1. RNA Seq Analysis Aaron Odell June 17 th 2014

2. Mapping Strategy A few questions you’ll want to ask about your data… - What organism is the data from? - Protocol Used… (single-end,paired-end)? - Computation Resources available? These will all play a factor in your mapping strategy

3. What Organism? Splicing… NOYES Bowtie, BWA, etc… Tophat2, GSNAP, STAR, etc… Annotation Set can help improve mapping results if available Published Paper: Systematic evaluation of spliced alignment programs for RNA-seq data

4. Protocol? Stranded? NOYES Expression over regions of overlapping Genes that are on opposing strands is ambiguous.. Strand specific transcripts can be distinguished. Make Sure desired mapping program supports you’re strand specificity.

5. Computational Resources? Example: 6 Samples of human RNA seq data ~50 Million reads a piece Want to map with Tophat2 ~3,000 – 6,000 compute hours (125- 250 days for just mapping) NOT Viable on local machine. Must find an alternative…

6. Example Data Set Organism = Mouse Protocol = Paired End, Stranded Two samples – Ethanol (treated) – Saline (untreated) Questions – Find differentially expressed genes and isoforms

7. Mapping – Fastq  SAM/BAM Assemble Transcripts Merge Transcripts Differential Expression Tests Visualization (We’re going to skip this part)

8. Analysis Strategy Mapping (Tophat2) Gene/Isoform abundance (Cufflinks) Differential Expression (CuffDiff)

9. For now… Log into vieques cp –r /projects/sreadgrp/homeworkfiles/chr1_RNA /Users/username/ Change to “SCRIPTS” directory and edit the tophat pbs scripts. Change “odella” to your username Load the following modules tophat_2.0.6 samtools_0.1.16 bowtie_bowtie2-2.0.2 cufflinks_2.1.1 From /Users/username/chr1_RNA/SCRIPTS directory submit ethanol_tophat.pbs and saline_tophat.pbs

10. Change to ethanol_tophat directory and run “samtools flagstat accepted_hits.bam” to get alignment statistics Samtools index accepted_hits.bam for viewing in igv