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Old 04-14-2011, 02:59 PM   #1
vebaev
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Default miRNA read counts regarding DE

Hi,
I'm using several software to annotate known miRNAs from deep-seq. Regarding differential expression, most of the software point only miRNAs present in all the samples and their fold change.

I was thinking what about for examples if some miRNA are present in my control replicas (2) and have read counts, and in the other 2 samples replicas this miRNA is not present at all?
Does it mean something or in DE analysis should be considered miRNAs presented on some level in all samples?

Thanks!
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Old 04-17-2011, 10:27 PM   #2
Simon Anders
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What kind of software are you talking about? If you want to test for differential expression of miRNA between conditions, use the same tools as used for mRNA, i.e., DESeq, edgeR or BaySeq. Neither of these has any problem if counts are zero for some samples.
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Old 04-18-2011, 04:22 AM   #3
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I mean when I got that some miRNA are zero in one sample and a specific reads in other sample should I look them at all? Is it biologically meaningful when in one sample is zero?
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Old 04-18-2011, 07:04 AM   #4
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what is the number of reads in the other samples ?
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Old 04-19-2011, 03:05 AM   #5
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I have checked and the other reads are quite low, so I'm planning to discard these entries
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Old 04-19-2011, 03:11 AM   #6
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If you use a tool like DESeq, edgeR, BaySeq, you don't need to worry about this at all; the tools will judge themselves whether counts are too low to be significant; and for this, there it makes no difference whether the count is just very low or actually zero
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Old 04-19-2011, 03:17 AM   #7
vebaev
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Yes, I'm exploring for example DESeq, but I have problems creating the tabular input format for my 4 samples (2 controls and 2 conditions).
I do not know that to use to create it, should be tabular with readcounts, but miRNAs corresponding reads should be indentified first....so Im confused...what should I pipe into DESeq?

Can you recommend me a tutorial or documentation, because I found all the things for DESeq with protein coding seqs where are more complex?
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Old 04-19-2011, 03:20 AM   #8
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the input of DESeq is a data.frame ( column : sample, row : miRNA )

ex:
Code:
         Sample1     Sample2         Sample3
miR-1      10              20              15
miR-2      100             30              109
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Old 04-19-2011, 03:22 AM   #9
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Thanks I will try that and post here my progress
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Old 04-19-2011, 03:25 AM   #10
vebaev
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PS
should I worry how to list columns (order) sample1 2 3 , as they are 2 controls and 2 conditions?
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Old 04-19-2011, 03:28 AM   #11
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no

you've to read the DESeq doc : http://www-huber.embl.de/users/anders/DESeq/
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Old 04-19-2011, 03:29 AM   #12
vebaev
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Thanks will do that now
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Old 04-19-2011, 05:01 AM   #13
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as a total number of reads for library it should be considered total number of mapped reads (sum of all mIR counts in column) or total number of reads from sequencing?
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Old 04-27-2011, 12:31 AM   #14
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Quote:
Originally Posted by vebaev View Post
as a total number of reads for library it should be considered total number of mapped reads (sum of all mIR counts in column) or total number of reads from sequencing?
same question
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Old 04-27-2011, 12:48 AM   #15
vebaev
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From what I have read I have end up with this - the total number is probably the total bumber of mapped reads, please correct me if I'm wrong!
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Old 04-27-2011, 12:59 AM   #16
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For DESeq, you don't need this. Just use 'estimateSizeFactors'.
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Old 05-19-2011, 11:52 AM   #17
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Quote:
Originally Posted by NicoBxl View Post
same question
also same question!
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