Exercises for you to try:
There is a different database for microRNA that we’ve analysed this dataset against, called MirGeneDB. MirGeneDB is a database of manually curated microRNA genes that have been validated and annotated as initially described in Fromm et al. 2015 and Fromm et al. 2020. MirGeneDB 2.1 includes more than 16,000 microRNA gene entries representing more than 1,500 miRNA families from 75 metazoan species and published in the 2022 NAR database issue.
The output of the MirGeneDB analysis can be found at /work/training/2024/smallRNAseq/runs/run3_MirGeneDB, if you want to practice editing the R scripts we’ve given you to get the same plots as above for this analysis (in preparation for you doing it for your own data).
Precomputed results from session 6:
We ran the small RNA seq samples against the MirGeneDB database and the results can be found at:
| Code Block |
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/work/training/2024/smallRNAseq/runs/run3_MirGeneDB/results/mirna_quant/edger_qc/mature_counts.csv /work/training/2024/smallRNAseq/data/human_disease/metadata_microRNA.txt |
Let’s create a “DESeq2” folder and copy the files needed for the statistical analysis:
| Code Block |
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cp $HOME/workshop/2024-2/session6_smallRNAseq/scripts/transpose_csv.py $HOME/workshop/2024-2/session6_smallRNAseq/runs/run2_human_MirGeneDB/DESeq2 cp $HOME/workshop/2024-2/session6_smallRNAseq/data/metadata_microRNA.txt $HOME/workshop/2024-2/session6_smallRNAseq/runs/run2_human_MirGeneDB/DESeq2 cp /work/training/2024/smallRNAseq/runs/run3_MirGeneDB/results/mirna_quant/edger_qc/mature_counts.csv $HOME/workshop/2024-2/session6_smallRNAseq/runs/run2_human_MirGeneDB/DESeq2 cd $HOME/workshop/2024-2/session6_smallRNAseq/runs/run2_human_MirGeneDB/DESeq2 |
To transpose the initial “mature_counts.csv” file do the following:
| Code Block |
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python transpose_csv.py --input mature_counts.csv --out mature_counts.txt |
Differential expression analysis using RStudio
Run analysis script in RStudio
a. Open RStudio
b. Create a new R script ('File'->'New File'-> ‘R script’)
c. Hit the save button and save this file in the working directory you created above (H:\workshop\2024-2\session6_smallRNAseq\runs\run2_human_MirGeneDB\DESeq2). Name the R script ‘DESeq2.R’.
d. Copy and paste the entire script from the code window below into your R script.
Step 1: LOAD PACKAGES
Step 2: