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Update - 3-3-15

Update - 3-3-15

Mar 03, 2015
Update 3/3/15:

Now that I'm finished traveling, finding time for this should be less of a problem.

I have now acquired all necessary bisulfite-seq data (1) with the exception of the ago4 knockout genetic background. It should exist, but wasn't from the same publication. Still looking for that. I also have downloaded the reference genome for Arabidopsis thaliana (TAIR 10) to which the sequencing reads will be mapped.

With regard to the actual data analysis, I have installed bowtie/bismark on my home computer and finally got them to work over the weekend. Now I will need a good test dataset, so I don't need to work with the whole genomes just to see if analyses will fail. There are several loci which I use for testing methylation in the lab, including the retrotransposon AtSN1 and the gene FWA. There are others as well. My test dataset will consist of several of these genes' sequences which are affected by RdDM. I will then be able to test my strategy on the smaller dataset before scaling up to the whole genome. Ideally I want to include a locus that we know will become hypermethylated in at least one of these genetic backgrounds as well.

I also found out that the bismark program I will be using for the analysis of bisulfite-converted reads is actually already implemented in iPlant. It required me to log in again after selecting the High Performance Computing category in the Discovery Environment. So, that will save me some trouble, I believe.

Next Up:
  1. Create a test dataset.
  2. Run the test dataset through Bismark and see if it displays the expected output for my RdDM deficient mutants at known RdDM-targeted loci.
  3. Evaluate the output to figure out what sort of scripting I will need for file conversion
Difficulties:

Aside from the availability of the AGO4 knockout BS-seq data, nothing has really been a problem. That data should exist, I would think. Hopefully I'll be able to locate it shortly.

References:
  1. Wierzbicki AT, Cocklin R, Mayampurath A, et al. Spatial and functional relationships among Pol V-associated loci, Pol IV-dependent siRNAs, and cytosine methylation in the Arabidopsis epigenome. Genes Dev. 2012;26(16):1825-36.