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Forum: Illumina/Solexa 07-06-2018, 12:36 AM
Replies: 2
Views: 451
Posted By fkrueger
You should be able to use Trim Galore for this...

You should be able to use Trim Galore for this which will try to identify the adapter sequence for you. The command for adapter and quality trimming is simply:

trim_galore --paired file1 file2
Forum: Bioinformatics 06-29-2018, 12:27 AM
Replies: 1
Views: 555
Posted By fkrueger
Hi Hedi86, for RRBS the command would be: ...

Hi Hedi86,

for RRBS the command would be:

trim_galore --rrbs --paired r1.fastq.gz r2.fastq.gz

Which version of Trim Galore were you using (the latest one is v0.5.0,...
Forum: Bioinformatics 12-24-2017, 12:09 PM
Replies: 1
Views: 718
Posted By fkrueger
I suggested the option of using one of several...

I suggested the option of using one of several BS-SNP callers (Bis-SNP, MethylExtract or BS-SNPer) via email, so I am hoping one of them will prove useful.
Forum: Bioinformatics 11-28-2017, 05:55 AM
Replies: 7
Views: 956
Posted By fkrueger
yes, that's correct.

yes, that's correct.
Forum: Bioinformatics 11-28-2017, 12:46 AM
Replies: 7
Views: 956
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, 04:26 AM
Replies: 7
Views: 956
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, 01:11 AM
Replies: 641
Views: 147,542
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, 07:05 AM
Replies: 641
Views: 147,542
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, 04:09 AM
Replies: 2
Views: 543
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, 04:39 AM
Replies: 641
Views: 147,542
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, 01:51 AM
Replies: 641
Views: 147,542
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, 01:51 AM
Replies: 3
Views: 784
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, 02:53 AM
Replies: 641
Views: 147,542
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, 12:58 AM
Replies: 641
Views: 147,542
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, 01:43 AM
Replies: 1
Views: 593
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, 05:15 AM
Replies: 10
Views: 1,170
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, 04:42 AM
Replies: 10
Views: 1,170
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, 04:12 AM
Replies: 10
Views: 1,170
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, 11:52 AM
Replies: 10
Views: 1,170
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, 02:15 AM
Replies: 10
Views: 1,170
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, 02:45 PM
Replies: 7
Views: 1,206
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, 05:20 AM
Replies: 7
Views: 1,206
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, 12:33 PM
Replies: 641
Views: 147,542
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, 07:32 AM
Replies: 641
Views: 147,542
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, 01:51 PM
Replies: 641
Views: 147,542
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...
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