It all depends on the options you used from Bismark. Here is the relevant information regarding your outputs from the docs:
Strand-specific methylation output files (default): As its default option, the bismark_methylation_extractor will produce a
strand-specific output which will use the following abbreviations in
the output file name to indicate the strand the alignment came from:
OT – original top strand CTOT – complementary to original top
strand OB – original bottom strand CTOB – complementary to
original bottom strand Methylation calls from OT and CTOT will be
informative for cytosine methylation positions on the original top
strand, calls from OB and CTOB will be informative for cytosine
methylation positions on the original bottom strand. Please note that
specifying the --directional (the default mode) option in the Bismark
alignment step will not report any alignments to the CTOT or CTOB
As cytosines can exist in any of three different sequence contexts
(CpG, CHG or CHH) the bismark_methylation_extractor default output
will consist of 12 individual output files per input file (CpG_OT_...,
CpG_CTOT_..., CpG_OB_... etc.).
Context-dependent methylation output files (--comprehensive option):
If strand-specific methylation is not of interest, all available
methylation information can be pooled into a single context-dependent
file (information from any of the four strands will be pooled). This
will default to three output files (CpG-context, CHG-context and
CHH-context), or result in 2 output files (CpG-context and
Non-CpG-context) if --merge_non_CpG was selected (note that this can
result in enormous file sizes for the non-CpG output).
Both strand-specific and context-dependent options can be combined
with the --merge_non_CpG option.
This option may be used for PBAT-Seq libraries (Post-Bisulfite Adapter
Tagging; Kobayashi et al., PLoS Genetics, 2012). This is essentially
the exact opposite of alignments in 'directional' mode, as it will
only launch two alignment threads to the CTOT and CTOB strands instead
of the normal OT and OB ones. Use this option only if you are certain
that your libraries were constructed following a PBAT protocol (if you
don't know what PBAT-Seq is you should not specify this option). The
option --pbat works only for FastQ files and uncompressed temporary
Thus, keep in mind that the use of
--directional will have an effect on which strand information is kept. Further, what is likely happening here, is that you used the
--pbat option since you are only seeing CTOT and CTOB outputs.
Regarding non CG context, refer to the "Context-dependent methylation output files" /
--comprehensive option. There are additional parameters you may consider for non-CG context (EDIT - to complement: unless you specified the option
bismark_methylation_extractor, then your bedGraph should only have CpGs. If you do not specify this, but use
--comprehensive, you can generate bedGraphs for the other contexts separately by using the
Thank you very much for the answer. Now I am clear about question 1. Are there any comments about question #2?
Yes, look at my last paragraph, I even added an edit shortly after posting it to make things clear.
I saw it. Thank you very much!
Also, as an FYI, don't post a comment to your own question in the answer section of this forum. Just add it to the