WGBS Analysis Part 29

Running randomization tests on mox

I initially tried running the randomization tests on the R Studio server, but encountered several timeouts. I decided to run an R script through a SLURM script, like I’ve tried previously.

Editing code

First, I removed my randomization test code from my DML identification script, and copied my code into a new randomization test script. I trimmed down the randomization test code to only include the 75% difference for female samples and 99% difference for indeterminate samples in calculateDiffMeth. I also pointed to the revised script in this SLURM script, and modified my output text file name so I could easily find it.

Then I ran the SLURM script! Of course I ran into package installation issues. When loading methylKit, R could not find the GenomicRanges package. Thinking this had something do to with the ~/.Renviron file I modified for the R Studio server, I set a new path for my R packges: /gscratch/srlab/rpackages. I realized soon after that this wouldn’t solve the problem, since methylKit and all its associated packages are nested in my user directory: /gscratch/home/yaaminiv/R/x86_64-pc-linux-gnu-library/3.6/! In a build node, I went down the rabbit hole of packages that needed to be explicitly loaded, and ended up with the following list to get methylKit loaded successfully:

require(BiocGenerics, lib.loc = "/gscratch/home/yaaminiv/R/x86_64-pc-linux-gnu-library/3.6/")
require(S4Vectors, lib.loc = "/gscratch/home/yaaminiv/R/x86_64-pc-linux-gnu-library/3.6/")
require(IRanges, lib.loc = "/gscratch/home/yaaminiv/R/x86_64-pc-linux-gnu-library/3.6/")
require(GenomeInfoDb, lib.loc = "/gscratch/home/yaaminiv/R/x86_64-pc-linux-gnu-library/3.6/")
require(GenomicRanges, lib.loc = "/gscratch/home/yaaminiv/R/x86_64-pc-linux-gnu-library/3.6/")
require(methylKit, lib.loc = "/gscratch/home/yaaminiv/R/x86_64-pc-linux-gnu-library/3.6/")

Another mox error

Once I cleared this hurdle, I encountered another one. My randomization test was not running because the sample ID information was not formatted correctly. I thought this was strange becaus I ran this code on the R Studio server without running into this error. In any case, I looked at the output file and saw that the sample ID information was saved as factors, and not characters. After creating the sampleMetadataFem dataframe with information, I converted the factors to characters:

sampleMetadataFem <- data.frame("sampleID" = c("1", "3", "4",
                                               "6", "7", "8"),
                             "slide-label" = c("02-T1", "06-T1", "08-T2",
                                               "UK-02", "UK-04", "UK-06"),
                             "pHTreatment" = c(rep(1, times = 3),
                                               rep(0, times = 3))) #Create metadata table
sampleMetadataFem$sampleID <- as.character(sampleMetadataFem$sampleID) #Convert to character format

I did this for the indeterminate samples too, since I’d likely run into the same error! When I ran my revised script, it started cranking through. Then I encountered other errors I hadn’t seen before:

Screen Shot 2021-05-17 at 5 48 46 PM

I posted this discussion to get feedback, and started running the randomization test without the mc.cores argument to see if I could reproduce the error. Two hours after running the test without mc.cores in calculateDiffMeth, I still hadn’t encountered that error. I wonder if these errors could have contributed to the R Studio server crashing when I used mc.cores previously. In any case, I’m not sure how I can run these tests now…

Going forward

  1. Run methylKit randomization test
  2. Update mox handbook with R information
  3. Obtain relatedness matrix and SNPs with EpiDiverse/snp
  4. Identify overlaps between SNP data and other epigenomic data
  5. Write methods
  6. Write results
  7. Identify significantly enriched GOterms associated with DML
  8. Identify methylation islands and non-methylated regions
  9. Determine if larval DNA/RNA should be extracted
Written on May 17, 2021