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FelixKrueger / Bismark

A tool to map bisulfite converted sequence reads and determine cytosine methylation states
http://felixkrueger.github.io/Bismark/
GNU General Public License v3.0
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directional and nondirectional #694

Closed iranianuser closed 3 months ago

iranianuser commented 3 months ago

Hi Felix I finished bismark alignment and extraction to the end. and I got the results but I didn't know anything about directional and nondirectional so I set the default option (directional) for bismark alignment. My data was bisulfite-seq (WGBS) and now I read somewhere that bisulfite-seq libraries are non-directional. Is it true? and should I do my analysis again? does it affect the results???

iranianuser commented 3 months ago

I want to do DMR analysis next....

FelixKrueger commented 3 months ago

Generally, it is very important to know they kind of sequencing and appropriate trimming. Here are some recommendations. If you can link the FastQC HTML reports, your trimming command and the Bismark mapping result stats I can probably help you a bit more.

iranianuser commented 3 months ago

Hi I sent an email and attached what you mentioned.

thanks a lot

iranianuser commented 3 months ago

to this fkrueger@altoslabs.com‬

FelixKrueger commented 3 months ago

apologies, the email got stuck in the spam folder...

The data is definitely directional, as R1 is C-low and G-rich. Also, you've got a mapping efficiency of nearly 80%. If the data was non-directional, the expected maximum mapping efficiency would be 50% tops. Since there is a bias at the start of reads you may want to include some clipping there, using a command like:

trim_galore --paired --clip_r1 5 --clip_r2 5 *_1.fastq *_2.fastq

Other than that you should be good to go!

iranianuser commented 3 months ago

OK, it's great ... Your answers always make me happy I will try your suggestion