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Image Quantitation in Microarray Analysis

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					  Image Quantitation
in Microarray Analysis




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                     tomorrow...
Microarray analysis
   Array construction, hybridisation, scanning


   Quantitation of fluorescence signals

   Data visualisation


   Meta-analysis (clustering)


   More visualisation
Technical


                  pseudo-colour
sample
(labelled)            image




       probe
                  [image from Jeremy Buhler]
      (on chip)
Experimental design
   Track what’s on the chip
        • which spot corresponds to which gene

   Duplicate experimental spots
        • reproducibility

   Controls
    – DNAs spotted on glass
        • positive probe (induced or repressed)
        • negative probe (bacterial genes on human chip)
    – oligos on glass or synthesised on chip (Affymetrix)
        • point mutants (hybridisation plus/minus)
Images from scanner
   Resolution
    – standard 10m [currently, max 5m]
    – 100m spot on chip = 10 pixels in diameter

   Image format
    – TIFF (tagged image file format)
    – can be compressed
        • (eg. Lempel-Ziv-Welch:   ~ 5x compression)
    – 1cm x 1cm image at 16 bit = 2Mb (uncompressed)
    – other formats exist eg. SCN (used at Stanford University)

   Separate image for each fluorescent sample
    – channel 1, channel 2, etc.
Images in analysis software
   Typical experiment:
    –   “normal” state, Cy3-labelled sample (green)
    –   “perturbed” state, Cy5-labelled sample (red)
    –   hybridisation, then scanning
    –   overlay images  pseudo-colour image
    –   qualitative representation of results


         Image spot colour   Signal strength      Gene expression
         • yellow:           normal = perturbed   unchanged
         • green:            normal > perturbed   repressed
         • red:              normal < perturbed   induced
Quantitation process (1)
Accurate representation of signal for each spot
  and determine ratio channel1:channel2

   Determine spot boundary
    – construct grid (dimensions of array / spot size)
    – iterative process to find spots

   Measure signal
    – fluorescence
        • 8 bit = 256 shades
        • 16 bit = 65’536 shades
    – absolute output values vary from system to system
Quantitation process (2)
   Measure background
    – local (usually best)
    – selected region
    – selected spots / probes from different species

   Quality control
    – eg. fraction of pixels greater than background (ScanAlyze)
    – flag aberrant spots

   Determine ratio of signal strengths for each spot

           Ch1/Ch2 = (Ch1I-Ch1B)/Ch2I-Ch2B)
Normalisation
   Eliminate systematic variation

    – correct for
          • dye incorporation
          • print-tip effects
          • hybridisation efficiencies
          • etc.

   How?
   Use            STATISTICS!
Normalisation
   Simple example: green vs red


    Normgreeni = greeni     TotRed - TotRedB
                          TotGreen - TotGreenB
Normalisation
   More complexe:
    Within slide
       global (constant over the slide)
       spot intensity dependent (spot by spot)
       within print-tip group (group by group)
       scale (outlayers spread)
    Between slides
       paired-slides (dye swap)
    Multiple slides
       scale (slide outlayers spread)
Normalisation
Normalisation
   Example: Within slide print-tip group scaling




    Before scaling                    After scaling
Normalisation references
[statistics]
                       Normalization for cDNA
                           microarray data
                        Yang et al. (2001)
                          In preparation

            Statistical methods for identifying
        differentially expressed genes in replicated
               cDNA microarray experiments
                    Dudoit et al. (2000)
     Technical report #578 Berkeley Statistics Dept.

               Both PDFs are available from the web site of the course
Quantitation - problems
   Reference signal is close zero
    – channels ratio (Ch1/Ch2) tends to infinity


   Non-uniform background
    – “mean” background sometimes non-representative
        • bright particles
        • streaks on image

    – safer to use “median” (middle value)
        • less contribution by extreme values
Background problems
Background removal
Quantitation à la ScanAlyze

  signal




background
ScanAlyze output
 CH1I         ch1 intensity
 CH2I         ch2 intensity
 SPIX         number of pixels in spot
 CH1B         median intensity of the local background (recommended)
 CH2B         median intensity of the local background (recommended)
 CH1BA        mean intensity of the local background
 CH2BA        mean intensity of the local background
 BGPIX        number of background pixels

 Thus to calculate channel ratios:      Ch1   CH1I - CH1B
                                        --- = -----------
                                        Ch2   CH2I - CH2B
 Quality control:

 CH1GTB1      fraction of pixels in spot greater than background (CH1B)
 CH2GTB1      fraction of pixels in spot greater than background (CH2B)
 CH1GTB2      fraction of pixels in spot greater than 1.5 X background (CH1B)
 CH2GTB2      fraction of pixels in spot greater than 1.5 X background (CH2B)
 CH1EDGEA     mean magnitude of the horizontal and vertical Sobel edge vectors within spot 1
 CH2EDGEA     mean magnitude of the horizontal and vertical Sobel edge vectors within spot 2
HEADER   SPOT     GRID      TOP       LEFT      BOT       RIGHT    ROW       COL       CH1I       CH1B         CH1AB     CH2I       CH2B         CH2AB     SPIX
REMARK            S
         SOFTWAREcanAlyze
REMARK   SOFTVERS      2.24
REMARK   CH1 IMAGEDef143Cy3
REMARK   CH2 IMAGEDef143Cy5
REMARK   GRID FILEE:\MIcroarray demo examples\ScanAlyze\49_4.SAG
REMARK   DATE      16/08/99
REMARK   TIME      11:52:40
SPOT            1         1       138      119       154       135       1         1      12232          285       587      11467          486       556          208
SPOT            2         1       139      170       155       186       1         2      10301          299       546      10719          486       538          208
SPOT            3         1       139      221       155       237       1         3      14762          329       623      12740          475       534          208
SPOT            4         1       140      272       156       288       1         4       8881          301       522       7967          453       495          208
SPOT            5         1       141      324       157       340       1         5       9590          248       473       7744          415       456          208
SPOT            6         1       141      375       157       391       1         6      16150          268       580      10959          468       503          208
SPOT            7         1       142      426       158       442       1         7      36667          308       597      32749          536       582          208
SPOT            8         1       188      118       204       134       2         1       1125          311       512        854          476       516          208
SPOT            9         1       189      169       205       185       2         2       2440          287       509       3505          497       524          208
SPOT           10         1       189      220       205       236       2         3       1127          291       515        986          456       496          208
SPOT           11         1       190      272       206       288       2         4       1364          300       517       2393          440       479          208
SPOT           12         1       191      323       207       339       2         5        860          316       535        818          457       501          208
SPOT           13         1       191      374       207       390       2         6       2422          314       533       2488          548       588          208
SPOT           14         1       192      425       208       441       2         7       3685          270       520       3350          507       549          208
SPOT           15         1       238      117       254       133       3         1       1423          287       490       1556          499       549          208
SPOT           16         1       239      169       255       185       3         2       6571          274       526       6950          469       568          208
SPOT           17         1       239      220       255       236       3         3       2793          291       526       2886          497       535          208
SPOT           18         1       240      271       256       287       3         4       3309          295       518       3410          457       505          208
SPOT           19         1       241      322       257       338       3         5       7797          328       548       7062          572       619          208
SPOT           20         1       241      374       257       390       3         6      27862          340       575      30550          580       643          208
SPOT           21         1       242      425       258       441       3         7       2573          320       518       2272          543       578          208
SPOT           22         1       288      117       304       133       4         1       1154          292       519       4883          505       547          208
SPOT           23         1       288      168       304       184       4         2       2370          285       508       2022          485       584          208
SPOT           24         1       289      219       305       235       4         3       3166          295       517       7757          465       515          208
SPOT           25         1       290      270       306       286       4         4      10732          322       534       6458          485       522          208
SPOT           26         1       290      322       306       338       4         5      44583          383       680      46716          606       714          208
SPOT           27         1       291      373       307       389       4         6       5443          343       565       8866          590       637          208
SPOT           28         1       292      424       308       440       4         7      17012          347       553      29325          580       682          208
SPOT           29         1       338      116       354       132       5         1      32500          342       652      33559          503       587          208
SPOT           30         1       338      167       354       183       5         2      22714          312       547      23678          471       508          208
SPOT           31         1       339      218       355       234       5         3       2138          291       520       1857          443       473          208
SPOT           32         1       340      270       356       286       5         4       2361          331       532       1886          476       520          208
SPOT           33         1       340      321       356       337       5         5       3085          305       543       1619          584       786          208
SPOT           34         1       341      372       357       388       5         6       1222          364       566       1171          573       606          208
SPOT           35         1       342      423       358       439       5         7       3796          385       593       4295          626       677          208
SPOT           36         1       388      115       404       131       6         1       4385          304       545      14781          493       564          208
SPOT           37         1       388      167       404       183       6         2       8969          332       557      48525          487       580          208
SPOT           38         1       389      218       405       234       6         3       6398          289       494      16025          458       538          208
SPOT           39         1       390      269       406       285       6         4       8348          339       565       8107          490       554          208
SPOT           40         1       390      320       406       336       6         5      16648          348       630      16935          619       676          208
SPOT           41         1       391      372       407       388       6         6        988          343       565        812          558       593          208
SPOT           42         1       392      423       408       439       6         7       1347          340       570       1462          561       594          208
SPOT           43         1       438      115       454       131       7         1      17859          351       955      16601          501       890          208
SPOT           44         1       438      166       454       182       7         2       9499          292       502       9257          475       508          208
SPOT           45         1       439      217       455       233       7         3      34944          327       569      32059          457       636          208
SPOT           46         1       440      268       456       284       7         4       4143          329       542       5101          424       490          208
SPOT           47         1       440      320       456       336       7         5       1067          317       543       1271          575       606          208
SPOT           48         1       441      371       457       387       7         6        551          306       518        690          537       577          208
SPOT           49         1       442      422       458       438       7         7        514          254       500        598          542       577          208
SPOT           50         2       147      580       163       596       1         1      18086          251       513      13899          481       537          208
SPOT           51         2       148      631       164       647       1         2      28733          317       663      28563          457       582          208
SPOT           52         2       149      683       165       699       1         3       3060          299       502       3273          447       490          208
SPOT           53         2       150      734       166       750       1         4      10034          223       517       5959          407       552          208
SPOT           54         2       150      785       166       801       1         5       1945          227       449       1560          316       358          208
SPOT           55         2       151      836       167       852       1         6        594          196       434        426          333       363          208
Input files for Cluster
   Table: tab delimited text, 1 line/gene, 1 column/experiment

   Minimal table:




   Extended table:
Prepare data for Cluster
HEADER   SPOT     GRID      TOP       LEFT      BOT       RIGHT    ROW       COL       CH1I       CH1B         CH1AB     CH2I       CH2B         CH2AB     SPIX
REMARK   SOFTWAREcanAlyze
                  S                                                                                                                                                                HEADER   SPOT     GRID      TOP       LEFT      BOT       RIGHT    ROW       COL       CH1I       CH1B         CH1AB     CH2I       CH2B         CH2AB     SPIX
REMARK   SOFTVERS      2.24
                                                                                                                                                                                   REMARK            S
                                                                                                                                                                                            SOFTWAREcanAlyze




                                                                                                                                                                            Exp1
REMARK   CH1 IMAGEDef143Cy3




                                                                                                                                                                        
REMARK
REMARK
         CH2 IMAGEDef143Cy5
         GRID FILEE:\MIcroarray demo examples\ScanAlyze\49_4.SAG
                                                                                                                                                                                   REMARK   SOFTVERS      2.24
REMARK   DATE      16/08/99                                                                                                                                                        REMARK   CH1 IMAGEDef143Cy3
REMARK   TIME      11:52:40
SPOT            1         1       138      119       154       135       1         1      12232          285       587      11467          486       556          208              REMARK   CH2 IMAGEDef143Cy5
SPOT            2         1       139      170       155       186       1         2      10301          299       546      10719          486       538          208
SPOT            3         1       139      221       155       237       1         3      14762          329       623      12740          475       534          208              REMARK   GRID FILEE:\MIcroarray demo examples\ScanAlyze\49_4.SAG
SPOT            4         1       140      272       156       288       1         4       8881          301       522       7967          453       495          208
SPOT            5         1       141      324       157       340       1         5       9590          248       473       7744          415       456          208              REMARK   DATE      16/08/99
SPOT
SPOT
                6
                7
                          1
                          1
                                  141
                                  142
                                           375
                                           426
                                                     157
                                                     158
                                                               391
                                                               442
                                                                         1
                                                                         1
                                                                                   6
                                                                                   7
                                                                                          16150
                                                                                          36667
                                                                                                         268
                                                                                                         308
                                                                                                                   580
                                                                                                                   597
                                                                                                                            10959
                                                                                                                            32749
                                                                                                                                           468
                                                                                                                                           536
                                                                                                                                                     503
                                                                                                                                                     582
                                                                                                                                                                  208
                                                                                                                                                                  208
                                                                                                                                                                                   REMARK   TIME      11:52:40
SPOT
SPOT
                8
                9
                          1
                          1
                                  188
                                  189
                                           118
                                           169
                                                     204
                                                     205
                                                               134
                                                               185
                                                                         2
                                                                         2
                                                                                   1
                                                                                   2
                                                                                           1125
                                                                                           2440
                                                                                                         311
                                                                                                         287
                                                                                                                   512
                                                                                                                   509
                                                                                                                              854
                                                                                                                             3505
                                                                                                                                           476
                                                                                                                                           497
                                                                                                                                                     516
                                                                                                                                                     524
                                                                                                                                                                  208
                                                                                                                                                                  208
                                                                                                                                                                                   SPOT            1         1       138      119       154       135       1         1      12232          285       587      11467          486       556          208
SPOT           10         1       189      220       205       236       2         3       1127          291       515        986          456       496          208              SPOT            2         1       139      170       155       186       1         2      10301          299       546      10719          486       538          208
SPOT           11         1       190      272       206       288       2         4       1364          300       517       2393          440       479          208
SPOT           12         1       191      323       207       339       2         5        860          316       535        818          457       501          208              SPOT            3         1       139      221       155       237       1         3      14762          329       623      12740          475       534          208
SPOT           13         1       191      374       207       390       2         6       2422          314       533       2488          548       588          208
SPOT           14         1       192      425       208       441       2         7       3685          270       520       3350          507       549          208              SPOT            4         1       140      272       156       288       1         4       8881          301       522       7967          453       495          208
SPOT           15         1       238      117       254       133       3         1       1423          287       490       1556          499       549          208
SPOT           16         1       239      169       255       185       3         2       6571          274       526       6950          469       568          208
                                                                                                                                                                                   SPOT            5         1       141      324       157       340       1         5       9590          248       473       7744          415       456          208
SPOT
SPOT
               17
               18
                          1
                          1
                                  239
                                  240
                                           220
                                           271
                                                     255
                                                     256
                                                               236
                                                               287
                                                                         3
                                                                         3
                                                                                   3
                                                                                   4
                                                                                           2793
                                                                                           3309
                                                                                                         291
                                                                                                         295
                                                                                                                   526
                                                                                                                   518
                                                                                                                             2886
                                                                                                                             3410
                                                                                                                                           497
                                                                                                                                           457
                                                                                                                                                     535
                                                                                                                                                     505
                                                                                                                                                                  208
                                                                                                                                                                  208
                                                                                                                                                                                   SPOT            6         1       141      375       157       391       1         6      16150          268       580      10959          468       503          208
SPOT           19         1       241      322       257       338       3         5       7797          328       548       7062          572       619          208              SPOT            7         1       142      426       158       442       1         7      36667          308       597      32749          536       582          208
SPOT           20         1       241      374       257       390       3         6      27862          340       575      30550          580       643          208
SPOT           21         1       242      425       258       441       3         7       2573          320       518       2272          543       578          208              SPOT            8         1       188      118       204       134       2         1       1125          311       512        854          476       516          208
SPOT           22         1       288      117       304       133       4         1       1154          292       519       4883          505       547          208
SPOT           23         1       288      168       304       184       4         2       2370          285       508       2022          485       584          208              SPOT            9         1       189      169       205       185       2         2       2440          287       509       3505          497       524          208
SPOT           24         1       289      219       305       235       4         3       3166          295       517       7757          465       515          208
SPOT           25         1       290      270       306       286       4         4      10732          322       534       6458          485       522          208              SPOT           10         1       189      220       205       236       2         3       1127          291       515        986          456       496          208
SPOT           26         1       290      322       306       338       4         5      44583          383       680      46716          606       714          208
SPOT           27         1       291      373       307       389       4         6       5443          343       565       8866          590       637          208
                                                                                                                                                                                   SPOT           11         1       190      272       206       288       2         4       1364          300       517       2393          440       479          208
SPOT
SPOT
               28
               29
                          1
                          1
                                  292
                                  338
                                           424
                                           116
                                                     308
                                                     354
                                                               440
                                                               132
                                                                         4
                                                                         5
                                                                                   7
                                                                                   1
                                                                                          17012
                                                                                          32500
                                                                                                         347
                                                                                                         342
                                                                                                                   553
                                                                                                                   652
                                                                                                                            29325
                                                                                                                            33559
                                                                                                                                           580
                                                                                                                                           503
                                                                                                                                                     682
                                                                                                                                                     587
                                                                                                                                                                  208
                                                                                                                                                                  208
                                                                                                                                                                                   SPOT           12         1       191      323       207       339       2         5        860          316       535        818          457       501          208
SPOT           30         1       338      167       354       183       5         2      22714          312       547      23678          471       508          208              SPOT           13         1       191      374       207       390       2         6       2422          314       533       2488          548       588          208
SPOT           31         1       339      218       355       234       5         3       2138          291       520       1857          443       473          208
SPOT           32         1       340      270       356       286       5         4       2361          331       532       1886          476       520          208              SPOT           14         1       192      425       208       441       2         7       3685          270       520       3350          507       549          208
SPOT           33         1       340      321       356       337       5         5       3085          305       543       1619          584       786          208
SPOT           34         1       341      372       357       388       5         6       1222          364       566       1171          573       606          208              SPOT           15         1       238      117       254       133       3         1       1423          287       490       1556          499       549          208
SPOT           35         1       342      423       358       439       5         7       3796          385       593       4295          626       677          208
SPOT           36         1       388      115       404       131       6         1       4385          304       545      14781          493       564          208              SPOT           16         1       239      169       255       185       3         2       6571          274       526       6950          469       568          208
SPOT           37         1       388      167       404       183       6         2       8969          332       557      48525          487       580          208
SPOT           38         1       389      218       405       234       6         3       6398          289       494      16025          458       538          208
                                                                                                                                                                                   SPOT           17         1       239      220       255       236       3         3       2793          291       526       2886          497       535          208
SPOT
SPOT
               39
               40
                          1
                          1
                                  390
                                  390
                                           269
                                           320
                                                     406
                                                     406
                                                               285
                                                               336
                                                                         6
                                                                         6
                                                                                   4
                                                                                   5
                                                                                           8348
                                                                                          16648
                                                                                                         339
                                                                                                         348
                                                                                                                   565
                                                                                                                   630
                                                                                                                             8107
                                                                                                                            16935
                                                                                                                                           490
                                                                                                                                           619
                                                                                                                                                     554
                                                                                                                                                     676
                                                                                                                                                                  208
                                                                                                                                                                  208
                                                                                                                                                                                   SPOT           18         1       240      271       256       287       3         4       3309          295       518       3410          457       505          208
SPOT           41         1       391      372       407       388       6         6        988          343       565        812          558       593          208              SPOT           19         1       241      322       257       338       3         5       7797          328       548       7062          572       619          208
SPOT           42         1       392      423       408       439       6         7       1347          340       570       1462          561       594          208
SPOT           43         1       438      115       454       131       7         1      17859          351       955      16601          501       890          208              SPOT           20         1       241      374       257       390       3         6      27862          340       575      30550          580       643          208
SPOT           44         1       438      166       454       182       7         2       9499          292       502       9257          475       508          208
SPOT           45         1       439      217       455       233       7         3      34944          327       569      32059          457       636          208              SPOT           21         1       242      425       258       441       3         7       2573          320       518       2272          543       578          208
SPOT           46         1       440      268       456       284       7         4       4143          329       542       5101          424       490          208
SPOT           47         1       440      320       456       336       7         5       1067          317       543       1271          575       606          208              SPOT           22         1       288      117       304       133       4         1       1154          292       519       4883          505       547          208
SPOT
SPOT
               48
               49
                          1
                          1
                                  441
                                  442
                                           371
                                           422
                                                     457
                                                     458
                                                               387
                                                               438
                                                                         7
                                                                         7
                                                                                   6
                                                                                   7
                                                                                            551
                                                                                            514
                                                                                                         306
                                                                                                         254
                                                                                                                   518
                                                                                                                   500
                                                                                                                              690
                                                                                                                              598
                                                                                                                                           537
                                                                                                                                           542
                                                                                                                                                     577
                                                                                                                                                     577
                                                                                                                                                                  208
                                                                                                                                                                  208
                                                                                                                                                                                   SPOT           23         1       288      168       304       184       4         2       2370          285       508       2022          485       584          208
SPOT
SPOT
               50
               51
                          2
                          2
                                  147
                                  148
                                           580
                                           631
                                                     163
                                                     164
                                                               596
                                                               647
                                                                         1
                                                                         1
                                                                                   1
                                                                                   2
                                                                                          18086
                                                                                          28733
                                                                                                         251
                                                                                                         317
                                                                                                                   513
                                                                                                                   663
                                                                                                                            13899
                                                                                                                            28563
                                                                                                                                           481
                                                                                                                                           457
                                                                                                                                                     537
                                                                                                                                                     582
                                                                                                                                                                  208
                                                                                                                                                                  208
                                                                                                                                                                                   SPOT           24         1       289      219       305       235       4         3       3166          295       517       7757          465       515          208
SPOT           52         2       149      683       165       699       1         3       3060          299       502       3273          447       490          208              SPOT           25         1       290      270       306       286       4         4      10732          322       534       6458          485       522          208
SPOT           53         2       150      734       166       750       1         4      10034          223       517       5959          407       552          208
SPOT           54         2       150      785       166       801       1         5       1945          227       449       1560          316       358          208              SPOT           26         1       290      322       306       338       4         5      44583          383       680      46716          606       714          208
SPOT           55         2       151      836       167       852       1         6        594          196       434        426          333       363          208
                                                                                                                                                                                   SPOT           27         1       291      373       307       389       4         6       5443          343       565       8866          590       637          208
                                                                                                                                                                                   SPOT           28         1       292      424       308       440       4         7      17012          347       553      29325          580       682          208
                                                                                                                                                                                   SPOT           29         1       338      116       354       132       5         1      32500          342       652      33559          503       587          208
                                                                                                                                                                                   SPOT           30         1       338      167       354       183       5         2      22714          312       547      23678          471       508          208
                                                                                                                                                                                   SPOT           31         1       339      218       355       234       5         3       2138          291       520       1857          443       473          208
                                                                                                                                                                                   SPOT           32         1       340      270       356       286       5         4       2361          331       532       1886          476       520          208
                                                                                                                                                                                   SPOT           33         1       340      321       356       337       5         5       3085          305       543       1619          584       786          208
                                                                                                                                                                                   SPOT           34         1       341      372       357       388       5         6       1222          364       566       1171          573       606          208
HEADER
REMARK
         SPOT     GRID
                  S
         SOFTWAREcanAlyze
                            TOP       LEFT      BOT       RIGHT    ROW       COL       CH1I       CH1B         CH1AB     CH2I       CH2B         CH2AB     SPIX
                                                                                                                                                                                   SPOT           35         1       342      423       358       439       5         7       3796          385       593       4295          626       677          208
REMARK   SOFTVERS      2.24                                                                                                                                                        SPOT           36         1       388      115       404       131       6         1       4385          304       545      14781          493       564          208




                                                                                                                                                                            Exp2
REMARK   CH1 IMAGEDef143Cy3




                                                                                                                                                                        
REMARK   CH2 IMAGEDef143Cy5                                                                                                                                                        SPOT           37         1       388      167       404       183       6         2       8969          332       557      48525          487       580          208
REMARK   GRID FILEE:\MIcroarray demo examples\ScanAlyze\49_4.SAG
REMARK   DATE      16/08/99                                                                                                                                                        SPOT           38         1       389      218       405       234       6         3       6398          289       494      16025          458       538          208
REMARK   TIME      11:52:40
SPOT            1         1       138      119       154       135       1         1      12232          285       587      11467          486       556          208              SPOT           39         1       390      269       406       285       6         4       8348          339       565       8107          490       554          208
SPOT            2         1       139      170       155       186       1         2      10301          299       546      10719          486       538          208
SPOT            3         1       139      221       155       237       1         3      14762          329       623      12740          475       534          208              SPOT           40         1       390      320       406       336       6         5      16648          348       630      16935          619       676          208
SPOT
SPOT
                4
                5
                          1
                          1
                                  140
                                  141
                                           272
                                           324
                                                     156
                                                     157
                                                               288
                                                               340
                                                                         1
                                                                         1
                                                                                   4
                                                                                   5
                                                                                           8881
                                                                                           9590
                                                                                                         301
                                                                                                         248
                                                                                                                   522
                                                                                                                   473
                                                                                                                             7967
                                                                                                                             7744
                                                                                                                                           453
                                                                                                                                           415
                                                                                                                                                     495
                                                                                                                                                     456
                                                                                                                                                                  208
                                                                                                                                                                  208
                                                                                                                                                                                   SPOT           41         1       391      372       407       388       6         6        988          343       565        812          558       593          208
SPOT            6         1       141      375       157       391       1         6      16150          268       580      10959          468       503          208              SPOT           42         1       392      423       408       439       6         7       1347          340       570       1462          561       594          208
SPOT            7         1       142      426       158       442       1         7      36667          308       597      32749          536       582          208
SPOT            8         1       188      118       204       134       2         1       1125          311       512        854          476       516          208              SPOT           43         1       438      115       454       131       7         1      17859          351       955      16601          501       890          208
SPOT            9         1       189      169       205       185       2         2       2440          287       509       3505          497       524          208
SPOT           10         1       189      220       205       236       2         3       1127          291       515        986          456       496          208              SPOT           44         1       438      166       454       182       7         2       9499          292       502       9257          475       508          208
SPOT           11         1       190      272       206       288       2         4       1364          300       517       2393          440       479          208
SPOT           12         1       191      323       207       339       2         5        860          316       535        818          457       501          208              SPOT           45         1       439      217       455       233       7         3      34944          327       569      32059          457       636          208
SPOT           13         1       191      374       207       390       2         6       2422          314       533       2488          548       588          208
SPOT           14         1       192      425       208       441       2         7       3685          270       520       3350          507       549          208
                                                                                                                                                                                   SPOT           46         1       440      268       456       284       7         4       4143          329       542       5101          424       490          208
SPOT
SPOT
               15
               16
                          1
                          1
                                  238
                                  239
                                           117
                                           169
                                                     254
                                                     255
                                                               133
                                                               185
                                                                         3
                                                                         3
                                                                                   1
                                                                                   2
                                                                                           1423
                                                                                           6571
                                                                                                         287
                                                                                                         274
                                                                                                                   490
                                                                                                                   526
                                                                                                                             1556
                                                                                                                             6950
                                                                                                                                           499
                                                                                                                                           469
                                                                                                                                                     549
                                                                                                                                                     568
                                                                                                                                                                  208
                                                                                                                                                                  208
                                                                                                                                                                                   SPOT           47         1       440      320       456       336       7         5       1067          317       543       1271          575       606          208
SPOT           17         1       239      220       255       236       3         3       2793          291       526       2886          497       535          208              SPOT           48         1       441      371       457       387       7         6        551          306       518        690          537       577          208
SPOT           18         1       240      271       256       287       3         4       3309          295       518       3410          457       505          208
SPOT           19         1       241      322       257       338       3         5       7797          328       548       7062          572       619          208              SPOT           49         1       442      422       458       438       7         7        514          254       500        598          542       577          208
SPOT           20         1       241      374       257       390       3         6      27862          340       575      30550          580       643          208
SPOT           21         1       242      425       258       441       3         7       2573          320       518       2272          543       578          208              SPOT           50         2       147      580       163       596       1         1      18086          251       513      13899          481       537          208
SPOT           22         1       288      117       304       133       4         1       1154          292       519       4883          505       547          208
SPOT           23         1       288      168       304       184       4         2       2370          285       508       2022          485       584          208              SPOT           51         2       148      631       164       647       1         2      28733          317       663      28563          457       582          208
SPOT           24         1       289      219       305       235       4         3       3166          295       517       7757          465       515          208
SPOT           25         1       290      270       306       286       4         4      10732          322       534       6458          485       522          208
                                                                                                                                                                                   SPOT           52         2       149      683       165       699       1         3       3060          299       502       3273          447       490          208
SPOT
SPOT
               26
               27
                          1
                          1
                                  290
                                  291
                                           322
                                           373
                                                     306
                                                     307
                                                               338
                                                               389
                                                                         4
                                                                         4
                                                                                   5
                                                                                   6
                                                                                          44583
                                                                                           5443
                                                                                                         383
                                                                                                         343
                                                                                                                   680
                                                                                                                   565
                                                                                                                            46716
                                                                                                                             8866
                                                                                                                                           606
                                                                                                                                           590
                                                                                                                                                     714
                                                                                                                                                     637
                                                                                                                                                                  208
                                                                                                                                                                  208
                                                                                                                                                                                   SPOT           53         2       150      734       166       750       1         4      10034          223       517       5959          407       552          208
SPOT           28         1       292      424       308       440       4         7      17012          347       553      29325          580       682          208              SPOT           54         2       150      785       166       801       1         5       1945          227       449       1560          316       358          208
SPOT           29         1       338      116       354       132       5         1      32500          342       652      33559          503       587          208
SPOT           30         1       338      167       354       183       5         2      22714          312       547      23678          471       508          208              SPOT           55         2       151      836       167       852       1         6        594          196       434        426          333       363          208
SPOT           31         1       339      218       355       234       5         3       2138          291       520       1857          443       473          208
SPOT           32         1       340      270       356       286       5         4       2361          331       532       1886          476       520          208
SPOT           33         1       340      321       356       337       5         5       3085          305       543       1619          584       786          208
SPOT           34         1       341      372       357       388       5         6       1222          364       566       1171          573       606          208
SPOT           35         1       342      423       358       439       5         7       3796          385       593       4295          626       677          208
SPOT           36         1       388      115       404       131       6         1       4385          304       545      14781          493       564          208
SPOT           37         1       388      167       404       183       6         2       8969          332       557      48525          487       580          208
SPOT           38         1       389      218       405       234       6         3       6398          289       494      16025          458       538          208
SPOT           39         1       390      269       406       285       6         4       8348          339       565       8107          490       554          208
SPOT           40         1       390      320       406       336       6         5      16648          348       630      16935          619       676          208
SPOT           41         1       391      372       407       388       6         6        988          343       565        812          558       593          208
SPOT           42         1       392      423       408       439       6         7       1347          340       570       1462          561       594          208
SPOT           43         1       438      115       454       131       7         1      17859          351       955      16601          501       890          208
SPOT           44         1       438      166       454       182       7         2       9499          292       502       9257          475       508          208
SPOT           45         1       439      217       455       233       7         3      34944          327       569      32059          457       636          208
SPOT           46         1       440      268       456       284       7         4       4143          329       542       5101          424       490          208
SPOT           47         1       440      320       456       336       7         5       1067          317       543       1271          575       606          208




                                                           .
SPOT           48         1       441      371       457       387       7         6        551          306       518        690          537       577          208
SPOT           49         1       442      422       458       438       7         7        514          254       500        598          542       577          208
SPOT           50         2       147      580       163       596       1         1      18086          251       513      13899          481       537          208
SPOT           51         2       148      631       164       647       1         2      28733          317       663      28563          457       582          208
SPOT           52         2       149      683       165       699       1         3       3060          299       502       3273          447       490          208
SPOT           53         2       150      734       166       750       1         4      10034          223       517       5959          407       552          208
SPOT           54         2       150      785       166       801       1         5       1945          227       449       1560          316       358          208
SPOT           55         2       151      836       167       852       1         6        594          196       434        426          333       363          208




                                                           .
                                                           .
HEADER   SPOT     GRID      TOP       LEFT      BOT       RIGHT    ROW       COL       CH1I       CH1B         CH1AB     CH2I       CH2B         CH2AB     SPIX
REMARK            S
         SOFTWAREcanAlyze
REMARK   SOFTVERS      2.24




                                                                                                                                                                            ExpN
REMARK   CH1 IMAGEDef143Cy3




                                                                                                                                                                        
REMARK   CH2 IMAGEDef143Cy5
REMARK   GRID FILEE:\MIcroarray demo examples\ScanAlyze\49_4.SAG
REMARK   DATE      16/08/99
REMARK   TIME      11:52:40
SPOT            1         1       138      119       154       135       1         1      12232          285       587      11467          486       556          208
SPOT            2         1       139      170       155       186       1         2      10301          299       546      10719          486       538          208
SPOT            3         1       139      221       155       237       1         3      14762          329       623      12740          475       534          208
SPOT            4         1       140      272       156       288       1         4       8881          301       522       7967          453       495          208
SPOT            5         1       141      324       157       340       1         5       9590          248       473       7744          415       456          208
SPOT            6         1       141      375       157       391       1         6      16150          268       580      10959          468       503          208
SPOT            7         1       142      426       158       442       1         7      36667          308       597      32749          536       582          208
SPOT            8         1       188      118       204       134       2         1       1125          311       512        854          476       516          208
SPOT            9         1       189      169       205       185       2         2       2440          287       509       3505          497       524          208
SPOT           10         1       189      220       205       236       2         3       1127          291       515        986          456       496          208
SPOT           11         1       190      272       206       288       2         4       1364          300       517       2393          440       479          208
SPOT           12         1       191      323       207       339       2         5        860          316       535        818          457       501          208
SPOT           13         1       191      374       207       390       2         6       2422          314       533       2488          548       588          208
SPOT           14         1       192      425       208       441       2         7       3685          270       520       3350          507       549          208
SPOT           15         1       238      117       254       133       3         1       1423          287       490       1556          499       549          208
SPOT           16         1       239      169       255       185       3         2       6571          274       526       6950          469       568          208
SPOT           17         1       239      220       255       236       3         3       2793          291       526       2886          497       535          208
SPOT           18         1       240      271       256       287       3         4       3309          295       518       3410          457       505          208
SPOT           19         1       241      322       257       338       3         5       7797          328       548       7062          572       619          208
SPOT           20         1       241      374       257       390       3         6      27862          340       575      30550          580       643          208
SPOT           21         1       242      425       258       441       3         7       2573          320       518       2272          543       578          208
SPOT           22         1       288      117       304       133       4         1       1154          292       519       4883          505       547          208
SPOT           23         1       288      168       304       184       4         2       2370          285       508       2022          485       584          208
SPOT           24         1       289      219       305       235       4         3       3166          295       517       7757          465       515          208
SPOT           25         1       290      270       306       286       4         4      10732          322       534       6458          485       522          208
SPOT           26         1       290      322       306       338       4         5      44583          383       680      46716          606       714          208
SPOT           27         1       291      373       307       389       4         6       5443          343       565       8866          590       637          208
SPOT           28         1       292      424       308       440       4         7      17012          347       553      29325          580       682          208
SPOT           29         1       338      116       354       132       5         1      32500          342       652      33559          503       587          208
SPOT           30         1       338      167       354       183       5         2      22714          312       547      23678          471       508          208
SPOT           31         1       339      218       355       234       5         3       2138          291       520       1857          443       473          208
SPOT           32         1       340      270       356       286       5         4       2361          331       532       1886          476       520          208
SPOT           33         1       340      321       356       337       5         5       3085          305       543       1619          584       786          208
SPOT           34         1       341      372       357       388       5         6       1222          364       566       1171          573       606          208
SPOT           35         1       342      423       358       439       5         7       3796          385       593       4295          626       677          208
SPOT           36         1       388      115       404       131       6         1       4385          304       545      14781          493       564          208
SPOT           37         1       388      167       404       183       6         2       8969          332       557      48525          487       580          208
SPOT           38         1       389      218       405       234       6         3       6398          289       494      16025          458       538          208
SPOT           39         1       390      269       406       285       6         4       8348          339       565       8107          490       554          208
SPOT           40         1       390      320       406       336       6         5      16648          348       630      16935          619       676          208
SPOT           41         1       391      372       407       388       6         6        988          343       565        812          558       593          208
SPOT           42         1       392      423       408       439       6         7       1347          340       570       1462          561       594          208
SPOT           43         1       438      115       454       131       7         1      17859          351       955      16601          501       890          208
SPOT           44         1       438      166       454       182       7         2       9499          292       502       9257          475       508          208
SPOT           45         1       439      217       455       233       7         3      34944          327       569      32059          457       636          208
SPOT           46         1       440      268       456       284       7         4       4143          329       542       5101          424       490          208
SPOT           47         1       440      320       456       336       7         5       1067          317       543       1271          575       606          208
SPOT           48         1       441      371       457       387       7         6        551          306       518        690          537       577          208
SPOT           49         1       442      422       458       438       7         7        514          254       500        598          542       577          208
SPOT           50         2       147      580       163       596       1         1      18086          251       513      13899          481       537          208
SPOT           51         2       148      631       164       647       1         2      28733          317       663      28563          457       582          208
SPOT           52         2       149      683       165       699       1         3       3060          299       502       3273          447       490          208
SPOT           53         2       150      734       166       750       1         4      10034          223       517       5959          407       552          208
SPOT           54         2       150      785       166       801       1         5       1945          227       449       1560          316       358          208
SPOT           55         2       151      836       167       852       1         6        594          196       434        426          333       363          208
Software packages - quantitation
   ScanAlyze
    –   by Michael Eisen (Stanford University)
    –   quantitation of images
    –   no data visualisation
    –   free from http://rana.lbl.gov/

   ImaGene
    – BioDiscovery Inc.
    – quantitation and some data visualisation
    – demo from http://www.biodiscovery.com/

   plus many others - explore!
Making sense of raw data
   Difficult to see results in tabulated data

   Represent in graphical form


        Data visualisation
          examples
                 from
                     ImaGene and others. . .
Data visualisation - scatter plot
Data visualisation - M vs A




 log 2 R vs log2 G   M  log2 (R / G)
                     A  log2 ( RG)
Data visualisation - pie chart
ScanAlyze
 quick demo

				
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posted:3/14/2012
language:English
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