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Forum: Epigenetics 09-24-2019, 02:51 AM
Replies: 1
Views: 550
Posted By fkrueger
Dear Ali, Thanks for your kind words, and...

Dear Ali,

Thanks for your kind words, and for your thoughtful questions. As you will see below, I will probably not be able to give you a satisfactory answer to all questions you raised, but I...
Forum: Illumina/Solexa 09-13-2019, 05:52 AM
Replies: 1
Views: 317
Posted By fkrueger
In my opinion long single-end reads are a very...

In my opinion long single-end reads are a very good alternative to overlapping, redundant PE reads. Read processing should also be generally faster.

Just make sure that the reads are properly...
Forum: Bioinformatics 06-02-2019, 08:12 AM
Replies: 13
Views: 1,891
Posted By fkrueger
Don't worry, Bismark is taking care of all that...

Don't worry, Bismark is taking care of all that for you. Just run the alignments in paired-end mode, and use the BAM file further downstream. There is no obvious reason why you should be running...
Forum: Bioinformatics 05-29-2019, 12:43 AM
Replies: 13
Views: 1,891
Posted By fkrueger
I am afraid the situation doesn't appear to have...

I am afraid the situation doesn't appear to have changed much:

- the data you sent over appears to have been adapter trimmed already, so I can't really say much about what the really raw data...
Forum: Bioinformatics 05-28-2019, 04:00 AM
Replies: 13
Views: 1,891
Posted By fkrueger
Thanks for those. I'll be out this afternoon but...

Thanks for those. I'll be out this afternoon but will try to get back to you tonight or tomorrow.
Forum: Bioinformatics 05-28-2019, 02:02 AM
Replies: 13
Views: 1,891
Posted By fkrueger
If you run paired-end sequencing data on it's own...

If you run paired-end sequencing data on it's own as single-end reads, you need to specify --pbat for Read 2. I am sure this will bring the mapping efficiency up somewhat.

If you wanted you could...
Forum: Bioinformatics 01-18-2019, 01:24 AM
Replies: 136
Views: 41,272
Posted By fkrueger
Trim Galore tries to identify read-through...

Trim Galore tries to identify read-through adapter contamination, which is the kind of contaminant that will prevent your sequences from aligning at all, or might even cause mis-alignments. For the...
Forum: Bioinformatics 01-17-2019, 12:47 AM
Replies: 136
Views: 41,272
Posted By fkrueger
The quality trimming is performed by a sliding...

The quality trimming is performed by a sliding window approach across the read like the one that is used by BWA. Copied below is the text from the Cutadapt --help:

-q 3'CUTOFF ...
Forum: Bioinformatics 12-18-2018, 07:35 AM
Replies: 3
Views: 3,013
Posted By fkrueger
Yes, this is normal. Paired-end BAM files have...

Yes, this is normal. Paired-end BAM files have Read 1 and Read 2 on consecutive lines, so there is only one file per read pair.
Forum: Bioinformatics 11-20-2018, 01:49 AM
Replies: 1
Views: 495
Posted By fkrueger
Cutadapt can deal with your data, this is taken...

Cutadapt can deal with your data, this is taken from the Cutadapt help:

Colorspace options:
-c, --colorspace Enable colorspace mode
-d, --double-encode
...
Forum: Bioinformatics 10-04-2018, 07:16 AM
Replies: 649
Views: 170,644
Posted By fkrueger
Hi Amira, The reason for this is the overlap...

Hi Amira,

The reason for this is the overlap detection, and -removal, in paired-end files. What this simply does is to detect when R1 and R2 start overlapping, and as soon as this is the case the...
Forum: Bioinformatics 09-05-2018, 03:06 AM
Replies: 649
Views: 170,644
Posted By fkrueger
Just generally, in order to get reads for both...

Just generally, in order to get reads for both the forward and reverse strands you need to have a fairly good sequencing depth and have a library with a good complexity. While your sample 1 has...
Forum: Bioinformatics 09-04-2018, 06:59 AM
Replies: 13
Views: 1,891
Posted By fkrueger
Sorry for the late reply but I was travelling....

Sorry for the late reply but I was travelling. And no I don't think a second trim is necessary, the 20bp is an arbitrary cutoff anyway because sequences shorter than that will typically not map...
Forum: Bioinformatics 09-04-2018, 02:19 AM
Replies: 649
Views: 170,644
Posted By fkrueger
Hi Daisy, Were you expecting to see...

Hi Daisy,

Were you expecting to see imbalances in your library, i.e. was it some kind of target enrichment or (PCR) amplification library? Depending on what you expect from the library design you...
Forum: Bioinformatics 08-14-2018, 03:03 AM
Replies: 2
Views: 1,070
Posted By fkrueger
Hi Hedi, No, I'm afraid you canít say...

Hi Hedi,



No, I'm afraid you canít say that. The numbers reported are the overall numbers of methylation calls performed for the entire run, and have nothing to do with the number of genomic...
Forum: Bioinformatics 07-30-2018, 03:07 AM
Replies: 6
Views: 1,003
Posted By fkrueger
Not quite. A 0.04 fold-coverage means that - on...

Not quite. A 0.04 fold-coverage means that - on average - each position in the genome was covered 0.04 times. A 4-fold would be, well, 4.

This value is obviously not so meaningful if you used only...
Forum: Bioinformatics 07-30-2018, 02:13 AM
Replies: 6
Views: 1,003
Posted By fkrueger
It depends a little on which kind of statistics...

It depends a little on which kind of statistics you are interested in. If you really just want to get a value for a fold-coverage of your experiment you should probably import the (deduplicated)...
Forum: Bioinformatics 07-27-2018, 01:40 AM
Replies: 6
Views: 1,003
Posted By fkrueger
Bismark itself is merely the alignment tool, and...

Bismark itself is merely the alignment tool, and as such does not do any further analysis. I am sure there are several different options to choose from to assess sequence coverage, I can only...
Forum: Bioinformatics 07-26-2018, 03:07 AM
Replies: 13
Views: 1,891
Posted By fkrueger
Hi Hedi86, Thanks for providing the data. I...

Hi Hedi86,

Thanks for providing the data. I have now run a few preliminary tests, here is what I found (in no particular order):

- From the sequence composition plot, the data does not look...
Forum: Bioinformatics 07-25-2018, 06:06 AM
Replies: 13
Views: 1,891
Posted By fkrueger
Hi Hedi, Could you email...

Hi Hedi,

Could you email (felix.krueger@babraham.ac.uk) some unprocessed reads, e.g. 200000, to me in .gz format? I could then take a look and report back to you with what I find.

Cheers, Felix
Forum: Illumina/Solexa 07-06-2018, 12:36 AM
Replies: 2
Views: 1,329
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: 3
Views: 3,013
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: 1,219
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: 1,684
Posted By fkrueger
yes, that's correct.

yes, that's correct.
Forum: Bioinformatics 11-28-2017, 12:46 AM
Replies: 7
Views: 1,684
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*...
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