visualizing methylation levels at individual CpG sites

[petermchale]

When I run bedgraphtobigwig on this bedgraph file:

chr1    69590   69591   0.783515615800065
chr1    191549  191550  0.4875594150534202
chr1    259409  259410  0.7866381108397801
chr1    605419  605420  0.3955424153029119
chr1    633317  633318  0.11658784436577774
chr1    777871  777872  0.9435822827865346
chr1    777912  777913  0.9245702644897906
chr1    779896  779897  0.43717435961143386
chr1    803583  803584  0.451108712860254
chr1    817114  817115  0.1956207519829175

and visualize the resulting bigwig file in IGV (version 2.19.4), I get this visualization:

whereas I was expecting 10 bars, each 1bp wide. What am I doing wrong? Thanks.

[jackh726]

I'm curious if IGV is maybe using zoom data or something. I don't have the time to dig into this right now, but you could try passing --nzooms 0 and see what you get.

[petermchale]

This is what I get:

[jackh726]

So, that's potentially right? Not sure if IGV will display 1 BP regions at that resolution. If you zoom in, can you see them?

[petermchale]

No, you can't. In this image, I'm expecting to see a single bar of height 0.783515615800065 at chr1:69590 ...

[jackh726]

Interesting...thanks for hanging in, but one last thought: does 2bp-sized regions display? What about more? I just wonder if IGV renders such small regions weird.

[petermchale]

I changed the second record to encompass 2bps, and the third record to encompass 3bps. Still nothing.

chr1	69590	69591	0.783515615800065
chr1	191549	191551	0.4875594150534202
chr1	259409	259412	0.7866381108397801

[jackh726]

Okay, can you tell me exactly the command you're running? Running with --nzooms 0, I can't reproduce:

[petermchale]

bedgraphtobigwig --nzooms 0 208567320164_R08C01.unique.sorted.no_nulls.no_bounds_errors.sorted.head.bed /scratch/ucgd/lustre-labs/quinlan/data-shared/constraint-tools/reference/grch38/chromosome-sizes/hg38.chrom.sizes.sorted a.bw

[petermchale]

$ cat /scratch/ucgd/lustre-labs/quinlan/data-shared/constraint-tools/reference/grch38/chromosome-sizes/hg38.chrom.sizes.sorted
chr1    248956422
chr2    242193529
chr3    198295559
chr4    190214555
chr5    181538259
chr6    170805979
chr7    159345973
chr8    145138636
chr9    138394717
chr10   133797422
chr11   135086622
chr12   133275309
chr13   114364328
chr14   107043718
chr15   101991189
chr16   90338345
chr17   83257441
chr18   80373285
chr19   58617616
chr20   64444167
chr21   46709983
chr22   50818468
chrX    156040895
chrY    57227415

[petermchale]

$ bedgraphtobigwig --version 
bedgraphtobigwig 0.5.6

[jackh726]

Can you share your output bigwig?

And, can you try this bigwig in your IGV? https://www.dropbox.com/scl/fi/flj40nvg7gbjrwsx5nz4h/regions.bigWig?rlkey=iosxxv79f4dredz9y8rm6eo8x&st=ha7plj6b&dl=0

[petermchale]

Still nothing:

[petermchale]

Here's the bigwig I was using: https://www.dropbox.com/scl/fi/hrvtojv08ijcuy3wjbcu6/a.bw?rlkey=zmf4g34zmbzhl11io2da2s831&st=qo24cgab&dl=0

[jackh726]

So...something is weird with IGV here...what version are you using? I am using 2.15.4.

One thing: can you manually set your data range to 0-1? I don't think this is the issue, but certainly something to double check.

[petermchale]

I'm using 2.19.4. Try it.