Killifish Hypoxia RRBS Part 15

Methylation landscape information

I have two small tasks I need to accomplish: characterize the methylation landscape and generate genome feature tracks. Turns out these small tasks are big tasks that brought about more questions (#classic).

Methylation landscape

Characterizing the methylation landscape is fueled by a need to update the results section of my manuscript. To determine what methylation landscape information I needed, I reviewed Neel’s zebrafish preprint. Based on that results section, I needed global methylation percentages and the number of methylated and unmethylated CpGs for each treatment. I created this Jupyter notebook to get the information I needed.

The first thing I did was create a union bedGraph with sorted and filtered bedGraphs for each sample so I could count the number of CpGs with 10x-500x coverage in at least one sample. The metilene output only considered CpGs with data in all samples for DMR analysis, so I couldn’t just look at the number of CpGs from my all populations comparison. Which brought up a question…should I allow for missing values with --mis1 and --mis2 in BAT_summarize? Within populations, I don’t think it makes sense to allow for missing values since the sample size is small to begin with. But perhaps for the all populations comparison, I could account for missing values for a few samples at a CpG locus.

Next, I calculated global methylation level and the number of methylated and unmethylated CpGs. I set the cutoff for methylated CpGs as > 10% methylation rate, since I’m using that same cutoff to define a DMR. I need to run this by Neel since that information wasn’t in the preprint. I used this code to count CpGs in each category:

#Remove header
#Find CpGs with > 10% methylation
#Count number of CpGs
! tail -n+2 all-samples-averages.bedgraph \
| awk -F'\t' -v OFS='\t' '{if ($4 > .1) { print $26 }}' ${f} \
| wc -l

#Remove header
#Find CpGs with > 10% methylation
#Count number of CpGs
! tail -n+2 all-samples-averages.bedgraph \
| awk -F'\t' -v OFS='\t' '{if ($4 <= .1) { print $26 }}' ${f} \
| wc -l

I used this code to average methylation rate across all loci to get global methylation level:

#Calculate average methylation
! tail -n+2 all-samples-averages.bedgraph \
| awk '{ total += $26; count++ } END { print total/count }'

Interestingly, there were more unmethylated than methylated CpGs, and global methylation rate was ~20%. This was pretty consistent across populations and population x treatment combinations. My understanding of vertebrates is that they have higher levels of methylation. I wonder if accommodating missing loci would boost methylation rate, or if killifish just have lower constitutive levels of methylation?

Genome feature struggles

I went to download the GFF for the genome version (3.0.2) I was using from NCBI but realized I couldn’t! I also couldn’t get the RepeatMasker output. I was only able to download the GFF for the newest genome (4.1). Neel had the GTF associated with the genome version I was using on vortex, but it poses larger questions: 1) does Neel have a GFF for v.3.0.2 and 2) should I be using v.4.1 instead?

I decided to extract genome feature tracks from the GTF I had in this Jupyter notebook. I pulled out genes, exons, CDS, 5’ UTR, and 3’ UTR from the GTF. The GFF would have had lncRNA information as well, so that would be nice to have. When I tried using complementBed to generate a non-coding track, I encountered this error:

#Find the complement to the exon track (non-coding sequences)
#Create a BEDfile of IGV
!{bedtoolsDirectory}/complementBed \
-i Fundulus_heteroclitus-3.0.2.105-exon.gff \
-g mummichog.chrom.length \
> Fundulus_heteroclitus-3.0.2.105-nonCDS.bed

***** ERROR: illegal number ":primary". Exiting...

I wondered if this had anything to do with the file format, so I extracted the chromosome, start, and stop information and fed that into complementBed and still got the same error. I commented on this recently posted Github issue, and was shown that my length for the mitochondrial chromosome was “:primary!” Something must have gone wrong when I generated the genome file. I went to the NCBI genome page and opened the full sequence report to find that the mitochondrial sequence is 16,526 bp long. I updated the genome text file with this information and was able to create non-coding and intron tracks. I’m not sure how to proceed with flanking regions in killifish, and I don’t know how to extract CpG island information. Without this information, I can’t create an accurate intergenic region track, so I stopped there.

Visualizing DMR and genome features

The last thing I did was create an IGV session for DMR and genome feature tracks so I could see where DMR were located before I used bedtools to quantify overlaps. I added the DML bedGraphs and genome feature files to this IGV session. I need to create an OSF repo soon so I can use links instead of file paths!

Going forward

  1. Determine if I should use the new genome for analysis
  2. Ask Neel about missing values from group 1 and 2 for BAT_summarize
  3. Confirm methylated and unmethylated definitions
  4. Finish generating genome feature tracks
  5. Annotate DMR locations
  6. Update methods and results
  7. Try DMR identification with bismark and methylKit
  8. Start RNA-Seq analysis
  9. Create OSF repository for all intermediate files
Written on May 26, 2022