Did something change between versions of Limma for R 1.8.1 and 1.9.0 that I missed? Where did the NAs come from? These are the results from running topTable on four dye-swapped arrays. My code is identical in both cases. Thanks, Dave. Approximate R code (cut and pasted from Java): RG <- new("RGList", list(R=Rs,G=Gs,Rb=Rbs,Gb=Gbs)) RG$printer <- list(ngrid.r=max(genefile$V1),ngrid.c=max(genefile$V2),nspot.r=max(gen efile$ V3),nspot.c=max(genefile$V4)) controlgenes <- c("rbcL","LTP4","cab","RCA","XCP2","KLF5","beta-actin","blank") status <- as.character(genefile$V5 %in% controlgenes) weights <- modifyWeights(weights,status,values="TRUE",multipliers=0) MA <- normalizeWithinArrays(RG, method="printtiploess", layout = RG$printer, weights=weights) MA <- normalizeBetweenArrays(MA) RGnorm <- RG.MA(MA) fit <- lmFit(MA,design=designmatrix,ndups=3,spacing=3) eb <- eBayes(fit,proportion=0.01) topGenes <- topTable(eb,number=30, adjust.method="fdr",genelist=genenb, sort.by="B") R 1.8.1 and Limma 1.3.11 AA936757 3.719032040892476 13.29244868974321 21.273219140535428 1.7700675638573854E-8 16.41630333165485 AA683077 2.012539960953221 11.482499529443794 13.663709222466158 2.0625199090952707E-6 11.250663857920369 R60193 -1.3466735409962498 10.752153167818404 -13.03136976097657 2.4313326561267002E-6 10.685625239796412 AA495846 -1.6680548543855949 12.382884989290126 -12.671686948601973 2.5492658305820254E-6 10.351882638349387 AA488391 -1.4254635359938195 14.808395517282252 -10.843395066628524 1.283926471841737E-5 8.501052463671101 AA487582 -1.228055202475718 11.663677107858677 -10.178183283163676 2.227968350968346E-5 7.7559891264874485 AA115076 -0.9539621605022185 12.078999589916572 -9.624781911437797 3.621290380935091E-5 7.103310688538175 rbcL -0.7072592325606469 15.672906565723503 -9.374764679536662 4.270881833754837E-5 6.798070109334531 N92478 0.7189453707342979 12.281634742105913 8.549326128043308 1.0637336404591882E-4 5.741441090946004 R43483 1.202633856423655 12.863115098568308 8.239234808480374 1.35077439495208E-4 5.324064717014463 W65461 -0.8499390380452945 11.862250529587449 -8.171524696487337 1.35077439495208E-4 5.2313772459233325 R22625 1.007519719039666 11.685107805563577 8.148614344126274 1.35077439495208E-4 5.199887898229524 N64741 0.7156235702764399 11.809730661346153 7.870773633988644 1.8158032889909942E-4 4.812803441820608 cab -1.2941102587414737 15.680695154681269 -7.595232962116644 2.470103994514213E-4 4.419245595896798 H99588 0.8652153443867413 11.392510470124181 7.534377849323958 2.5114113484872455E-4 4.3310008628463175 AI091770 0.8504457738567389 12.106173602043109 7.237131332358541 3.600014660391982E-4 3.8929531152731576 N47099 -0.8114613642578669 12.662883264096203 -7.138898624042952 3.856946884800288E-4 3.745596346529296 AA927490 0.8516455736631379 12.848624380999476 7.091226324062262 3.856946884800288E-4 3.6736147292532495 H93335 0.7766571255106037 11.626933364317638 7.055574805801781 3.856946884800288E-4 3.6195822554773978 AA460756 -0.722318285156469 12.914573719569278 -7.037093214568916 3.856946884800288E-4 3.5915040918515695 H91651 0.8999563298639041 11.531392574985665 6.981365473171944 3.988349279240792E-4 3.5065580814100192 R98064 0.5788079495799782 13.92756842842436 6.925988294495901 4.022593487966753E-4 3.4217262940760174 N92519 -0.8817222351213301 12.325915022550825 -6.8959183292308674 4.022593487966753E-4 3.3754864306526473 H57309 1.0221437769371449 11.665260456460624 6.885789182466807 4.022593487966753E-4 3.3598824729822754 AA676254 0.6331758737992141 12.333616026625913 6.803062369233151 4.3712786030422087E-4 3.231913465384914 R70685 0.7174146630194426 12.522316848076509 6.7508474635412545 4.5473122234414076E-4 3.1506570801542066 AA156324 0.6274341213034178 13.95646992606678 6.709548771767108 4.6613320948945386E-4 3.0861213173329025 AA970865 0.7372368804313136 12.529532666637065 6.616778943118437 5.092549091370438E-4 2.940291066618914 AA426311 1.1933268346805304 12.384127923158252 6.604555005565974 5.092549091370438E-4 2.9209863477282267 AI266752 0.6269423513027547 12.315553383243532 6.454717305524467 6.20093863325188E-4 2.682661207506496 R 1.9.0 and Limma 1.6.2 AA936757 3.719032040892476 10.559981507974102 28.23882285130472 7.863145275660577E-10 19.413771711320685 H29557 -1.897133224001365 15.350252408436896 -19.413578403894515 3.988945258694179E-8 15.20366412339247 AA425823 -1.666448034534032 11.02998345932825 -15.292205566460252 4.790547716180304E-7 12.374499220492003 AA504505 -1.4474488966374865 12.078999589916572 -14.87358063161449 5.010456645004823E-7 12.042391815850303 R59304 1.544443491508911 11.453083271566156 13.271793476075693 1.3297285005387517E-6 10.67797369790008 R60193 -1.3466735409962498 10.7521531678184 -13.242098267228087 1.3297285005387517E-6 10.651167901354318 AA495846 -1.6680548543855949 12.38288498929012 -12.750002501701173 1.780703847793079E-6 10.19828768873374 AA487582 -1.228055202475718 12.65779688842052 -12.349291873690223 2.265897629733821E-6 9.816997544388586 H77652 0.9059438434642344 12.256507847750266 9.83023585045215 2.7275150855477153E-5 7.124830784102192 rbcL -0.7072592325606469 15.6729065657235 -9.751179795203823 2.7275150855477153E-5 7.031042906997234 N62259 0.7123986545109168 15.049128544130609 9.629646891234945 2.853790704567304E-5 6.885633360176124 AA404273 -0.9996886769470037 11.43235822647634 -9.389025706663572 3.198886319749833E-5 6.593265337263933 N59764 -0.7145132049203675 15.676890538969966 -9.36240960883919 3.198886319749833E-5 6.56055438319232 W92278 -1.1476019449939237 10.439293470618457 -9.327682420283907 3.198886319749833E-5 6.517762525489527 AA148641 -0.9404721200518092 15.082140123430541 -8.861667197571945 5.258261946783176E-5 5.930945556384787 N92478 0.7189453707342979 12.281634742105913 8.805175962280739 5.2876863355363295E-5 5.858183976942382 AI000633 1.6008005679752557 9.955996678736588 8.180663817805218 1.0879675440219568E-4 5.029306410490687 NA NaN 12.337598516303787 -8.109277965186818 1.0879675440219568E-4 4.93161742950074 H14569 -1.0380998956395908 11.531392574985661 -8.10822919035774 1.0879675440219568E-4 4.930177603648442 W96155 -0.572117857511056 14.869545562513855 -8.01482513126453 1.1280454960831163E-4 4.801407377034307 AI356395 -1.1000763326219425 9.452794366579658 -8.005509561604235 1.1280454960831163E-4 4.7885059099214935 H98218 0.6001708323940103 13.334379713309595 7.275588365011955 2.9398547598623775E-4 3.7435867091686763 cab -1.2941102587414737 11.46022414171325 -7.123140520806918 3.4972921801739885E-4 3.5166308112029494 R43483 1.202633856423655 11.014639250100172 6.857870675428175 4.933421851847506E-4 3.1143009120476144 NA NaN 10.896076797612105 6.798345329400819 4.937898133672889E-4 3.022712666695405 AA902249 -0.66593123879359 14.260402317937773 -6.792525490061204 4.937898133672889E-4 3.0137321220865587 RCA 2.4220685426497868 8.673579100397403 7.006012361156719 4.937898133672889E-4 2.96802105001 AI337373 0.9378765424156126 11.790798293433005 6.749319582910434 4.937898133672889E-4 2.9469169513263953 AA952925 -0.8110412337442618 13.950937078203266 -6.738004595584546 4.937898133672889E-4 2.929376916256361 AA933077 0.48901878798830556 15.501228055330621 6.726858105944119 4.937898133672889E-4 2.9120809607229425

The change-log for limma is at http://bioinf.wehi.edu.au/limma/changelog.txt. I can't think of any changes which I'd expect to make a difference. If you want to track down the reason you need to be more proactive yourself, by giving a reproducible example or at very least identifying which function introduced the difference. Gordon >Did something change between versions of Limma for R 1.8.1 and 1.9.0 that I >missed? Where did the NAs come from? These are the results from running >topTable on four dye-swapped arrays. My code is identical in both cases. > >Thanks, Dave. > >Approximate R code (cut and pasted from Java): > >RG <- new("RGList", list(R=Rs,G=Gs,Rb=Rbs,Gb=Gbs)) > >RG$printer <- >list(ngrid.r=max(genefile$V1),ngrid.c=max(genefile$V2),nspot.r=max(ge nefile$ >V3),nspot.c=max(genefile$V4)) > >controlgenes <- >c("rbcL","LTP4","cab","RCA","XCP2","KLF5","beta-actin","blank") > >status <- as.character(genefile$V5 %in% controlgenes) > >weights <- modifyWeights(weights,status,values="TRUE",multipliers=0) > >MA <- normalizeWithinArrays(RG, method="printtiploess", layout = RG$printer, >weights=weights) > >MA <- normalizeBetweenArrays(MA) > >RGnorm <- RG.MA(MA) > >fit <- lmFit(MA,design=designmatrix,ndups=3,spacing=3) > >eb <- eBayes(fit,proportion=0.01) > >topGenes <- topTable(eb,number=30, adjust.method="fdr",genelist=genenb, >sort.by="B") > > >R 1.8.1 and Limma 1.3.11 > >AA936757 3.719032040892476 13.29244868974321 >21.273219140535428 1.7700675638573854E-8 >16.41630333165485 > >AA683077 2.012539960953221 11.482499529443794 >13.663709222466158 2.0625199090952707E-6 >11.250663857920369 > >R60193 -1.3466735409962498 10.752153167818404 >-13.03136976097657 2.4313326561267002E-6 >10.685625239796412 > >AA495846 -1.6680548543855949 12.382884989290126 >-12.671686948601973 2.5492658305820254E-6 >10.351882638349387 > >AA488391 -1.4254635359938195 14.808395517282252 >-10.843395066628524 1.283926471841737E-5 >8.501052463671101 > >AA487582 -1.228055202475718 11.663677107858677 >-10.178183283163676 2.227968350968346E-5 >7.7559891264874485 > >AA115076 -0.9539621605022185 12.078999589916572 -9.624781911437797 >3.621290380935091E-5 7.103310688538175 > >rbcL -0.7072592325606469 15.672906565723503 >-9.374764679536662 4.270881833754837E-5 >6.798070109334531 > >N92478 0.7189453707342979 12.281634742105913 >8.549326128043308 1.0637336404591882E-4 >5.741441090946004 > >R43483 1.202633856423655 12.863115098568308 >8.239234808480374 1.35077439495208E-4 >5.324064717014463 > >W65461 -0.8499390380452945 11.862250529587449 >-8.171524696487337 1.35077439495208E-4 >5.2313772459233325 > >R22625 1.007519719039666 11.685107805563577 >8.148614344126274 1.35077439495208E-4 >5.199887898229524 > >N64741 0.7156235702764399 11.809730661346153 >7.870773633988644 1.8158032889909942E-4 >4.812803441820608 > >cab -1.2941102587414737 15.680695154681269 >-7.595232962116644 2.470103994514213E-4 >4.419245595896798 > >H99588 0.8652153443867413 11.392510470124181 >7.534377849323958 2.5114113484872455E-4 >4.3310008628463175 > >AI091770 0.8504457738567389 12.106173602043109 >7.237131332358541 3.600014660391982E-4 >3.8929531152731576 > >N47099 -0.8114613642578669 12.662883264096203 >-7.138898624042952 3.856946884800288E-4 >3.745596346529296 > >AA927490 0.8516455736631379 12.848624380999476 7.091226324062262 >3.856946884800288E-4 3.6736147292532495 > >H93335 0.7766571255106037 11.626933364317638 >7.055574805801781 3.856946884800288E-4 >3.6195822554773978 > >AA460756 -0.722318285156469 12.914573719569278 >-7.037093214568916 3.856946884800288E-4 >3.5915040918515695 > >H91651 0.8999563298639041 11.531392574985665 >6.981365473171944 3.988349279240792E-4 >3.5065580814100192 > >R98064 0.5788079495799782 13.92756842842436 >6.925988294495901 4.022593487966753E-4 >3.4217262940760174 > >N92519 -0.8817222351213301 12.325915022550825 >-6.8959183292308674 4.022593487966753E-4 >3.3754864306526473 > >H57309 1.0221437769371449 11.665260456460624 >6.885789182466807 4.022593487966753E-4 >3.3598824729822754 > >AA676254 0.6331758737992141 12.333616026625913 6.803062369233151 >4.3712786030422087E-4 3.231913465384914 > >R70685 0.7174146630194426 12.522316848076509 >6.7508474635412545 4.5473122234414076E-4 >3.1506570801542066 > >AA156324 0.6274341213034178 13.95646992606678 6.709548771767108 >4.6613320948945386E-4 3.0861213173329025 > >AA970865 0.7372368804313136 12.529532666637065 6.616778943118437 >5.092549091370438E-4 2.940291066618914 > >AA426311 1.1933268346805304 12.384127923158252 6.604555005565974 >5.092549091370438E-4 2.9209863477282267 > >AI266752 0.6269423513027547 12.315553383243532 >6.454717305524467 6.20093863325188E-4 >2.682661207506496 > > > >R 1.9.0 and Limma 1.6.2 > >AA936757 3.719032040892476 10.559981507974102 >28.23882285130472 7.863145275660577E-10 19.413771711320685 > >H29557 -1.897133224001365 15.350252408436896 -19.413578403894515 >3.988945258694179E-8 15.20366412339247 > >AA425823 -1.666448034534032 11.02998345932825 >-15.292205566460252 4.790547716180304E-7 12.374499220492003 > >AA504505 -1.4474488966374865 12.078999589916572 >-14.87358063161449 5.010456645004823E-7 12.042391815850303 > >R59304 1.544443491508911 11.453083271566156 13.271793476075693 >1.3297285005387517E-6 10.67797369790008 > >R60193 -1.3466735409962498 10.7521531678184 -13.242098267228087 >1.3297285005387517E-6 10.651167901354318 > >AA495846 -1.6680548543855949 12.38288498929012 >-12.750002501701173 1.780703847793079E-6 10.19828768873374 > >AA487582 -1.228055202475718 12.65779688842052 >-12.349291873690223 2.265897629733821E-6 9.816997544388586 > >H77652 0.9059438434642344 12.256507847750266 9.83023585045215 >2.7275150855477153E-5 7.124830784102192 > >rbcL -0.7072592325606469 15.6729065657235 -9.751179795203823 >2.7275150855477153E-5 7.031042906997234 > >N62259 0.7123986545109168 15.049128544130609 9.629646891234945 >2.853790704567304E-5 6.885633360176124 > >AA404273 -0.9996886769470037 11.43235822647634 >-9.389025706663572 3.198886319749833E-5 6.593265337263933 > >N59764 -0.7145132049203675 15.676890538969966 -9.36240960883919 >3.198886319749833E-5 6.56055438319232 > >W92278 -1.1476019449939237 10.439293470618457 >-9.327682420283907 3.198886319749833E-5 6.517762525489527 > >AA148641 -0.9404721200518092 15.082140123430541 >-8.861667197571945 5.258261946783176E-5 5.930945556384787 > >N92478 0.7189453707342979 12.281634742105913 8.805175962280739 >5.2876863355363295E-5 5.858183976942382 > >AI000633 1.6008005679752557 9.955996678736588 >8.180663817805218 1.0879675440219568E-4 5.029306410490687 > >NA NaN 12.337598516303787 -8.109277965186818 >1.0879675440219568E-4 4.93161742950074 > >H14569 -1.0380998956395908 11.531392574985661 -8.10822919035774 >1.0879675440219568E-4 4.930177603648442 > >W96155 -0.572117857511056 14.869545562513855 >-8.01482513126453 1.1280454960831163E-4 4.801407377034307 > >AI356395 -1.1000763326219425 9.452794366579658 >-8.005509561604235 1.1280454960831163E-4 4.7885059099214935 > >H98218 0.6001708323940103 13.334379713309595 7.275588365011955 >2.9398547598623775E-4 3.7435867091686763 > >cab -1.2941102587414737 11.46022414171325 -7.123140520806918 >3.4972921801739885E-4 3.5166308112029494 > >R43483 1.202633856423655 11.014639250100172 6.857870675428175 >4.933421851847506E-4 3.1143009120476144 > >NA NaN 10.896076797612105 6.798345329400819 >4.937898133672889E-4 3.022712666695405 > >AA902249 -0.66593123879359 14.260402317937773 >-6.792525490061204 4.937898133672889E-4 3.0137321220865587 > >RCA 2.4220685426497868 8.673579100397403 7.006012361156719 >4.937898133672889E-4 2.96802105001 > >AI337373 0.9378765424156126 11.790798293433005 >6.749319582910434 4.937898133672889E-4 2.9469169513263953 > >AA952925 -0.8110412337442618 13.950937078203266 >-6.738004595584546 4.937898133672889E-4 2.929376916256361 > >AA933077 0.48901878798830556 15.501228055330621 >6.726858105944119 4.937898133672889E-4 2.9120809607229425