Posted by & filed under Cgigas DNA Methylation.

Finally re-getting around to examing our MBD-array data in the context of RNA-seq data. Briefly we have three oysters that were heat shocked with samples taken prior to, and following. An MBD-array experiment was carried out where for each oyster we have information on 50bp genome features (selected probes on the array) whether the region was hypomethylated, hypermethylated or did not change.

Following heat shock approximately 10k differentially methylated features (value >/= 1.8) were identified in all oysters. For all oysters a majority of the features were hypomethylated. Specifically, for oysters #2, #4, and #6 the number of hypomethylated features was 7224, 6560, and 7645, respectively. When only features were considered where at least 3 adjacent probes were also differentially methylated the number of features (merged) for oysters #2, #4, and #6 was 112, 58, and 62, respectively. A majority of these features were hypomethylated (108, 48, and 53, respectively)

Oyster Hypo-methylated Hyper-methylated Hypo-3plus-merged Hypo-3plus-merged
2 7224 2803 108 4
4 6560 3587 48 10
6 7645 4044 53 9

The files with differentially methylated region (DMRs) as defined by value >/= 1.8 are name as 2014.07.02.6M_sig.bedGraph and have the following format (ie head output)

track type=bedGraph name=&quot;6M_sig&quot; description=&quot;6M_sig&quot; visibility=full color=100,100,0 altColor=0,100,200 priority=20
scaffold1   54599   54654   -1.38187662416007
scaffold1   162129  162191  1.85685479189849
scaffold1   163536  163586  -1.15032035523765
scaffold1   172654  172714  1.33561271440876
scaffold1   174287  174343  -1.62903936976887
scaffold1   178075  178128  1.42323539316231
scaffold1   178685  178740  1.30886296151914
scaffold1   184271  184330  -1.20699853451878
scaffold1   184661  184715  -1.61107459826899


As part of the data provided by the core facility we also obtained genome feature files where there were three or more adjacent signficant probes. The naming structure is 2014.07.02.4M_Hypo_3plusAdjactentProbes.gff with the file format:

scaffold100 804406  804960
scaffold1018    367127  367319
scaffold1018    367618  367674
scaffold1077    401205  401534
scaffold12  244080  244376
scaffold1324    356489  356781


To merge these adjacent features I ran bedtools merge. For example

bedtools merge -d 100 -i
&gt; ./analyses/2M_3plusmerge_Hypo.gff
wc -l ./analyses/2M_3plusmerge_Hypo.gff


These values are refered to in the above table as Hypo(Hyper)-3plus-merged and the file format:

 scaffold1737   485256  485565
scaffold1894    222981  223032
scaffold1894    223601  223653
scaffold1894    224777  224838
scaffold392 326519  326818
scaffold433 204898  205194
scaffold544 44676   44853
scaffold544 45028   45086
scaffold854 353009  353316


filenames: 6M_3plusmerge_Hypo.bed

This screenshot shows the different file types

It is important to note that the plusAdjactentProbes.gff files were identified scrictly based on adjacent prove regardless of distance, where the merged files are spatially constrained.

For now I will treat the 6 bed files shown above as the canonical DMRs, for analysis purposes, but I imagine this will change. Hopefully if my IPython notebooks are set up right, this should not be a problem.

These 9 files are available here

2014.07.02.2M_sig.bedGraph  4M_3plusmerge_Hyper.bed
2014.07.02.4M_sig.bedGraph  4M_3plusmerge_Hypo.bed
2014.07.02.6M_sig.bedGraph  6M_3plusmerge_Hyper.bed
2M_3plusmerge_Hyper.bed     6M_3plusmerge_Hypo.bed
2M_3plusmerge_Hypo.bed


IPython notebooks used to get here are here.