WGBS Analysis Part 34

Digging into enrichment results

I’m returning to the topGO analysis I did previously. One of Steven’s main concerns after looking at the enrichment results was that several enriched GOterms appeared to be related to sperm motility. Since I only used female samples, having terms related to sperm didn’t make a lot of sense. Additionally, I wrote and discussed my results in the context of significantly enriched GOterms, but didn’t tie back to the genes. So I’m going back to this R Markdown file to look at the enrichment results, annotations, and related genes.

Revising annotations

The first thing I noticed when I looked at my results spreadsheet was that I never actually added gene product annotations. To do this, I imported the list of gene products and transcript IDs I generated in this Jupyter Notebook. Then, I used merge to combine both spreadsheets and saved the file here.

When I used simplifyEnrichment to visualize the enrichment results, I created a dataframe that clustered my enriched GOterms by semantic similarity. I wanted to append the cluster information to my spreadsheet so I could use that to understand the different gene functions. Cluster 1 is related to cilia and motility, while clusters 2-5 are related to development. It’s possible that clusters 2-5 are really a supercluster instead of four clusters with one enriched GOterm each. Looking at my dataframe after merging the product information with the clusters, I saw I was missing information for cluster 4. It was at this point that I realized I should have used left_join when combining dataframes (now and upstream). It’s likely that the GOterms related to the fourth cluster didn’t have any transcript annotations from the annotation table I generated, so those rows were being dropped with merge.

I went back to the top of my script and proceeded to use left_join instead of merge. A couple of significant GO IDs had topGO annotations, but were not present in my manual blastx annotations. I thought it would be good to keep the GOterms associated with these GO IDs only present in the topGO database. I also wanted to pull the transcript IDs associated with these enriched GOterms. I used genesInTerm to extract the genes of interest, converted it into a dataframe, then extracted the transcript IDs so they were on separate rows:

sigRes.allBPGenes <- genesInTerm(allGOdataBP, whichGO = allRes.allBP$GO.ID[1:10]) #Extract genes associated with each GOterm from topGO's annotations sigRes.allBPGenes <- as.data.frame(vapply(sigRes.allBPGenes, paste, collapse = ",", character(1L))) #Collapse all transcript IDs into one column and convert into a dataframe. GO IDs will be the rownames colnames(sigRes.allBPGenes) <- c("transcript") sigRes.allBPGenes$GOID <- rownames(sigRes.allBPGenes) #Convert row names to a separate column rownames(sigRes.allBPGenes) <- 1:nrow(sigRes.allBPGenes) #Convert row names to numeric values sigRes.allBPGenes <- separate_rows(sigRes.allBPGenes, transcript, sep = ",") #Separate transcripts so each transcript is on a separate row 

I used left_join to match this information with topGO enrichment scores and GO IDs. As expected, every GO ID had at least one corresponding transcript. Then, I used left_join based on GO ID and transcript to add my manual annotations and methylKit information. At this step, I realized that the GO IDs not present in my manual annotations did not get any corresponding methylKit information, even though there was a transcript ID. I tried for a while to think of a way to complete the annotation process, but got stuck and figured if those cells had N/A values, then I would know those were the GOterms only present in the topGO database. Finally, I appended the product and cluster information to all rows and saved the output here. I repeated this process with the CC GOterms and found an overlap between the genes in each list! I didn’t look at cluster information, since there was only one cluster determined by simplifyEnrichment. I saved the spreadsheet with product information here.

Going forward

  1. Look into unannotated GOterms and potential nesting
  2. Understand function of genes with enriched GOterms
  3. Determine if there’s a bias towards hyper- or hypomethylated genic DML with enriched functions
  4. Report mc.cores issue to methylKit
  5. Perform randomization test
  6. Update mox handbook with R information
  7. Determine if larval DNA/RNA should be extracted

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