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Forum: Bioinformatics 11-28-2017, 06:55 AM
Replies: 7
Views: 616
Posted By fkrueger
yes, that's correct.

yes, that's correct.
Forum: Bioinformatics 11-28-2017, 01:46 AM
Replies: 7
Views: 616
Posted By fkrueger
When you have both paired-end (PE) and single-end...

When you have both paired-end (PE) and single-end (SE) alignments I would methylation extract the files separately (the methylation extractor should auto-detect what to do), and then use the CpG*...
Forum: Bioinformatics 11-22-2017, 05:26 AM
Replies: 7
Views: 616
Posted By fkrueger
Hi Tsutsui, In a case like yours Read 1...

Hi Tsutsui,

In a case like yours Read 1 seems to be absolutely fine, and your library is directional, so that looks all fine. If I had to guess what the reason for the low mapping efficiency in PE...
Forum: Bioinformatics 10-22-2017, 02:11 AM
Replies: 641
Views: 137,281
Posted By fkrueger
Hi Alan, Thanks for making the files...

Hi Alan,

Thanks for making the files available. I have just tried to run your files with the following command line:

bismark2bedGraph --dir output_dir -o test --scaffold *G10G06*

And it...
Forum: Bioinformatics 10-20-2017, 08:05 AM
Replies: 641
Views: 137,281
Posted By fkrueger
Hi there, Could you please update to the...

Hi there,

Could you please update to the latest version (0.19.0; https://github.com/FelixKrueger/Bismark/releases) to see if the problem is still there? If you are still seeing the same problem...
Forum: Bioinformatics 10-20-2017, 05:09 AM
Replies: 2
Views: 307
Posted By fkrueger
Hi Heidi86, I think the answer to your...

Hi Heidi86,

I think the answer to your question is fairly simple: Bismark coverage files or the CpG methylation call files contain methylation data (as single-base calls), while SAM, BAM or sorted...
Forum: Bioinformatics 10-04-2017, 05:39 AM
Replies: 641
Views: 137,281
Posted By fkrueger
Hi Amira, Your understanding is now spot-on!...

Hi Amira,

Your understanding is now spot-on! For the last point: For paired-end reads we always use the sum of the alignment scores (AS) and sum of the next best alignment scores (XS) of both...
Forum: Bioinformatics 10-03-2017, 02:51 AM
Replies: 641
Views: 137,281
Posted By fkrueger
Hi Amira, I think that reads that produce...

Hi Amira,

I think that reads that produce more that one valid alignment should be reported based on Bowtie2 default mode (not all alignments, only one). How does having the same number of lowest...
Forum: Sample Prep / Library Generation 08-10-2017, 02:51 AM
Replies: 3
Views: 648
Posted By fkrueger
Hi aleichter, I'd be happy to take at one...

Hi aleichter,

I'd be happy to take at one of the samples to see if I can find any of the typical suspects in the data. Emailing of a subset of say 500K sequences (gzipped) is typically sufficient...
Forum: Bioinformatics 08-08-2017, 03:53 AM
Replies: 641
Views: 137,281
Posted By fkrueger
We tend to use SeqMonk for this purpose...

We tend to use SeqMonk for this purpose (https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/). It is a very fast and powerful genome viewer and quantitation tool. Some examples can be found...
Forum: Bioinformatics 07-28-2017, 01:58 AM
Replies: 641
Views: 137,281
Posted By fkrueger
Erm, what would you like to compare exactly?...

Erm, what would you like to compare exactly? SeqMonk can certainly do a number of things, I suggest you follow the guidelines and practical of this methylation analysis course:...
Forum: Bioinformatics 06-29-2017, 02:43 AM
Replies: 1
Views: 394
Posted By fkrueger
Hi Juulluu, Just briefly before answering...

Hi Juulluu,

Just briefly before answering your question, have you trimmed off 7-8bp from the 5 end of reads as is recommended for TruSeq libraries? If not I would recommend doing so, please see...
Forum: Bioinformatics 05-26-2017, 06:15 AM
Replies: 10
Views: 702
Posted By fkrueger
I couldn't tell why this would be so. In my first...

I couldn't tell why this would be so. In my first message yesterday I included the [ MAIL ] tags, maybe this could be seen as malicious and/or spam and thus flag the institute IP as a potential...
Forum: Bioinformatics 05-26-2017, 05:42 AM
Replies: 10
Views: 702
Posted By fkrueger
Excellent, sorry for being so ranty! :P I...

Excellent, sorry for being so ranty! :P

I tried three different computers (PC/Mac) on site yesterday, tried posting from my phone (on Eduroam but on site as well) but then had to wait until I was...
Forum: Bioinformatics 05-26-2017, 05:12 AM
Replies: 10
Views: 702
Posted By fkrueger
I just replied this via email: Hi David, ...

I just replied this via email:

Hi David,

Thanks for the sequences and the other update on SeqAnswers.

I had a look at your sequencing files, and came to the same conclusions. Both files...
Forum: Bioinformatics 05-25-2017, 12:52 PM
Replies: 10
Views: 702
Posted By fkrueger
Sorry for the slow reply, it seems that I am not...

Sorry for the slow reply, it seems that I am not allowed to post anymore when I am at work, trying from home now...

i again,

this is where it is getting confusing, I just had to draw this out...
Forum: Bioinformatics 05-25-2017, 03:15 AM
Replies: 10
Views: 702
Posted By fkrueger
There are a couple of things that could affect...

There are a couple of things that could affect the mapping efficiency, I'll list a few here:

- depending on the amplification strategy you might have to use --non_directional for mapping. This...
Forum: General 05-22-2017, 03:45 PM
Replies: 7
Views: 799
Posted By fkrueger
The kind of adapter contamination you want to get...

The kind of adapter contamination you want to get rid off is the read-through kind, where you get a piece of fragment that then continues to read into the adapter. These are all taken care of by Trim...
Forum: General 05-22-2017, 06:20 AM
Replies: 7
Views: 799
Posted By fkrueger
In the vast majority of cases when people want to...

In the vast majority of cases when people want to remove multiple different adapter it turns out that they do not actually want to do that. If you ran standard sequencing (e.g. TruSeq, Sanger iTag...
Forum: Bioinformatics 05-12-2017, 01:33 PM
Replies: 641
Views: 137,281
Posted By fkrueger
I am not quite sure if I understand your question...

I am not quite sure if I understand your question here to be honest.


chr11 113509 113509 100 4 0

This example line means that for the position 113509 on chromosome 11 you had 4 methylation...
Forum: Bioinformatics 05-12-2017, 08:32 AM
Replies: 641
Views: 137,281
Posted By fkrueger
You should probably look at the coverage file...

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...
Forum: Bioinformatics 05-11-2017, 02:51 PM
Replies: 641
Views: 137,281
Posted By fkrueger
A typical command to extract methylation calls...

A typical command to extract methylation calls and get a coverage file is:

bismark_methylation_extractor --bedGraph --buffer_size 10G file_bismark.bam

Do you have any problems running this...
Forum: Bioinformatics 04-24-2017, 08:36 AM
Replies: 641
Views: 137,281
Posted By fkrueger
The score min function is linear (L), the first...

The score min function is linear (L), the first number is offset (which will be added to the final score which is determined by the number of mismatches, Ns, or insertions and deletions in the read),...
Forum: Bioinformatics 04-18-2017, 02:04 AM
Replies: 132
Views: 30,560
Posted By fkrueger
Very short reads generally don't tend to align...

Very short reads generally don't tend to align uniquely in bisulfite-seq mapping because the three letter alignment allows more ambiguous alignments. In that sense the shortness of reads sorts itself...
Forum: Bioinformatics 04-10-2017, 02:20 PM
Replies: 641
Views: 137,281
Posted By fkrueger
The alignment line is truncated, the methylation...

The alignment line is truncated, the methylation call is not quite long enough and the read does not contain information about the read or genome conversion which is required by the methylation...
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