# Perform DE analysis We will now perform differential gene expression analysis using R. {% file src="/files/-M4U-7aZZdBgs6fYisNl" %} R script (run\_edgeR.R) {% endfile %} Download the above R script, save in the **LSLNGS2015** folder, unzip and run ``` cd ~/LSLNGS2015 Rscript --vanilla run_edgeR.R ``` Output shown in the terminal ``` Loading required package: limma Loading required package: methods null device 1 null device 1 null device 1 ``` ## View tabular results The R script output and save the results of the analysis into 2 files: `DE_analysis.gene.txt` and `DE_analysis.transcript.txt`. `head DE_analysis.gene.txt` ``` chr left right gid score strand name source biotype logFC logCPM F PValue FDR 1 14403 29570 ENSG00000227232 . - WASH7P havana unprocessed_pseudogene 2.89008492452335 2.97744129628027 103.370995316555 8.24483666908638e-09 3.265037563718e-08 1 89294 133723 ENSG00000238009 . - RP11-34P13.7 ensembl_havana lincRNA 11.5139234165179 1.60559878078354 167.167165147925 2.10113367749614e-10 1.2064846331368e-09 1 135140 135895 ENSG00000268903 . - RP11-34P13.15 havana processed_pseudogene 5.06459149590687 0.0881194042027241 42.478504740963 6.16479106039061e-06 1.38838943956985e-05 1 184922 200322 ENSG00000279457 . - FO538757.1 ensembl protein_coding 1.69721957446631 1.80636207182208 24.1093251919636 0.000103795172528207 0.000194091404654342 1 257863 297502 ENSG00000228463 . - AP006222.2 havana lincRNA 13.0394746095367 3.07489541107415 477.124570057853 3.59556529227458e-14 4.97461947310632e-13 1 629639 630683 ENSG00000225630 . + MTND2P28 havana unprocessed_pseudogene 3.13735915488644 4.44862764583624 199.762650496711 3.1768017645391e-10 1.76596074752208e-09 1 631073 632616 ENSG00000237973 . + MTCO1P12 havana unprocessed_pseudogene 0.443696840509747 4.12363294925077 6.13472027306747 0.02307860917183 0.0308376479322919 1 633695 634376 ENSG00000248527 . + MTATP6P1 havana unprocessed_pseudogene 1.34550546639341 5.77825431060693 3.37645905964982 0.191889704720111 0.224260991075299 1 725884 778626 ENSG00000228327 . - RP11-206L10.2 havana transcribed_unprocessed_pseudogene 1.88938948596173 2.34383806326781 33.6517600212232 1.78166537736188e-05 3.74464140783175e-05 ``` `head DE_analysis.transcript.txt` ``` chr left right tid score strand name source biotype logFC logCPM F PValue FDR 1 34553 36081 ENST00000417324 . - FAM138A havana lincRNA 14.7287711778192 4.74754988064348 855.407406314551 9.10858850487461e-05 0.00433065266019644 1 110952 129173 ENST00000471248 . - RP11-34P13.7 ensembl_havana lincRNA -11.6703832090145 1.69559510020159 212.015384690327 0.000721411986621592 0.0087655978717093 1 135140 135895 ENST00000494149 . - RP11-34P13.15 havana processed_pseudogene 0.335112637932721 0.296069849428322 0.0990772814490298 0.768783599380141 0.826519388419821 1 165888 168767 ENST00000491962 . - RP11-34P13.13 havana processed_transcript 0.653389858107978 2.37876611393755 2.69735804942855 0.176126586222385 0.266941473371366 1 257863 264733 ENST00000442116 . - AP006222.2 havana lincRNA 2.10853347357073 1.82097762397731 30.5633854683598 0.00528769030910118 0.0209971307641988 1 258567 259024 ENST00000450734 . - RPL23AP21 havana processed_pseudogene 1.85301838291288 3.06998220640272 13.5659404366886 0.0212839694345138 0.0554117574914843 1 266360 297502 ENST00000424587 . - AP006222.2 havana lincRNA -10.8844390575141 0.915559268435426 124.294800531017 0.00158219973633488 0.0119137545202454 1 347981 348366 ENST00000458203 . - RPL23AP24 havana processed_pseudogene 1.76650999454457 1.51923976505446 7.9898205742989 0.0477136055714843 0.0994641817520193 1 357382 359681 ENST00000441866 . - RP5-857K21.15 havana lincRNA 0.260927151869554 0.438550143910703 0.120976329938423 0.745572526693337 0.807497648960747 ``` ## Download PDFs and view plots ### Multidimensional scaling (MDS) plot MDS plot shows clear separation of the GM12878 versus K562 samples. ![MDS plot](/files/-M44VzB7WJXQFoU8VfQI) ### Mean-Difference (MD) Plot per sample The `plotMD()` from **limma** generate mean difference plot displaying the log fold changes and average expression values for each gene (or M versus A plot or MA plot). #### Gene-level plots ![Gene-level MD plot](/files/-M44VzB99pTMECYUfpTn) #### Transcript-level plots ![Transcript-level MD plot](/files/-M44VzBB6i5gApweZ_LB) ### Mean-Difference (MD) Plot, between 2 cell lines The differentially expressed genes (adjusted *P* = 0.05) are highlighted in red. ![Smear Plot](/files/-M44VzBDcL1kbg7abS0P) --- # Agent Instructions: Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. 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