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Aim:
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| Table of Contents |
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Aim:
Identify statistically significant (FDR < 0.05) differentially expressed genes. Visualise results with PCA plots, heatmaps and volcano plots.
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Go to https://rvdi.qut.edu.au/
Click on ‘VMware ‘VMware Horizon HTML Access’Access’
Log on with your QUT username and password
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To access this count table:
Go to the/sandpit/demo/run3_full_pipeline/ W:\training\rnaseq\runs\run3_RNAseq\results folder that contains the results from running the nfcore/rnaseq pipeline. The output folders from task 3 look like this:
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The expression count file that we are interested is salmon.merged.gene_counts.tsv Let's see the content of the file by printing the top lines using the following command (in PuTTy):
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head salmon.merged.gene_counts.tsv |
the above command will print:
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gene_id gene_name CD49fmNGFRm_rep1 CD49fmNGFRm_rep2 CD49fmNGFRm_rep3 CD49fpNGFRp_rep1 CD49fpNGFRp_rep2 CD49fpNGFRp_rep3 MTEC_rep1 MTEC_rep2 MTEC_rep3
ENSMUSG00000000001 Gnai3 2460 2395 2749 2686 3972 4419 7095 4484 6414
ENSMUSG00000000003 Pbsn 0 0 0 0 0 0 0 0 0
ENSMUSG00000000028 Cdc45 43 57 55 79 87.999 89 1241 830 1041.999
ENSMUSG00000000031 H19 2 0 1 17.082 24 16.077 200 139 145.604
ENSMUSG00000000037 Scml2 8 8 16 23 29.001 29 69 57 67
ENSMUSG00000000049 Apoh 1 0 2 1 2 0 0 0 2
ENSMUSG00000000056 Narf 522 496 539 368 457 538 1939 1483 1734
ENSMUSG00000000058 Cav2 1352.999 1349 1371.999 2684.001 4370 4386 6018.999 3429 5501
ENSMUSG00000000078 Klf6 4411 3492 4500 3221 3989 4637 3812 2741 3558 |
Now let’s copy the ‘salmon.merged.gene_counts.tsv’ file to your laptop/desktop using the file finder.
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Now let’s find the full path to the ‘salmon.merged.gene_counts.tsv’ file:
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Windows:
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Mac:
cd /folder/that/contains/feature_counts/
pwd
Rstudio:
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Open Rstudio, go to the top bar a click on “Session” → “Select working directory: → “Choose directory”
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salmon.merged.gene_counts.tsv
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head salmon.merged.gene_counts.tsv |
Differential Expression Analysis using DESeq2
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a. First create a new folder , on your desktop, Documents, etcin H:\workshop\RNAseq . Call it something informativesuitable, such as ‘DE_analysis_workshop’
b. Create a sub folder here called ‘data’. This is where your two data files will be stored
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