Pr 2 of 2: Add Microarray Pathway Analysis - GSEA example #347

cbethell · 2020-11-05T02:59:51Z

Analysis Purpose

This PR closes issue #284

Pull Request Stage

This is a Refined PR - needs review of details and polishing

The draft PR relevant to this PR is #339.

Strategy

This PR iterates on open PR #345 by adding the GSEA and visualization steps to the microarray GSEA example analysis.

I copied over the steps from draft PR #339, incorporating the relevant feedback from there.

Therefore, this PR includes:

The implementation of clusterProfiler::GSEA() -- per this comment on the relevant draft PR, some research into the function led me to the current implementation in this PR as it seems that fgseaMultilevel() is the recommended approach to use and the default for clusterProfiler::GSEA() when the nPerm argument is not provided (info gathered partly from the fgsea docs and partly from the warning message below):

Also probably worth noting, when attempting to run fgseaSimple() and/or fgseaMultilevel() (without using the clusterProfiler wrapper), 0 results were returned (could be an error in my implementation however).

To answer the question re the pvalue cutoff on the draft PR, the p-value cutoff (FDR in this case) is an arbitrary cutoff which is now equal to 0.05 in this PR based on what seems to be standard practice according to various docs (including but not limited to Toward a gold standard for benchmarking gene set enrichment analysis and clusterProfiler: an R Package for Comparing Biological Themes Among Gene Clusters).
The decision to create the pre-ranked gene vector based on the t-statistic rather than log fold change was explored based on this comment from the draft PR and eventually made based on what is recommended in Discovering statistically significant pathways in expression profiling studies and the explanation from a biostars forum which says "Try to understand how they relate to the question you're interested in e.g. if you're most interested in effect size then the fold change is what you should use but if you're more interested in statistical significance then look for one of the statistics taking into consideration the assumptions they make e.g. t-test" as I believe we are encouraging users to look at statistical significance here.
Visualizations of the GSEA results using enrichplot::gseaplot() was included in this PR as well as they were in the draft PR
Note that I also set the seed in this PR as I noticed that the results were slightly changing and realized that I did indeed this important step.

Concerns/Questions for reviewers:

Note that at line 317, I left a prompt to START REVIEW HERE as this is the point where PR 1 of 2 (#345) ends and the new content being added in this PR begins (I will remove the prompt once #345 is merged).

Overall, do the decisions included in this PR make sense?

Does the context re GSEA() (the goal of it and the implementation) make sense?
Do the prompts seem sufficient for users?

Analysis Pull Request Check List (roughly in order):

Content checks

All {{BLANKS}} have been replaced with the correct content.
Sources are cited
Seed is set (if applicable)

Formatting Checks

Removed any manual numbering of sections.
Removed any instances of chunk naming.
Comments and documentation are up to date.
All links have been checked and are properly formatted.

Add datasets to S3

Added data and metadata files to S3.

Docker/Snakemake rendering components

Added the .html link to the navigation bar. (Done in PR 1 of 2 Pr 1 of 2: Add Microarray Pathway Analysis - GSEA example #345)
Any not yet added packages needed for this analysis have been added to the Dockerfile and it successfully builds.

* Adding in some style with css * Use css magic * Try making the navbar blue * Add survey link * Make font smaller * Need a comma * Change to normalizePath * normalizepath separate step references.bib * Move references.bib to component folder * Made ccs modifications, added logo file Made changes to css/navbar.html Tried to add the logo but it but it cuts out and not sure how to make it decent. * Resolve render-notebooks.R conflict * Remove testing html from file diff * uncommented mobile nav Co-authored-by: dvenprasad <[email protected]>

@dvenprasad

* Adding in some style with css * Use css magic * Try making the navbar blue * Add survey link * Make font smaller * Need a comma * Change to normalizePath * normalizepath separate step references.bib * Move references.bib to component folder * Update github actions to reflect staging branch (#311) * Update github actions to reflect staging branch * Add libglpk40 to Dockerfile * Make it gh-pages-stages! * Remove dockerfile change that should have been on its own all along * Does this work? * Declare a uses * Switch how env is declared * Force it to run so we can test it * try no curly brackets * What's up with the branch * Move to bash if instead * Need quotes? * forgot a `then` * Try dollar signs * Doesn't like the `.`? * Use curly brackets * Try ${GITHUB_REF} * Try ${BRANCH_NAME} * try ${GITHUB_REF#refs/*/} * use jashapiro suggestion * Change to base ref * Change back to `github.ref` * Get rid of PR `on:` * Try another test * Docker dep fix: Add lib package 40 thing that clusterprofiler needs (#316) * Add lib package 40 thing that clusterprofiler needs * Try adding options(warn = 2) * Test if options(warn =2) means it breaks like it should * Revert "Test if options(warn =2) means it breaks like it should" This reverts commit d9f688f. * Revert "Try another test" This reverts commit 845cf1a. * Add google analytics to renderings (#314) * Try adding google analytics * Add to header using includes * temporary file snuck in there * Restore master version so they aren't in the review * Let's call an html file and html file * Docker dep fix: Add lib package 40 thing that clusterprofiler needs (#316) * Add lib package 40 thing that clusterprofiler needs * Try adding options(warn = 2) * Test if options(warn =2) means it breaks like it should * Revert "Test if options(warn =2) means it breaks like it should" This reverts commit d9f688f. * Only push if we are in master. For simplicity, we will now run this even if the dockerfile hasn't changed. * Add test target * test staging workflow with this branch * back to latest tag * Try separate push step * change tags to test push * Revert "change tags to test push" This reverts commit 6a38574. * Remove this branch from triggers * Push staging, retag and push master Okay, so the branch name is now inaccurate, but that is fine... * Made ccs modifications, added logo file Made changes to css/navbar.html Tried to add the logo but it but it cuts out and not sure how to make it decent. * Resolve render-notebooks.R conflict * Remove testing html from file diff * uncommented mobile nav * Update scripts/render-notebooks.R * Add some issue templates (#319) * Add some rough draft issue templates * Incorporate cbethell review * Get rid of `Other` labels that aren't useful * Update diagrams showing how microarray/RNA-seq work (#326) * Mechanics for CSS file and navbar add feedback URL (#303) * Adding in some style with css * Use css magic * Try making the navbar blue * Add survey link * Make font smaller * Need a comma * Change to normalizePath * normalizepath separate step references.bib * Move references.bib to component folder * Made ccs modifications, added logo file Made changes to css/navbar.html Tried to add the logo but it but it cuts out and not sure how to make it decent. * Resolve render-notebooks.R conflict * Remove testing html from file diff * uncommented mobile nav Co-authored-by: dvenprasad <[email protected]> * Update microarray and RNAseq overview figures - add context re figures - change .jpg to .png for consistency * Revert "Mechanics for CSS file and navbar add feedback URL (#303)" This reverts commit 8b81fdd. * update links to diagrams * @dvenprasad updated figure spacing * add the right updated figure * replace section of link to figures with updated commit id * incorporate @cansavvy's suggested changes Co-authored-by: Candace Savonen <[email protected]> Co-authored-by: dvenprasad <[email protected]> Co-authored-by: Joshua Shapiro <[email protected]> Co-authored-by: dvenprasad <[email protected]> Co-authored-by: Chante Bethell <[email protected]>

fix merge conflicts

cansavvy

I think this looks great! I just have some wording suggestions.

I floated an idea about moving some of the "how this is calculated" info close to where the plots are since I think the plots illustrate that well and visuals can be helpful for absorbing the ideas. Let me know what you think.