Hi Roger, I am not the author of flowCore but I believe the ellipsoid gate constructor takes covariance matrices since this is used in the Gating-ML specification. Mathematically, this is a nice way to specify ellipsoids in multidimensional space. I have posted a simple spreadsheet that allows you to convert the representation of an ellipse by half-axes, rotation and centre point to covariance matrix, mean (= centre point), and distance square (=1 for this case). Please follow this link for download: http://sourceforge.net/projects/flowcyt/files/Gating-ML/Gating- ML%201.5/EllipseCalculations.xls/download I hope this addresses your issues; let me know if further help is needed. Unfortunately, I'll have to leave it up to the flowCore/flowViz authors to get back to you with your other questions. Cheers, Josef > Message: 6 > Date: Mon, 13 Sep 2010 15:51:55 +0100 > From: Roger Leigh<rleigh at="" codelibre.net=""> > To: bioconductor at stat.math.ethz.ch > Subject: [BioC] FlowCore/FlowViz issues > Message-ID:<20100913145155.GW6128 at codelibre.net> > Content-Type: text/plain; charset=utf-8 > > Hi, > > [Apologies if this is delivered twice; my initial mail didn't > appear to be accepted for several hours, and I didn't see any > failure message; does the list object to GPG signatures?] > > I've just started to use FlowCore/FlowViz to analyse some of my > flow cytometry data, and ran into a few problems. I'm hoping > that you might be able to point me in the right direction! > > I've been very pleased with it so far, and have got some nice > plots and stats out of it, but I'm sure I'm doing some things > very inefficiently and/or incorrectly! > > [I'm using R version 2.11.1 (2010-05-31) on x86_64-pc-linux-gnu > (Debian GNU/Linux) with current Bioconductor packages)] > > > 1) Gating with an ellipsoidGate > > cov<- matrix(c(400000000, 0, 0, 0.08), ncol=2, > dimnames=list(c("FS.Lin", "SS.Log"), c("FS.Lin", "SS.Log"))) > mean<- c("FS.Lin"=32000, "SS.Log"=2.8) > cells<- ellipsoidGate(filterId="CellGate", .gate=cov, mean=mean) > > I want to select my cells using an ellipsoid gate on forward- and side- > scatter plots. In the above situation, they lie in a region where > FS=32000?10000 and log??(SS)=2.8?0.5. However, the values in the > covariance matrix don't match the dimensions; is there any explanation > regarding how to construct a covariance matrix from the actual > dimension I want (I got the above by trial and error until it fitted > nicely--I'm afraid I know little about these matrices). > > In some plots I'd also like to rotate the ellipse, but I'm not sure > how to put this into the matrix, if that's the way to do things. > Is this possible? > > Is there an alternative constructor to create a gate from real > dimensions? > > > > Many thanks for all your help, > Roger > > -- > .''`. Roger Leigh > : :' : Debian GNU/Linux http://people.debian.org/~rleigh/ > `. `' Printing on GNU/Linux? http://gutenprint.sourceforge.net/ > `- GPG Public Key: 0x25BFB848 Please GPG sign your mail. > > >

Dear Josef, Many thanks, the speadsheet was most helpful! I did try creating a covariance matrix from separate scaling and rotation matrices (below), but this didn't work, I suppose because they are too simple--operating in Euclidean space rather than Mahalanobis space. cov.matrix.simple <- function (a, b, angle) { theta <- angle * (pi/180) R <- matrix(c(cos(theta), -sin(theta), sin(theta), cos(theta)), byrow=TRUE, ncol=2) S <- matrix(c(a, 0, 0, b), byrow=TRUE, ncol=2) S%*%R } Using your spreadsheet, I've created a macro to create a covariance matrix in R from a, b and an angle, which I've verified to match the results shown in your spreadsheet: cov.matrix <- function (a, b, angle) { theta <- angle * (pi/180) c1 <- ((cos(theta)^2)/a^2) + ((sin(theta)^2)/b^2) c2 <- sin(theta) * cos(theta) * ((1/a^2) - (1/b^2)) c3 <- ((sin(theta)^2)/a^2) + ((cos(theta)^2)/b^2) m1 <- matrix(c(c1, c2, c2, c3), byrow=TRUE, ncol=2) m2 <- solve(m1) m2 } This appears to work well when rotation is not involved. However, as soon as I try to add rotation, it blows up: library(flowCore) library(flowViz) library(flowUtils) cov.matrix <- function (a, b, angle) { theta <- angle * (pi/180) c1 <- ((cos(theta)^2)/a^2) + ((sin(theta)^2)/b^2) c2 <- sin(theta) * cos(theta) * ((1/a^2) - (1/b^2)) c3 <- ((sin(theta)^2)/a^2) + ((cos(theta)^2)/b^2) m1 <- matrix(c(c1, c2, c2, c3), byrow=TRUE, ncol=2) m2 <- solve(m1) m2 } d <- read.FCS("0isotype.fcs", alter.names=TRUE) d <- transform(d, `SS.Log` = log10(`SS.Log`)) # No rotation cov <- cov.matrix(20000, 0.4, 0) colnames(cov) <- c("FS.Lin", "SS.Log") rownames(cov) <- c("FS.Lin", "SS.Log") mean <- c("FS.Lin"=40000, "SS.Log"=2.8) cells <- ellipsoidGate(filterId="CellGate", .gate=cov, mean=mean) print(cov) pdf("test.pdf", width=8, height=8, pointsize=12) print(xyplot(`SS.Log` ~ `FS.Lin`, d, filter=cells, xlab="FS", ylab=expression(log[10]~(SS)))) dev.off() # Now repeat with a 20 degree rotation... cov <- cov.matrix(20000, 0.4, 20) colnames(cov) <- c("FS.Lin", "SS.Log") rownames(cov) <- c("FS.Lin", "SS.Log") mean <- c("FS.Lin"=40000, "SS.Log"=2.8) cells <- ellipsoidGate(filterId="CellGate", .gate=cov, mean=mean) print(cov) pdf("test-rotate.pdf", width=8, height=8, pointsize=12) print(xyplot(`SS.Log` ~ `FS.Lin`, d, filter=cells, xlab="FS", ylab=expression(log[10]~(SS)))) dev.off() I've put the source data, script and results at: http://www-users.york.ac.uk/~rl522/flowcore-test/ http://www-users.york.ac.uk/~rl522/flowcore-test/test.pdf http://www-users.york.ac.uk/~rl522/flowcore-test/test-rotate.pdf You can see that while an ellipse is drawn correctly in the first instance, when we add a 20 degree rotation, it's completely screwed. I'm not sure if this is due to the extreme differences in the x and y dimensions, which alter the length of a and b, or for some other reason. Many thanks, Roger On 14/09/2010 18:00, Josef Spidlen wrote: > Hi Roger, > I am not the author of flowCore but I believe the ellipsoid gate > constructor takes covariance matrices since this is used in the > Gating-ML specification. Mathematically, this is a nice way to specify > ellipsoids in multidimensional space. > > I have posted a simple spreadsheet that allows you to convert the > representation of an ellipse by half-axes, rotation and centre point to > covariance matrix, mean (= centre point), and distance square (=1 for > this case). Please follow this link for download: > http://sourceforge.net/projects/flowcyt/files/Gating-ML/Gating- ML%201.5/EllipseCalculations.xls/download > > I hope this addresses your issues; let me know if further help is needed. > > Unfortunately, I'll have to leave it up to the flowCore/flowViz authors > to get back to you with your other questions. > > Cheers, > Josef > >> Message: 6 >> Date: Mon, 13 Sep 2010 15:51:55 +0100 >> From: Roger Leigh<rleigh at="" codelibre.net=""> >> To: bioconductor at stat.math.ethz.ch >> Subject: [BioC] FlowCore/FlowViz issues >> Message-ID:<20100913145155.GW6128 at codelibre.net> >> Content-Type: text/plain; charset=utf-8 >> >> Hi, >> >> [Apologies if this is delivered twice; my initial mail didn't >> appear to be accepted for several hours, and I didn't see any >> failure message; does the list object to GPG signatures?] >> >> I've just started to use FlowCore/FlowViz to analyse some of my >> flow cytometry data, and ran into a few problems. I'm hoping >> that you might be able to point me in the right direction! >> >> I've been very pleased with it so far, and have got some nice >> plots and stats out of it, but I'm sure I'm doing some things >> very inefficiently and/or incorrectly! >> >> [I'm using R version 2.11.1 (2010-05-31) on x86_64-pc-linux-gnu >> (Debian GNU/Linux) with current Bioconductor packages)] >> >> >> 1) Gating with an ellipsoidGate >> >> cov<- matrix(c(400000000, 0, 0, 0.08), ncol=2, >> dimnames=list(c("FS.Lin", "SS.Log"), c("FS.Lin", "SS.Log"))) >> mean<- c("FS.Lin"=32000, "SS.Log"=2.8) >> cells<- ellipsoidGate(filterId="CellGate", .gate=cov, mean=mean) >> >> I want to select my cells using an ellipsoid gate on forward- and side- >> scatter plots. In the above situation, they lie in a region where >> FS=32000?10000 and log??(SS)=2.8?0.5. However, the values in the >> covariance matrix don't match the dimensions; is there any explanation >> regarding how to construct a covariance matrix from the actual >> dimension I want (I got the above by trial and error until it fitted >> nicely--I'm afraid I know little about these matrices). >> >> In some plots I'd also like to rotate the ellipse, but I'm not sure >> how to put this into the matrix, if that's the way to do things. >> Is this possible? >> >> Is there an alternative constructor to create a gate from real >> dimensions? >> >> >> >> Many thanks for all your help, >> Roger >> >> -- >> .''`. Roger Leigh >> : :' : Debian GNU/Linux http://people.debian.org/~rleigh/ >> `. `' Printing on GNU/Linux? http://gutenprint.sourceforge.net/ >> `- GPG Public Key: 0x25BFB848 Please GPG sign your mail. >> >> >