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Forum: RNA Sequencing 07-25-2014, 05:02 AM
Replies: 3
Views: 2,607
Posted By Alex234
Hi vas72985, I have tried to get around this...

Hi vas72985,

I have tried to get around this problem by only counting uniquely mapping
reads (with no mismatches) that map to intergenic repetitive elements,
although this probably is excluding...
Forum: RNA Sequencing 05-02-2014, 11:09 AM
Replies: 0
Views: 1,544
Posted By Alex234
Strange edgeR BCV plots - any ideas?

Hello,

To measure expression of repetitive elements in my samples I mapped my reads to a .fa file containing one copy of each type of repetitive element, then ran the HT-Seq-edgeR pipeline on them...
Forum: RNA Sequencing 12-10-2013, 12:35 PM
Replies: 3
Views: 2,557
Posted By Alex234
Thanks dpryan - when you say 'large fold-change',...

Thanks dpryan - when you say 'large fold-change', what sort of magnitude do you mean?

My data has a high degree of variability, I think because some of it comes from different strains, and...
Forum: RNA Sequencing 12-10-2013, 10:02 AM
Replies: 3
Views: 2,557
Posted By Alex234
PCA / MDS analysis useful if only small number of genes (e.g. 1-20) diff. expressed?

All in the title really - if a condition only resulted in a change of expression of only a small number of genes (e.g. 1-10), would you expect to see that on a PCA / MDS plot?


Thanks

Alex
Forum: Bioinformatics 11-29-2013, 09:43 AM
Replies: 12
Views: 10,240
Posted By Alex234
Hi, I don't suppose you could send me a link...

Hi, I don't suppose you could send me a link where I could download the public older versions of Repbase?

Thanks

Alex
Forum: RNA Sequencing 11-28-2013, 04:44 AM
Replies: 0
Views: 1,548
Posted By Alex234
Repeat masker - how to use it for estimating repeat expression levels?

Hello,

I have read about people using repeat masker files to actually look at expression of repeat elements (e.g. in RNA-Seq analysis), but could anyone briefly tell me how to do this? Or with...
Forum: RNA Sequencing 11-27-2013, 09:22 AM
Replies: 3
Views: 2,607
Posted By Alex234
Measuring expression of repetitive elements?

Hello, I would simply like to asses the expression levels of all types of repetitive elements in my RNA-Seq samples.

I assume that these are not being aligned in the first place by TopHat2?
...
Forum: RNA Sequencing 09-03-2013, 02:14 AM
Replies: 5
Views: 2,490
Posted By Alex234
Thanks dpryan - which program are these...

Thanks dpryan - which program are these repeatmasker tracks in?
Forum: RNA Sequencing 08-30-2013, 03:17 AM
Replies: 5
Views: 2,490
Posted By Alex234
Hi, (a) Yes both conditions gave equal...

Hi,

(a) Yes both conditions gave equal quality and quantity of reads, but I'm not sure how to check the quality of the reads - HTSeq converted them to 'counts' (1 count = 1 read?)

(b) Thanks,...
Forum: RNA Sequencing 08-30-2013, 03:11 AM
Replies: 7
Views: 2,374
Posted By Alex234
Thanks for the advice, do you think it would be...

Thanks for the advice, do you think it would be better to exclude the Black6 samples from the experiment and just focus on comparing KO and WT samples of the same strain?

I.e. would the...
Forum: RNA Sequencing 08-30-2013, 02:28 AM
Replies: 1
Views: 2,362
Posted By Alex234
Limited number of independent clones - how many 'independent' biological replicates?

Hello, I am comparing transcription in KO and WT ES cell lines and am basically worried about pseudoreplication.

If a single cell line is split into two, and then they undergo many passages...
Forum: RNA Sequencing 08-30-2013, 02:22 AM
Replies: 5
Views: 2,490
Posted By Alex234
Decreased transcription of short intergenic patches - a common artefact?

Hello, I've compared transcription between a WT and KO ES cell line by RNA-Seq using DESeq and a GTF file assembled from cufflinks and have found a decrease in the transcription of many short...
Forum: Bioinformatics 08-27-2013, 05:35 AM
Replies: 8
Views: 7,231
Posted By Alex234
Thanks, that did remove some, but not all, of the...

Thanks, that did remove some, but not all, of the error lines. And couldn't these be important sequences that we are grepping?

Alex
Forum: RNA Sequencing 08-23-2013, 02:26 AM
Replies: 0
Views: 1,278
Posted By Alex234
Changes in Retroelement expression with TopHat2-HTSeq-DESeq workflow?

Hi, was just wondering whether TopHat-HTSeq-DESeq is an appropriate workflow for finding changes in retroelements / other repetitive elements in a mouse KO vs WT cell line?

Are repetitive...
Forum: RNA Sequencing 08-22-2013, 04:36 AM
Replies: 11
Views: 4,021
Posted By Alex234
I thought it was part of DESeq - dpryan...

I thought it was part of DESeq - dpryan introduced it to me:
Forum: RNA Sequencing 08-22-2013, 04:31 AM
Replies: 11
Views: 4,021
Posted By Alex234
Thanks - if I re-load DeSEQ 1 from bioconductor,...

Thanks - if I re-load DeSEQ 1 from bioconductor, will it update the copy on my system?

Is there any advantage of plotMA over plotDE anyway?


Thanks

Alex
Forum: RNA Sequencing 08-21-2013, 02:28 AM
Replies: 11
Views: 4,021
Posted By Alex234
Great, both worked - re: the vsd data (the...

Great, both worked - re: the vsd data (the numbers are counts?): can/should this be used in the same way as the original counts data to look at DEGs (since the problem of 0 counts seems to have been...
Forum: RNA Sequencing 08-21-2013, 01:08 AM
Replies: 11
Views: 4,021
Posted By Alex234
Hi, got a related problem now where the...

Hi, got a related problem now where the scatterplot of ordinary vs moderated log ratios in DESeq seems to be wrong - the points seem to be upside down (picture attached and lines of code below)
...
Forum: RNA Sequencing 08-20-2013, 07:36 AM
Replies: 11
Views: 4,021
Posted By Alex234
Thanks, I'm still not sure what was wrong with...

Thanks, I'm still not sure what was wrong with plotMA, but the plotDE line you gave me works fine!


Thanks

Alex
Forum: RNA Sequencing 08-20-2013, 06:15 AM
Replies: 11
Views: 4,021
Posted By Alex234
Contradictory log-fold-change figures from DESeq?

Hello, I have made a couple of figures in DESeq, the first should plot genes with adjusted p-value <0.05 in red with >0.05 genes in grey, and was made with:

plotMA(res,
+ col =...
Forum: RNA Sequencing 08-19-2013, 12:31 PM
Replies: 7
Views: 2,374
Posted By Alex234
Thanks, that was really helpful. My concern now...

Thanks, that was really helpful. My concern now is that in my second PCA (the one with four points), the two ko points seem to be very far apart, with no genetic or strain differences - is this...
Forum: RNA Sequencing 08-19-2013, 10:39 AM
Replies: 7
Views: 2,374
Posted By Alex234
Thanks for the advice - from the sound of it we...

Thanks for the advice - from the sound of it we cannot know for sure what the principal component is, only how large the effect of the first and second ones are? What do you think the other pc could...
Forum: RNA Sequencing 08-19-2013, 09:35 AM
Replies: 7
Views: 2,374
Posted By Alex234
Mouse ES cell strain differences and PC1 / PC2 figure from DESeq

Hi, I'm looking at transcription in WT and K/O ES cells of a particular gene - I have three biological replicates of each genotype - two of each are from 129 mouse ES cells and the other one of each...
Forum: RNA Sequencing 08-16-2013, 04:04 AM
Replies: 2
Views: 1,373
Posted By Alex234
Thanks, I'll give it a go!

Thanks, I'll give it a go!
Forum: RNA Sequencing 08-15-2013, 09:55 AM
Replies: 2
Views: 1,373
Posted By Alex234
Analysing RNA-Seq from Scratch?

Hello, I am trying to find DE genes under two different conditions (I have three replicates for each). So far I have followed the TopHat->Cufflinks->Cuffdiff workflow, but the found some of the genes...
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