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Forum: Bioinformatics 02-02-2013, 09:49 AM
Replies: 10
Views: 1,857
Posted By billstevens
Hello community! I have 3 conditions and 5...

Hello community!

I have 3 conditions and 5 total replicates. So my DOF is 15 samples - 3 conditions = 12. Therefore I am using the gene-est only for estimating my dispersions.

cds <-...
Forum: Bioinformatics 11-05-2012, 01:33 PM
Replies: 30
Views: 15,306
Posted By billstevens
Hi guys, So I'm wondering about DEseq and...

Hi guys,

So I'm wondering about DEseq and the old and new methods for accounting for variability. I see that the new methods uses the regression line as default variability for the gene, unless...
Forum: Bioinformatics 11-02-2012, 11:30 AM
Replies: 13
Views: 2,635
Posted By billstevens
DESeq questions

Hey guys,

So I just got my new runs. They all look great. I can clearly see the difference between biological replicates and condition. Unfortunately, they don't group ANYWHERE near where my...
Forum: Bioinformatics 11-01-2012, 05:58 PM
Replies: 30
Views: 15,306
Posted By billstevens
Also, as a follow-up DESeq question, can DESeq do...

Also, as a follow-up DESeq question, can DESeq do comparisons between 3 conditions? From the vignette, I can see it can do multi-factor testing with library type and condition. But my library-types...
Forum: Bioinformatics 11-01-2012, 02:10 PM
Replies: 30
Views: 15,306
Posted By billstevens
Hi guys, I'm trying to plotPCA with DESeq, but it...

Hi guys, I'm trying to plotPCA with DESeq, but it says it only has a maximum of 12 colors. Is there a way to get around this? I tried just using arrayQualityMetrics, but that just gave me a PDF...
Forum: Bioinformatics 10-20-2012, 11:30 AM
Replies: 17
Views: 21,534
Posted By billstevens
Hi Simon, I wasn't sure how they meant to do...

Hi Simon, I wasn't sure how they meant to do this, but the point was to essentially disregard some reads to get all of conditions to have roughly the same amount of reads before running DESeq. Have...
Forum: Bioinformatics 10-16-2012, 12:41 PM
Replies: 17
Views: 21,534
Posted By billstevens
I have a question about normalization and...

I have a question about normalization and downscaling:

I have 7 samples with the following number of aligned reads, based on samtools flagstat
Sample 1a: 50M, Sample 1b: 45M, Sample 2a: 95 M,...
Forum: Bioinformatics 10-15-2012, 04:20 PM
Replies: 6
Views: 1,860
Posted By billstevens
Papers with just RNA-Seq data

Hi guys,

So I'm a grad student who really wants to graduate. Truly, that is all I want. I am tired of being here. I badly need a publication to do so.

I have five biological replicates of...
Forum: Bioinformatics 10-15-2012, 08:36 AM
Replies: 16
Views: 5,030
Posted By billstevens
Thirded! Anyone else? Solutions?

Thirded! Anyone else? Solutions?
Forum: Bioinformatics 10-09-2012, 02:48 PM
Replies: 6
Views: 1,204
Posted By billstevens
Also, sorry, does anyone know how to filter more...

Also, sorry, does anyone know how to filter more than one file at a time with Fastq?
Forum: Bioinformatics 10-09-2012, 07:56 AM
Replies: 6
Views: 1,204
Posted By billstevens
bump? Sorry for the simple question. I wasn't...

bump? Sorry for the simple question. I wasn't filtering, but I went to a workshop that said you should always filter/trim, and I just wanted to have one more confirmation.
Forum: Bioinformatics 10-08-2012, 03:03 PM
Replies: 6
Views: 1,204
Posted By billstevens
Sorry, very basic, very quick question. I have...

Sorry, very basic, very quick question. I have very good 100bp PE data (Illumina Phred Scores ~33). That usually holds all the way through from base 1 to 100. However, there are some reads that...
Forum: Bioinformatics 10-06-2012, 10:58 AM
Replies: 8
Views: 2,062
Posted By billstevens
Do you need to upgrade to Tophat 2? I got good...

Do you need to upgrade to Tophat 2? I got good alignment with Tophat 1.4 and Bowtie 1, and I'm wondering if I should upgrade. If it can only do one core at a time, I definitely won't.
Forum: Bioinformatics 09-26-2012, 08:19 AM
Replies: 23
Views: 24,236
Posted By billstevens
Thanks. One very challenging aspect is to go...

Thanks.

One very challenging aspect is to go from quantifying differential RNA to quantifying differential protein. I'm using epithelial cells, and I have a few popular cytokines that have...
Forum: Bioinformatics 09-24-2012, 03:29 PM
Replies: 23
Views: 24,236
Posted By billstevens
So I used SPIA, and I came up with a couple very...

So I used SPIA, and I came up with a couple very interesting pathways. In the pathways, there are some genes that are fold changes of 1.3, 1.5 and some genes that are fold changes over 2. There are...
Forum: Bioinformatics 09-23-2012, 05:53 PM
Replies: 23
Views: 24,236
Posted By billstevens
Thanks Wallysb01, that's what I figured. But...

Thanks Wallysb01, that's what I figured. But here's the question, what is "real power"? I have 4-5 biological replicates, and I will be doing it in triplicate (5 technical triplicates, and I just...
Forum: Bioinformatics 09-23-2012, 03:17 PM
Replies: 7
Views: 1,625
Posted By billstevens
bump? Simon? Wolfgang? Bueller?

bump?

Simon? Wolfgang? Bueller?
Forum: Bioinformatics 09-23-2012, 03:15 PM
Replies: 23
Views: 24,236
Posted By billstevens
dpryan, that's good advice. I want to input my...

dpryan, that's good advice. I want to input my results into SPIA, and a couple of pretty cool pathways pop up, but some of thse are fold changes of 1.3 or so (very highly expressed, which is why...
Forum: Bioinformatics 09-23-2012, 07:58 AM
Replies: 21
Views: 3,755
Posted By billstevens
Yes, I definitely have replicates. If I...

Yes, I definitely have replicates.

If I only kept log2foldchange set at 2, I would lose the majority of my genes, and DESeq becomes much less powerful.

I guess the fundamental question I'm...
Forum: Bioinformatics 09-22-2012, 08:11 PM
Replies: 21
Views: 3,755
Posted By billstevens
Haha, this might have been the best thing I've...

Haha, this might have been the best thing I've seen on SeqAnswers.

There's also DEXSeq, which does splicing, so at least you can have some consistency in your method. Whatever you do, don't...
Forum: Bioinformatics 09-22-2012, 12:21 PM
Replies: 23
Views: 24,236
Posted By billstevens
About qPCR validation, my DESeq run calls many...

About qPCR validation, my DESeq run calls many genes that have a differential expression difference as low as 20%, significantly differentially expressed. While I appreciate the power of DESeq, I...
Forum: Bioinformatics 09-21-2012, 11:31 AM
Replies: 7
Views: 1,625
Posted By billstevens
Sorry, I think I should have asked that question...

Sorry, I think I should have asked that question more explicitly. How can we deal with the bias in counting methods in the gene enrichment?
Forum: Bioinformatics 09-19-2012, 01:10 PM
Replies: 7
Views: 1,625
Posted By billstevens
What I imagine is that it matters to actually...

What I imagine is that it matters to actually call something differentially expressed because if longer genes give off higher counts, then DESeq requires a lower threshold of fold change to call it...
Forum: Bioinformatics 09-18-2012, 06:04 PM
Replies: 7
Views: 1,625
Posted By billstevens
DESeq with SPIA

Hello all,

I've been using SPIA (Signal Pathway Impact analysis), and I think its great. I'm surprised I don't see it up on the forum more often, and I thought I would start this thread, one to...
Forum: Bioinformatics 09-18-2012, 03:50 PM
Replies: 13
Views: 2,635
Posted By billstevens
Wow, that's a very interesting idea I hadn't...

Wow, that's a very interesting idea I hadn't considered. The thing is, like you said WT1 clusters in the wrong area and I harvested these replicates by number together (all the 1s, 2s and 3s). ...
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