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  • DESeq question

    Hi Simon (and others),

    Quick question about DESeq. So far it looks like a nice approach! But it was giving me some somewhat strange (although possible reasonable) numbers...so I wanted to check....

    Sometimes the values estimated for baseMeanA and baseMeanB differ in the direction and the extent of change from the original raw counts. Is this expected? These baseMeans are meant to be 'corrected' estimates of read counts for the conditions in question? If I am getting such differences between the raw read counts and the baseMeans, is this simply a reflection of the strength of the dataset (I am running without biological reps). If so, thats fine (I think). But just making sure all is well..

    For example...
    Code:
    Counts_Novel	Counts_Familiar	DESeq-id	DESeq-baseMean	DESeq-baseMeanA	DESeq-baseMeanB	DESeq-foldChange
    141	139	28	140.176915	133.0658319	147.2879981	1.106880677
    143	136	30	139.5312055	134.9532905	144.1091204	1.067844436
    Is this normal?

  • #2
    Hi Chris

    If, by "strength", you mean sequencing depth, yes.

    In order to make the samples comparable, differences in sequencing depth (a.k.a. library size) have to be addressed. If sample A is sequenced 20% deeper than sample B, then 120 counts in sample A should be considered as equal in expression strength to 100 counts in B.

    The function 'estimateSizeFactors' estimates the sequencing depth, and with 'sizeFactors', you can print out the estimates. In the example just given, you might get size factors 1.2 and 1.0 for A and B (or another pair of numbers with the ratio 1.2:1).

    To get the 'baseMean' value, all count values are transformed to the "base scale" (or "common scale" in the paper) by dividing them with the appropriate size factor.

    I've explained our scheme to estimate the size factors a while ago in this post.

    Simon

    Comment


    • #3
      counts deseq

      hi simon,

      nope, thats not what i meant by 'strength', but it makes much much more sense...

      thanks!

      Comment


      • #4
        Hi Simon,

        I am trying to install DESeq package on Mac OS X 10.4 and Bioconductor 2.10.
        But I keep getting this error message:

        $ R CMD install /Users/lilyanamargaretha/Desktop/DESeq
        * installing to library ‘/Library/Frameworks/R.framework/Resources/library’
        * installing *source* package ‘DESeq’ ...
        ** libs
        ** arch - i386
        gcc -arch i386 -std=gnu99 -I/Library/Frameworks/R.framework/Resources/include -I/Library/Frameworks/R.framework/Resources/include/i386 -I/usr/local/include -fPIC -g -O2 -c pval.c -o pval.o
        cc1: error: unrecognized command line option "-arch"
        make: *** [pval.o] Error 1
        ERROR: compilation failed for package ‘DESeq’
        * removing ‘/Library/Frameworks/R.framework/Resources/library/DESeq’
        * restoring previous ‘/Library/Frameworks/R.framework/Resources/library/DESeq’

        Any idea how to fix it?

        Thanks!

        Comment


        • #5
          Hi M!x

          Originally posted by m!x View Post
          Hi Simon,
          I am trying to install DESeq package on Mac OS X 10.4 and Bioconductor 2.10.
          But I keep getting this error message:

          [...]
          cc1: error: unrecognized command line option "-arch"
          This is a most peculiar error. Your C compiler does not accept the '-arch' option. However, this is a standard option of GCC (and, if I am not completely confused, has been so since long before thre release of Tiger). Hence, something in your Xcode installation seems to be utterly broken.

          Could you please type in a terminal the commands "gcc -v" and "which gcc" and send me the output (by private mail to anders(at)embl(dot)de, in order to not clog the forum with details)?

          Cheers
          Simon

          Comment


          • #6
            Hi Simon,

            I finally managed to install DESeq package and ran it pretty well.
            Thanks!

            Comment


            • #7
              Hi Simon,

              I am trying to use DESeq. After step-

              cds <- newCountDataSet( countsTable, conds)

              I am getting this error-

              Error in round(countData) : Non-numeric argument to mathematical function

              Any suggestions? Thanks in advance.

              Comment


              • #8
                Originally posted by ikumar2 View Post
                cds <- newCountDataSet( countsTable, conds)

                I am getting this error-

                Error in round(countData) : Non-numeric argument to mathematical function
                It probably means that your countsTable contains non-numeric data, e.g. string with gene names. It should contain only numbers. Make sure to cut away any columns with annotation data and/or move them to the row / column names.

                Simon

                Comment


                • #9
                  DESeq installation doubts...

                  Originally posted by Simon Anders View Post
                  Hi M!x



                  This is a most peculiar error. Your C compiler does not accept the '-arch' option. However, this is a standard option of GCC (and, if I am not completely confused, has been so since long before thre release of Tiger). Hence, something in your Xcode installation seems to be utterly broken.

                  Could you please type in a terminal the commands "gcc -v" and "which gcc" and send me the output (by private mail to anders(at)embl(dot)de, in order to not clog the forum with details)?

                  Cheers
                  Simon
                  Hello Simon!

                  Hope you can give me a hand about your program.

                  I started a session in R 2.10.1 and typed the following to load DESeq:

                  > source("http://www.bioconductor.org/biocLite.R")
                  > biocLite("DESeq")
                  Using R version 2.10.1, biocinstall version 2.5.10.
                  Installing Bioconductor version 2.5 packages:
                  [1] "DESeq"
                  Please wait...

                  Warning message:
                  In getDependencies(pkgs, dependencies, available, lib) :
                  package ‘DESeq’ is not available

                  Do you recognize the warning message? I think it may have not installed DESeq after all, 'cause R doesn't seem recognize the function "newCountDataSet".

                  Sorry if this is too basic but just started with R and DESeq. Any pointers will be appreciated.

                  Daniel

                  Comment


                  • #10
                    Originally posted by dgu View Post
                    I started a session in R 2.10.1 and typed the following to load DESeq:

                    > source("http://www.bioconductor.org/biocLite.R")
                    > biocLite("DESeq")
                    Using R version 2.10.1, biocinstall version 2.5.10.
                    Installing Bioconductor version 2.5 packages:
                    [1] "DESeq"
                    Please wait...

                    Warning message:
                    In getDependencies(pkgs, dependencies, available, lib) :
                    package ‘DESeq’ is not available

                    Do you recognize the warning message? I think it may have not installed DESeq after all, 'cause R doesn't seem recognize the function "newCountDataSet".

                    Sorry if this is too basic but just started with R and DESeq. Any pointers will be appreciated
                    It says: "package ‘DESeq’ is not available". So, of course, you haven't installed it. The Bioconductor web server had occasional hick-ups recently, so maybe it really was not available for a while. Please try again, and, if it still does not work, follow the manual installation instructions here.

                    Simon

                    Comment


                    • #11
                      Bioconductor versions for DESeq

                      Hi dgu and Simon,

                      DESeq was introduced into Bioconductor at release 2.6. You are trying with release 2.5, that's why you get "package ‘DESeq’ is not available". Bioconductor releases are synchronised with R releases, so Bioconductor 2.6 goes with R 2.11. The recommended approach is to install R >= 2.11 and try with "biocLite" again, as you described. See also http://www.bioconductor.org -> Getting Started.

                      Nothing to do with server availability, which as far as I can tell has been fine.

                      Wolfgang Huber
                      Wolfgang Huber
                      EMBL

                      Comment


                      • #12
                        Originally posted by Wolfgang Huber View Post
                        Hi dgu and Simon,

                        DESeq was introduced into Bioconductor at release 2.6. You are trying with release 2.5, that's why you get "package ‘DESeq’ is not available". Bioconductor releases are synchronised with R releases, so Bioconductor 2.6 goes with R 2.11. The recommended approach is to install R >= 2.11 and try with "biocLite" again, as you described. See also http://www.bioconductor.org -> Getting Started.

                        Nothing to do with server availability, which as far as I can tell has been fine.

                        Wolfgang Huber
                        Thanks, Wolfgang!

                        Indeed, I downloaded R 2.11.1 and the problem was taking care of. Thanks!

                        Comment


                        • #13
                          Originally posted by Simon Anders View Post
                          It says: "package ‘DESeq’ is not available". So, of course, you haven't installed it. The Bioconductor web server had occasional hick-ups recently, so maybe it really was not available for a while. Please try again, and, if it still does not work, follow the manual installation instructions here.

                          Simon
                          Hi Simon!

                          Thanks for your reply. I sorted the problem out using the newest version of R.

                          I am going through each step of differential gene expression analysis from the DESeq manual. It is great and very well explained.

                          I had one question about Fig. 2 (pg 7). I managed to get the "smoothScatter" function to work, but the regression fit (in red) doesn't want to come out. Below is the script:

                          ## Fit from the local regression
                          lines(log10(fittedBaseVar)~log10(baseMean),diagForYC[order(diagForYC$baseMean),],col="red")

                          The plotting windows kinda freezes indefinitely and I get the following message:

                          Error in plot.xy(xy.coords(x, y), type = type, ...) :
                          plot.new has not been called yet

                          I've rewritten the script many times with no success. Do you see anything wrong?

                          Thanks in advance for any pointers!

                          Daniel

                          Comment


                          • #14
                            Hi Daniel,

                            maybe you have closed the plotting windows after calling 'smoothScatter'? The 'lines' function expects that a plot is already displayed so that the axes ares set.

                            Simon

                            Comment


                            • #15
                              Thanks for the prompt answer, Simon!

                              Indeed... that was the problem. I was closing the window.

                              R is great. It is just not THAT straightforward, if you know what I mean.

                              Thanks again for your help. Do you have any updates on your DESeq paper?

                              Cheers,
                              Daniel

                              Comment

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