limma-voom example for RNA-seq w/o quantile normalization (#95)

* Change differential expression notebook to reflect technology * Update the DGE README to reflect RNA-seq addition * Background for RNA-seq DGE * Fixed a comment * Add RNA-seq data * limma-voom * Hey apparently that volcano plot step didn't work * Improve documentation * Remove plot print out * Typo * Update in response to PR comments * Few more typos + add'n doc * Update differential-expression/rnaseq_DGE.Rmd Co-Authored-By: Candace Savonen <[email protected]> * Update differential-expression/rnaseq_DGE.Rmd Co-Authored-By: Candace Savonen <[email protected]> * Rerun to update HTML
AlexsLemonade · Aug 6, 2019 · 9a3d032 · 9a3d032
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diff --git a/differential-expression/README.md b/differential-expression/README.md
@@ -1,7 +1,15 @@
-# Refine.bio Example Workflow: Differential expression
+# refine.bio Example Workflow: Differential expression
 
-[This notebook](https://alexslemonade.github.io/refinebio-examples/differential-expression/gene_DE.nb.html)
-takes data and metadata from refine.bio and identifies genes that are differentially expressed between two groups.
+refine.bio includes both microarray and RNA-seq experiments. 
+The methods for performing differential gene expression (DGE) analysis for each technology differ.
+In this module, we include simple two-group comparison examples for each.
+
+#### Contents
+
+* [**Microarray notebook**](https://alexslemonade.github.io/refinebio-examples/differential-expression/microarray_DGE.nb.html): takes microarray data and metadata from refine.bio and identifies genes that are differentially expressed between two groups.
+* [**RNA-seq notebook**](https://alexslemonade.github.io/refinebio-examples/differential-expression/rnaseq_DGE.nb.html): takes RNA-seq data without quantile normalization from refine.bio and identifies genes that are differentially expressed between two groups. Read more about skipping quantile normalization [here](http://docs.refine.bio/en/latest/main_text.html#skipping-quantile-normalization-for-rna-seq-experiments) in our documentation.
+* [**GenePattern differential expression analysis**](#differential-expression-analysis-with-genepattern): [GenePattern](https://cloud.genepattern.org/gp/pages/login.jsf) modules can be run via a GUI.
+To use refine.bio data with GenePattern, you will need to change the format as described in this section.
 
 ## Requirements and usage
 

diff --git a/differential-expression/data/SRP078441.tsv b/differential-expression/data/SRP078441.tsv
diff --git a/differential-expression/data/metadata_SRP078441.tsv b/differential-expression/data/metadata_SRP078441.tsv
@@ -0,0 +1,17 @@
+refinebio_accession_code	experiment_accession	refinebio_age	refinebio_cell_line	refinebio_compound	refinebio_disease	refinebio_disease_stage	refinebio_genetic_information	refinebio_organism	refinebio_platform	refinebio_race	refinebio_sex	refinebio_source_archive_url	refinebio_source_database	refinebio_specimen_part	refinebio_subject	refinebio_time	refinebio_title	refinebio_treatment
+SRR3895749	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	peripheral blood	peripheral blood		CBF77-Blood-ASXL2	
+SRR3895742	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	bone marrow	bone marrow		CBF124-BM-ASXLwt	
+SRR3895743	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	bone marrow	bone marrow		CBF234-BM-ASXLwt	
+SRR3895746	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	bone marrow	bone marrow		CBF370-BM-ASXL2	
+SRR3895745	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	peripheral blood	peripheral blood		CBF369-Blood-ASXL2	
+SRR3895748	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	bone marrow	bone marrow		CBF54-BM-ASXLwt	
+SRR3895738	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	bone marrow	bone marrow		41267-BM-ASXL2	
+SRR3895744	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	bone marrow	bone marrow		CBF337-BM-ASXLwt	
+SRR3895741	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	bone marrow	bone marrow		49060-2010-BM-ASXLwt	
+SRR3895739	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	bone marrow	bone marrow		45565-BM-ASXL1	
+SRR3895736	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	bone marrow	bone marrow		37582-BM-ASXLwt	
+SRR3895740	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	peripheral blood	peripheral blood		48549-Blood-ASXL2	
+SRR3895735	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	bone marrow	bone marrow		35450-BM-ASXL2	
+SRR3895747	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	peripheral blood	peripheral blood		CBF376-Blood ASXL2	
+SRR3895734	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	bone marrow	bone marrow		08462-BM-ASXL1	
+SRR3895737	SRP078441							HOMO_SAPIENS	Illumina HiSeq 2500 (IlluminaHiSeq2500)				SRA	bone marrow	bone marrow		40810-BM-ASXLwt	
diff --git a/differential-expression/gene_DE.nb.html b/differential-expression/gene_DE.nb.html
diff --git a/differential-expression/gene_DE.Rmd → differential-expression/microarray_DGE.Rmd b/differential-expression/gene_DE.Rmd → differential-expression/microarray_DGE.Rmd
@@ -1,5 +1,5 @@
 ---
-title: "Differential Expression Analysis"
+title: "Differential Expression Analysis: Microarray"
 author: "ALSF CCDL - Candace Savonen"
 output:   
   html_notebook: 
@@ -9,8 +9,7 @@ output:
 
 *Purpose*: This notebook takes data and metadata from refine.bio and identifies 
 differentially expressed genes. 
-This script is very generally applicable to pre-processed RNA-Seq or
-microarray data.
+This script is generally applicable to microarray data.
 
 ## 1) Install libraries
 This script uses the bioconductor R package limma to identify differentially 
@@ -23,7 +22,7 @@ studies.” Nucleic Acids Research, 43(7), e47.
 
 ```{r Install limma}
 if (!("limma" %in% installed.packages())) {
-  # Install ComplexHeatmap
+  # Install limma
   BiocManager::install("limma", update = FALSE)
 }
 ```
@@ -121,9 +120,6 @@ qq.plot <- qqt(fit$t, pch = 16, cex = 0.2); abline(0,1)
 png(file.path("plots", "qqplot_GSE71270.png"))
 qqt(fit$t, pch = 16, cex = 0.2); abline(0,1)
 dev.off()
-
-# Print out plot here
-qq.plot
 ```
 
 Make a volcano plot and save to png
@@ -134,11 +130,8 @@ voc.plot <- volcanoplot(fit, main = "Volcano Plot")
 
 # Save plot to png
 png(file.path("plots", "volcano_plot_GSE71270.png"))
-voc.plot
+volcanoplot(fit, main = "Volcano Plot")
 dev.off()
-
-# Print out the plot here
-voc.plot
 ```
 
 ## 6) Write statistics to output results file

diff --git a/differential-expression/microarray_DGE.nb.html b/differential-expression/microarray_DGE.nb.html
diff --git a/differential-expression/plots/qqplot_GSE71270.png b/differential-expression/plots/qqplot_GSE71270.png
diff --git a/differential-expression/plots/volcano_plot_GSE71270.png b/differential-expression/plots/volcano_plot_GSE71270.png
diff --git a/differential-expression/plots/volcano_plot_SRP078441.png b/differential-expression/plots/volcano_plot_SRP078441.png