation

5. First things first
Log onto vieques and load all modules we will need to run analysis successfully

6. module load commands module load python_2.7.3
module load samtools_0.1.16
module load numpy_1.6.1
module load pysam_0.8.1
module load bowtie_bowtie
module load tophat_2.0.6
module load htseq_0.6.1

7. Lets begin…
cp -r /projects/sreadgrp/RNA /Users/username/
Edit /Users/username/RNA/SCRIPTS/ILS_E_tophat_samtools.pbs in text editor
Change all “odella” to your actual username
Example /Users/odella/  /Users/your_username/
Change #PBS –M your_ _address to your actual address (or if you don’t want the then do nothing)
From the /Users/username/RNA/SCRIPTS directory qsub ILS_E_1_tophat_samtools.pbs
Have half the class copy. Then wait a minute or two. Then have to other half the class do the copy

8. While we wait… Edit /Users/username/RNA/SCRIPTS/ILS_E_htSeqCount.pbs
Change all “odella” to your actual username
htSeqCount manual
Once /Users/username/RNA/SCRIPTS/ILS_E_tophat_samtools.pbs has run to completion, qsub /Users/username/RNA/SCRIPTS/ILS_E_htSeqCount.pbs

9. For those of you who are bored…
Read through the tophat and htseq manuals
Take a look at various files created by our mapping/quantification pipeline.
Tophat manual explains what all these files are and where they come from
Run this command from /Users/username/RNA/ILS_E directory
samtools flagstat ILS_E_sorted.bam
Do you understand the output? Search the internet or ask someone if you don’t

10. Time to visualize! Copy bam and genome files to student directory
cp /Users/username/RNA/ILS_E/ILS_E_sorted.bam /projects/sreadgrp/student/username/ILS_E_sorted.bam
cp /Users/username/RNA/ILS_E/ILS_E_sorted.bam.bai /projects/sreadgrp/student/username/ILS_E_sorted.bam.bai
cp /User/username/RNA/GENOME/chr19.fasta /projects/sreadgrp/student/username/chr19.fasta
cp /User/username/RNA/GENOME/chr19.fasta.fai /projects/sreadgrp/student/username/chr19.fasta.fai
cp /User/username/RNA/GENOME/chr19.gtf /projects/sreadgrp/student/username/chr19.gtf

11. Load the approriate files
Load genome from file
Chr19.fasta
Load from file
Chr19.gtf
ILS_E_sorted.bam

12. Look at ILS_E_htSeq.txt
Count the number of reads you see mapping in IGV over any given gene and compare to the htSeqCount value
Does it make sense?