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Old 04-25-2017, 01:46 AM   #621
pig_raffles
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Excellent, thank you for the very clear explanation
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Old 05-11-2017, 01:39 PM   #622
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Quote:
Originally Posted by Ahra View Post
hello. I am a newbie using bismark. I'm not a bioinformation and also i don't know much about NGS alignment, especially how to running alignment programs. But, i could use bismark thanks to kindly well made bismark user guide book.

i ran bismark and got the report file and sam file. so to get the position of methylC, i ran bismark_methylation_extractor especially used --bedGraph option to see methlylation %.

According to user guide book,

A typical command including the optional bedGraph --counts output could look like this:
bismark_methylation_extractor -s --bedGraph --counts --buffer_size 10G s_1_sequence.txt_bismark.sam


my data is paired-end so i edited little that one. like this:

bismark_methylation_extractor -p --no_overlap --comprehensive --CX
--bedGraph --counts --buffer_size 10G s_1_sequence.txt_bismark.sam


but, i couldn't get additional information about <chromosome> <start position> <end position> <methylation percentage>

my result was like thins

Bismark methylation extractor version v0.7.11
HWI-D00111:9926ADACXX:7:1101:1567:2165_1:N:0:CTTGTA/1 - Vr08 7498192 x
HWI-D00111:9926ADACXX:7:1101:1567:2165_1:N:0:CTTGTA/1 - Vr08 7498173 x
HWI-D00111:9926ADACXX:7:1101:1567:2165_1:N:0:CTTGTA/2 - Vr08 7498096 x
HWI-D00111:9926ADACXX:7:1101:1567:2165_1:N:0:CTTGTA/2 -

how can i get the methylation percentage information and run further bedGraph2Cytosines process!

i am looking forward anyone's reply
I have the same question as yours. Did you solve your problem? Then which command should I use the generate the methylation percentage?
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Old 05-11-2017, 01:51 PM   #623
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A typical command to extract methylation calls and get a coverage file is:

Code:
bismark_methylation_extractor --bedGraph --buffer_size 10G file_bismark.bam
Do you have any problems running this command, and if so can you post details?
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Old 05-12-2017, 07:04 AM   #624
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Quote:
Originally Posted by fkrueger View Post
A typical command to extract methylation calls and get a coverage file is:

Code:
bismark_methylation_extractor --bedGraph --buffer_size 10G file_bismark.bam
Do you have any problems running this command, and if so can you post details?
Hi, Thanks fkrueger! I ran your code. The following is the head what I have from the bedgraph file:

track type=bedGraph
chr11 110189 110190 100
chr11 110211 110212 100
chr11 113464 113465 100
chr11 113508 113509 100
chr11 113509 113510 100
chr11 113524 113525 100
chr11 113525 113526 100
chr11 123420 123421 100
chr11 123449 123450 100
So my question is: Is there all of the probe having 100% methylation percentage? That is a little weird....
By the way, I have another question: If I have paired samples, can I compare the methylation percentage with bismark? Thanks!
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Old 05-12-2017, 07:32 AM   #625
fkrueger
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You should probably look at the coverage file because this will also tell you how many counts you saw methylated or unmethylated. If you see 100% then I would suspect you saw only a single call for this position, which in this case happened to be methylated.

To compare different samples we tend to use SeqMonk, a lightweight but fast and powerful genome browser and analysis tool. Here are some presentations to about what methylation analysis in SeqMonk looks like. https://www.bioinformatics.babraham....ing.html#bsseq

Best, Felix
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Old 05-12-2017, 10:03 AM   #626
twotwo
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Quote:
Originally Posted by fkrueger View Post
You should probably look at the coverage file because this will also tell you how many counts you saw methylated or unmethylated. If you see 100% then I would suspect you saw only a single call for this position, which in this case happened to be methylated.

To compare different samples we tend to use SeqMonk, a lightweight but fast and powerful genome browser and analysis tool. Here are some presentations to about what methylation analysis in SeqMonk looks like. https://www.bioinformatics.babraham....ing.html#bsseq

Best, Felix
Hi, Felix,
Thanks for your quick answer. Here is the head of the coverage file. Does that mean that I should merge the data (get all the information for one SNP) and get the methylation percentage?


chr11 110190 110190 100 1 0
chr11 110212 110212 100 2 0
chr11 113465 113465 100 1 0
chr11 113509 113509 100 4 0
chr11 113510 113510 100 1 0
chr11 113525 113525 100 2 0
chr11 113526 113526 100 1 0
chr11 123421 123421 100 1 0
chr11 123450 123450 100 1 0
chr11 123849 123849 100 5 0
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Old 05-12-2017, 12:33 PM   #627
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I am not quite sure if I understand your question here to be honest.

Code:
chr11 113509 113509 100 4 0
This example line means that for the position 113509 on chromosome 11 you had 4 methylation calls in total that were methylated (in the entire dataset), and 0 calls that were unmethylated. This translates into a 100% methylation percentage at this position (column 4). Also, the positions here are simply cytosines in the genome but not SNP.

Just to remind you this this the format:

Code:
The coverage output looks like this (tab-delimited, 1-based genomic coords):
============================================================================================================================================

<chromosome>  <start position>  <end position>  <methylation percentage>  <count methylated>  <count non-methylated>
I hope this helps.
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Old 05-12-2017, 12:43 PM   #628
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Thank you very much!
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