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Goal: Use RNA-seq to compare expression between oysters (n=3) pre and post heat shock.

Based on IPlant Collaborative Tutorial

Tophat is a specialized alignment software for RNA-seq reads that is aware of splice junctions when aligning to a reference assembly.

1) Click Apps from DE workspace and select TopHat2-SE. Use search bar.

2) Under ‘Analysis Name’ leave as defaults for make any changes.

3) Under Input data for FASTQ files add six fastq.gz files localed in austral-data with prefixes 2M, 2M-HS, 4M, 4M-HS, 6M, 6M-HS.

4) Under Reference Genome for ‘Provide a reference genome file in FASTA format’ select /austral-data/Crassostrea_gigas.GCA_000297895.1.24.dna_sm.toplevel.fa

5) For Reference Annoations add the GTF file /austral-data/Crassostrea_gigas.GCA_000297895.1.24.gtf

6) Click Launch Analyses and monitor the status of you job.

7) Once complete….

• Note this takes ~10 hours

Cufflinks assembles RNA-Seq alignments into a parsimonious set of transcripts, then estimates the relative abundances of these transcripts based on how many reads support each one, taking into account biases in library preparation protocols. A detailed manual can be found at http://cufflinks.cbcb.umd.edu/manual.html.

2) For Input Data add the six bam files from the bam subdirectory of the TopHat2 output.

3) Under Reference Sequence use custom option select /austral-data/Crassostrea_gigas.GCA_000297895.1.24.dna_sm.toplevel.fa

4) For Reference Annoations add the GTF file /austral-data/Crassostrea_gigas.GCA_000297895.1.24.gtf

5) Click Launch Analyses and monitor the status of you job.

• Note this takes ~2 hours

#### Task 3: Merge all Cufflinks transcripts into a single transcriptome annotation file using Cuffmerge2

Cuffmerge merges together several Cufflinks assemblies. It handles also handles running Cuffcompare. The main purpose of this application is to make it easier to make an assembly GTF file suitable for use with Cuffdiff. A merged, empirical annotation file will be more accurate than using the standard reference annotation, as the expression of rare or novel genes and alternative splicing isoforms seen in this experiment will be better reflected in the empirical transcriptome assemblies.

1) Open the Cuffmerge2 app. Under ‘Input Data’, browse to the results of the cufflinks analyses (abovee) and add the 6 gtf files located in the gtf subdirectory.

2) For Reference Annoations add the GTF file /austral-data/Crassostrea_gigas.GCA_000297895.1.24.gtf

3) Under Reference Sequence use custom option select /austral-data/Crassostrea_gigas.GCA_000297895.1.24.dna_sm.toplevel.fa

4) Click Launch Analyses and monitor the status of you job.

#### Task 4: Compare expression analysis using CuffDiff2

Cuffdiff is a program that uses the Cufflinks transcript quantification engine to calculate gene and transcript expression levels in more than one condition and test them for significant differences. http://cufflinks.cbcb.umd.edu/manual.html

1) Open Cuffdiff2. For ‘Input Data’ Sample 1 Name enter Pre adn add three bam file from Tophat analysis..

For Sample 2 enter post and add other three bam files …

2) Next, open the Reference Annotations section and add a custom reference annotation file using the merged_with_ref_ids.gtf file from the cuffmerge output folder.

3) Click Launch Analyses and monitor the status of you job.