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Michal Lulu ▴ 100
@michal-lulu-5533
Last seen 9.7 years ago
Hi, At first I'd like to explain that my RNAseq experiments involve RNAi depletions of RNA decay factors, so one should expect to see more upregulations. The libraries are ribodepleted, paired end and stranded. After mapping with tophat I use HTSeq/DESeq combo to discover DE genes (among tophat genes.gtf) Using raw counts I run DESeq on default settings and I get certain numer of signif. DE genes, approx. 1:1 up- to down-regulated. Everything seems fine though my MA plots are a bit skewed (example attached), there is a clear slope suggesting that more upregulation of genes of lower expression. Should I worry about this? I also tried to compare DESeq normalization with normalization to spike-ins present in the libraries, but the size factors assigned by DESeq seems much more accurate; although it's unclear why ? https://www.dropbox.com/s/lkq9clcc7nu8qma/analysis.pdf Finally, I turned to normalization that is not recommended by authors but some people do this: http://jura.wi.mit.edu/bio/education/hot_topics/rnaseq/ *rnaseqde_dec2011*.*pdf* Strangely, when I introduce pseudocounts, which in principal should not affect analyzes that much, they actually do. New upregulated hits appear and most downregulations disappear, importantly most of the upregulated enriched already existing clusters in GO. This suggests they may be real. Please note this is my second RNAseq analysis, so I'm really fresh and I would appreciate a lay explanation :) Cheers, Michael [[alternative HTML version deleted]]
RNASeq Normalization GO DESeq RNASeq Normalization GO DESeq • 1.2k views
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wang peter ★ 2.0k
@wang-peter-4647
Last seen 9.7 years ago
did u removed the rRNA reads? try it shan
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yes after mapping i counted reades by htseq against tophat genes.gtf which does not contain rRNA genes On Fri, Oct 5, 2012 at 3:03 AM, wang peter <wng.peter@gmail.com> wrote: > did u removed the rRNA reads? try it > shan > [[alternative HTML version deleted]]
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did u check the read quality? and clean the adapter sequences? shan
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yes and yes does it look really bad in your opinion? I also compared results from with mapping of forward read only, same trend the only thing I can come up with is difference in the library size, as these replicates have the largest size factors, 1.6 and 1.4 i just tried a local fit when estimating dispresion and looks a bit better, but still not perfect On Fri, Oct 5, 2012 at 8:16 PM, wang peter <wng.peter@gmail.com> wrote: > did u check the read quality? > and clean the adapter sequences? > shan > [[alternative HTML version deleted]]
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