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Forum: Epigenetics 08-13-2020, 11:31 PM
Replies: 37
Views: 36,399
Posted By fkrueger
Hi Anton, The methylation rate in Unknown...

Hi Anton,

The methylation rate in Unknown context is really only for your information, but as these calls cannot be assigned a specific context they are just discarded in the methylation...
Forum: Epigenetics 07-13-2020, 09:00 AM
Replies: 37
Views: 36,399
Posted By fkrueger
Hi Iraia, I don't think you should start...

Hi Iraia,

I don't think you should start aligning the entire file until you know exactly what is the best way to do it. You are absolutely welcome to send me a few reads.

If you are on Linux or...
Forum: Epigenetics 07-06-2020, 08:29 AM
Replies: 37
Views: 36,399
Posted By fkrueger
Hi there, The mm10 and GRCm38 genomes should...

Hi there,

The mm10 and GRCm38 genomes should be exactly the same sequence, there are only some minor differences (e.g. chromosomes are called chr1, chr2 and not 1, 2 etc, chrM instead of MT and so...
Forum: Bioinformatics 01-31-2020, 04:00 AM
Replies: 4
Views: 741
Posted By fkrueger
That sounds like you are working with paired-end...

That sounds like you are working with paired-end data. In that case the Read 1 and Read 2 will be ends of the fragment you are sequencing, and they may well be further apart. In this case it doesn't...
Forum: Bioinformatics 01-31-2020, 12:54 AM
Replies: 4
Views: 741
Posted By fkrueger
Hi Hedi, Every alignment that is reported by...

Hi Hedi,

Every alignment that is reported by Bismark are unique hits (ambiguosly aligning reads are discarded, and not-aligning reads - well - don't align at all). So you can simply look at the...
Forum: Epigenetics 09-24-2019, 02:51 AM
Replies: 1
Views: 5,684
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: 1,167
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: 2,506
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: 2,506
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: 2,506
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: 2,506
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: 46,499
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: 46,499
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: 5,629
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: 735
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: 187,882
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: 187,882
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: 2,506
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: 187,882
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: 1,430
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: 1,325
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: 1,325
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: 1,325
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: 2,506
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: 2,506
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
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