Question

duplicate correlation of 4 "within array replicates" limmaGUI

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Gordon Smyth 52k

@gordon-smyth

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WEHI, Melbourne, Australia

Dear Christine, duplicate correlation in limma is only for replicate spots which are regularly spaced. The spacing value is the number of spots between replicates. Your value of 21 cannot be correct on an array with 16 columns and 11 rows, and that is what limmaGUI is telling you. You haven't actually told us how the 4 replicates for each mir are arranged on the arrays. If your replicates really are regularly spaced, then limma commands readGAL() and getLayout(guess=TRUE) will probably tell you what the spacing is. Best wishes Gordon > Date: Wed, 8 Oct 2008 20:07:09 +0200 > From: "Christine Voellenkle" <christine.voellenkle at="" gmail.com=""> > Subject: [BioC] duplicate correlation of 4 "within array replicates" > limma gui > To: bioconductor at stat.math.ethz.ch > Message-ID: > <b184b04b0810081107o1c98ab67hee91b203524835c8 at="" mail.gmail.com=""> > Content-Type: text/plain > > Dear all, > > I perform 2 color hybridization, using microRNA and Exiqon slides. > slide design: 4 replicates of 1 array > each array consits of 8 subarrays, > each subarray of 16 columns and 11 rows > each mir has 4 replicates > > For analysis of the data I use limma GUI, to obtain the p-values of > differentially expressed mirs I carried out loess normalization > (backgroundsubstraction normexp, offset=10) and subsequently applied the > Linear model fit, which works, as long as I treat each replicate like a > single mir. > I would like to now also about the correlation of the replicates, so I > entered as replicate number 4 and as distance 21 (according to > "Bioinformatics+ computional statistics using R and Bioconductor" the > distance corresponds to the number of spots that lies betweeen the > replicates). > But I get the following message: > > "Error in dim(M)<- c(spacing,ndups, ngroups, nslides) > dims[product 5628] do not match the length of object [5632]" > > I rechecked the gal file and recounted on the image, 21 is the correct > number (considering the column-distance, using the number of spots of row > distance results in dims[product 0]). > I read the limma guide and checked also the examples on the limma gui > homepage, but it's always either no replicate or only 2 replicates next to > each other. > Do you know a solution? > > Also another question: > IF one day I manage to do the duplicate correlation and given that I would > use weights to identfy features with the flag "bad", how will the software > handle this if one or more replicates are flagged bad? will it exclude the > flagged bad replicate from the correlation? > If yes, is there a way to put a minimum limit of 2 unflagged spots per mir, > so that in case of only 1 unflagged replicate of a mir to exclude this mir > from analysis? > > Thank you a lot in advance! > Greetings, Christine > > > > > > > > > Dr. Christine V?llenkle, Ph.D. > Research Laboratories-Molecular Cardiology > I.R.C.C.S. Policlinico San Donato > Via R. Morandi, 30 > 20097 S. Donato M.se (MI) Italy > Phone: +39 02 52774 683 (lab) > +39 02 52774 533 (office) > Fax: +39 02 52774 666 > email: christine.voellenkle at gmail.com

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ADD COMMENT • link 16.2 years ago • updated 16.1 years ago Gordon Smyth 52k

score 0 · Answer 1 · 2008-10-14

Dear Gordon, Excuse me for the last mail, I simply replied, I didn't realise that I was sending it to your personal email. Thank you a lot for your fast answer! I attached a schema of the slide-design, also with an example of the 4 replicates, hoping that like this I could explain it sufficiently. For my understanding the replicates are regularly arranged. As example mir 16, which we use as control mir, its 4 replicates can be found in block 5, block 13, block 21 and block 29, always with the same coordinates: column 9, row 3. If I count along the column there are 21 spots between the replicates (NOT counting the replicates), if I count along rows there are 127 spots between the replicates, but also this leads to an error message. I could not imagine another way to count the distance. I followed your advice, to see if limma tells me the distance using the comand getLayout, but as spacing he tells me not available/missing value $ngrid.r [1] 8 $ngrid.c [1] 4 $nspot.r [1] 11 $nspot.c [1] 16 $ndups [1] 4 $spacing [1] NA attr(,"class") [1] "PrintLayout" > I wondered, since limma recognizes the 4 replicates $ndups [1] 4 if it is enough to tell him spacing 1, I tried it, but it doesn't work, in the top table list gives a weired result, there are mir missing and it's shrinking the 4 replictes into 2 for every mir. What would you advice? Thanks a lot, Christine -- Dr. Christine V?llenkle, Ph.D. Research Laboratories-Molecular Cardiology I.R.C.C.S. Policlinico San Donato Via R. Morandi, 30 20097 S. Donato M.se (MI) Italy Phone: +39 02 52774 683 (lab) +39 02 52774 533 (office) Fax: +39 02 52774 666 email: christine.voellenkle at gmail.com

score 0 · Answer 2 · 2008-10-15

Dear Christine, If your arrays truly consist of four subarrays, with each subarray having identical probes in the same arrangement, then the spacing between duplicates is 1/4 of the total number of spots, i.e., spacing <- nrow(MA)/4 According to your previous email, your data has 5632 rows, so the spacing is 5632/4=1408. Note that each of your meta blocks has 32 spots, so the spacing represents the eight metablocks making up one subarray, that is 8*32=1408. The limma documention tells you that spacing represents the number of rows in the data object or GAL file separating the replicate arrays. This is not the same as the number of rows or columns which you count in a straight line on the image, as you seem to be assuming. I'm sorry that getLayout() didn't correctly deduce the spacing for you. The function was written mainly with ndups=2 in mind. Best wishes Gordon ----------- original message --------------- [BioC] duplicate correlation of 4 "within array replicates" limmaGUI Christine Voellenkle christine.voellenkle at gmail.com Tue Oct 14 11:04:29 CEST 2008 Dear Gordon, Excuse me for the last mail, I simply replied, I didn't realise that I was sending it to your personal email. Thank you a lot for your fast answer! I attached a schema of the slide-design, also with an example of the 4 replicates, hoping that like this I could explain it sufficiently. For my understanding the replicates are regularly arranged. As example mir 16, which we use as control mir, its 4 replicates can be found in block 5, block 13, block 21 and block 29, always with the same coordinates: column 9, row 3. If I count along the column there are 21 spots between the replicates (NOT counting the replicates), if I count along rows there are 127 spots between the replicates, but also this leads to an error message. I could not imagine another way to count the distance. I followed your advice, to see if limma tells me the distance using the comand getLayout, but as spacing he tells me not available/missing value $ngrid.r [1] 8 $ngrid.c [1] 4 $nspot.r [1] 11 $nspot.c [1] 16 $ndups [1] 4 $spacing [1] NA attr(,"class") [1] "PrintLayout" > I wondered, since limma recognizes the 4 replicates $ndups [1] 4 if it is enough to tell him spacing 1, I tried it, but it doesn't work, in the top table list gives a weired result, there are mir missing and it's shrinking the 4 replictes into 2 for every mir. What would you advice? Thanks a lot, Christine -- Dr. Christine Vllenkle, Ph.D. Research Laboratories-Molecular Cardiology I.R.C.C.S. Policlinico San Donato Via R. Morandi, 30 20097 S. Donato M.se (MI) Italy Phone: +39 02 52774 683 (lab) +39 02 52774 533 (office) Fax: +39 02 52774 666 email: christine.voellenkle at gmail.com