[akramdi]

Hi Amira,

For C in CpG context you need to use only the Cs methylated and unmethylated in CpG context, and not all Cs found in total. So here it would be:

Total methylated C's in CpG context = (43871230/106252080)*100 = 29.2%.

Cheers, Felix

Thank you for the quick reply!

Okay, so it's relative to the context. In your illustration, I think you meant:

Total methylated C's in CpG context = (43871230/ (106252080+ 43871230) )*100 = 29.2%.

Thanks again,
Amira

[fkrueger]

Hi Amira,

For C in CpG context you need to use only the Cs methylated and unmethylated in CpG context, and not all Cs found in total. So here it would be:

Total methylated C's in CpG context = (43871230/106252080)*100 = 29.2%.

Cheers, Felix

[akramdi]

Hi Felix,

I have recently started using Bismark. While going through the detailed log, I could not get the same pourcentages of methylated cytosines (before and after extraction) as the one shown in the report. Here's my cytosine methylation report before extraction to illustrate:

Final Cytosine Methylation Report
=================================
Total number of C's analysed: 928829076

Total methylated C's in CpG context: 43871230
Total methylated C's in CHG context: 16835845
Total methylated C's in CHH context: 25330412
Total methylated C's in Unknown context: 58

Total unmethylated C's in CpG context: 106252080
Total unmethylated C's in CHG context: 136818631
Total unmethylated C's in CHH context: 599720878
Total unmethylated C's in Unknown context: 408

C methylated in CpG context: 29.2%
C methylated in CHG context: 11.0%
C methylated in CHH context: 4.1%
C methylated in Unknown context (CN or CHN): 12.4%

For me, Total methylated C's in CpG context = (43871230/928829076)*100 = 4.72% (in the report, I see 29.2%)

What am I missing??

Cheers,

Amira

[fkrueger]

Hi Felix,

I have four lanes of data for each biological sample. Should I add them together before trimming and Bismark? Or is it better to do the trimming and Bismark run on the individual lane of reads? Thanks.

Regards,
BBM

For me personally merging them before mapping is the preferred way because this enables you to do the deduplication a single sample and it is just more convenient overall. If time is of the essence you could also align them separately and merge them before the deduplication, but that is really a matter of taste (also if you have paired-end sequences and use samtools merge for merging BAM files you need to make sure to use samtools sort -n before trying to deduplicate because samtools sort does not guarantee to keep mates together).

[bbm]

Hi Felix,

I have four lanes of data for each biological sample. Should I add them together before trimming and Bismark? Or is it better to do the trimming and Bismark run on the individual lane of reads? Thanks.

Regards,
BBM

[fkrueger]

Hi Felix,
I'm having a special need. I indexed each alignment in my BAM file as an extra column. Is it feasible to also print this information into the .txt file when I run bismark_methylation_extractor? Basically I want to know which alignment each cytosine is from, and group the cytosines from the same alignment together.

Cheers,
Youyou

Hi Youyou. The methylation extractor output still has the read ID printed in each line, so at this stage it is still possible to tell which C came from which read. If you proceed to the bedGraph stage or beyond this information will be lost unfortunately.

[chxu02]

Hi Felix,
I'm having a special need. I indexed each alignment in my BAM file as an extra column. Is it feasible to also print this information into the .txt file when I run bismark_methylation_extractor? Basically I want to know which alignment each cytosine is from, and group the cytosines from the same alignment together.

Cheers,
Youyou

[bbm]

Hmm, when you type:

/usr/local/apps/bowtie2/bowtie2-build

on the command line you need to see the bowtie2 indexing options. Does that that happen? If not you need to supply the exact path to where the executable is...

I see, let me try

[fkrueger]

Hmm, when you type:

/usr/local/apps/bowtie2/bowtie2-build

on the command line you need to see the bowtie2 indexing options. Does that that happen? If not you need to supply the exact path to where the executable is...

[bbm]

I'm trying to do the genome conversion with this line:

bismark_genome_preparation --bowtie2 --verbose /gpfs_share/hlibyar/hlibyar/genome_files/ --path_to_bowtie /usr/local/apps/bowtie2/



"...
Step II - Genome bisulfite conversions - completed


Bismark Genome Preparation - Step III: Launching the Bowtie 2 indexer
Please be aware that this process can - depending on genome size - take several hours!

Preparing indexing of CT converted genome in /gpfs_share/hlibyar/hlibyar/genome_files/Bisulfite_Genome/CT_conversion/
Parent process: Starting to index C->T converted genome with the following command:

/usr/local/apps/bowtie2/bowtie2-build -f genome_mfa.CT_conversion.fa BS_CT

Can't exec "/usr/local/apps/bowtie2/bowtie2-build": No such file or directory at /usr/local/apps/bismark/v0.14.5/bismark_genome_preparation line 163, <IN> line 3352281.
Preparing indexing of GA converted genome in /gpfs_share/hlibyar/hlibyar/genome_files/Bisulfite_Genome/GA_conversion/
Child process: Starting to index G->A converted genome with the following command:

/usr/local/apps/bowtie2/bowtie2-build -f genome_mfa.GA_conversion.fa BS_GA

(starting in 10 seconds)
Can't exec "/usr/local/apps/bowtie2/bowtie2-build": No such file or directory at /usr/local/apps/bismark/v0.14.5/bismark_genome_preparation line 178, <IN> line 3352281."

Is there sth wrong with my bowtie path?
Thanks.

[VC87]

Hi!Can anyone explain to me the differences between bisulfite-seq, WGBS, RRBS and COBRA library reads?thanks in advance!

[fkrueger]

Right, I believe it is now fixed, the latest version can be downloaded here: https://github.com/FelixKrueger/Bismark/issues/18

[biocomputer]

I'm using 0.14.4:

$ bismark2bedGraph --version


Bismark Methylation Extractor Module -
bismark2bedGraph

Bismark Extractor Version: v0.14.4
Copyright 2010-15 Felix Krueger, Babraham Bioinformatics

https://www.bioinformatics.babraham.ac.uk/projects/bismark/

[fkrueger]

When I try to run bismark2bedGraph from a directory that doesn't directly contain the input file it fails to find the input file, even though I've specified the path to the input file. For example, if I'm in the directory that contains the input file and use this command it works properly (note that I've taken the output from the methylation extractor and split it by chromosome for batch processing but the same thing happens if I use the original unsplit file):



If I move up a directory and run this command it doesn't work:



The programs ends with:

I have just been looking into this and can't reproduce the error. I then went back to the Release Notes and found this one for version 0.14.0:

bismark2bedGraph: Fixed path handling for cases where the input files were given with path information and an output directory had been specified as well

Did you by any chance run an older version than that?

[MagdalenaZ]

Hi,

yep, when I benchmarked that code I get same results if I include the SE or not.

I'm a bit keen to also use SE reads, as I after adapter trimming get improved mapping results, but some 3% of reads are after adapter trimming unpaired. I guess I could chuck them away, but I'll have a go with your suggestion:
to merge them in bismark2bedGraph.

Every little helps ..... :-)

Many thanks for your rapid reply!