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  • Analyzing RNA seq in R

    Hi,
    In my experiment i did 3 replicates for 2 conditions.(in total 6 samples)
    I am interesting in a group of 16 genes that when i checked them in the Genome Browser i found that they have low expression (the number of counts is ~5-20 or even less in general conditions).
    In total i have 32,300 genes in my data.
    i used rsem to find counts to my data.

    The "i" is replicates for the first condition where the i* is the replicates for second condition

    name i i* i i* i i*
    A 92 119 378 201 116 57
    B 2 0 2 1 0 1
    C 20 12 28 28 22 16
    D 5 3 13 0 2 3
    E 0 0 8 4 3 0
    F 1 0 1 0 5 0
    G 1 0 1 0 5 0
    H 1 1 0 0 1 0
    I 341 655 939 470 529 389
    J 341 655 939 470 529 389
    K 9 12 13 9 10 5
    L 1003 1268 2729 1039 1196 929
    M 2 2 16 8 2 4
    N 3 9 20 3 6 5

    As we can see by eyes, there is no a strict rule(at list i didnt found one..)

    When i used DESeq to find differential expression between the 2 condition, i had a table of all the genes of my data.
    At the end, i was looking for significant in those genes , but i didnt find.

    i used rsem as:
    rsem-calculate-expression -p 20 --paired-end R1_001.fastq R2_001.fastq reference out &

    i used DESeq as:
    conditions <- c("1","2","1","2","1","2")
    cds <- newCountDataSet(x,conditions)
    cds <- estimateSizeFactors(cds)
    cds <- estimateDispersions(cds,method="per-condition",sharingMode="maximum",fitType="local")
    res <- nbinomTest(cds,condA="1",condB="2")

    i am wondering if i know that i am interesting in genes with low counts, do i have to do something different in my commands? (both rsem & DESeq)
    i am also know that the different between the condition should be around X 2 in the expression of the genes for most of our genes of interest. (we did some wet tests with pcr )

    I will really appreciate any recommend

    Thanks,
    Pap
    Last edited by papori; 03-04-2012, 01:40 AM.

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