Microaaray in R, Agilent two colour

Question

[monika.stefanska]

Hi, I am super strugling with Agilent two colour microarray. I am moving forward, but super slowly. Now I am in the possession of a decent script where i have to upload Spottype file. All the files for this array I finally found but when i upload spottype file i get an error.

So what I see is below.

target=readTargets("Targets.txt" )

data=read.maimages(files = target, source ="agilent", green.only = F, columns = list(G="gMedianSignal",Gb="gBGMedianSignal",R="rMedianSignal",Rb="rBGMedianSignal"),

               annotation = c("ProbeName","SystematicName")  )

plotMD(object = data) spottypes=readSpotTypes(file = "Spots3.txt", check.names=FALSE)

Error in scan(file = file, what = what, sep = sep, quote = quote, dec = dec, : line 32133 did not have 20 elements

I even tried to delete this line but the error cames up all the time. Can someone please help me? Maybe I am uploading wrong file time as this Spottype file?

[ATpoint]

Is this your or published data? This all seems overly complicated to me. Just read the CEL files into R and normalize with RMA as suggested in the limma manual.

[monika.stefanska]

This is my data but the script was given to me by someone else. Could you share with me the code to do it or limma manual?

[ATpoint]

Ok, but what data do you have? CEL files?

https://www.bioconductor.org/packages/devel/bioc/vignettes/limma/inst/doc/usersguide.pdf

[monika.stefanska]

txt files I have, Thanks ATpoint, I have followed this limma guidelines and finally start to understand My txt files starts with something like that

 YPE    text    text    text    integer text    float   float   text    integer text    text    integer integer integer integer float   float   float   float   float   float   text    text    text    text    text    text    text    text    text    text    text    integer integer integer
FEPARAMS    Protocol_Name   Protocol_date   Scan_ScannerName    Scan_NumChannels    Scan_Date   Scan_MicronsPerPixelX   Scan_MicronsPerPixelY   Scan_OriginalGUID   Scan_NumScanPass    Grid_Name   Grid_Date   Grid_NumSubGridRows Grid_NumSubGridCols Grid_NumRows    Grid_NumCols    Grid_RowSpacing Grid_ColSpacing Grid_OffsetX    Grid_OffsetY    Grid_NomSpotWidth   Grid_NomSpotHeight  Grid_GenomicBuild   FeatureExtractor_Barcode    FeatureExtractor_Sample FeatureExtractor_ScanFileName   FeatureExtractor_ArrayName  FeatureExtractor_ScanFileGUID   FeatureExtractor_DesignFileName FeatureExtractor_ExtractionTime FeatureExtractor_UserName   FeatureExtractor_ComputerName   FeatureExtractor_Version    FeatureExtractor_IsXDRExtraction    FeatureExtractor_ColorMode  FeatureExtractor_QCReportType
DATA    GE2_107_Sep09 (Read Only)   29-Sep-2009 17:04   Agilent Technologies G2600D SG11410002  2   09-03-2021 09:05:26 3   3   65afd1a0-abd5-4625-a7f1-d0b3589997c8    2   072363_D_F_20150612 12-Jun-2015 00:00   1   1   384 164 36.6617 31.745  0   18.3309 30  30  hg19:GRCh37:Feb2009 257236320035_1_1        D:\Skany\SG11410002_257236320035_S002.tif   SG11410002_257236320035_S002    65afd1a0-abd5-4625-a7f1-d0b3589997c8    072363_D_F_20150612.xml 03-Sep-2021 11:48:42    Admin   DATASYSTEM  10.7.3.1    0   1   0
*
TYPE    float   float   float   integer integer float   float   float   integer integer float   float   float   float   float   float   integer float   float   float   integer float   float   integer integer float   float   float   integer float   float   integer integer integer integer integer float   float   float   integer integer integer integer integer float   float   float   integer float   float   float   float   float   float   float   integer integer float   float   integer integer float   float   float   float   float   float   float   float   float   float   float   float   float   float   integer float   float   float   float   integer float   float   float   float   integer float   float   float   float   float   float   integer float   float   float   float   float   float   integer float   float   float   float   integer float   float   float   float   float   float   float   integer float   float   float   integer float   float   float   float   float   float   float   float   integer integer integer float   float   float   integer float   float   float   float   integer float   float   float   float   float   float   text    text    float   float   float   float   float   float   float   float   float   float   float   float   float   float   float   float   float   float   integer integer integer integer float   float   integer text    integer integer float   float   float   float   boolean float   float   float   boolean float   boolean float   boolean float   boolean float   boolean float   boolean float   boolean float   boolean float   boolean float   boolean float   boolean float   boolean
STATS   gDarkOffsetAverage  gDarkOffsetMedian   gDarkOffsetStdDev   gDarkOffsetNumPts   gSaturationValue    rDarkOffsetAverage  rDarkOffsetMedian   rDarkOffsetStdDev   rDarkOffsetNumPts   rSaturationValue    gAvgSig2BkgeQC  rAvgSig2BkgeQC  gAvgSig2BkgNegCtrl  rAvgSig2BkgNegCtrl  gRatioSig2BkgeQC_NegCtrl    rRatioSig2BkgeQC_NegCtrl    gNumSatFeat gLocalBGInlierNetAve    gLocalBGInlierAve   gLocalBGInlierSDev  gLocalBGInlierNum   gGlobalBGInlierAve  gGlobalBGInlierSDev gGlobalBGInlierNum  rNumSatFeat rLocalBGInlierNetAve    rLocalBGInlierAve   rLocalBGInlierSDev  rLocalBGInlierNum   rGlobalBGInlierAve  rGlobalBGInlierSDev rGlobalBGInlierNum  gNumFeatureNonUnifOL    gNumPopnOL  gNumNonUnifBGOL gNumPopnBGOL    gOffsetUsed gGlobalFeatInlierAve    gGlobalFeatInlierSDev   gGlobalFeatInlierNum    rNumFeatureNonUnifOL    rNumPopnOL  rNumNonUnifBGOL rNumPopnBGOL    rOffsetUsed rGlobalFeatInlierAve    rGlobalFeatInlierSDev   rGlobalFeatInlierNum    AllColorPrcntSat    AnyColorPrcntSat    AnyColorPrcntFeatNonUnifOL  AnyColorPrcntBGNonUnifOL    AnyColorPrcntFeatPopnOL AnyColorPrcntBGPopnOL   TotalPrcntFeatOL    gNumNegBGSubFeat    gNonCtrlNumNegFeatBGSubSig  gLinearDyeNormFactor    gRMSLowessDNF   rNumNegBGSubFeat    rNonCtrlNumNegFeatBGSubSig  rLinearDyeNormFactor    rRMSLowessDNF   DyeNormDimensionlessRMS DyeNormUnitWeightedRMS  gSpatialDetrendRMSFit   gSpatialDetrendRMSFilteredMinusFit  gSpatialDetrendSurfaceArea  gSpatialDetrendVolume   gSpatialDetrendAveFit   rSpatialDetrendRMSFit   rSpatialDetrendRMSFilteredMinusFit  rSpatialDetrendSurfaceArea  rSpatialDetrendVolume   rSpatialDetrendAveFit   gNonCtrlNumSatFeat  gNonCtrl99PrcntNetSig   gNonCtrl50PrcntNetSig   gNonCtrl1PrcntNetSig    gNonCtrlMedPrcntCVBGSubSig  rNonCtrlNumSatFeat  rNonCtrl99PrcntNetSig   rNonCtrl50PrcntNetSig   rNonCtrl1PrcntNetSig    rNonCtrlMedPrcntCVBGSubSig  gCtrleQCNumSatFeat  gCtrleQC99PrcntNetSig   gCtrleQC50PrcntNetSig   gCtrleQC1PrcntNetSig    geQCMedPrcntCVBGSubSig  geQCSig2BkgLow1 geQCSig2BkgLow2 rCtrleQCNumSatFeat  rCtrleQC99PrcntNetSig   rCtrleQC50PrcntNetSig   rCtrleQC1PrcntNetSig    reQCMedPrcntCVBGSubSig  reQCSig2BkgLow1 reQCSig2BkgLow2 gNegCtrlNumInliers  gNegCtrlAveNetSig   gNegCtrlSDevNetSig  gNegCtrlAveBGSubSig gNegCtrlSDevBGSubSig    rNegCtrlNumInliers  rNegCtrlAveNetSig   rNegCtrlSDevNetSig  rNegCtrlAveBGSubSig rNegCtrlSDevBGSubSig    gAveNumPixOLLo  gAveNumPixOLHi  gPixCVofHighSignalFeat  gNumHighSignalFeat  rAveNumPixOLLo  rAveNumPixOLHi  rPixCVofHighSignalFeat  rNumHighSignalFeat  NonCtrlAbsAveLogRatio   NonCtrlSDevLogRatio NonCtrlSNRLogRatio  eQCAbsAveLogRatio   eQCSDevLogRatio eQCSNRLogRatio  AddErrorEstimateGreen   AddErrorEstimateRed TotalNumFeatures    NonCtrlNumUpReg NonCtrlNumDownReg   eQCObsVsExpLRSlope  eQCObsVsExpLRIntercept  eQCObsVsExpCorr NumIsNorm   ROIHeight   ROIWidth    CentroidDiffX   CentroidDiffY   NumFoundFeat    MaxNonUnifEdges MaxSpotNotFoundEdges    gMultDetrendRMSFit  rMultDetrendRMSFit  gMultDetrendSurfaceAverage  rMultDetrendSurfaceAverage  eQCLowSigName1  eQCLowSigName2  gNonCtrl50PrcntBGSubSig rNonCtrl50PrcntBGSubSig gCtrleQC50PrcntBGSubSig rCtrleQC50PrcntBGSubSig gMedPrcntCVProcSignal   rMedPrcntCVProcSignal   geQCMedPrcntCVProcSignal    reQCMedPrcntCVProcSignal    gOutlierFlagger_Auto_FeatB_Term rOutlierFlagger_Auto_FeatB_Term gOutlierFlagger_Auto_FeatC_Term rOutlierFlagger_Auto_FeatC_Term gOutlierFlagger_Auto_BgndB_Term rOutlierFlagger_Auto_BgndB_Term gOutlierFlagger_Auto_BgndC_Term rOutlierFlagger_Auto_BgndC_Term OutlierFlagger_FeatChiSq    OutlierFlagger_BgndChiSq    GriddingStatus  IsGoodGrid  NumGeneNonUnifOL    TotalNumberOfReplicatedGenes    gPercentileIntensityProcessedSignal rPercentileIntensityProcessedSignal ExtractionStatus    QCMetricResults gNonCtrlNumWellAboveBG  rNonCtrlNumWellAboveBG  UpRandomnessRatio   DownRandomnessRatio UpRandomnessSDRatio DownRandomnessSDRatio   GridHasBeenOptimized    gNegCtrlSpread  rNegCtrlSpread  Metric_IsGoodGrid   Metric_IsGoodGrid_IsInRange Metric_AnyColorPrcntFeatNonUnifOL   Metric_AnyColorPrcntFeatNonUnifOL_IsInRange Metric_gNegCtrlAveBGSubSig  Metric_gNegCtrlAveBGSubSig_IsInRange    Metric_gNegCtrlSDevBGSubSig Metric_gNegCtrlSDevBGSubSig_IsInRange   Metric_rNegCtrlAveBGSubSig  Metric_rNegCtrlAveBGSubSig_IsInRange    Metric_rNegCtrlSDevBGSubSig Metric_rNegCtrlSDevBGSubSig_IsInRange   Metric_gNonCntrlMedCVBkSubSignal    Metric_gNonCntrlMedCVBkSubSignal_IsInRange  Metric_rNonCntrlMedCVBkSubSignal    Metric_rNonCntrlMedCVBkSubSignal_IsInRange  Metric_gE1aMedCVBkSubSignal Metric_gE1aMedCVBkSubSignal_IsInRange   Metric_rE1aMedCVBkSubSignal Metric_rE1aMedCVBkSubSignal_IsInRange   Metric_absE1aObsVsExpCorr   Metric_absE1aObsVsExpCorr_IsInRange Metric_absE1aObsVsExpSlope  Metric_absE1aObsVsExpSlope_IsInRange
DATA    10  10  0.868459    65536   777384  10  10  0.910604    65536   776738  152.027 6.52897 1.57599 1.05361 96.4645 6.19676 0   19.4322 29.4322 1.49345 62741   29.4322 1.49345 62741   0   10.125  20.125  0.635364    62578   20.125  0.635364    62578   15  148 0   235 10  88.6255 568.863 62814   18  186 0   398 10  53.5643 283.553 62774   0   0   0.033346    0   0.482724    0.965447    0.0344822   17949   17180   38.271  53.7961 22710   21694   35.3838 67.7605 0.919396    0.834444    0.692178    2.24168 1.00001 1.06942 43.196  0.459627    0.941464    1.00025 0.959101    21.3935 0   249.478 35.8444 27.9565 12.6699 0   547.775 12.0714 8.85366 17.5704 0   17160   3153.52 270.27  11.705  30.1874 9.99977 0   249.605 60.8636 13.675  15.8394 1.56301 1.3541  302 30.5131 1.95478 -2.68338    1.86033 308 10.6829 1.0063  -0.711531   0.87484 0.0754891   1.48279 0.0553578   65  0.104548    1.14102 0.0543065   26  0.591873    0.159822    5.52338 2.25946 0.0538244   52.8605 85.7912 35.3838 62976   8272    5516    0.390809    -2.25946    0.829155    3162    3.70105 6.34867 0.610044    0.493244    62062   0   0.00914634  0.0856678   0.119994    0.999414    1.00501 (+)E1A_r60_n11  (+)E1A_r60_a97  2.61284 0.591803    3120.5  49.6505 11.8696 10.9339 7.07311 12.0157 782.439 398.974 56.936  8.44681 85.1788 59.1514 34.5735 9.479   2.3431  1.288   0   1   19  58201   198.578 188.442 1   ExtractionEvaluate  10732   13269   1.05468 2.19546 0.235026    0.419171    0   6.2539  2.67541 1   1   0.033346    1   -2.68338    1   1.86033 1   -0.711531   1   0.87484 1   12.6699 1   17.5704 1   11.705  1   15.8394 1   0.829155    0   0.390809    0
*
TYPE    integer integer integer integer integer text    text    float   float   float   float   float   float   float   float   float   float   float   float   float   float   float   boolean boolean boolean boolean boolean boolean boolean boolean boolean boolean boolean float   float   boolean boolean boolean boolean float   float   float
FEATURES    FeatureNum  Row Col SubTypeMask ControlType ProbeName   SystematicName  PositionX   PositionY   LogRatio    LogRatioError   PValueLogRatio  gProcessedSignal    rProcessedSignal    gProcessedSigError  rProcessedSigError  gMedianSignal   rMedianSignal   gBGMedianSignal rBGMedianSignal gBGPixSDev  rBGPixSDev  gIsSaturated    rIsSaturated    gIsFeatNonUnifOL    rIsFeatNonUnifOL    gIsBGNonUnifOL  rIsBGNonUnifOL  gIsFeatPopnOL   rIsFeatPopnOL   gIsBGPopnOL rIsBGPopnOL IsManualFlag    gBGSubSignal    rBGSubSignal    gIsPosAndSignif rIsPosAndSignif gIsWellAboveBG  rIsWellAboveBG  SpotExtentX gBGMeanSignal   rBGMeanSignal
DATA    1   1   1   260 1   GE_BrightCorner GE_BrightCorner 514.623 294.725 4.691757995e-001    7.503510691e-002    4.032862392e-010    1.935085e+004   5.700013e+004   1.940236e+003   5.700066e+003   354 2388    26  21  5.556107e+000   3.069040e+000   0   0   0   0   0   0   0   0   0   0   0   307.034 2311.05 1   1   1   1   27.2919 26.6938 20.8402
DATA    2   1

[Gordon Smyth]

CEL files are an Affymetrix format they and are only produced by Affy arrays. Agilent arrays have spots in instead of probe-sets. The Agilent Feature Extraction software produces text files in the format shown by OP, which are read by read.maimages() with source="agilent".

Answer 1

[Gordon Smyth]

If you have Agilent arrays all of the same design, then you read them into R by

library(limma)
target <- readTargets("Targets.txt" )
data <- read.maimages(target, source ="agilent")

The error message you give seems to be coming from readSpotTypes (the input file seems to be wrong) but there is no need to use that function for Agilent arrays. All the required annotation information is already in the Agilent scanner files.