Image Quantitationin Microarray Analysis

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Microarray analysis Array construction, hybridisation, scanning

Quantitation of fluorescence signals

Data visualisation

Meta-analysis (clustering)

More visualisation

Technical

probe(on chip)

sample(labelled)

pseudo-colourimage

[image from Jeremy Buhler]

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 10m [currently, max 5m]– 100m 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

Normalization for cDNAmicroarray data

Yang et al. (2001)

In preparation

Statistical methods for identifyingdifferentially 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

[statistics]

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 outputCH1I 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 - CH1BCh1 CH1I - CH1B

--- = -------------- = -----------Ch2 CH2I - CH2BCh2 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 SOFTWAREScanAlyze

REMARK SOFTVERS 2.24

REMARK CH1 IMAGEDef143Cy3

REMARK CH2 IMAGEDef143Cy5

REMARK GRID FILE E:\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

Minimal table:

Extended table:

Table: tab delimited text, 1 line/gene, 1 column/experiment

Prepare data for Cluster Exp1

HEADER SPOT GRID TOP LEFT BOT RIGHT ROW COL CH1I CH1B CH1AB CH2I CH2B CH2AB SPIX

REMARK SOFTWAREScanAlyze

REMARK SOFTVERS 2.24

REMARK CH1 IMAGEDef143Cy3

REMARK CH2 IMAGEDef143Cy5

REMARK GRID FILE E:\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

HEADER SPOT GRID TOP LEFT BOT RIGHT ROW COL CH1I CH1B CH1AB CH2I CH2B CH2AB SPIX

REMARK SOFTWAREScanAlyze

REMARK SOFTVERS 2.24

REMARK CH1 IMAGEDef143Cy3

REMARK CH2 IMAGEDef143Cy5

REMARK GRID FILE E:\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

HEADER SPOT GRID TOP LEFT BOT RIGHT ROW COL CH1I CH1B CH1AB CH2I CH2B CH2AB SPIX

REMARK SOFTWAREScanAlyze

REMARK SOFTVERS 2.24

REMARK CH1 IMAGEDef143Cy3

REMARK CH2 IMAGEDef143Cy5

REMARK GRID FILE E:\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

HEADER SPOT GRID TOP LEFT BOT RIGHT ROW COL CH1I CH1B CH1AB CH2I CH2B CH2AB SPIX

REMARK SOFTWAREScanAlyze

REMARK SOFTVERS 2.24

REMARK CH1 IMAGEDef143Cy3

REMARK CH2 IMAGEDef143Cy5

REMARK GRID FILE E:\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

Exp2

... ExpN

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

log2R vs log

2G

€

M = log2 (R / G)

€

A = log2 ( RG)

Data visualisation - pie chart

ScanAlyzequick demo