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Forum: Epigenetics 06-16-2014, 07:53 PM
Replies: 1
Views: 3,398
Posted By EpiBrass
I often see this too in my libraries. I'm...

I often see this too in my libraries. I'm preparing from Epitect bisulphite kit and library prep with Epicentre EpiGnome kit. What I do is just headcrop the first 16 bases using trimmomatic. Have you...
Forum: Epigenetics 06-16-2014, 07:18 PM
Replies: 15
Views: 8,536
Posted By EpiBrass
Hi BFM, I used Trimmomatic to trim for...

Hi BFM,

I used Trimmomatic to trim for adapters and quality (collect orphaned reads). Imported this into CLC Genomics server to remove chloroplast aligning reads and duplicate reads (again collect...
Forum: Bioinformatics 01-27-2014, 08:55 PM
Replies: 1
Views: 1,020
Posted By EpiBrass
Count C versus read length

Hi,

I've had a good look through the bioinformatics thread but as far as I can see nobody has asked this question yet. I am using bisulphite sequencing in plants (CpG, CHG & CHH contexts).

I...
Forum: Bioinformatics 12-23-2013, 04:00 PM
Replies: 18
Views: 2,265
Posted By EpiBrass
What is your current quality cut off? I wouldn't...

What is your current quality cut off? I wouldn't go below Phred20.

I'm going to assume your "13/15/20 millions" are different samples which can't be pooled?
Forum: Bioinformatics 12-23-2013, 03:56 PM
Replies: 18
Views: 2,265
Posted By EpiBrass
It depends on what genome you're using, how long...

It depends on what genome you're using, how long your reads are and whether they're paired-end or single-end and how even the coverage is. If you're dealing with say a wheat genome (17 GB) then I'd...
Forum: Epigenetics 12-10-2013, 02:45 PM
Replies: 15
Views: 8,536
Posted By EpiBrass
Hi, Just to let you all know, I managed to...

Hi,

Just to let you all know, I managed to sort out all the trimming issues (thanks for the advice!). I've attached a couple of images so you can see the difference it has made since the original...
Forum: Epigenetics 11-26-2013, 03:58 PM
Replies: 15
Views: 8,536
Posted By EpiBrass
Thanks for the heads up on FastQC only doing a...

Thanks for the heads up on FastQC only doing a subset, I had no idea. As far as I understand CLC uses the whole thing... although I could be wrong. I'll give trim-galore a go and see what the...
Forum: Epigenetics 11-25-2013, 05:37 PM
Replies: 15
Views: 8,536
Posted By EpiBrass
After doing a FastQC with the exported data it...

After doing a FastQC with the exported data it appears that something is going wrong during the QC in CLC. The Qscores start dropping down below 15 within the first 20 bp... This is really strange...
Forum: Epigenetics 11-25-2013, 01:44 PM
Replies: 14
Views: 7,292
Posted By EpiBrass
Hi Evelien, The combination was quite...

Hi Evelien,

The combination was quite straight forward. My input DNA was from plant leaf tissue freshly extracted using a Qiagen DNeasy kit (final elution in DNase/RNase free ultra pure water)....
Forum: Epigenetics 11-20-2013, 07:06 PM
Replies: 15
Views: 8,536
Posted By EpiBrass
I do expect a lower CG % in a whole genome...

I do expect a lower CG % in a whole genome bisulphite library, although much greater than ~1% as there are large genomic regions which are hypermethylated in plants (e.g. transposable elements).
...
Forum: Epigenetics 11-20-2013, 01:04 PM
Replies: 15
Views: 8,536
Posted By EpiBrass
QC

Thanks for the reply Devon.

I have attached the three images I was referring to in my original post. They are based on 16.5 million paired end reads from a single run, post trim for quality...
Forum: Epigenetics 11-19-2013, 09:03 PM
Replies: 15
Views: 8,536
Posted By EpiBrass
MiSeq & Bisulphite Sequencing

Hi,

Is there anyone else out there who are using the MiSeq (or HiSeq) platform for performing paired end whole genome bisulphite (aka bisulfite) sequencing? I'd like something to compare my QC...
Forum: Epigenetics 11-07-2013, 03:49 PM
Replies: 5
Views: 2,188
Posted By EpiBrass
Smile Thanks for that. Definitely a helpful...

Thanks for that. Definitely a helpful clarification! I think what I'll do is a couple of technical reps (a) as well as assess biological variation (c) from a couple of individuals.
Forum: Epigenetics 11-07-2013, 01:26 PM
Replies: 5
Views: 2,188
Posted By EpiBrass
The aim of this is to develop a standard...

The aim of this is to develop a standard methylation profile with which I can later use as a reference to compare back to for methylation studies using different tissues at different time points...
Forum: Epigenetics 11-06-2013, 02:43 PM
Replies: 14
Views: 7,292
Posted By EpiBrass
Hi, I have recently used a mix of kits...

Hi,

I have recently used a mix of kits successfully: Qiagen EpiTect Bisulphite Kit followed by EpiGnome Methyl-Seq kit and then sequenced on MiSeq. All very easy and straightforward.

Hope that...
Forum: Epigenetics 11-06-2013, 02:33 PM
Replies: 5
Views: 2,188
Posted By EpiBrass
Methylation Study

Hi,

I'm a PhD student and currently working on building up a Methylome of a plant species. I have a plant line which has been inbred for 9 generations to ensure homozygosity. I would like to know...
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