Load samples and transcriptome

1. Go to usegalaxy.org and log in.

2. Click here to access a shared history on Galaxy. Click the “Import this History” button on the upper left. In the pop-up dialog, choose to copy all files included hidden and deleted files.

3. Then refresh your Galaxy homepage. On the right History panel, click on the “double arrows” icon to switch to the history called Copy of S. elizabethae RNAseq fastq. Rename this history to something that make sense to you. You will perform all analyses in this History.

Quality control: Trimmomatic

4. Use the Trimmomatic tool with the following parameters:

• “Single-end or paired-end reads?”: Paired-end (as collection)
• “Select FASTQ dataset collection with R1/R2 pair”: xx: eliz_paired
• “Perform initial ILLUMINACLIP step?”: Yes
• “Adapter sequences to use”: TruSeq3 (paired-ended, for MiSeq and HiSeq)
• In “Trimmomatic Operation”:
• One operation should be there already.
• “Select Trimmomatic operation to perform”: Sliding window trimming (SLIDINGWINDOW)
• Click “Insert Trimmomatic Operation”
• “Select Trimmomatic operation to perform”: Cut bases off end of a read, if below a threshold quality (TRAILING)
• Click “Insert Trimmomatic Operation”
• “Select Trimmomatic operation to perform”: Cut bases off start of a read, if below a threshold quality (LEADING)
• Click “Insert Trimmomatic Operation”
• “Select Trimmomatic operation to perform”: Drop reads with average quality lower than a specific level (AVGQUAL)
• “Minimum length of reads to be kept”: 25
• Click “Insert Trimmomatic Operation”
• “Select Trimmomatic operation to perform”: Drop reads below a specified length (MINLEN)
• “Minimum length of reads to be kept”: 50
• “Output trimmomatic log messages?”: Yes
• Click
  
  

5. You can proceed to the next steps while Trimmomatic is still running.

6. There will be three new items in your history. Rename the output “Trimmomatic on collection XX: paired” as fastqc_cleaned by clicking the item and then the pen icon.

Remapping on the raw transcriptome

7. Use the Align reads and estimate abundance tool with the following parameters:

• “Transcripts”: Elizabethae_Assembly_Trinity.fasta
• “Paired or Single-end data?”: Paired
• “Left/Forward strand reads”
• Click the “Multiple datasets” button
• Click on the Folder button at the right
• Type to Search: paired_1
• Select the 6 files: Trimmomatic on ..._paired_1.fq.gz (R1 paired)
  by holding the Ctrl key and clicking the file.
• “Right/Reverse strand reads”
• Click the “Multiple datasets” button
• Click on the Folder button at the right
• Type to Search: paired_2
• Select the 6 files; Trimmomatic on ..._paired_2.fq.gz (R2 paired)
• “Strand specific data”: Yes
• “Abundance estimation method”: Salmon
• Click

8. When the analysis is done, rename the 6 items in History that end with “…isoforms counts”

· Click in the information panel (i icon) to check which samples were used for each History item.

o Look under “Tool Parameters” then “Left/Forward strand reads”

· Based on the samples, click the pencil icon and rename the datasets as either Q1, Q2, Q3, W1, W2, or W3.

Merge the mapping tables and compute normalizations

9. Use the Build expression matrix tool with the following parameters:

• “Abundance estimates”: Q1, Q2, Q3, W1, W2, W3
  (hold the Ctrl key and click, note that there will be some lag)
• “Abundance estimation method”: Salmon

Differential Expression (DE) Analysis

10. Use the Differential expression analysis tool with the following parameters:

• “Expression matrix”: Build expression matrix … (raw counts)
• “Sample description”: sample_description.txt
• “Differential analysis method”: DESeq2

Download results

11. When the analysis is done, click the ‘i’ in the result “Differential expression results”.

12. In the middle panel, scroll down to “Job Output”.

13. Click on the green dataset next to “Differential expression results on”.

14. Click “0: input.matrix.Q_vs_W.DESeq2”, then click the “disk” icon to download this table.

15. Rename the file to “input.matrix.Q_vs_W.DESeq2.txt”.

16. Also click the green dataset next to “Differential expression plots on”.

17. Click “0: input.matrix.Q_vs_W.DESeq2.DE_results.MA_n_Volcano”, then click the “disk” icon to download this pdf file.

18. We will examine these files in the next lab.