Extracting Methylation Data

Question

[Quest96]

Hello. I was analyzing some data obtained from bisulfite sequencing and was trying to look at the site of methylation for all of the amplicons. I initially used Interactive Genome Viewer (IGV) to see the sites of methylation. However, I was not sure how to actually obtain the data for the site of methylation for the CG, CHG, and CHH sites of interest. I wanted to take the data for the sites of methylation on each read and create a graph from the comparing and contrasting the different sites of methylation. What program would be recommended to obtain the methylation data from the sequences? Thank you.

Answer 1

[WouterDeCoster]

I've had good experiences with https://github.com/dpryan79/MethylDackel

[Quest96]

I'm new to coding so I had a bit of trouble getting MethylDackel to work. I used R Studio as my IDE and downloaded the prerequisites (GSS, htslib, and libbigwig) mentioned on the page along with the actual MethylDackel files. I then tried to do the compilation/installation steps they mentioned, but it seems to run into a fatal error whenever I inputted those commands. It can't seem to find sam.h. To clarify I put all the files in different folders and then tried to do this. I also tried putting the htslib and libbigwig folders in MethylDackel, but that also didn't work. Not sure how to proceed. Do you have any insight?

To be specific I was doing these commands:

git clone https://github.com/dpryan79/MethylDackel.git
cd MethylDackel
make LIBBIGWIG="/some/path/to/libBigWig.a"
make install prefix=/some/installation/path

And that third command was where I had my issues.

[WouterDeCoster]

but it seems to run into a fatal error whenever I inputted those commands

It would be rather useful if you would share that error.

[Quest96]

[User@rstudio MethylDackel]$ make LIBBIGWIG=~/MethylDackel/libBigWig.a
echo '#define VERSION "0.5.1"' > version.h
cc -c -Wall -g -O3 -pthread  -IlibBigWig common.c -o common.o
In file included from common.c:1:0:
MethylDackel.h:4:24: fatal error: htslib/sam.h: No such file or directory
 #include "htslib/sam.h"
                        ^
compilation terminated.
make: *** [common.o] Error 1

[Devon Ryan]

Your life will be much easier if you just install it with conda.

[Quest96]

Ok. So if I were doing the installation with Conda what steps would I take in terms of installation. Would I still use R Studio?

[arturtjaro]

The third line is meant to be filled in with the actual path to the libBigWig.a static library file. You'll need to:

1. clone the libBigWig repo with git clone https://github.com/dpryan79/libBigWig.git
2. cd into the directory
3. run make. This should generate the libBigWig.a file.
4. Run the above commands as you were doing, but providing the path to the libBigWig.a file, and a real installation path in the last line

[Devon Ryan]

Or just install via conda and not have to bother with any of this.