From acf0e9df1c9cfd12e81d3acc0eadaaa0d6d4510a Mon Sep 17 00:00:00 2001 From: Joshua Shapiro Date: Mon, 17 Jun 2024 17:15:50 +0000 Subject: [PATCH 1/4] Change GSVA to new syntax --- .../03-gene_set_variation_analysis.Rmd | 60 ++++++++++--------- 1 file changed, 32 insertions(+), 28 deletions(-) diff --git a/pathway-analysis/03-gene_set_variation_analysis.Rmd b/pathway-analysis/03-gene_set_variation_analysis.Rmd index fb38a4ca..aee74738 100644 --- a/pathway-analysis/03-gene_set_variation_analysis.Rmd +++ b/pathway-analysis/03-gene_set_variation_analysis.Rmd @@ -5,7 +5,7 @@ output: toc: true toc_float: true author: CCDL for ALSF -date: 2020 +date: 2024 --- ## Objectives @@ -148,20 +148,24 @@ The output is a gene set by sample matrix of GSVA scores. ### Perform GSVA ```{r run_gsva} -gsva_results <- gsva(rnaseq_mat, - hallmarks_list, - method = "gsva", - # Appropriate for our transformed data on log2-like scale - kcdf = "Gaussian", - # Minimum gene set size - min.sz = 15, - # Maximum gene set size - max.sz = 500, - # Compute Gaussian-distributed scores - mx.diff = TRUE) +gsva_results <- gsva( + gsvaParam( + exprData = rnaseq_mat, + geneSets = hallmarks_list, + # Minimum gene set size + minSize = 15, + # Maximum gene set size + maxSize = 500, + # Kernel for estimation + # Gaussian for our transformed data on log2-like scale + kcdf = "Gaussian", + # enrichment score is the difference between largest positive and negative + maxDiff = TRUE + ) +) ``` -**Note: the `gsva()` documentation says we can use `kcdf = "Gaussian"` if we had RNA-seq log-CPMs, log-RPKMs or log-TPMs, but we would use `kcdf = "Poisson"` on integer counts.** +**Note: the `gsvaParam()` documentation says we can use `kcdf = "Gaussian"` if we had RNA-seq log-CPMs, log-RPKMs or log-TPMs, but we would use `kcdf = "Poisson"` on integer counts.** ```{r gsva_peek} # Let's explore what the output of gsva() looks like @@ -230,18 +234,16 @@ random_gene_sets <- random_gene_sets |> Run GSVA on our dataset with the same parameters as before, but now with random gene sets. ```{r random_gsva, live = TRUE} -random_gsva_results <- gsva(rnaseq_mat, - random_gene_sets, - method = "gsva", - # Appropriate for our transformed data on - # log2-like scale - kcdf = "Gaussian", - # Minimum gene set size - min.sz = 15, - # Maximum gene set size - max.sz = 500, - # Compute Gaussian-distributed scores - mx.diff = TRUE) +random_gsva_results <- gsva( + gsvaParam( + exprData = rnaseq_mat, + geneSets = random_gene_sets, + minSize = 15, + maxSize = 500, + kcdf = "Gaussian", + maxDiff = TRUE + ) +) ``` Now let's make a plot to look at the distribution of scores from random gene sets. @@ -254,9 +256,11 @@ random_long_df <- random_gsva_results |> # Gene set names are rownames tibble::rownames_to_column("gene_set") |> # Get into long format - tidyr::pivot_longer(cols = -gene_set, - names_to = "Kids_First_Biospecimen_ID", - values_to = "gsva_score") |> + tidyr::pivot_longer( + cols = -gene_set, + names_to = "Kids_First_Biospecimen_ID", + values_to = "gsva_score" + ) |> # Remove the .version added by make.names() dplyr::mutate(gene_set = stringr::str_remove(gene_set, "\\..*")) |> # Add a column that keeps track of the gene set size From 1cb1613841e745158e6678f36e1ce07b6b75cab3 Mon Sep 17 00:00:00 2001 From: Joshua Shapiro Date: Mon, 17 Jun 2024 17:21:16 +0000 Subject: [PATCH 2/4] Add a little explanatory paragraph, plus parallelism --- pathway-analysis/03-gene_set_variation_analysis.Rmd | 11 +++++++++-- 1 file changed, 9 insertions(+), 2 deletions(-) diff --git a/pathway-analysis/03-gene_set_variation_analysis.Rmd b/pathway-analysis/03-gene_set_variation_analysis.Rmd index aee74738..abcbb998 100644 --- a/pathway-analysis/03-gene_set_variation_analysis.Rmd +++ b/pathway-analysis/03-gene_set_variation_analysis.Rmd @@ -147,6 +147,9 @@ The output is a gene set by sample matrix of GSVA scores. ### Perform GSVA +The main work for GSVA is done by the `gsva()` function, which can actually do a variety of different enrichment score calculations. +To specify that we want the algorithm used by [Hänzelmann _et al._ (2013)](https://doi.org/10.1186/1471-2105-14-7), we will pass as the first argument a call to the `gsvaParam()` function, which is where we will put our data, gene sets, and other parameters specific to that algorithm. + ```{r run_gsva} gsva_results <- gsva( gsvaParam( @@ -161,7 +164,9 @@ gsva_results <- gsva( kcdf = "Gaussian", # enrichment score is the difference between largest positive and negative maxDiff = TRUE - ) + ), + # Use 4 cores for multiprocessing + BPPARAM = BiocParallel::MulticoreParam(4) ) ``` @@ -242,7 +247,9 @@ random_gsva_results <- gsva( maxSize = 500, kcdf = "Gaussian", maxDiff = TRUE - ) + ), + # Use 4 cores for multiprocessing + BPPARAM = BiocParallel::MulticoreParam(4) ) ``` From aef1d229d653dcee57b7dd8e275856264ea08720 Mon Sep 17 00:00:00 2001 From: Joshua Shapiro Date: Mon, 17 Jun 2024 17:26:03 +0000 Subject: [PATCH 3/4] Add back testing of pathway notebooks --- scripts/render-live.sh | 6 +++--- 1 file changed, 3 insertions(+), 3 deletions(-) diff --git a/scripts/render-live.sh b/scripts/render-live.sh index 7252b4a1..2cb07fc4 100644 --- a/scripts/render-live.sh +++ b/scripts/render-live.sh @@ -41,9 +41,9 @@ files=( # machine-learning/02-openpbta_consensus_clustering.Rmd # machine-learning/03-openpbta_PLIER.Rmd # machine-learning/04-openpbta_plot_LV.Rmd - # pathway-analysis/01-overrepresentation_analysis.Rmd - # pathway-analysis/02-gene_set_enrichment_analysis.Rmd - # pathway-analysis/03-gene_set_variation_analysis.Rmd + pathway-analysis/01-overrepresentation_analysis.Rmd + pathway-analysis/02-gene_set_enrichment_analysis.Rmd + pathway-analysis/03-gene_set_variation_analysis.Rmd ) for file in ${files[@]} do From e927eb85a86cdd4a5fab2d82c922887ecafb8b26 Mon Sep 17 00:00:00 2001 From: Joshua Shapiro Date: Mon, 17 Jun 2024 18:33:33 +0000 Subject: [PATCH 4/4] add figure to scRNA-seq-advanced --- 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