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Old 04-09-2014, 10:26 PM   #1
TheSeqGeek
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Default Normalizing by Library Amount

I got my Seq data back and processed with bow tie and htseq and I have my mapped gene counts... in my library I see that this is how much DNA was used for sequencing by the staff

615 ng ---- Sample 1
423 ng ---- Sample 2
426 ng ----- Sample 3


Can I divide the total mapped reads by these numbers to get a kind of weighted normalization?

What would be the danger of doing that?
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Old 04-10-2014, 06:17 AM   #2
crazyhottommy
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Normalized to the total mapped reads would be more reasonable.
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Old 04-10-2014, 06:35 AM   #3
NicoBxl
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use DESeq or edgeR ( in R ) to normalize and check for DE genes
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Old 04-10-2014, 06:38 AM   #4
TheSeqGeek
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Quote:
Originally Posted by NicoBxl View Post
use DESeq or edgeR ( in R ) to normalize and check for DE genes
I use DESeq2 and I print out the normalized values then do a ttest of my own but my pvalues are nothing like in DESeq2.

I use this code to print out normalized values.

normalizedCounts <- t( t(counts(dds)) / sizeFactors(dds) )
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Old 04-10-2014, 06:41 AM   #5
NicoBxl
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why do you use a ttest and not DESeq test (based on negative binomial) ? RNA-Seq data do not follow a normal distribution.

for normalized count, you can use also : normalizedCounts <- counts(dds,normalized=TRUE)
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Old 04-10-2014, 06:44 AM   #6
TheSeqGeek
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Quote:
Originally Posted by NicoBxl View Post
why do you use a ttest and not DESeq test (based on negative binomial) ? RNA-Seq data do not follow a normal distribution.
Well there we go... I guess DESeq2 doesn't perform ttest...

Can you reference some quality sources to get familiar with negative binomial theory
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Old 04-10-2014, 06:49 AM   #7
NicoBxl
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read DESeq paper : http://genomebiology.com/2010/11/10/R106 and also DESeq vignette that is pretty well done.
or maybe edgeR paper also
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