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MAIA 2.7 User Manual

1. elv t x 1 Sp 1249 NoName Ratio Characteristic TypicgH70 1 99 9 This Quality Determination 1 D 4 3402 9402 Symmetry 3402 ISymmetry T 3402 CVRatios 3402 Select the toggle button aea e 5 ul ee 1 ABackground 3402 Manual Quality MIENNE pn Control acon Regression Plot Left Click selects the B soo spot Cy5 vs Cy3 intensity plot for the selected spot Regress ion Plot Block Independent see page Shift x v Shift Y Image Alignment s 2 Default w t TEE 5 ar DLL diit in D 1 T 018 e Bini GI ICCEIECCIISIIEIES ES a Iz 16483 4742 Y 403 X 370 Bl Y 1 X 1 Sp Y 12 X 9 Ho ID Localization 6 5625 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 42 Institut Curie Selected Spot 2 MAIA27 D Images Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help 6 amp Bim poco MOS oM ONE x Byix spQiz X9 NoName Ratio Characteristic Typical 0 195 99 995 This Quality Determination 1 DurbinWatson T 1 4 Contamination 3402 Diameter 3402 GSymmetry 3402 ISymmetry T 3402 hd iC Ratios 3402 Spot can be zoomed using t 5 5 either the Zoom spinner p se Signal 3402
2. Image Alignment Block Independent V save Default sv goi PR t leeosss MAAL eee y r JOODocsl L jeleloiel 1452 483 FIRA Bl Y X j xi Ho ID Localization 6 562 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 39 Institut Curie Localization Settings P MAIA27 D images Microarray CGH First rawdata0 20805 020805FR1087_cy3 bin File Run Options Window Help do Wim rco HOS Oo BR OIX Bx spi x No Name Show Hide Main Grid Acteristic Typical 0 1 This Quality rmination n J rbinwatson Contamination 3402 Diameter Symmetry 3402 ISymmetry 3 9402 iC Ratios 3402 Several settings that may 77 wl influence the localization fese poen Signal 3402 procedure are available at f LT EE ignment Quantfication Local Quaity the Menu Item Value Default E le nter Spot Volume 0 2 0 2 OptionslAnalysis Ease DDDBI Fiter on B b 0 2 s 2 0 2 0 Options Alt O tab z 1 0 1 a Localization See next page for details Image Alignment Block Independent Default v 1452 483 FIRA BI X Pap 91 7 Ho ID Localization 6 5625 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1
3. Microarray block inde Spot column coordinate within the block Spot row coordinate within the block Clone ID P MAIA27 D Images Wicroarray CGH Fir trawdata0 2080510 2080 Clone Name Fis Rin Options Window Help X coordinate of the spot center in pixels dl f Y coordinate of the spot center in pixels Y amp im Pc WE eo uu ONE Diameter of the spot Dia Ratio Rgn Ratio Ratio of Means Log2 of Segmentation Ratio Log Ratio User defined quality value Manual io 7 m n Flag of the bad spots 1 Flag lse 518 9 44 748 0 68 1 Pagg tiamo EAD oP eg L RU pata j Overall quality value Overall Quality i Coefficient of determination of the linear regression Determination User can define which LU E Dp BM em Corresponding quality parameter Q Determination Durbin Watson parameter of the linear regression residuals DurbinWatson fields and 1n which order fBackground ahi Corresponding quality parameter Q DurbinWatson Amount of flagged out contamination pixels Contamination should be pre sented in the RR 0 79 RS 0 772 Corresponding quality parameter Q Contamination Diameter of the spot Diameter output file select the Corresponding quality parameter Q Diameter z 1 Geometrical symmetry Symmetry Menu Item 2 Pa Corresponding quality parameter Q GSymmetry PL Intensity symmetry ISymmetry z 3 Options lOutput Format Corresponding quality parameter Q ISymmetry Coefficient of var
4. Characteristic Typical 0 195 99 9 This Quality e T co9Qooo0 eo0c0e8 Determination 0 93 0 05 1 0 99 0 99 eeenvesoooes L i EPI eee e o Durbinwatson 1 35 2 36 1 71 0 77 sae L HH ee H sse EHE REREN P t 6000000000002 000 Contamination p 11 B 0 58 800668006 III 6 86 944 o5 p ss 890996 0 558 0 52 0 81 299 1 i s ese _ 0 3 2 65 0 29 0 78 oo jo 4 b 0 14 o3 3 87 0 09 0 95 Quality characteristics of the spots wd 508 78 459 712 73 514 06 0 94 1820 68 10 5483 5 229 1 RR 0 766 RS 0 762 See next page for details eeeeeees ee000Ge e Quality 0 58 gt Manual A a 2 Reset Manual E Novikov and E Barillot Image Alignment An algorithm for automatic AIT Y Shift x 0 2 evaluation of the spot quality maT in two color DNA microarray experiments BMC BOIT Bioinformatics 2005 6 293 1408 459 Y 514 X 15 Bl Y 5 X Sp X uantification 2 265 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 64 Institut Curie Quality Characteristics in Detail Coefficient of determination CD of the linear regression indicates the degree of linear relationship between the intensities in Cy3 and Cy5 channels For higher quality spots relatively high values of determination coefficient 1 are expected Muc
5. 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 40 Institut Curie Localization Settings in Detail Inter Spot Volume represents Filter on Borders defines roughly the ratio of the filtering properties at the edges inter spot gap to the inter spot of the array Higher this value distance mn less sensitive the algorithm to Ano yes topHone the bright regions at the edges ern i Alignment Quantification Local Quality of the array Value Default Inter Spot Volume 0 2 0 2 Filter on Borders 0 2 0 2 Regularity Weight 2 0 2 0 ae Grid Refine Range 0 1 0 1 ee Regularity Weight controls Grid Refine Range defines contribution of the regularity Ok the range related to the components with respect to inter spot distance for the the intensity component in final grid lines adjustments the regularity parameter With the weight equals to 0 the regularity components will be ignored The default values of these parameters are suitable for a broad variety of experimental designs E Novikov and E Barillot A noise resistant algorithm for grid finding in microarray image analysis Machine Vision and Applications 2006 17 337 345 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 41 Institut Curie Spot Selection 2 MAIA27 D Images Wicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help 6 amp Nim rocho MM 4 e BE O fe x
6. color Bl Y 1 X 1 Sp Y 10 X 10 Ho ID Localization 6 562 5 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 31 Institut Curie Brief Help on Manual Correction Brief help on the manual correction possibilities is available at the Menu Item HelplManual Grid Info 11 Oct 06 P MAIA27 D images Microarray CGH First rawdata0 20805 020805FR1087_cy3 bin File Run Options Window Help 6 eco MES o mlm o DEAR E BI Y X 2 Qn Characteristi 0 1 99 995 Quality Make Selection Shift Double Click Select all Grids Shift Click Select a Grid Shift Home End PgUp PgDn Select and iterate through the Grids Ctrl Click Select a Line Ctrl Home End PgUp PgDn Select and iterate through the Lines Alt Click Select a Cut Alt Home End PgUp PgDn Select and iterate through the Cuts Click without Shift Ctrl lt Clear selection Shift Ctrl Alt Delete Clear selection Move Selection by Pixel Shift Ctrl lt Mouse Dragging Move Selection by Pixel Shift Ctrl lt Arrows Up Down Right Left Move Selected Grid by Spot Shift Ctrl Arrows Up Down Right Left UnDo Moving Ctr1l zZ ReDo Moving Ctrl4Shift 4Zz il 3 ERS Save Default w E E iit 18 qual 0189 agii Fal E 452 483 Sp Y X No 10 Local
7. Fit Limits 0 171 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 83 Quality Plot with Replicated Spots 9 MAIA27 D lmages Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d ME pco HOS o He ORERE Bl vi0 X 0 Sp v 9 X 7 NoName 99 995 This Quality coQco o eense8o G eo0c0esce oeeo LJ S Pseeee een ope geooses CAOOCCOHEBEOEEO 2000 bohm 0 cDeooe T le 86 Each dot represents a replicate with the overall quality value at y axis and ratio variation coefficient Lu es Manual 0 85 7 CV of the replicates at x mcm axis Image Alignment oeooeoe e eeneeos Block Independent V 7 5 Shift X 0 2 9 wsxis shifty 04 L4 114444 CPLLL TIRE See next page for details 0 22 Quali Quality Plot PILLILE OGSOECOeCCeOD engece eeoeecneeoeo eeceneee 075 Ratio CV lt 445 504 Bl Y X Sp Y Xi Ho ID Fit Limits 0 171 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 3 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 84 Institut Curie Spot Quality Fit The weights w for the overall quality parameter Q see page can be estimated using replicated spots on the same array or over a set of r
8. for changing both limits d H DWS D AB and or typical Hd H ze ee Quality 0 58 o e II oe value D AB S wewa 1 is s EE 2 Reset Manual s 00 008 ee t O00 s HE a9 Image Alignment e 4 te e Block Independent V LIII Hj o 3 ee ee Limit adjustment should BR cg x s w Shifty 018 be continued until all gt Defaut v 1 Y55 X 13 spots visually classified as zt x1 7 HE EHRTHHEHRHEHEE bad spots are flagged 1428 484 Y 190 X 20 out Bl Y 0 X 0 Sp Y 15 X 0 t Quantification 2 2655 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 71 Institut Curie Default Limits P MAIA27 D lmages Wicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d ME pco ENEN 4 e dm ORDO MISX e 123 Bizx2 senxa N Ratio 7 d Charact 0 1 99 9 Contamination 0 Diameter LEETE CEKA EE e20 008280 Secocoeceedesd 000600690 DEIIIIII C Ratios The default limits can be restored using the Init Limits button from the een Toolbar or the Menu Item the table header RunlInit Limits iur Goose Ctrl F9 boose Tes ecoo eo LE L ONES 0000000000 H The default limits for each Suy 05 l MEEIDEHTHHHHRES z E Manual AZ ooeoooo o quality characteristic are the Lees E
9. Ho Name Ratio Typical 0 1 99 995 This Quality 1 4 9402 3402 oS NEM n 3402 Array Design To start image m oem processing array design p eckoround j UR Default v New Delete Change Name pma 3402 GAL File should be properly Regression Pict o defined use the Array Design button from the 8 Block Design Distance Between Replicas Toolbar or select the rien V tE Menu Item Blocks in Blocks OptionslArray Design E a EN B Spots in Spots Alt A Y 21 212 Y os Image Alignment Spot Diameter Replicas See next page for details used 105 pixels X v m UE i 1445 501 Y 352 X 546 Bl Y 5 X X Ho ID Design 1 1 4 4 21 21 10 0 0 0 0 1 1 1 Currently used Array Design 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 19 Institut Curie Array Design in Detail One may use several microarray designs under different names to be able to switch quickly from one design to another Amount of sub arrays blocks per sub array and spots per block in Y and X directions of the array ee Spot diameter may be used as a prior value in spot localization and spot quantification procedures 11 Oct 06 Array Design Design Default GAL File C GAL Block Design SubArrays Y Blocks Y Spots Spot Diameter C used
10. Image Alignment Block Independent V Shift x 0 28 v Shift Y 018 No Name Ratio v 9 97 ll 3 97 E LE LETTIE EI CIIIEI ia oS oti A IN L EG ITE fon 13 FE Ea a8 E IX LA ITI E DE E E 3 E I ZIEL a 3i LITE LI Js su III E m d c C A s E Li Je Las H LEE EH ZEN EO goap 86 56 NIII NS AE LII EEL TERREN Save Defaut v 1 Quit 012 _ 12473 1693 Y 96 X 275 Bl Y 0 X 1 l Sp 7 X 0 Ho ID Ho Hame Quantification 2 2655 Design 1 1 4 4 22 21 10 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 74 Institut Curie Groups of Spots for Manual Qualification 9 MAIA27 D lmages Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help Groups of spots can be ER CNET INN A selected for manual siv 1 x 2 Sp 0 X 17 Mo Name EE Sess Characteristic Typical 0 199 99 995 This Quality ai T M 5 j ENSESE M qualification gt b ted dede C UT Contamination O0 o m b s s Diameter amp ss o ba o oi ie 4 Spots can be added into wb P mi the group one by one eek ees bs TTTTITITT TT oe j j pe rr a 8b 6444442014 Shift Left Click or E eeceeegeeseee 066 several at once C
11. the Fit Limits button Boro x0 op Characte from the Toolbar or by ertt ILI the Menu Item RunlFit PEE E ES i 0295595 i Pi x 1 i Pe 604 Limits Ctrl F10 Diameter 6 86 be 26 4s pz ur TDA 33 vert alate H eto CVRatios des HE as i Quality fit estimates the 38 Sis MH H limits of the quality a characteristics such that M Manuali 0 858 7 Reset Manual o the spot overall quality is TELIA H Block Independent aligned along the user z defined quality curve D mis J sev og 0 22 Qua 0 25 Quality Plot soscot ecoeo Sestetetoctoco t ee epeceted RRRPPPMPS HAE e L4 4444 oto tbootttooocccce SPST 44444424 eves L4 4 4 Before fitting it is advisable to restore the default limits the Init Limits button from the Toolbar or the Menu Item oo os 10 15 20 25 30 In Manual RunUnit Limits Ctrl F9 eo8eetttoo Tod ERES P GE R PER eoceecoe 1471 527 Y 37 X 257 Bl Y X pki Ho ID Fit Limits 0 171 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 81 Institut Curie Fix the Limits Certain limits can be fixed so that they are not changed by the fit The fixed quality characteristics are shown in italics 11 Oct 06 File Run Options Window Help d d MH Palo wo ee O RT IRL o e Bl vi0 X 0 Sp v 9 7 NoName C
12. The selected replicates are highlighted on both arrays The first array 021026 Manual Reset Manual Image Alignment Block Independent V T e e e o I Default 3l OC gt IE du EB 3 bre OCEA eje gog lt EE lolalelal REIR DEDE 0 2 2 Quatt 0 25 EEE z 1310 395 Ho ID Design 1 1 4 4 22 21 0 0 0 0 0 1 1 3 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 122 Institut Curie Image Simulator MAIA27 Experiment 1 bin File Run Options Window Help i d d m reo HAs o ele O a Bi X Sp Y Xi Ho Hame haracteristic Typical 0 195 99 995 This Quality Determination 1 DurbinWatson 4 Contamination 3402 Diameter 3402 GSymmetry 3402 ISymmetry 3402 1 CVRatios 3402 To open Image Simulator M Window select the Menu E TR ignal ni Item RunlSimulator F Regression Plot AEE j X Ho ID eee l Design 1 1 4 4 21 21 10 0 0 0 0 1 1 1 nnii 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 123 Institut Curie Main Simulator Window To start simulations press the Run Simulations button from the Toolbar or select the Menu Item FilelRun Simulations F5 To simulate an image the following parameters should be defined Image 0 stands for Cy3 ima
13. 11 Oct 06 P MAIA27 D lmages Wicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d ME eco HAS ommum ONERE w e Bvo x0 sp v 9 X 7 NoName 99 9 o Contamination 0 0 Diameter 16 86 1 19 C Ratios Quality Manual Image Alignment Block Independent Shift x o00eese00 200000806 eeooocosesed eas000c0ncese00 LLLI ad seouuwvs 200 oooseeese eeeDooccese eotenese0e enpeoseeeose reo 0 25 0 50 T2 LLITITIPITIII 0e00e0esede eo oceceesoe IIIIITI eGece000 5880 etreo G0eseGe0ee800 16060 00 0 008800 860 SGe0neeseoeonee 2e0e0800080880 DEME LEJ Seee00e0001880 eeecoe rece eeeznose oce ottteceeccecottt 0000000000 ececee eseateseoe 200 WALIZIPIILJ Ratio CV 1436 547 Bl Y X Sp Y Xi Ho ID Y 7 X 617 Fit Limits 0 75 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 3 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 88 Optimize the Quality Limit P MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help b b Wim eco BE 4 E OREBHIX e 14 B v0 X0 sp s x7 L LASE EEE I EI RT HMTTT e eosecoete r48 p 92 KELLEDE LEET EL e20800 SGeceesoeoned 26
14. 7 and for each sector the gs of pixels belonging to the spot N i 1 8 and to the circle Np i 1 8 is counted Then the quality characteristic is defined as GS DIN NA N For ideal circular spots GS must approach 0 whereas highly un circular spots should give relatively high GS values q GS exp GS Intensity symmetry of the spot is defined as IS XII I I where I i 1 8 are the mean intensities for the same 8 sectors and J is the mean intensity for the whole spot A spot may have perfect circular shape but within this circle very bright or dark and highly concentrated groups of pixels originated from the pieces of dust or other contamination may occur q IS exp IS Coefficient of variation of two ratio estimates CVR 2 RR RSW RR RS Despite the differences in the estimation the variation between the two obtained ratios RS and RR should be as small as possible Large variation would indicate a problematic spot q CVR exp CVR Uniformity of the background around the spot i e along the grid lines separating neighborhood spots is defined as UB YIB BI B where B i 1 8 are the mean intensities in 8 sectors of the grid line around the spot and B is the mean intensity for the whole grid line around the spot Extremely small values may be due to relatively bright contamination around the spot large variability in the background or merged neighborhood spots q UB exp UB A
15. D 1 9402 3402 3402 Under mouse b Bu 9402 coordinates of the block i E p pe 3402 Bl spot Sp clone ID I oeer and clone Name NII Clone IDs and clone names are available from GAL files i Sg oko os abo Cy3 Mag Image Eas Block Independent V 1448 515 Ho ID Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 24 Institut Curie Spot Localization Output Main Grid P MAIA27 D Images Microarray CGH First rawdata0 20805 020805FR1087_cy3 bin File Run Options Window Help d d BE Pco MB 6S OIN x Bl Y X EE T Ho Hai p bie p Determination DurbinWatson Contamination lt a zi COUOEBEBBBBBE x E 1m p Using the Toolbar buttons n sala lelaelels se eleele tata NS REN d E y mu BERR BBA is T IDEBBBODO Show eae s const 2 or Show Hide Adjusted 6 Mud R MEE co ede m BEHREEEE grid one can mask either of two spot localization grids e el e e IE j9 IL nosed lelol e Main Grid is shown ar IESES ETCTCTI au fe ioiniiin z est eal tee ee 2 OOS SB00000000 0 Sele elerelesreters gite DEHBEBREROPERREIRIE l iiiki Isieisisissisie e Lokissisese sess ogi O0OQOCOCODI g lel IDICIDCOI AA e Block I
16. Histogram of Ratios Cikamiri b 2500 00 k 12500 00 Diameter z M A 2000 00 L 1500 00 ip T6000 The Toolbar button aiz a a inno 3 so0000 Show Hide Bad ia LM abd M J t 0 00 mii Spots allows one to Ass c CESS UO MAUCSR AH C33 Rx GEO EN d 5 000 10 000 15 000 20 000 show hide bad spots on oon ee 2g h z 1 c i Counts Ratio rCy5 Cy3 the diagnostic p ots Linear Log2 O Linear Log2 Linear O Log2 Linear Log2 M A Plot Ratio vs Intensity If the button is on all spots are used to build up the diagnostic plots and SP 6 00 bad spots are indicated in ees EE 2 085 orange Manual 4B 8 If the button is off only good Image Alignment S spots are used to build up the amp 31000 diagnostic plots wl shirt x 2 145 00 lt 3 Shift Y 041 2 2 500 5 000 7 500 10 000 g Y 7 Xi zd Save Defaut Cy3 Cy5 In M 0 05 Quali 022 E 0 25 50 75 Ratio Cy3 Cy5 Bl Y 0 X Q Sp Y 1 Xj O Fit Limits 0 828 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 log2 Cy3 Cy5 2 Linear Log2 liner Log2 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 98 Institut Curie Spot Selection on Diagnostic Plots 2 MAIA27 D mgeecWicrnarraw CGH Firet rawdataN 20 RO 5VI20RORER1OR7 cw3 hin DEAR Fie Run Options PESMIM ETTI ei Sibi x uw Quality Histograms Results Plots 3000 00 j
17. See next page for details Quality characteristics of the selected spot Estimation of the signal and background 11 Oct 06 P MAIA27 D images Microarray CGH First rawdata0 20805 020805FR1087_cy3 bin File Run Options Window Help Mm pco MES oe IGI mm x Ol 11 Bi 1 1 Sp 12 9 No Mame Characteristic Typical 0 1 99 9 This Quality Cluster I 0 71 1 1 Contamination a 748 0 6 0 7 10 72 0 41 0 78 0 14 0 95 475 58 s 492 04 0 84 2270 07 E e ji Quality 0 6 Manual Back 492 04 0 00 hatin A 18 Reset Manual Signal 4890 8 9 0 00 0 25 0 50 075 1 00 1 25 Signal CV lt D Images Microarray CGH Firstir ve Bl Y 15 X 1 Sp Y 12 X 9 Ho ID Y 404 X 369 uantification 1 516 s Desion 1 1 4 4 22 21 40 0 0 0 0 1 1 3 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie Quality Characteristics Cluster C is the ratio of the diameter of the largest cluster of bright pixels of the spot to the diameter of the spot For low intensity spots segmentation procedure may identify many non intersecting pixel clusters with the average intensity somewhat higher than the background level The parameter C is expected to be low for such spots g C C Spot contamination is a number of out ranged pixels with the int
18. ae sud e ae Ctrl Home Ctrl End Ctrl PgUp Ctrl PgDn Move Selection by Pixel s ssssose Ctrl Drag ae e E je oOgooooo Ct r 1 f 4 lt Block Independent Clear Selection Mouse Click Ctrl Del Upon selection the line changes the J 1490 514 color BI Y 1 X 1 Sp Y 9 X 11 Ho ID Localization 6 562 s pesion 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 30 Institut Curie Manual Correction of the Adjusted Grid 3 MAIA2T D Images Microarray CGH First rawdata0 20805 020805FR1087_cy3 bin File Run Options Window Help If a separation cut Bm sa ME Mm OR between the Bx Sb X3 NoName Ratio Characteristic Typical 0 195 99 995 This Quality neighborhood spots is lcs Durbinwatson T erroneous one can Contamination perform manual Diameter GSymmetry correction of the selected CVRatios cut position RBackground ABackground Signal Regression Plot Select a cut and iterate through the cuts Alt Left Click Alt Home Alt End Alt PgUp Alt PgDn Move Selection by Pixel Alt Drag Image Alignment Alt 7 434 Block Independent 7 Clear Selection Mouse Click Ctrl Del EN L ogia Upon selection the cut changes the 11956 1744 Y 383 X 380
19. i 020805FR321673_dapitit 020805FRE24_cy3tit A P o20805FR326671_cy3tit 020805FR824_cy5 tif i 1 1 E d e _cy5tit _dapiti fields and in which order He ection pe should be presented in the o ns ll ie suicida Filename 020805FR1087_cy3_res tt output file select the a mesora urs Menu Item f OptionslOutput Format so Hic 0 00 0 25 0 50 075 Alt F Signaicv ms Bl Y 5 X Sp Y X Ho ID Fit Limits 1 484 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 3 See next page for details 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 143 Institut Curie Output Table Format Description of the field non editable Editable name of the field to be appeared in the output file Order specifies the sequence of the fields If this field is empty the corresponding field is not included in the output file Include all fields Exclude all fields Restore previous set of fields 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 Output Format Institut Curie Overall Quality i Cluster Q Cluster LL Contamination Q Contamination Diameti Q Diameter Symmetry RBackground ABackground ABackground Q ABackground Signal 144 Batch Processing and Global Quality Analysis To run batch processing Batch Processing and one have to define Global Quality Analysis actions to be applie
20. O 3402 Regression Plot 1 00 0 75 2 amp 0 50 0 25 0 00 T r a r 0 00 0 25 0 50 0 75 1 00 Cy3 Le JL 1 Image Alignment Block Independent V rL IET AI v Ir EH IEE M 2 Default sv LUDUM Tere IDEO 1 15 018 19 9 616 JOD OOCISIBIBI LEES 1452 483 Y 3 X 131 BI Y X Sp X Ho ID Ho Hame Localization 6 5625 MAIA 2 7 Manual Copyright C 2005 2006 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 35 Institut Curie Manual Correction Hints If spot localization is satisfactory there is no need to perform grids in block lines refinement and cuts refinement This is already done by the spot localization procedure However if grids were misplaced and manual correction has been performed then either of grids in block or lines refinement or cuts refinement may be necessary It depends on the manual correction If the main grid is misplaced only the external lines of the grid i e the first and last lines of the main grid can be adjusted and the grids in block will put all the other internal grid lines in between the external grid lines If internal lines of the main grid are misplaced then only these lines can be corrected and the lines refinement puts them in the refined positions Finally if cuts of the adjusted grid are wrong then after their manual correction the cuts refinement can be performed 11 Oct 0
21. box or the mouse wheel Regression Plot 5 000 6 000 Brightness and contrast are copied from the whole image window so that the spot appearances are consistent f image Alignment Contrast and Block Independent V Shift x om Brightness controls can e sav o be used to further adjust Le orn J pr brightness and contrast of E 013 mier 16483 4742 Y 403 X 370 the selected spot Bl Y 1 X 1 Sp Y 12 X 9 Ho ID Localization 6 562 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 43 Institut Curie Image Alignment 2 MAIA2T D Images WWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help There may be relative shift 7 77 as pce MB Mila o Rima x between the Cy3 and Cy5 sex Tsozx9 nonme p images The performance o e an e a of the quantification VON m procedures can be a increased if the two p BE images are aligned oe Use the Image Sos p po Alignment button from the Toolbar or the Menu ME Item RunlImage dr steal Alignment Ctrl F7 to ie us align images vlo Jins Image Alignment Block Independent V The shift value may be the ae T slelelsle i Paigaieige same for all blocks on the Save Defaut v
22. 1 00 LJ 0 75 2 E LJ 0 25 9 LJ 2 0 00 0 00 0 25 2 50 0 75 Tee c Cy3 o LJ o ILS JL 1 oA E 9 Image Alignment DIEI eeece 9099090 92 v lt gt Y 0 X 0 ig 0 18 11531 1285 Y 400 X 60 BI Y X Sp X Ho ID Ho Hame Design 1 1 4 4 21 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 17 Manual Pixel Flagging II Ctrl Left Click within a contour effaces this contour Double click on the image effaces all contours 11 Oct 06 9 MAIA27 D lmages Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help Bu co E EI 2 Bl i x Ssp nx NoName 99 9 This Quality Characteristic Typical 0 1 Determination 0 Durbinwatson 1 4 Contamination 3402 3402 492 3402 8402 3402 3402 3402 Regression Plot Image Alignment eeceeeereoerese eocernece LLLI 12224 2026 Bl Y X 3 413 X 141 Design 1 1 4 4 21 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 18 Array Design 2 MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin DEAR File Run Options Window Help 6 6 m o HI 4 cB O Alea X Bl f X Sp Y X
23. 2005 2006 Institut Curie 14 Color Swap By default green color is used for the Cy3 image and red color for the Cy5 image This assignment can be inverted by the Menu Item FilelSwap Colors 11 Oct 06 2 MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help Em P Go JAE DR BI Y x Sp Y X Characteristic Typical 0 195 99 9 This Quality Determination 0 DurbinWatson o M 1 Contamination 8492 3402 3402 3402 psn2 3402 3402 3402 Regression Plot Image Alignment Cs Seeceeososeececteece ee e009 1431 518 Y 120 X 4 Design 1 1 4 4 21 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 15 Image Zoom Image can be zoomed using either the Zoom spinner box or the mouse wheel Negative values of the zoom indicate contraction positive values indicate stretching Original image is obtained with either 1 or 1 zoom Zoom does not influence the analysis 11 Oct 06 2 MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help ld Nw iG Q0 i er s EE amp DAR Bee Sp Characteristic Typical 0 1 99 9
24. 3402 3402 Select a grid and iterate through the grids Shift Left Click Shift Home Shift End Shift PgUp Shift PgDn Move Selection by Pixel Shift Drag Shift 74a Move Selection by Spot Shift Ctrl 72 43 Clear Selection Mouse Click Ctrl Del Regression Plot oe ee BSOOCS00000000mEm B SSO000008EB0000050 EEREEENMAEEREEE EE IBGCODOODOpOOgOOoODaadanmaagnn RSSeSe Li ies fels etetelotsisisl Tt ttt ty Image Alignment Block Independent V Default sil 01 Ji BEER EB HHHBEBBEBE iaip dios ol Inn sss m BB GB CHEESE uo i BIEBCOCCOCCOOOOOOOOHOOS 1146 870 Y 363 X 382 Upon selection the grid changes the Bl Y 1 X 1 Sp Y 8 X 10 Ho ID color 11 Oct 06 Localization 6 5625 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 28 Manual Correction of the Main Grid Multiple Grids Selection TS ImagesWMicroarray CGH First rawdata020805 020805FR1087_ ce bin File Run Options Window Help d d ME pco MOS M ORF x BI Y X Sp Y Xt No Hame Ratio Characteristic Typical 0 1 99 9 This Quality Determination DurbinWatson Contamination Several grids can be selected using Shift Left Click ele ele eleleio E jel Ir
25. 508 78 459 712 73 516 13 0 92 p Signal 1820 68 10 5483 5 15 1 Contour one can control RR 2083 R50 37 whether the spot contours are visible Cy3 Quality 0 48 Manual AB Reset Manual Image Alignment Block Independent V eee v Shift Y 018 lt gt 7 save Default w Y55 X 13 swe 12 Quait 0 18 n 12270 2210 Y 371 X 339 Bl Y 1 X 1 Sp Y 9 X 6 Ho ID Quantification 2 2655 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 57 Institut Curie Background Contours Using the Toolbar button Show Hide Background Contour one can control whether the background contours are visible Segmentation procedure creates two contours pixels within the spot contours are used to estimate the signal Sc 55 4 pixel outside the background contours are used to estimate the background Bc Bc and pixels that are between the two contours are ignored 11 Oct 06 2 MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d ME pco HAS o He OR Ge lex DR BI Y 1 x 1 Sp 12 9 NoName Characteristic Typical 0 1 99 9 This Quality Determination 0 98 0 05 0 99 0 99 DurbinWatson 1 35 0 51 j2 6 Contamination e i le 68 0 94 1 51 o s4 ees 0 29 0 7
26. Alt 0 5 Back N S gt Radius 2 5 Density Restore Defaults Shift X Shift Y 10000 1000 553 1000 65535 To save the generated images in the TIFF files use the Export Image button from the Toolbar or the Menu Item FilelExport Image Alt b NoiseToSignal 0 1 NoiseType Proportional Seed 123455 Only single page TIFF files are currently supported Simulation finished 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 132 Institut Curie Artificial Images Model for a spot The generated spots must have more or less circular contours in the horizontal projection and relatively sharp edges in the vertical projection ee Ee Te Tee Dl where x and y are the coordinates of the center of the spot r is its approximate radius and is the fluorescence intensity in the center of the spot in the Cy3 color channel Fluorescence intensity in the Cy5 color channel is defined as fos Y Rf eG y where R is the ratio of the test and control samples for each spot The coordinates x and y the radius r and the ranges for x and y for each spot cell are defined from the user established array layout The intensity parameters and R should also be provided by the user Nonspecific hybridization results in an additional component 5 in the detected fluorescence intensity JEY fi x y B The number of non specific molecules contributing into each scanned fluorescenc
27. Manual 0 51 Found Clone Reset Manual Image Alignment Cancel Restore Defaults Block Independent v T PET T4 g Sx 022 lt gt Shift Y 012 t Y 7 x 12 Save Default v ENT 0 05 2 Qualt 028 dde et wis i 1448 540 Y 0 X 317 Bl Y 5 X X Ho ID Fit Limits 0 828 s Design 1 1 4 4 22 21 10 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 108 Institut Curie Batch Processing To run batch processing a list of actions to be applied to files in the batch should be defined SO Set Options SL Spot Localization IA Image Alignment SQ Spot Quantification FL Fit Limits SA Save Analysis The batch processing can be started using the Run Batch button from the Toolbar or the Menu Item Run Run Batch F5 11 Oct 06 EEk MAIA27 Barch ucfaulte MAIA Fie Model Run Options Help b sols i so F sa Visible D Images Micros ray Q20805FR1087 cy3 tiF D20805FR326671 cy D20805FR9216 cy3 tif Design 1 1 4 4 21 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie After the first processing images with the obtained results grid parameters settings etc are saved on the disk in the internal binary format experiment files If the program is unable
28. Signal 1820 68 10 5483 5 415 1 RR 0 83 RS 0 837 5 000 mE Quality 0 48 Manual AT Reset Manual Image Alignment Block Independent V Shift x 022 Shift Y 042 lt gt 7 Save Defaul w 1 Y55 X 13 Anm 12 Quit 018 16806 5313 Y 402 X 371 BI Y 1 X 1 Sp Y 12 X 9 Ho ID Ho Name Quantification 2 2655 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 55 Institut Curie Ratio Estimation in Detail Segmentation Ratio This approach is based on isolation of the spot pixels from the background pixels surrounding the spot Once this is done the quantification procedure is fairly straightforward one can compose the following ratio Scys Beys AS Scys Boys where S cys Cy 3 is the mean estimate of the intensity within the contoured spot in the Cy5 Cy3 channel and Bes Be j is the mean estimate of the background level in the Cy5 Cy3 channel Mean estimates are known to be more precise but they can be very much affected by the outliers Since regression filtering eliminates outliers we can safely use mean estimates for the spots Regression Ratio In this approach a ratio can be represented as a slope of the linear regression line of the pixel intensities in say Cy5 channel versus Cy3 channel The main advantage of this method is tha
29. 2005 2006 62 Institut Curie Quantification Settings in Detail Visible spots may have Spot pixels with excessively several more or less well high or low intensity with defined intensity levels respect to majority of the spot Intensity Levels specifies how FRAPS nine pixels are discarded The many such levels should be Localization Algnment Quantfication admissible range is defined as identified at the spot Spots x EN ER TELE median of spots pixels will be segmented at the ters Levels i n n inter quartile distance of highest level of intensity Signal Outlier 0 01 0 01 the spot pixels 1 35 where n Regression Top Outlier 0 01 0 01 2 l pV and p is a user Regression Side Outlier 0 1 0 1 a defined Signal Outlier confidence limit This filterin Outlier Limit Top Sides UM procedure is appropriate for i defines critical p values of the the spots with large amount of F statistics in the detection of pixels the pixel outliers selected from the top of the intensity ranges and from the sides of the linear regression fit The default values of these parameters are suitable for a broad variety of experimental designs 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 63 Institut Curie Quality Characteristics 2 MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help b gt WE eco MAS OHM ORDO MM o 11 BI Y 2 X 2 spv uxe
30. 9 97 Il E 97 1 99 995 This Quality LETI DEEI EET 1 1 19 17 8 7 48 0 92 Contamination 0 Diameter 16 86 eGeceee0 ceo SGe0n0eesuoeoned 2ece080e080880 PT 3759 0 02 1 Ctrl Left Clicks followed by Ctrl Right Click create the contour on the Quality Seso0eesee06o8 plot The replicates that are ne TE esseesesess 2 cesececcettt within the contour are rera 2 8 Image Alignment M t highlighted on the image PIN THHPEIS i shftx 0 28 4 ebecnese e ti 1 Y 5 X 16 Shift Y 04 Ej 20900000000000 e e Several contours in usur BB 3 sgeseseeseces 4 a Y ind wo o ee LJ different parts of the Quality Plot selected 6 s Sssssssceesess BE E graph can be created EHE E E i To efface contours click E H 0 00 CLA 6 THEME of the graph 0 00 025 O50 075 1 00 125 Ratio CV f 385 441 Y 504 X 6 BI Y X Sp X Ho ID Ho Name Fit Limits 0 75 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 3 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 87 Institut Curie Manual Qualification of the Selected Spots The same manual quality value can be assigned to all selected spots using either the right button popup slider or the spinner Manual The checkbox Selected should be on
31. Array Design can be completely specified using GAL files Axon Instruments Inc Correct image resolution mkm pixel should also be provided Des ose nass Distance Between Replicas In SubArrays 18 y oli x in Blocks is v of Tes Relative coordinates of the replicated spots it defines the position of the replicated spot with respect Replicas 10 ixels Y f P a current one in Spots 28 Y of Ka Amount of the replicated spots in the Y and X directions MAIA 2 7 Manual Copyright C 2005 2006 20 Institut Curie Spot Localization To start spot localization or grid finding use the Spot Localization button from the Toolbar or select the Menu Item RunlSpot Localization Ctrl F6 For automatic grid generation it is advisable to ensure relatively broad external margins distances from the edges of the array to the spotting area 11 Oct 06 File Run Options Window Help d d Hm Pilea Ha a E 020805 020805FR108 b El lo E SE a lo L 4 8x2 spzx2 No Name Ratio v 9 97 18 Characteristic Typical 0 1 94 9 This Quality Determination An x DurbinWatson j 4 H Contamination 0 3402 Diameter lo a402 lt B GSymmetry p 3402 ISymmetr D Be Cura D 402 Rgfckaround D 93402 e Background 84 o Signal
32. HX O 1 1 To save the results of quantification and quality Bl Y 1 X 1 Sp Y 12 9 No Name D Images Microarray CGH Firstir d m Characteristic Typical 0 1 99 9 This Quality gs analysis use the Save cuter pep n LET Contamination o 0 6 o 1 Analysis button from fe xm ps 07 GSymmetry 0 0 7 0 92 the Toolbar or the Menu E a h EE Ln 1 RBackground 0 03 0 14 0 98 Item FilelSavelAnalysis ABackground 475 58 441 3 492 04 0 84 Ctrl S Signal 2270 07 9898 439 1 Quality 0 64 Save in rawdata020805 f Manual AS 2 020805FR1087_cy3tit 020805FR356970_cy5 tit e 02080SFR9216_dapitit The results are saved as a Reset Manual ms 020805FR1087_cyS it 020805FR358970_dapitit M documents O2080SFRI087 depitif 020805FR368813_cy3 tit table in the text file EAE Baaai r rente E 020805FR1528 7_cy3tit 020805FR368813_cy5 tif i Back 492 04 i 020805FR1528 7 cySiif 020805FR368813_dapitit importable into Microsoft E 020805FR1528 7_dapitit 020805FR5212_cy3tif Bureau i o20805FR2155_cy3 it 020805FR5212_cy5 tit Excel i l o20805FR2155 cySiif O20805FRS212 depitit i 020805FR2155_dapitit 020805FR6159 cy3tif 020805FR321679_cy3tif 020805FR6159_cy5 tit 020805FR321679_cy5 tif 020805FR6159_dapittit User can define which KT
33. Ho Hame This Quality Determination Durbinwatson Contamination ground p402 ABackground Signal 3402 3402 Regression Plot v i gt Eu i Image Alignment Ratio 1453 560 Bl Y X Ho ID Y 36 X 828 Design 1 1 4 4 21 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 16 Manual Pixel Flagging I Groups of bad pixels can be flagged out using the Lasso selection tool The Manual Pixel Flagging toggle button should be selected Ctrl Left Clicks create the contour Ctrl Right Click closes the contour Flagged pixels are converted into the background pixels in spot localization and into the saturated pixels in spot quantification 11 Oct 06 9 MAIA27 D lmages Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help PiG Q 6 db Bm 8 Ee s oo e All SE a lo L 4 XC No Hame Ratio Sall 9 99 9 This Quality 1 LI L Sx Contamination 3402 Diameter 3402 lt GSymmetry D 8402 ISymmetry D Be CVRatios D 492 A RBackground o 3402 3 ABackground 840 o Signal o 3402 Regression Plot
34. Manual Copyright C 2005 2006 85 Institut Curie Fit the Limits 9 MAIA27 D lmages Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help The quality limits are fitted Viale M mITSCNN A oA On PERE using the Fit Limits Lee LIIS Characteristic Typical 0 195 99 995 This Qualit button from the Toolbar or E aaa the Menu Item RunlFit Contamination 0 4 93 0 1 Limits Ctrl F10 Diameter 686 119 zs 49 0 92 C Ratios Quality fit estimates the limits of the quality Quality 0 92 characteristics such that Manual 0857 n Reset Manual the spot overall quality 1S Image Alignment a aligned along the user 2 eee oa Hu defined quality curve emis sty o1 E 0 22 Qualt 0 25 2 Quality Plot Before fitting it is advisable to restore the default limits the Init Limits button from the Toolbar or the Menu Item 025 oso oys 100 125 M Ratio CV RuniInit Limits Ctrl4 F9 1457 517 Y 6 X 201 Bl Y X Sp Y X Ho ID Fit Limits 0 75 s Design 1 1 4 4 22 21 10 0 0 0 0 1 1 3 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 86 Institut Curie Bad Replicates File Run Options Window Help d d ME pco HAS He ON Be e 14 Bl Y 0 X 0 Sp Y 9 X 7 No Name Ratio v
35. Saved shift is selected 11 Oct 06 2 MAIA27 D Images Wicroarray CGH First rawdata0 208051020805FR1087 cy3 bin File Run Options Window Help 6 db Bim eco MOS eB OI x BI Y 1 X 1 Sp 12 9 Mo Name Characteristic Typical 0 1 99 995 This Quality Determination DurbinWatson 1 o 4 Contamination Diameter lu 3402 3402 Symmetry ISymmetry 3402 3402 CVRatios RBackground 3402 3402 ABackground 3402 Signal 3402 Regression Plot d lt Veo Kalo 1 a M gt v Image Alignment Block Independent V Sait x 1 28 Shift 0 7 018 Ratio JL isis 9t LI ele elololsielsisieleieio lolololol 1373 407 Y 561 0 Bl Y X Sp Y Xt Ho ID mage Alignment 0 578 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 50 Follow Up Grid Refinement Once the two images are aligned additional grid refinement may be needed image alignment slightly shifts the spots so that the border between the spots may not be correct any more Use the Lines Refinement button from the Toolbar or the Menu Item RunlLines Refinement Image alignment is important in order to increase the efficiency of the linear regressio
36. Shift Double Left Click S50 EET BEES selects all grids on the image Image Alignment Block Independent EEESeSsoseissesseas d ICICISIESI e aMisssucooocassoeaas noon t leeeses sisi ooo v Jeet eteesseoee et HH BH II duties LLL EE OZONO lt gt Default v BBEBEBHGSEEBHRHBRSORO lelelelolo DDOCOCGOODODCDBBBDCDO Y 0 X 0 mm Sse Hetet ie leleia CEECECBBRD arete terete sisle 014 5 1 En BEBEBEECBEBOB 1440 492 Y 369 X 630 je ee Bl Y 1 X 2 Sp Y 9 X 9 Ho ID Localization 6 5625 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 29 Institut Curie Manual Correction of the Main Grid Line Movements 2 MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help The line separations can Me sa MH mm om be corrected in the main WR 503 nomme Rao Characteristic Typical 0 195 99 995 This Quality grid _ petermination dd buuncweren T ol e o DHBHHORGSE ls aate d iit telsaleetalateltele DBDEDD Lelie e jeje lei lel lol 3402 3402 3402 3402 opgong je Beke ast Regression Plot Select a line and iterate through s smear the lines cecal OO500 Bud ut EBER Ctrl Left Click
37. array 021007 11 Oct 06 File Run Options Window Help MAIA27 D lmages Microarray CGH FirstWuplicates 1077 02 d d BE Poo HAS OE OIN D I Is d BIY X Spix NoName Ratio v 9 95 I 6 1 95 18 Characteristic Typical 0 195 99 995 This Quality Determination 0 93 0 58 n Eur Durbmwatson 1 35 aa Contamination 0 0 9 5 Diameter 5 23 3 02 94 AR d lee iC Ratios 0 01 0 0 37 Regression Plot 0 00 0 25 0 50 0 75 1 00 Cy3 v e JL 18 Manual 02 Reset Manual Image Alignment Block Independent V s ud zz Save Default w Ratio 0 22 Quai 0 25 2 CEEI T 2 T E sul po cm EX HESSE T I 1 1 Ec amm AE RAPA SEE FANS ae a EEEBREBEDEBEDEEE elatolol agate IRRGREIIEEREBEPREBBEMEE SS Y 0 X 0 Bl Y X Sp Y X Ho ID Ho Hame Design 1 1 4 4 22 21 0 0 0 0 0 1 1 3 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 121 The Selected Bad Replicates II 2 MAIA27 D Images Wicroarray CGH FirstWuplicates D1077 021026FR1077_cy3 bin DAR File Run Options Window Help d d ME Pco HES mE OIN E is x BIY X Sp i NoName Ratio Characteristic Typical 0 1 99 9 This Quality Determination 0 98 58 1 Contamination D 9 5 Diameter 5 23 3 02
38. eee ton p eee Diameter 3402 a 3 dineti i M GSymrgetry 3402 e eeceeete TITEL fmetry 3402 Ryfackground 3402 Use the Array Design pane SN Design Signal 3402 Default button from the Toolbar or select the Menu Item eat THE OptionslArray Design jeseseseo ese III ITI T Alt A Block Design Distance Between Replicas Aey TITTET SubArrays In SubArrays M Y Y v su gt Blocks in Blocks Y 0 x 0 Y 4 Y T Quality Plot Spots in Spots Y Y Spot Diameter Replicas IT IT C used E Y EIIIIIN 117217 TELLI Array Design is Ld HUE equivalent to the Two Color Image Case E nom Design 1 1 4 4 21 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 137 Institut Curie Spot Localization C MAIA27 D images Microarray CGH F File Run Options Window Help d amp Hm Pica BI X SPOX Characteristic Typical 0 1 Cluster Contamination j To start Spot Localization or grid finding use the Spot Localization button from the Toolbar or select the Menu Item RunlSpot Localization Ctrl F6 ant the BBOBooOOOCNENENEBENEEENN cv 444 Y 12 X 191 BI Y X Sp Y X Ho ID Ho H
39. i 2500 00 ipe 80 00 2000 00 ae symmetry Jos 1500 00 40 00 Ctrl Left Clicks followed P 1000 00 RA gt 20 00 by Ctrl Right Click on 500 00 sis i the diagnostic plots select aR ho ozs oso O78 1 WE X7 w 3 the spots to be Determination log2 Contamination Counts Determination Counts Contamination Linea Log2 Linear Log2 Linear O Log2 O Linear Log2 highlighted on the image 500 00 400 00 300 00 Diameter log2 CVRatios Counts Diameter Counts C Ratios Linear O Log2 Linear O Log2 Linear O Log2 O Linear Log2 Y 490 X 313 Bl Y 1 X Fit Limits 0 828 s Desian 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 99 Institut Curie Manual Qualification of the Selected Spots File Run Options Window Help d d HE rc HAG e He OI EB E e e 14d Bo x0 sp v 9 X 7 NoName Ratio x 9 I 10 97 Characteristic Typical 0 1 99 9 This Quality 05 X 3 1 Y T m 1 05 0 Contamination D o ju Jo f Diameter 6 86 lo 9 44 7 48 0 68 o o s CVRatios aot 49 looz p 99 Using the spinner EE pe t T c6 ogeeeccceao Manual one can assign TERM a quality value to the RR 0 79 RS 0 772 b e 2 500 NE selected spots 2 2 000 7 5 B P zt 1 0
40. is a user decision which depends on the image and EPUM ANUS NN user demands D 0 25 050 075 100 125 Ratio CV f 000 LLEOLI eotecece 0 05 Qual Quality Plot IIIS oottt IARE eevee ee eocceecoo LELE LE lt 423 523 Bl Y X Sp Y X Ho ID it Limits 0 828 s Design 1 1 4 4 22 21 10 0 0 0 0 1 1 3 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 91 Institut Curie Quality Settings Several settings that may influence the quality analysis are available through the Menu Item OptionslAnalysis Options Alt O tab Local Quality See next page for details 11 Oct 06 P MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help fb ME eco HAS e MR O INE EE ES e 1 d Eek Bl vofi0 Sp v 9 X 7 NoName Ratio v 9 97 Charactfristic Typical 0 1 99 9 This Quality 0 9 amination 0 0 05 jo 1 f neter 6 86 5 42 7 48 0 72 CVRatios Analysis Options e Value Default Quality 0 66 sae Salus p 05 TU i ED Limi 0 2 0 1 Low Percentile 0 1 0 1 soe onere High Percentile 109 9 99 9 Block Independent V v six 0 25 n YF X 12 Shift Y i 012 lt gt Ok cancel Restore Defaults 0 05 Quali 02
41. o 3402 Regression Plot ra 1 00 v 0 75 2 amp 2 50 0 25 0 00 E 0 00 0 25 0 50 0 75 1 00 Cy3 IL e J 3 1 Image Alignment v lt gt Y 0 X 0 18 ug 1520 859 Y 0 X 110 BI Y X Sp X Ho ID Ho Hame Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 21 Terminate Processing Any processing can be stopped by pressing the Stop button on the Toolbar or selecting the Menu Item RunlStop Ctrl F5 11 Oct 06 File Run Options Window Help b Hm HE g olmal aa sx lS L 4 BIY x Sp Y X No Name Ratio vi 9 97 1 Characteristic Jf Typical 0 1 99 9 This Quality Determinatj b h Durbiny tson o f Cop 4mination 0 g402 ameter lo 3402 GSymmetry D 8402 ISymmetry 3402 CVRatios p pez RBackground D 3402 Background 840 Signal 3402 Regression Plot 1 00 0 75 o amp 0 50 0 25 0 00 M 0 00 0 25 0 50 0 75 1 00 Cy3 vL 12 Image Alignment lt gt Y 0 X 0 LE 407 462 Y 1 x32 BI Y X Sp X Ho ID Ho Hame Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 22 Spot Localization Output Typical result of the spot localization two grids are impo
42. or the Menu Item RunlSpot Quantification Ctrl F8 Image Alignment Block Independent V six 0 22 Sz ve Zo cm w Shifty 0 15 EE 4 CDPEEB s 2 Default w Q BBA e o rski sis DEDBEEEE ME Cc EEBBEEEEHESR Ltt 02224000 000525 55029 isl lsels slelelolelelolol 451 489 Y 11 X 144 Bl Y 5 X X Ho ID Localization 0 687 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 54 Institut Curie Ratio Estimation Linear regression plot for the selected spot Two ratio estimates RR is based on the slope of the linear regression RS is based on the segmentation of the spot area See next page for details 11 Oct 06 Spots are contoured MAIA27 D lmages Microarray CGH First rawdata0 20805 020805FR1087_cy3 bin File Run Options Window Help d d ME pco HAG u OIN IEI xo 1 4d Bl v 4 1 Sp 12 9 NoName Ratio v 0 97 If 26 97 r Characteristic Typical 0 1 99 9 This Quality H i Determination 0 98 0 05 0 99 0 99 DurbinWatson 1 35 p 51 2 36 1 29 0 48 Contamination 0 0 11 0 1 Diameter lese 0 9 44 ees 0 94 GSymmetry b p pss rst jose Symmetry 0 3 o 2 65 0 29 10 77 CYRatios 001 0 49 bo i RBackground 0 14 5 03 8 87 o 12 0 93 ABackground 508 78 459 712 73 516 13 0 92
43. p 95 RR 0 79 RS 20 772 2 500 MEE a 2 000 LES 5 18 P 74 1 000 pt aa soo e 500 1 000 1 500 2 000 2 500 3 000 Cy3 oy 9 J amp 10 2 Quality 0 68 A Manual 19 Reset Manual Image Alignment dependent SI ShftX TAR lt gt Shi 012 t vini Save Default In M 0 05 2 Quali 022 Diagno io B 2 Quality Histograms Results Plots 3000 00 7 2500 00 4 2000 00 eo o E E Z 1500 00 2 o o 1000 00 500 00 0 00 0 00 0 25 0 50 0 75 1 00 Determination log2 Contamination Counts Determination Counts Contamination Linea Log2 Linear O Log2 Linear Log2 O Linear Log2 800 00 mun x 700 00 400 00 i 2 600 00 RE 500 00 300 00 S 400 00 8 sl Exc o H AE 200 00 t 200 00 100 00 n i 100 00 v r 0 00 0100 pane Maarn St 2 45 20 15 10 5 0 5 Diameter log2 CVRatios Counts Diameter Counts r CyRatios Linear Log2 Linear Log2 Linear O Log2 O Linear Log2 Bl Y 0 X 0 Sp Y 1 X1 Fit Limits 0 828 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 95 Quality Histograms 2 MAIA27 D lmagesWicroarray CGH First rawdgtaN 208051020 RO 5ER40R7 cv3 hin File Run Options Window Help 5 Diagnostics amp e Mm eco BEeof Quality Histograms Results Plots 300000
44. se so 712 73 514 06 0 94 percentiles for each i 5489 5 pao characteristic over all spots on the current array The percentiles can be modified directly in the table header oo ee IJ ee LJ ee000Ge e0c00600 oe Quality 0 58 PILIIIJ H Manual 12 Reset Manual r Image Alignment eeoeeo oeee LEAL LLEI Quality characteristics of uim the selected spot we E Shift D lt 1408 459 Y 514 X 15 Bl Y X Sp X uantification 2 265 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 66 Institut Curie Quality Parameter Each quality characteristic is rescaled into the corresponding marginal quality parameter e 0 1 J See page The minimal quality value from a set of marginal quality parameters is taken as an overall quality value See next page for details 11 Oct 06 2 MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help So He Pclo HE 6 HE ORDO Hm 11 B amp zx2 Spey 1 4 Ratio vi 9 77 Il 97 99 9 This Quality 3 99 0 99 Durbinwatson 1 35 51 2 36 1 71 0 7 Contamination D o i b pss Diameter lese 0 9 44 7 05Ao a5 haracteristic Typical 0 195 Determinatio
45. to find such a file it opens up the original image and applies the default settings which can be defined via different items of the Menu Options Data Options Analysis Options Colors Array Design and Output Format 109 Modify Batch Settings Typically all arrays from the batch are of the same array design and have the same settings One may want to define modify these settings before further processing This can be done using the items of the Menu Options Data Options Analysis Options Colors Array Design and Output Format Description of the current Array Design 11 Oct 06 aBa S MAIA27 Batch defaults MAIA File Model Run Options Help gt NM solsL ia sa FL SA Visible D Images microarray Q20805FR1087 cy3 tif ges Microarray D20805FR326671 cy Aages Microarray Q20805FR9216 cy3 tiF Design 1 1 4 4 21 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie Yet another possibility to modify settings is to open visualize one of the images and modify settings for that image Then the Menu Item FilelSet Batch Options of the Main Processing Window will send the new settings into the Batch Processing Window See page 110 Apply Setting to the Batch To send the modified settings to all images of the batch one needs to I run the batch with the SO SL IA SQ FL SA task Se
46. v 397 437 Y 569 X 2 BI Y X Sp Y Xt Ho ID mage Alignment 0 578 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 48 Institut Curie Default Shift 2 MAIA27 D Images Wicroarray CGH First rawdata0 208051020805FR1087 cy3 bin File Run Options Window Help d Nim ec MBS o B O SH x BI Y 1 1 Sp 12 9 Mo Name Characteristic Typical 0 195 99 995 This Quality Determination 0 1 DurbinWatson o 4 Contamination lo 3402 Diameter 0 3402 Symmetry 3402 ISymmetry T 3402 ec 99 CVRatios 3402 The Shift combo box is keea 5 used to switch between m epe different shift values Regression Plot Default shift is selected B so pe 1 000 p i Note the difference in the MET ms linear regression plot as v 9 NEJ 10 compared to the Zero shift O N 4 Image Alignment Black Independent vV Mx 022 eee v J lt gt Shift 012 Default v 1 Y 5 X 13 018 1 v 381 388 Y 566 X 0 BI Y X Sp Y X Ho ID Image Alignment 0 578 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 49 Institut Curie Saved Shift The Shift combo box is used to switch between different shift values
47. 0 2 B 020805FR321679_cy3tit O20805FR6159 cy5 tit ios Ctrl S Cy3 Mes documente 020805FR321679_cy5tif i 020805FR6159_dapi tif 020805FR321679_dapitif amp O20805FR824_cy3 tif oy 9 amp 10 Quality 0 68 43 020805FR326671_cy3tif 020805FR824_cy5 tif Q20805FR326871 cy5 tif ie 020805FR824_dapitif The results are saved as a tcd 0 51 Poste de travail a 020805FR326671_dapitif 020805FR9216_cy3 tit table in the text fi le 020805FR356970_cy3 tif E 020805FR9216_cy5 tit i O20805FR1087 cy3 rest t importable into Microsoft Exi qoo avers reese Shift X 025 Files of type Files Excel D lt gt t Y 7 x 12 Save Default v 0 05 2 Qualt 022 tttttttt 448 540 Y 0 X 317 Bl Y X Ho ID Fit Limits 0 828 s Design 1 1 4 4 22 21 10 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 103 Institut Curie Output Format Output File Format Description Column Title
48. 00 t a 500 e j B E j i o TIS 500 1 000 Tego 2 000 2 500 3 000 ses 4 3 D 09 090 al Qua 00 x v JG 10 2 Qua 0 68 s Manual 0 512 o Y Reset Manual o 5 t 1 26 51 76 Image Alignment Selected Block Independent Vv E Shift 0 25 TP 228 Sat v LIIS O o0 gt Shift Y 012 9000 6 t Y 7 x 12 Save Default v mM o052 Quit 02 TII 00 Lt 1454 522 Yr 15 X 428 BI Y X Sp X Ho ID Ho Hame Fit Limits 0 828 s 11 Oct 06 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 100 Change the Quality Limits P MAIA27 D lmages Microarray CGH First rawdatal 20 RN Ain NRNSERANRT cv3 hin File Run Options Window Help 2 Diagnostics d mm P cfa BEES ef a Tel E BI Y 0 X 0 SpQ9 x7 Ho Name lees 7 2 Qualty Histograms Results Plots 1 05 Ec 3000 00 2500 00 2000 00 vi 1500 00 5 i 2 o For each used quality 1000 00 TT characteristic the limits 500 00 fh T can be adjusted in the nr 0 25 0 50 0 75 vas 1 2 3 4 Q d Determination log2 Contamination Quality Histograms Counts Determination r Counts Contamination linear OLog2 Linea O Log2 Linear Log2 Olinear Log2 Shift Left Click Move ee 500 00 Shift Right Click 400 00 transfers a typical va
49. 00 ae wt ee o FilelSavelExperiment EN cr E 533 i 3 500 L o o P Ctrl W in the 500 1000 aig 2 500 3 000 d e s H HH H e LI o eoo o internal binary format to zoia E 7 HII f 3 8 be able to restore it using we 05 M Ssssscocssssss S EHE h M It Manual be ng DERE gt gt zs t e enu em mage Alignment e ee ae 5 Block Independent v FilelLoadlExperiment sux 022 H o lt gt Shift 042 Ctrl R to oe reanalyze the data ww J 9005S quit J 028 z iii al 1448 540 Y 0 X 317 BI Y X Sp X Ho ID Ho Hame Fit Limits 0 828 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 106 Institut Curie Set Batch Options Using the Menu Item FilelSet Batch Options all settings from the Main Processing Window can be sent to the Batch Processing Window to be applied to the other images from the same batch 11 Oct 06 File Run Options Window Help d d ME Pclo BEeONEOINEEBERS e 11 Bl Y 0 X 0 Characteristic sos x Typical 0 195 99 995 Thi 1 05 No Name is Quality Contamination 0 0 11 0 Diameter 16 86 5 18 9 44 7 48 1 0 68 C Ratios 0 01 3 49 0 02 0 99 RR 20 79 RS 20772 Ratio vi 9 77 tROO5 e068 209900444000 eeeves oooesoe SeP
50. 022 Counts Diameter r Counts CVRatios i Linear Log2 Linear Log2 Linear Log2 O Linear Log2 100 00 Bl Y X Fit Limits 0 828 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 102 Institut Curie Save the Results 2 MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d ME pco HAG HR oIINIEJs e L4 Bio Sp 9 7 NoName Ratio 9 97 CharagAristic Typical 0 1 99 9 This Quality Contamination 0 0 t Diameter 6 86 b 18 7 48 0 68 E CYRatios 0 01 To save the results of po quantification and quality d swen enero J d cim 1 ud z E 020805FR1087 cy31if O20805FR356970 cy51if E 020805FR9216_dapitit analysis use the Save RR 0 79 RS 0 772 a o20805FR1087 cySiif 020805FR356970 dapi if eni 20805FR1087_dapitit 020805FR368813_cy3 tif Analysis were button from xis O20805FR1528 7 cy3iif O20805FR368813 cy5if z 020805FR1528 7_cy5 tif 220805FR368813 depitif the Toolbar or the Menu 20805FR1528 7_dapitit 020805FR5212_cy3tit p a 20805FR2155 cy3 tif 020805FR5212_cy5 tif Item FilelS avel Analysis i 020805FR2155 cy5 tit 020805FR5212 dapitit A 020805FR2155_dapitit 020805FR6159_cy3 tif 500 1000 1 500 2 00
51. 08060860888 e809e8080608 DID ILITITTTT To optimize the position of the Quality Limit press the button Quality Limit a e Tt Ali t o A special procedure searches E h eseoooeeo o Block Independent gt lt E H e for the limit value such that Y sx 025 e ee Iit t t e j al e the number of replicates in the T H E o oe e Bottom Left Top Right 99000000909 2 ee essoo0o0cnceseseooso LJ ee quadrants of the quality plot NINH tH should be as small as E ssecosocess oe possible whereas in the S oso lg iN fix Bottom Right Top Left quadrants as big as o arr wrr MT n 8 TEMERE possible EMEN 1400 451 Y 463 X 17 Bl Y X Sp Y X it Limits 0 75 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 3 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 89 Institut Curie Optimize the Ratio CV Limit Using the button Ratio CV Limit the correspondent limit is set into a value ensuring the best exponential approximation for the cloud of replicates black dots 11 Oct 06 P MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help o B Pc KM 4688 OR DO EIS Bl Y 0 X 0 Sp 9 7 Ho Name o Contamination 0 Diameter 16 86 3 78 LEDI TEEI EE eGe08000 5880 S
52. 102 Quality 0 58 Manual Ag B Reset Manual m W Image Alignment Block Independent V Shift x 0 23 w Shift 018 lt gt el Save Defaul w E rex ime 12 Quit 0 18 1456 510 Y 513 X 574 BI Y X Sp X Ho ID Ho Hame Quantification 2 2655 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 39 All Pixel Regression Outliers P MAIA27 D lmages Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d Me pco EIE o He OR LS Te Bvzx2 sp v i x 4 NoName Ratio v Characteristic Typical 0 1 99 9 This Quality Determination 0 98 0 05 10 99 0 99 DurbinWatson 1 35 0 51 Contamination o Diameter lse o gazos pss GSymmetry o A 558 losz 0 81 isymmetry Jed o s po pm CVRatios oo 0 4 b i Using the Toolbar button o he km bor ox Show Hide Outlier ABackground 508 78 459 712 73 514 06 0 94 Signal 1820 68 10 5483 5 229 1 Pixels one can visualize AA OTRS OTER all pixel regression outliers Quality 0 58 Manual 12 Pixel regression outliers Reset Manual Image Alignment Block Independent Shift x 022 v Shift v 012 5 Y Save Defaul w Y 5 X 13 1 Quai Y 514 X 553 Bl Y 5 X Quantifi
53. 27 D Images Wicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help do d Bm ico KEEN e WEE O me x Bl Y X T Sp Y Characteristic Typical 0 1 3p 99 9 Ho Hame This Quality Determination DurbinWatson 1 4 Contamination Diameter 3402 3402 Symmetry ISymmetry 3402 3402 CVRatios RBackground T 3402 e402 ABackground Signal 3402 3402 Regression Plot Image Alignment Block Independent Default sil Show Hide Main Grid 1452 rz Na Bl Y X Ho ID Localization 6 5625 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 38 Save Restore Grids 2 MAIA27 D Images Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d Hm rc iB amp He OIN Bx Spx NoName Characteristic Typical 0 195 99 995 This Quality Determination 1 DurbinWatson 4 Contamination 3402 Tl b ene 3402 3402 3402 The generated grid can be saved on the disk using SUMA the Menu Item Fass FilelSavelGrid to be able to apply it using the Menu Item FilelLoad Grid to analyze other images with the similar design
54. 5535 1000 65535 Image shift in pixels in vertical direction NoiseToSignal NoiseType Seed 0 1 Proportional 123455 Peqgeaee oe eee oe m Simulation finished Non integer pixel shifts are possible 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie Additive Statistical Noise File Run Help Noise to signal level for mera the additive statistical Layout Margins noise This noise is UU aw finally added to each B pem roe pixel of the array Spots Interval Blacks Interval UO 1512 y x 20 Y Spot Radius Position SD Mean 3 SD Spots 13 Blocks Model for the standard mum deviation of the additive pots 0 5 Back N S Radius 2 5 S Density noise It can be constant Me proportional to signal or NES uc proportional to the square N wm mo me root of signal Seed for random number generator selection 1 as a seed will initiate the random generator with iseLOSignal 04 auto matic ally Or NoiseType Proportional Seed 123455 randomly chosen seed imation ished 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 131 Institut Curie Export of the Generated Image File Run Help To send the generated 5 96 images in the Main Processing Window ida use the Send Data re l i button from the Toolbar of Blocks terval or the Menu Item FilelSend Data Spots 1 Blocks Dust
55. 6 MAIA 2 7 Manual Copyright C 2005 2006 36 Institut Curie GAL Grid Generation If Array Design is specified using GAL file spot localization grid can be generated from this file use the Menu Item RunlGAL Grid 11 Oct 06 2 MAIA27 D Images Wicroarray CGH First rawdata0 208051020805FR1087 cy3 bin File Run Options Window Help d d m eco HE S Nm OI amp Brux Sp vi3 NoName Show Hide Main Grid Characteristic Typical 0 1 99 9 Quality Determination 1 DurbinWatson T 4 Contamination 3402 3402 3402 902 9402 3402 3402 Regression Plot Image Alignment Block Independent V v gil gt Save Defaul w E 2000 L leies 012 Jl 452 483 BI Y X Sp Y X Ho ID NESLNUISE Localization 6 562 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 37 Find ID If GAL file contains the IDs and Names for the spotted clones all spots representing interesting clones can be found use the Find ID or Find Name text fields to search for the clones Found spots will be highlighted The searching procedure supports regular expressions The upper and lower case letters are distinguished If spots are not found the search field is highlighted by red 11 Oct 06 2 MAIA
56. 7 s 349 0 01 RBackground 0 14 0 03 3 87 0 12 0 93 ABackground 508 78 459 712 73 516 13 0 92 Signal 1820 68 10 5483 5 415 1 RR 0 83 RS 0 837 Quality 0 48 Manual 13 a Reset Manual Image Alignment Block Independent V Shift x 022 Shift Y 018 Default v 12224 2559 Bl Y 15 X 1 l Sp Y 9 X 8 Ho ID Y 371 X 357 Quantification 2 265 5 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 58 Pixel Regression Outliers Pixel regression outliers 11 Oct 06 File Run Options Window Help KHEN 4 o he OREO Mex oO 1 4 d d OH pel B2 x2 sp x 4 NoName Characteristic Typical 0 1 99 9 This Quality Determination 0 93 0 05 1 0 99 0 99 Durbinwatson 1 35 o 51 36 1 71 0 77 Contamination D o fit b jose Diameter lese 0 9 44 o5 0 85 Symmetry b p 5 58 Jose p i ISymmetry 0 3 2 65 0 29 0 78 C Ratios 0001 fo B4 b i RBackground 0 14 0 03 3 87 0 09 0 95 Background 508 78 459 al 1820 68 10 712 73 514 06 0 94 5483 5 229 1 RR 0 766 RS 0 762 4 000 3 500 2 3 000 m rs cl 2 500 m 2 000 adi 1 500 ait 1000 we 500 1 000 2 000 5 000 4 000 Cy3 v
57. BAL DELES E AEETI BE AN ae e Sas ae maos or se e 07 ed LEITET 1 Using the Toolbar button EENEEEE c222 000000 H c TE TELLI es PUO ERU a p rd 4 6 b 4 79 Selection user qualified RR IJ8RS 0066 TETELE ee qeagaoa ON spots can be visualized H 28 o g 500 T apo 2 Oo E ia zz eee bl ees aoo These spots are signed by E Le dI S600 eso eee ie de uo ais ao esoo eee eee a dot in the left upper o TT A II eee eee ae LE E ERSS eas d corner of the spot area a oe n EP T7 T7 ees it anual 0 137 TTT e ee e8 eee LE E E Reset Manual PS NE ds m US EEE E EBEN rd 3050 S ong Image Alignment 2 3m dh eco e9 80D0 ba p E TITTET eee eee ch ES 6000 ZIILII ge soo lt gt ave er aul 3 LJ Lees onn 099090 09080000888000 nm 1 Quai 012 em I 1 1441 494 Y 259 X 757 BI Y X Sp Y X Ho ID Ho Hame Quantification 2 265 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 TI Institut Curie Quality Plot P MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help amp d Me ec HA o5 OIN e l1 B vri x2 spQo x12 Ratio LU EELEE Characteristic Typical 0 195 99 995 This Quality EEE E ESES EKEKCEEE AIRLINE omen ESER
58. Characteristic Typical 2500 00 2000 00 Quality Histograms panel contain histograms of the used quality zd zw 025 050 075 ch aract eri sti Cs su zb E Determination log2 Contamination DE Counts Determination Counts Contamination lt Linea Log2 Linear Log2 Linear Log2 O Linear Log2 Blue vertical lines a son 00 800 00 correspond to the typical 500 1 000 arg 2 500 3 000 700 00 on te d 600 00 value and 0 1 and 99 9 10 Quy 0 68 500 00 300 00 Manual 19 9 400 percentiles from the por oa quality table 25e canem 200 00 Block Independent v 100 Uli sx 0 28 lt shitty 018 i 3 x A 9 T5532 ove a Diameter log2 C VRafios ni 05 Counts Diameter Counts CVRatios as Liner Log2 Linea O Log2 Linear Log2 Olinear Log2 100 00 0 00 Y 0 00 o Bl Y 0 X 0 Fit Limits 0 828 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 96 Institut Curie Results Plots C MAIA27 D lmages Microarray CGH First rawdata0 20 RN SiN NRNSFRANRT cu hin _ BER File Run Options Window Help ecu Diagnostics DER d d He pch MEE 4 61 fal ce BI Y 0 0 Sp 9 X 7 Mo Name Lae MM Characteristic Typical 0 1 99 9 Th
59. Copyright C 2005 2006 Institut Curie 12 Green Channel MAIA27 D images Wicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d dH m co E st oc HER Hello xp x Bx Spex UNS HEN Characteristic Typical 0 1 99 9 Determination DurbinWatson Contamination Select the green dot Cy3 to visualize the image colored in green Regression Pic Image Alignment 443 524 Y 0 X 202 Design 1 1 4 4 21 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 13 Red Channel Select the red dot Cy5 to visualize the image colored in red 11 Oct 06 C MAIA27 D lmages Wicroarray CGH Fir File Run Options Window Help d Hi m Pco E H O eE SE e ow A BIY SPX NoName Characteristic Typical 0 1 99 9 This Quality Determination 1 DurbinWatson Im n Contamination lo 3402 Diameter lo 5402 GSymmetry lp Baga ISymm try lo laanz i Cvratios D 402 RBackground lo l3402 n Background lo 3402 Signal lo 4o Regression Plot 1440 554 Yee x 153 Ho Hame Bl Y X Sp Y Xi Ho ID Design 1 1 4 4 21 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C
60. Copyright C 2005 2006 Institut Curie Two Color Image Analysis 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie Data Import Settings To define the format of the microarray image files select the Menu Item OptionslData Options Alt D For multi page TIFF specify the pages for the Cy3 and Cy5 images 11 Oct 06 7 MAIA27 Batch defaults MAIA File Model Run Options Help w amp gt M SO SL IA SQ FL SA Visible Data Options Image Import Title MultiPage TIFF Page for Cy3 Page for Cy5 Design 1 1 4 4 21 21 10 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie Two options are available i Cy3 and Cy5 TIFF images are packed into one multi page TIFF file checked ii Cy3 and Cy5 TIFF images are stored in separate files unchecked File Name Selection Use the Toolbar button New Experiment or the Menu Item FilelNew Experiment Ctrl I to select microatray images 29 11 Oct 06 DE MAIA27 Batch defaults MAIA File Model Run Options Help eee b M Look in SO SL IA SQ FL SA Visible 2 Mes documents r cents 3 bit LJ Mes documents y Poste de travail rawdata020805 020805FR1087 _cy3 tif i 020805FR1528 7_cy3 tif i 020805FR2155_cy3 tif 020805FR321679_cy3 tif 020805FR326671_cy3tit i 020805FR356970_cy3 tif 020805FR368813_
61. E Slide up the bars b pea 20222002 separating the panels and open up the quality plot 1o8 o 6 6 e666 ERTES gt E EEEE E I IIIFIL BEXIIIIII BEXIFZITILT eefhesce TEDE LLL ELIET q E 43 Reset Manual r Image Alignment D E 2 e x cae GEIL XE E gv E EN Block Independent Shift x 0 2 Shift Y 012 eg QO LECTIONI REL Be Sas oa Beto E EISE EE No 12 Quit 012 Quality Plot EEEIEE HEELE EE E LE E EEE E E EKOL Benen EE mel EE E IE E E TEE amp EEIE EEEE LE E EE ES C SSEGQSECSAETOADI B BB EEG S OQCS8SSSABstOadcrcrcsesegaasee o TOR ROR EE TE fomekomemen TETELE E TTTTTTTITITO IIR 08 0000 ITE LE E L5 E L Q LEA E Ev Ly SEE E FrEE 1 25 1 50 1 75 In Manual 1436 480 Y 270 X 712 Bl Y 1 X 2 Sp Y 0 X 17 Quantification 2 2655 Design 11 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 78 Institut Curie Selected Spots for Quality Analysis 5 MAIA27 D MmagesWicroarrayACGHYFirstWrawdata02080510 20805FR1087 cy3 m File Run Options Window Help d d ME pco HOS o He Om I lel ox Bl v 0 0 sp v 9 x 7 NoNa
62. Gecneeuoeonee 20e0800 080880 oc00 09090900090 T6 T Quality Manual Image Alignment Block Independent Shift x Shift Y Reset Manual o0000006 os 0000 ecosces CELA ELLEI 000000000 0 50 075 1 00 Ratio CV I11111 11171 ELIT TI EDITTITT LIII a4 117 eceeeeetoo HIITIIT eeefoonecese ee0aceee0 eceesdecec esecoeseceo eeencseeo0d rode tee eseateseaeeoosood CELETTE eeeeeceoceocese 1408 451 Bl Y X Y 251 X 1 Sp Y X Ho ID it Limits 0 75 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 3 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 90 New Quality Plot P MAIA27 D lmages Wicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d ME eco HAS eim ONERE w e Bl vi0 X 0 Sp v 9 X 7 NoName Ratio 9 CobbbO 00 eooos 000 eee Contamination 0 0 1 000 Diameter l6 54 r4s 10 72 A somewhat more stringent quality curve is applied IILIIIILI L ILIIIIII Quality Manual Ratio CV Limit and Quality xxu Limit are used to generate the pres ideal quality curve The r decreasing rate of this curve characterizes how we are strict with respect to the spots quality If this curve decays rapidly one can 6000 expect that a lot of spots will be flagged out This
63. MAIA MicroArray Image Analysis Version 2 7 User Manual Copyright C 2005 2006 Institute Curie All rights reserved MAIA download page Author s Eugene Novikov Institut Curie E mail 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie Installation MATIA can be downloaded from the MAIA download page MAIA runs on Windows platforms 95 98 Me NT 2000 XP and needs the Java Runtime Environment JRE to be installed Click MAIA Setup 2 7 exe to start the MAIA 2 7 installer and follow the instructions MATIA 2 7 installation creates a Curie MAIA 2 7 folder in the list of Programs of the Windows Start menu This new folder contains the following entries MAIA 2 7 starts Microarray image analysis software User Manual is a user manual pdf file Uninstall MAIA will remove MAIA from your computer Installation procedure may also create a MAIA icon on your Desktop Installation procedure asks about the default size of the JVM Java Virtual Machine memory allocation pool It is recommended to set it as large as possible but not larger than the amount of available RAM 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 2 Institut Curie Batch Processing Window The Menu Model allows one to select the image analysis model 7 MAIA27 Batch defaults MAIA lis e Muss Run Options H 2 d amp v Pair ModellMAIA Pair ModellMAIASingle 11 Oct 06 MAIA 2 7 Manual
64. N o CRAs looi 5 49 lo CEREIIILI ZEE eee E All spots from the inal TUTTE TTT M e selection can be assigned Sooo oeeee 9 9 8 EELE IN Ner LJ a the same quality value esocs E g a ona o 6 dg a i amp Cece ae E s E Manual Quality x100 L D Pe aE sooo rl Le ick within a eid 26 51 Ctrl Left Click with r e contour effaces this contour e oman or 3u z z Shift Left Click inverts the EE b Sods eecooe TM selection of the spot Image Alignment 4 ja eeocoo e bd d a s on Double click on the image Block Independent v ipa effaces all contours all sv 018 5 Y Save Defaul w OT te o es 1 quai 018 LA E NS CESE E E Eg in pant 1460 516 Y 232 X 526 Bl Y 0 X 2 Sp Y 20 X 0 Ho ID Quantification 2 2655 Design 1 1 4 4 22 21 10 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 76 Institut Curie Manual Selection Markers 9 MAIA27 D images Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help amp d d ME pco HAS e us Om B Sis SAE a02 Sp v 0 17 NoName Ratio E Characteristic Typical 0 19 5 99 995 This Quality d wos on om SEE HH LIE EE ENSE lo Contamination DO b fu Diameter 686 b ba p E EREE EE PRESIESE 0 1 eee Goes im T311111
65. Position SD Spats 13 Blocks 38 j Dust 0 5 Back N S gt Radius Standard deviation of the positions of the spots and blocks with respect to the ideal alignment Larger SD value larger deviation of the positions of the spots blocks NoiseToSignal 01 from the ideal spot block alignment NoiseType Proportional Seed 123455 Simulation finished 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 126 Institut Curie Spot Characteristics Rate of the bright visible spots on the array 0 no visible spots are generated 1 all spots are visible File Run Help gt 0 amp Layout Margins Bottom bo Spots x 2 Y Spots Interval x 155 MN f Spot Radius 3 SD Image Blocks x Blocks Interval x 20 Y Position SD Spots 1 Blocks Dust Spots 0 5 Back WIS Radius 2 5 Density Restore Defaults Shift X Shift Y 10000 1000 1000 65535 Maximal spot intensity in the Cy3 and Cy5 color channels i e fluorescence intensity in es the center of the spot Seed 125455 Simulation finished 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 127 Institut Curie Non Specific Hybridization File Run Help Average intensity of non E wr specific hybridization in Layout the Cy3 and Cy5 color pu Bos channels Spots Bocs x zie v x Spots Interval Blocks Interval x 20 Y Position SD Spots 1 Block
66. Quality Plot BRA en aait anm em man nn an dREERERERE RR 0 25 0 50 0 75 1 00 125 Ratio CV f 1423 523 Bl Y 5 X Sp Y X Ho ID Y 7 X 84 Fit Limits 0 828 s Design 1 1 4 4 22 21 10 0 0 0 0 1 1 3 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 92 Quality Settings in Detail CV Limit is a characteristic Q Limit is the limit such that the value of the user defined spots with the overall quality ideal quality curve values below this limit will be Analysis Options indicated by a cross Value Default 0 05 1 0 0 2 0 1 0 1 0 1 99 9 99 9 Low and High Quality Percentile establishes the oir values of the quality characteristics in the sorted lists of the quality characteristics built up based on the results for all spots from the array that will be displayed in the corresponding fields of the quality table 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 93 Institut Curie Diagnostic Plots The Menu Item Run Diagnostics F3 opens up the window with different diagnostic plots 11 Oct 06 P MAIA27 D lmages Wicroarray CGH First rawdgtaN 20 RNIN INRNAERANRT cv3 hin File Run Options Window Help Characteristic Typical Contamination 500 1 000 1 500 2 000 2 500 3 000 Cy3 Quality 0 68 v o je 105 Manual 19 Reset Manual r Ima
67. R Fie Run Options Window Help bfo Mim rco HIE 4 o He OR DO Bim xo 4 Biz K2 Sp 1 x 4 NoName Ratio x Charactfristic Typical 0 1 99 9 This Quality p 98 pos 1 p99 0 99 jas fost 236 171 0 77 0 0 li b ss 6 86 p 9 44 7 05 pss 0 D ss ps2 pa I5ymmetry 0 3 p pes 0 29 0 78 4 C Ratios oot D 49 o f Several settings that may Ta influence the Rasckaround 508 78 59u 712 73 514 060 94 Analysis Options Signal 1820 68 10 5483 5 229 1 eer if 1 Localization Align Local Qualit quantification procedure RA 0766 RS 0762 Quay 4 000 Title Value Default lable f th ie i are avallable irom tne corel Intensity Levels ho 1 V 2 500 T ans Signal Outlier 0 01 0 01 Menu Item e Regression Top Outlier 0 01 0 01 3036 m Regression Side Outlier 0 1 0 1 Options Analysis EB Options Alt A tab uie dis Quantification SIT Quat Jose Manual AT Reset Manual Image Alignment Cancel Restore Defaults See next page for details Block Independent V Shift x 0 22 v Shift Y 018 lt gt save Default w Y55 X 13 inme 12 Quit 0188 s 1451 526 Y 514 X 553 BI X Sp Y X Ho ID Quantification 2 2655 Design 1 1 4 4 22 21 10 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C
68. RREUR corresponding percentiles Image Alignment oSesescoe over all spots on the array See bein i D E Shift x 0 2 H4 The percentage is defined in sv 012 e LJ o a tooo lt 1420 459 3 242 X 6 Bl Y 5 X Sp X I Quantification 2 2655 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 72 Institut Curie Manual Qualification of the Selected Spot Using the mouse right button or the spinner Manual any spot can be assigned a certain value from the interval 0 1 which can further be used as an additional parameter of quality If the user defined quality value is below the Quality Limit the corresponding spot will be crossed 11 Oct 06 9 MAIA27 D lmages Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d ME pco HAS o He OREBE e Box1 sp v 4 x7 NoName Ratio v Characteristic Typical 0 195 99 995 This Quality 0 05 Contamination 0 jo 0 1 Diameter 86 0 5 86 0 71 E C Ratios 0 11 0 93 a 2 LJ RR 0 893 P 1 037 i tv E ill Ww n Gene TEST rA ph ae E I Tu NE Xie faktas 500 550 600 650 700 750 800 poem en v e EE Quality 071 4 lanual 0052 T Reset Manual Image Alignment m Bx TET L i241 DI m A Block Indep
69. S laara emo array Block Independent 5006055 2549225 57 is on or specific for each 16483 4742 Y 403 X 370 block Block Independent is BUNAN PEGS Ho Hame Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 Localization 6 5625 off 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 44 Institut Curie Image Alignment Output 2 MAIA27 D Images Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help 6 amp Bim poco MOS oH ONE x BI Y 1 1 Sp 12 9 Mo Name Characteristic Typical 0 1 99 9945 Quality Determination 1 DurbinWatson 4 Contamination 3402 3402 3402 902 Relative shift in pixels ec in the horizontal X and ee vertical Y directions Sa between the Cy3 and Cy5 images This shift is visualized only for the selected spot and not for the whole image Tgag Alignment BlockNind gendent v Shift x 02 v Shift Y 012 lt gt Save Default w Y 5 X 13 012 1477 563 Y 8 X 399 BI Y X Sp Y X Ho ID mage Alignment 0 578 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 45 Institut Curie Manual Adjustment of the Image Alignment 3 MAIA27 D Images Wicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File R
70. al value x for each quality characteristic w log Q log q x or a gory 2 where q x is the scaled quality parameter calculated for x The critical value x sets up the limit such that if a certain characteristic i exceeds this limit the correspondent quality parameter g x will become lower than Q The experimental quality parameters q are obtained from the quantification procedure whereas the weights w or the critical values x are yet unknown The problem of spot quality analysis is therefore converted into the problem of weights w estimation which can be solved only if additional information is provided for example from the replicated spots or user expertise E Novikov and E Barillot An algorithm for automatic evaluation of the spot quality in two color DNA microarray experiments BMC Bioinformatics 2005 6 293 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 68 Institut Curie Bad Spots P MAIA27 D lmages Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin DER Fie Run Options Window Help Switch on the toolbar bear Jir button Show Hide MAA PYLA Characteristic Typical 0 1 99 9 This Quality Quality Markers Determination 0 98 pos p 99 0 99 DurbinWatson 1 35 lost 236 1 71 0 77 H Contamination 0 B lass eee Diameter 6 86 SF
71. ality value x axis is the coefficient of variation CV of the signal of the replicates The quality limits are initialized using the Init Limits button from the Toolbar or the Menu Item RunlInit Limits Ctrl F9 Then they are fitted using the Fit Limits button from the Toolbar or the Menu Item RunlFit Limits Ctrl F10 11 Oct 06 P MAIA27 D lmages Microarray CGH First rawdata0 20805 020805FR1087_cy3 bin File Run Options Window Help o Him vce MM eB Riso mx o 1 3 BI Y 1 X DE Sp Y 12 X 9T No Name s D Images Microarray CGH Firstiy Characteristic Typical 0 1 99 9 This Quality Cluster I 0 67 1 1 ji Contamination o os o i Diameter 6 36 451 9 76 7 48 p GSymmetry o 12 35 0 7 0 92 Isymmetry 32 o izs 041 0 64 RBackground 0 14 p 03 12 95 0 14 0 98 ABackground 475 58 441 3 605 95 492 04 0 84 l Signal 2270 07 428 Quality 0 64 2 Quali 0252 Quality Plot 0 00 aar Mimp nmn tends m t t 000 025 O50 O75 100 125 lt Signal CV IE x BI Y X Sp Y X Ho ID Fit Limits 1 484 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 3 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie Save the Results P MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options window Help o ta rco BEL 4 eM mE
72. ame ocaetion 6 1715 pese tuh i220 10 0 0 0 0 1 1 3 L j 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 138 Institut Curie Spot Quantification To start Spot Quantification use the Spot Quantification button from the Toolbar or the Menu Item RunlSpot Quantification Ctrl F8 Note that the Image Alignment button from the Toolbar as well as the Menu Item RunlImage Alignment Ctrl F7 do not show up for One Color Image Analysis 11 Oct 06 P MAIA27 D images Microarray CGH First rawdata0 20805 020805FR1087_cy3 bin File Run Options Window Help d Hoe Ple o BEES Bl Y X 3 T Sp Y X T Ho Name P AImageslMicroarrayVCGHiFirstlr Characteristic Typical 0 195 99 995 This Quality J luster a 0 71 1 c p P Diameter 6 96 lo 93 GSymmetry o Symmetry j Z 3 9 RBackground 944 0 03 ABackaroupe 475 58 M22 Sig 2270 07 9 64 LS JEJ 19 Contamination D Manual Reset Manual vi ul gt 0 0 Signal 1 Quatt Quality Plot rM es ET 0 25 0 50 075 1 00 1 25 lt Signal CV x Bl Y X Sp Y X No ID Quantification 1 5165 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 3 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie Spot Quantification Output Quality characteristics of the spots
73. bsolute level of background AB calculated in the proximity of each particular spot AB max Bc s Be is compared to the typical level of the local background for a given array Large deviations from the typical state may indicate the presence of the contamination areas which are larger than the size of the spot q AB exp 1 AB T if AB gt T and q AB exp AB T y 1 if AB T where T is the typical background level Signal S is defined as min Se Bey sScy3 Be where Scys Soy3 i is the mean estimate of the intensity within the contoured spot in the Cy5 Cy3 channel and Bo Bey 3 is the mean estimate of the background level in the Cy5 Cy3 channel q S 1 if S gt T and q S exp S T 1 if S lt T where T is the typical signal For the purposes of further quality analysis functions q rescale quality characteristics to fit the range between 0 bad spot and 1 good spot 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 65 Institut Curie Quality Table 2 MAIA27 Mmages Microarray CGH First rawdata0 20805 020805FR1087_cy3 bin File Run Options Window Help Typical median value 6 Me pce HH amp eHH ORME 1 3 for each characteristic awaKa mie Characterisl Typical 0 1 99 9 This Qualit over all spots on the xx Ht i f lt LET IA current array HE bo xy lo 85 5069 sz p 81 0 29 0 78 0 0 1 96 and 99 9 M bw fom
74. cation 2 2655 Design 1 1 4 4 22 21 10 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 60 Institut Curie Decorations for the Selected Spot 9 MAIA27 D lmages Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d ME pco HW 4 o m m ORF Oe B2 x2 sp x 4 NoName Ratio vi Characteristic Typical 0 1 99 9 This Quality Determination 0 98 0 05 1 0 99 0 99 Durbinwatson 1 35 51 236 171 Contamination ha Diameter le 86 GSymmetry 0 Symmetry lo 3 Using the Toolbar button CVRatios fi RBaderf nd 014 0 03 3 87 0 09 0 95 Show Hide Inset WBackground 508 78 459 712 73 514 06 0 94 Signal 1820 68 10 5483 5 229 n Decorations one can TARO 4 000 control whether the eed wo ean contour pixel outliers and B 000 grid lines are visible for Ee the selected spot Quality 0 58 Manual 12 Reset Manual Image Alignment Block Independent V Shift x 022 v Shift Y 042 5 Y Save Defaul w OE be es Y 513 X 567 Bl Y X X Ho ID Quantification 2 2655 Design 1 1 4 4 22 21 10 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 61 Institut Curie Quantification Settings MAIA27 D lmagesWicroarray CGH First rawdata0 20805 020805FR1087_cy3 bin DE
75. ction of the Grids If the generated grids are corrupted manual correction can be applied select the toggle button Manual Grid Correction All manual corrections of the grids can be undone Ctrl Z implements step by step UnDo and Ctrl Shft Z step by step ReDo 11 Oct 06 3 MAIA27 D Images Wicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d Wim rco MOS His olm x No Hame Ratio Characteristic Typical 99 9 Quality Determination 1 DurbinWatson Hi 7 4 Contamination 3402 D 9402 3402 3402 902 9402 3402 3402 Regression Plot Image Alignment Block Independent V Default w 359 446 Bl Y X X Ho ID Localization 6 562 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 27 Manual Correction of the Main Grid Grid Movements C MAIA27 D lmages Microarray CGH First rawdata020805 020805FR1087_cy3 bin The selected grid can be a amp amp Hm Pico File Run Options Window Help i I e oM O Ie x shifted on the discrete nE x Spt Characteristic Typical 0 1 No Hame o This Quality number of spot Determination rows columns or moved smoothly over the image 9402
76. cy3 tif 020805FR5212_cy3 tif 020805FR6159_cy3 tif 020805FR824_cy3 tif i 020805FR9216_cy3 tif File name 020805FR1087_cy3 tif Open v Cancel Files of type TIFF Cy3 532 Images MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie When single page TIFF files are used File Browser shows up only Cy3 file names The correspondent Cy5 file name will be downloaded automatically In this case filenames for the pair of Cy3 and Cy5 images must differ only by the suffix cy3 or 532 for Cy3 images and cy5 or 635 for Cy5 images For multi page TIFF filenames can be arbitrary Multiple File Name Selection Using the Toolbar One can select g i 1 button New MAIA27 Batch defaults MAIA See several filenames at Exp eriment or the File Model Run Options Help l once Menu Item FileINew O gt W sosiasor sa Experiment Ctrl N i visible more files can be added into the table Look in rawdata020805 020805FR1528 7 cy3if Mes amp 020805FR2155 cy3 tif documents E 020805FR321679_cy3tif snae 020805FR326671 cy3tit 3 amp 020805FR356970_cy3tif i 020805FR368813 cy3 tif Bureau i 020805FR5212 cy3 tif Ir E i 020805FR6159_cy3tif C i 020805FR824 cy3tif Mes documents 3e Man ILI sen i m File name 120805FR321679_cy3 tif 020805FR9216_cy3 ti Open Poste de travail File
77. d arrays into the global quality analysis window 114 Importing Experiments Global quality analysis can be performed assuming that the selected arrays are either replicates or not If they are replicates then all locally replicated spots from different arrays are combined and a unique overall quality value and a unique ratio CV are calculated for each replicated clone If the selected arrays are not replicates then local spot replicates from different arrays are treated independently in the overall quality plot In this case to have local spot replicates is essential for quality analysis 11 Oct 06 2 Global Analysis l Replicated Characteristic Determination i Typical Normalized 1 DEAR Quality DurbinWatson 4 Contamination 340282346638 340282346638 340282346638 340282346638 340282346638 340282346638 340282346638 eojojojojojojojojo j o 340282346638 Quality Plot MAIA 2 7 Manual Copyright C 2005 2006 0 5 Ratio CV Institut Curie If the selected arrays are replicates then before combining locally replicated spots from different arrays into a unique overall quality value and a unique ratio CV one may want to align arrays so that the averaged log ratio is equivalent for all arrays in the selection 115 Results Downloaded The quantification re
78. d to ELUYU E Me titer are equivalent to the all files in the batch File Model Run Options Help X Two Color Image Case amp b W so sL sor sa SO Set Options VC EE Visible SL Spot Localization D Images Micros ray O21007FR1077_cy3 tif SQ Spot Quantification D Images MicySarray 021026FR1077 cy3 tif FL Fit Limits SA Save Analysis The batch processing can be started using the Run Batch button from the Toolbar or the Menu Item Runl Run Batch F5 Design 1 1 4 4 21 21 10 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 145 Institut Curie Image Simulator DER Fie Run Help The parameters of the ey Image Simulator is Layout Margins Image 0 equivalent to the Two Bottom 30 Top 30 Left 30 Right Color Image Case uq us Spots Interval x 15 Y Spot Radius Position 5D Mean 3 Spots 1 Blocks Image Dust 0 5 Back N S 1 Radius 2 5 Density Restore Defaults One image is simulated Image Dust Shift X Shift Y Image soo 65535 0 NoiseToSignal 0 1 NoiseType Proportional Seed 123455 Simulation Finished 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 146 Institut Curie
79. e background around the spot i e along the grid lines separating neighborhood spots is defined as UB YIB BI B where B i 1 8 are the mean intensities in 8 sectors of the grid line around the spot and B is the mean intensity for the whole grid line around the spot Extremely small values may be due to relatively bright contamination around the spot large variability in the background or merged neighborhood spots q UB exp UB Absolute level of background AB calculated in the proximity of each particular spot is compared to the typical level of the local background for a given array Large deviations from the typical state may indicate the presence of the contamination areas which are larger than the size of the spot q AB exp 1 AB T if AB gt T and q AB exp AB T 1 if AB lt T where T is the typical background level Signal S is defined as a difference between the mean estimate of the intensity within the contoured spot and the mean estimate of the background level q S 1 if S gt T and q S exp S T 1 if S lt T where T is the typical signal For the purposes of further quality analysis functions q rescale quality characteristics to fit the range between 0 bad spot and 1 good spot MAIA 2 7 Manual Copyright C 2005 2006 141 Institut Curie Quality Analysis Quality analysis is equivalent to the Two Color Image Case Quality plot y axis is the overall qu
80. e pixel is a random value B Br ogBiG where B and o are the user defined average and noise to signal ratio of nonspecific fluorescence intensity in the color channel i and G is a gaussian random variable with zero mean and unit standard deviation Dust is represented by randomly distributed over the array more or less bright clusters of pixels which can hardly be distinguished from the spots We apply the same profile for the dust clusters as for the spots d x y L exp l X X J y Xa H X X l Y Yed j T r Y LA where x 4 and y_ are the coordinates of the center of a dust cluster r is its approximate radius and 7 is the intensity in the center of the cluster All these parameters are random variables We use uniform distributions for r in the interval 0 r and J in the interval 0 where r and J are user provided maximal dust cluster radius and maximal dust intensity respectively We also assume that the coordinates of the centers of dust clusters x and y are uniformly distributed over the array Statistical laws of the dust characteristics can generally be different for two channels i Cy3 Cy5 Finally one has to define the number or density of the dust clusters on the array The general model for the microarray image takes the form N M fi Y 3 fa y Bj 9 da x y k l k l where N is the number of spots and M is the number of dust clusters Statistical noise is finally added to
81. each pixel of the image fi f G y o05y G where o x y is the standard deviation of the pixel noise o x y can be i constant ii proportional to signal or iii proportional to the square root of signal The type of statistical noise as well as its quantitative characteristics is defined by the user E Novikov and E Barillot A robust algorithm for ratio estimation in two color microarray experiments Journal of Bioinformatics and Computational Biology 2005 3 1411 1428 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 133 Institut Curie One Color Image Analysis Very much similar to Two Color Image Analysis 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 134 Data Import Settings To define the format of Two options are the microarray image available files select the Menu S MAIA27 Batch defaults MAIA 1 TIFF images are Item Options Data File Model Rum Options Help B packed into one multi Options Alt D H y b W so sL sort sa page TIFF file visible checked ii TIFF images are stored in separate files EE unchecked O For multi page TIFF specify the page for the image to be analyzed Design 1 1 4 4 21 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 135 Institut Curie Main Processing Window Another image can be downloaded using the Load Data button from the Toolbar or the M
82. eeseoonopes CAOCOCCOCESECBO 8000000 seeeceneoes eoece nec 9 e 0 L 2 500 2 000 o amp 1 500 att al 2 1 000 wes an soo i i P i4 500 1 000 1 500 2 000 2 500 3 000 Cy3 L m 9 J amp 1042 Quality 0 68 e _ LJ Manual 0 512 Reset Manual gt Image Alignment 4 Block Independent V w Sx 02 gt Shift Y 012 t Y 7 x 12 Save Default v mM 0 05 Quit 022 2 at 1448 540 Y 0 X 317 BI Y X Sp X Ho ID Ho Hame Fit Limits 0 828 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 107 Colors 2 MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin DEAR File Run Options Window Help fo ME rco HAS o He OI Me BIY ko Sp v 9 x 7 Mo Mame Ratio Z Charsyferistic Typical 0 1 99 9 This Quality 0 rum 0 lo 0 ji less 515 7 48 0 68 To change the color of l some elements of the n izati Selected arid localization and sare quantification outputs use TOS Spot Contours mme 7 3 Back Contours the Menu Item 8 Saturated Pixels E m Bad Spots 3l i 5 OptionslColors Selected Loca LOETTETTETTETOETTH xxiii cya Selected Spot Manual Selection Quality 0 68 Outliers
83. endent v Shift X w shitty Save Defaut v lt gt Y 5 X 13 nm E Aj Qualit j 028 012 012 UR RE m 4 1 af EL eo 3 IIILIIITIEI EI CIIIBEI aa ie li Lis o gt A T5 LEM L LE os PE T Em TELLI ee 6 E EE Tn m r3 1 E T es amo EE LLL Ne MEN e Manual Quality x100 d II es mH E EIJ 2 LE Lf E EE LI I E Ein ANII i oy LI 1445 553 Bl Y X Sp Y X Ho ID Quantification 2 2655 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 73 Manual Spot Characterization Negative values available in the Manual spinner are not considered as quality values and can be used for additional spot characterization The Reset Manual button sets the manual parameter for all spots on the array in 1 11 Oct 06 9 MAIA27 D images Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d OH Pclo HE ee HH 2 RS m e e l3 Box spQrexo Characteristic Typical 0 1 99 9 This Quality 0 05 Contamination 0 o 1 jo 1 Diameter 6 86 lo 9 44 5 86 0 71 CVRatios 001 0 49 jot p 93 Manual I Reset Manual
84. ensity equal to 216 1 N q N 1 N S where S is the size of the correspondent spot i e the number of pixels within the spot contour Diameter of the spot D 2 S z Since it is hard to impose a priory an exact ideal value for the diameter the median diameter over all spots on the array is taken as a typical one Spots with exceptionally small or large diameters should normally be penalized q D exp T D if D gt T and q D exp Tp D if D lt T where T is the typical diameter Geometrical symmetry parameter measures deviation of the contoured spot from the ideal circle Both the real spot and the ideal circle are divided into 8 sectors pie slices defined as k7 4 k 1 7 4 k 0 7 and for each sector the number of pixels belonging to the spot N i 1 8 and to the circle Np i 1 8 is counted Then the quality characteristic is defined as GS DIN N N For ideal circular spots GS must approach 0 whereas highly un circular spots should give relatively high GS values q GS exp GS Intensity symmetry of the spot is defined as JS XII I I where I i 1 8 are the mean intensities for the same 8 sectors and J is the mean intensity for the whole spot A spot may have perfect circular shape but within this circle very bright or dark and highly concentrated groups of pixels originated from the pieces of dust or other contamination may occur q IS exp IS 11 Oct 06 Uniformity of th
85. enu Item FilelLoadlIData Ctrl O For the new images image file format i e multi page TIFF versus single page TIFF can be changed using the Menu Item OptionslData Options Alt D 11 Oct 06 g 7 CLIAE MAIJ D images Wicroarray CGH Fii File Run Options Window Help Charactfristic Typical 0 1 99 9 This Quality Cluster 1 ewe s 1 lt gt B Yi0 0 eeeee ELLET eee eeecceeoe RII o eee 646 Be Pco M4 fH TS IG Se E 13 E s Sp Y X T Ho Hame D Images Microarray CGH Firstir O95 Jl 20 95 1S HATAAIAAIAAAS eee eee 1g 01g e Seeeereseoo 868000 e e ee800c0ceeeeseeee 8 88888808 Quality Plot 1 00 0 75 Sos SSS ne eee ee ee ne Pree T T T ee ee ee ee o 0 25 0 00 0 00 0 25 0 50 0 75 100 es 391 Y O X 25 BI Y X Sp X Ho ID Ho Hame MAIA 2 7 Manual Copyright C 2005 2006 Design 1 1 4 4 21 21 0 0 0 0 0 1 1 1 Institut Curie 136 Array Design 2 MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help 66 m Pco I 4 TEES Bl Y X Sp Y X Ho Hame D Images Microarray CGH Firstir Characteristic Typical 0 1 99 9 This Quality Cluster 1 4 Contaminat n 3402 e eee
86. eplicated arrays The high quality spots belonging to the same replicate are expected to demonstrate very close to each other ratio value Relatively big difference between the observed ratios in the same replicate will signal that some of the spots from this replicate are irregular To formalize this approach we first define the quality value for the replicate Q min nl Qyj 1 where k enumerates the replicates n is the number of spots in a replicate and Qy is a spot quality value given by Eq 1 page Substituting Eq 1 page into 3 yields Q min imin ldg h where dy is the i th scaled quality parameter of the j th replicated spot in the k th replicate The weights w can be determined as the parameters ensuring the best fit of the obtained experimental quality values Q versus V to the user defined ideal quality curve f V where V is the ratio variation coefficient in the k th replicate f V defines how fast the overall quality of the replicates must decrease with the increase of the ratio variation The shape of the user defined quality curve f V should demonstrate monotonic decay We always use the exponential function f V exp V V and in this case only the expected typical ratio variation coefficient V must be predefined E Novikov and E Barillot An algorithm for automatic evaluation of the spot quality in two color DNA microarray experiments BMC Bioinformatics 2005 6 293 11 Oct 06 MAIA 2 7
87. ft can be saved using EE o o Signal O 3402 the button Save and Regression Plot used for comparison with ca e the automatically M generated Default and Id zu Zero 0 shift values ES SEE vN JE 102 M z N Image Alignment Block Independent V shift x v 12 8 Shift Y 07 lt Bi 3 save Saved v 1 SEIT I Es 1 392 454 Y 597 X 8 Bl Y X X Ho ID mage Alignment 0 578 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 47 Institut Curie Zero Shift 2 MAIA27 D Images Wicroarray CGH First rawdata0 208051020805FR1087 cy3 bin File Run Options Window Help 6 amp Bim eco MOS o BH ONSE x BI Y 1 1 Sp 12 9 Mo Name Ratio Characteristic Typical 0 195 99 995 This Quality Determination 1 DurbinWatson 4 Contamination 3402 Diameter 3402 Symmetry 3402 ISymmetry 3402 ec 29 CVRatios 3402 The Shift combo box is em ackground 3402 used to switch between petro MEM Signal 3402 different shift values i Regression Plot Zero shift is selected v 6 Ne 102 Image Alignment Black Independent vV NX oi zm s lt gt Shift 02 Save j 0 v 3 5 X 13 mcm 012 JE i69 ei 1 MB jelelolololsielsieleleleiollolololo
88. ge Image 1 stands for Cy5 image 11 Oct 06 File Run Help gt a 0 Layout Margins Bottom 308 Spots x Ans Spots Interval EKEN 15 Spot Radius Mean E 3i Image Spots Back NIS 30 Riot 30 Blocks x Blocks Interval x 20 x Position SD amp Spots oR Blocks E 08 Dust Radius 2 Denity 012 Back 5000 Dust Shift X Shift Y 0 Restore Defaults 65535 5000 65535 NoiseToSignal NoiseType Seed 0 01 Constant 1111 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie Array Layout I The distance in pixels from the edges of the bw array to the spotting area 4 xm Spots x Spots Interval Blocks Interval x P x 20 SS ee IRR Mean Spots 1 A7 Blocks Amount of spots per block and amount of blocks on the array 2 5 Density Restore Defaults 1000 65535 Distance in pixels between the spots in the blocks Distance in pixels between the blocks pes wu te Seed 123455 Simulation finished 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 125 Institut Curie Array Layout II File Run Help Mean and standard E pm 9 deviation of the Spot Layout Margins Radius ees If SD gt 0 spots will be generated Spots J Bees with randomly selected around is Mean radius Blocks Interval x 20 x
89. ge Alignment Block Independent V Shift x 028 Shift Y 012 Save Default v 0 05 2 Quali 022 M gt Ore Heine Jn M ac 2500 00 2000 00 1500 00 1000 00 500 00 0 00 0 00 0 25 Counts Liner Log2 0 50 0 75 Determination Determination Linear Log2 Counts Linear Log2 log2 Contamination Contamination Olinear Log2 800 00 700 00 600 00 500 00 400 00 300 00 200 00 100 00 0 00 0 Counts Linear Log2 Diameter Diameter Linear OLog2 500 00 400 00 300 00 200 00 100 00 0 00 Counts Linear O Log2 log2 CVRatios r CVRatios Olinear Log2 Bl Y 0 X Q Sp Y 1 X1 DEAR Ft Limits 0 828 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 94 Diagnostic Plots Layout The Columns spinner defines number of graphs in the rows and the Row height slider defines the height of the graphs panels Graphs can be shown in linear or log scales 11 Oct 06 D lmage oarra awdg File Run Options Window Help d d Mim pch MES oi Bl Y 0 X 0 Sp Y 9 X 7 Ho Name Characteristic Typical 0 1 99 9 This Quality 05 Contamination 0 11 t Diameter 6 86 o 144 7 48 0 68 CVRatf s jot 49 oo2
90. h lower values would point on either strong contribution of statistical noise which normally characterizes low level or absent spots or presence of a relatively bright but non correlated contamination q CD CD Durbin Watson statistic DWS controls the presence of first order autocorrelation in the residuals of the linear regression fit It ranges from 0 to 4 0 meaning positive correlation and 4 negative correlation DWS 2 leads to the conclusion that the residuals are uncorrelated and the model is appropriate Large departures from 2 suggests that this spot can not be modeled in terms of simple linear regression q DWS 1 IDWS 2V2 Spot contamination is a number of aberrant pixels within the spot contours flagged out by the filtering procedure N q N I N S where S is the size of the correspondent spot i e the number of pixels within the spot contour Diameter of the spot D 2 S z Since it is hard to impose a priory an exact ideal value for the diameter the median diameter over all spots on the array is taken as a typical one Spots with exceptionally small or large diameters should normally be penalized q D exp T D if D gt T and q D exp Tp D if D lt T where T is the typical diameter Geometrical symmetry parameter measures deviation of the contoured spot from the ideal circle Both the real spot and the ideal circle are divided into 8 sectors pie slices defined as kz A k4 1 z 4 k 0
91. haracteristic Typical 0 1 99 9 This Quality Determination 0 98 0 4 1 0 97 0 96 Contamination 0 4 8 1 ma 8 25 725 v Fixed Graos U5 5 Bag noz 10 99 Allde 4 i i All Fixed Ear 2225 71 1 All Free ji l 10 Quality 0 12 Manual 0 85 Reset Manual Image Alignment Block Independent v Ratio x ter Rd tttdotd ottoe eee i i PSSST OB Pte eee te 9959 Hatte oBo4R o toote 6444444454404 POPSEPIII OSS POFSSVSFESSS OF9 COFFE VISSSSVIOSIS SOSVSSS SPO OCLSESS Le 90 O 444 tede ed H T r 5 stx 022 geese 2 1 Y 5 X 16 Shift Y 012 HT TM e Sactoceeaee z In M 0227 Quat 025y tttocecessesete Quality Plot b obobc obsess an 2222727090489 0000 0cddodt 066000000 040 0 75 N e eevee S 0 50 4 2290 e 0 25 z toe di X H HM 0 00 ON Y er totttttttttt t 5 Op 00 0 5 10 15 20 25 30 In Manual 1389 453 BI Y X Sp Y X Ho ID Fit Limits 0 171 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 82 Quality Analysis Using Replicated Spots On this image three replicated spots are placed as neighbors in a row This is defined by the Array Design dialog c
92. iation of the regression and segmentation ratios C Ratios Alt F SEL A DOR Siain kisia 73 BO ODE Corresponding quality parameter Q C Ratios En Uniformity of the background around the spot RBackground Qualty 0 68 Corresponding quality parameter Q RBackground I 3 Background intensity maximal of Cy3 Cy5 ABackground See next page for details onus 051g Corresponding quality parameter Q ABackground Spot intensity Background intensity minimal of Cy3 CyS Signal r Image Alignment Corresponding quality parameter Q Signal 2 Block Independent Mean spot intensity Cy3 F532 Mean Hi PET Mean spot intensity Cy5 F635 Mean I Median spot intensity Cy3 F532 Median sv d o1 Median spot intensity Cy5 F635 Median Save Defaul w Standard deviation of spot intensity Cy3 F532 5d Standard deviation of spot intensity Cy5 F635 Sd Number of spot pixels Cy3 F532 Pixels Number of spot pixels Cy5 F635 Pixels Bl Y X Sp X Mean background intensity Cy3 B532 Mean Fit Limits 0 828 s J Mean background intensity Cy5 p635 Mean v 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 104 Institut Curie Output Table Format in Detail Description of the field non editable Editable name of the field to be appeared in the output file Order specifies the sequence of the fields If this field is empty the corresponding field is not included in the output file Include all fields Exclude all fie
93. is 2 Quality tistngrams RESUME PERS Histogram of Ratios Scatter Plot Contamination lo 2500 00 12500 00 Diameter ps 68 d 10000 00 i 1500 00 Se R 6 Results Plots panel Ae 6000 09 contain 600 00 eU 5 l lOS E 0 00 0 00 prey Histogram of ratios 45 50 25 00 25 50 75 2 500 5 000 7 500 10 000 12 500 Scatter plot muet log2 Ratio cya E uM us Counts Ratio rCy5 Cy3 M A plot a Linear Log2 O Linear Log2 Linear Log2 Linear Log2 Ratio vs Intensity plot M A Plot Ratio vs Intensity Cy3 Quality 0 68 Manual AG Reset Manual r Image Alignment log2 Cy5 Cy3 w Sx lt gt shifty 01 t vixi Save Default 024 2 500 5 000 Cy3 Cy5 Ratio Cy3 Cy5 log2 Cy3 Cy5y2 linear Log2 liner Log2 Bl Y 0 X 0 Sp Y 1 X1 is Fit Limits 0 828 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 97 Institut Curie Diagnostic Plots with All Spots 2 MAIA27 D lmages Wicroarray CGH FirstirawdgtaN 208051020 RO 5ER10R7 cv3 hin File Run Options Window Help 2 Diagnostics d d HE pco MEE 4 Bv 0 X0 Sp v 9 7 NoName Characteristic Typical 0 1 99 9 This Ky l Quality Histograms Resuksi Pots
94. ization 6 5625 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 32 Institut Curie Main Grid Refinement Find Grids in Blocks 2 MAIA27 D Images Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d Hm rco iB 4 He OI 8x Spx o Hame Characteristic Typical pA This Quality Determination DurbinWatson Contamination Manual correction can be done only for the borders of the blocks The other internal lines of the grids are found automatically using the Grids in Blocks button from the Toolbar or the Menu Item RunlGrids in Blocks yis Block Independent V J I A fs ir g Default w eoe PR 7l sjele les leise j r mM JOUODDODUI Bees 1452 483 FIRA Bl Y X Pap RES Ho ID Localization 6 562 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 33 Institut Curie Main Grid Refinement Lines Refinement When the main grid is almost good further refinement procedure will try to place the grid lines more precisely use the Lines Refinement button from the Toolbar or the Menu Item RunlLines Refinement 11 Oct 06 3 MAIA27 D Images Wicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Wind
95. lData Options Alt D 391 442 Y 0 X 30 BI Y X Sp X Ho ID Ho Hame Design 1 1 4 4 21 21 40 0 0 0 0 1 1 1 Pixel intensities Pixel coordinates 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 11 Institut Curie Image Visualization Settings 9 MAIA27 D lmages Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d Be rco Has ea amp o HE X Bx sb 5 xX NoName Characteristic Typical 0 1 99 9 This Quality Determination Durbinwatson 0 1 4 Contamination Diameter 13402 183402 G5ymmetry ISymmetry p peo 3402 CVRatios Contrast and RBackground lo jo 3402 o 3402 Brightness controls can Background Signal lo 8492 o 3402 be used to adjust brightness and contrast of the images Brightness and contrast can be adjusted either independently for each color channel the button All Images is off or simultaneously for all Regression Plot Image Alignment channels the button AII 0 123 liz Ratio eee eeqeoesooescrseece eececccese Images is on 1582 549 Sp Y X Y 1 X 320 11 Oct 06 Design 1 1 4 4 21 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual
96. lds Restore previous set of fields 11 Oct 06 Output Format MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie i poera gn Ratio atio of Means og Ratio ormalize verall Quality Determination Determination Durbinwatson DurbinWatson ontamination Contamination iameter Diameter Symmetry GSymmetry ymmetry ISymmetry c Ratios C Ratios RBackground Q RBackground ABackground Q ABackground 532 Median 635 Median 532 Pixels F635 Pixels Lr ho P 0 y E E o es ae e SSS PEN SSS See EE a a PO aes CEPI a A sl ae e E E E T O O i as 105 Save the Experiment Experiment File e AIA D age oarra awdata0 20805 0 20805FR108 b PBS Fie Run Options Window Help d d ME pco EIN 4 He ON B Miele e L3 B vox0 spv 9 7 NoName Ratio v 9 97 I dx 97 12 Characteristic Typical 0 195 99 995 This Quality Ha Contamination D o i ocece Diameter Bas 5 18 p 44 7 48 pes HIE oaeeo LEKET The whole experiment CVRatios p or b 349 0 02 0 99 HEN IJ E cooo results parameters grid 1 EN and other settings can be RR 0 79 RS 0 772 BE A eocese saved on the disk using d 2 000 g the Menu Item S 1 5
97. lick the Array Design button from the Toolbar or select the Menu Item OptionslArray Design Alt A After changing the design the Init Limits button from the Toolbar or the Menu Item Runinit Limits Ctrl F9 can be used to restore the default limits 11 Oct 06 P MAIA27 D MmagesMWicroarrayYCGHW irstirawdata0208051020805FR1087 cy3 bin File Run Options Window Help amp d d MH palo HE 4 o HE O INE DES e 14 DR Bl Y 0 X 0 Sp Y 9 X 7 Ho Name Typical 0 1 99 9 This Quality 0 4 ontamination 0 jo i 0 j Diameter 6 86 6 15 7 48 0 12 iC Ratios Quality gt Manual 0 85 ES Reset Manual Image Alignment Block Independent V v lt gt Shift x D 2 E EE V 5 X 16 shifty 012 Se In M 0 22 Qual 0 25 2 Quality Plot a 15 20 25 In Manual Ratio vi 9 97 Ob Oe tot Py ers or Pees eee ee4 Array Design 24 y g Design Default GAL File C GAL Block Design SubArrays Spot Diameter C used 102 t t0 a REPAS S66 BA oh Dn dh Distance Between Replicas In SubArrays Y oy in Blocks Y E in Spots Replicas pixels Y SPT SVS 41144 Stoves kd eve POPSESIIII OSS 99S OFFS SS e bt oto PORTS tottttttttt oP e i4 4424 44444 SISSISSOOLSESS 1428 476 Bl Y X Y 114 X 8
98. lue Fus eum o5 aes or a limit into a new i 200 00 location of the qualit Image Alignment q y Block Independent V 100 00 histogram sx 023 v 0 00 lt gt shitty 0 14 N k B S 1 YEZ X12 d Save Defaut I Diameter log2 CVRatios nm 05 gt Diameter Counts C Ratios Bo Bl Y X Fit Limits 0 828 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 101 Institut Curie Save the Selected Plot P MAIA27 D lmages Wicroarray CGH FirstirawdetaN 208051020 RO 5ER10R7 cv3 hin File Run Options Window Help dd Bm slo B v 0 A ce Characteristic Typical 99 995 Quality Histograms Results Plots Cl c X na Contamination ane Diameter n 2000 00 1500 00 4 Any diagnostic plot can 1000 00 be saved as an image file 500 00 tif jpg gif bmp formats E Determination log2 Contamination Counts Determination r Counts Contamination Linear Log2 liner OLog2 Linear Log2 Olinear Log2 500 00 500 1 000 1 500 2 000 2 500 3 000 Cy3 400 00 n 9 amp 10 2 Quality 0 68 300 00 Manual 0 51 E r Image Alignment Block Independent v 2 sx 02 EN v EM UNE 0 00 lt z shey oaa o 20 15 0 6 1 YEZ X12 d Save Defaut I Diameter log2 CVRatios 0 05 2 Qualit
99. me Ratio Characteristic Typical 0 195 99 995 This Quality bs T 688 Contamination 0 p m p p E re E Diameter se o 44 748 0 57 a E a EE S Y EIL E LEET a LES pt pth aded LJ Qoe ra ee D EELEE User assigns quality values 07 15 9e ee c ALLE eooceesese RUIT ALLL ecesseece eeeeee 0 1 to some a s IIEZI ae a LEEREN representative spots These nen TIT esee 000000 values z are converted as gi cse os Oe Su 00o JIIITI Manual a L i a eog I In z to create the x axis Reset Manus e008 TELET Image Alignment F4 rd E a e of the quality plot Fick reependnt TT TITELE T i satx 022 E SFL E E y axis the overall quality f m 222 Wa o50 parameter ETF e seer LI Quality Plot e 7 C a oa gt E s LALELE I PESES a ee Visually qualified spots are WAMIIZITIIIIII Ll ma marked by a dot in the upper i i i eeeseeeeeseacsase o LT A E 1 0 1 5 20 d 4 left corner of the spot area In Manual Se VEO tot Bl Y X Pap RES Ho ID Quantification 2 2655 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 79 Institut Curie Quality Curve File Run Options Window Help d d HE Pclo Egoi Bvo x0 sp v 9 X 7 NoName 99 995 i Quality WES T IEEELANdJELl Contamination 0 o ooo 1 ONG amp 6seees amp amp sddesl5 Diame
100. n 0 98 m Symmetry D b 5 58 942 o 81 Symmetry 0 3 0 2 65 0 29 0 78 C Ratios oo i Bf bo i RBackground 0 14 03 53 87 0 09 0 95 Background 508 78 326 712 73 514 06 0 94 Signal 1820 69 10 5483 5 229 1 R 0 766 RS 0 762 4 000 r u EP ost 7 3 000 4 000 o Cy3 L e 2 e Quality 0 58 fe e Manual EJ gt Reset Manual Image Alignment LEAL LLEI eocece Block Independent V Shift x 0 23 Shift D M LAAALULAALAL 01 Jie e29 008280 Secocoeceedesd ooeccera omen cok 2 2 2eeceeso e 8 LJ 6 L s LJ 9 L IIIETI eooee LJ enopegeoose 0000 beh ooe 1408 459 Bl Y X Sp Y Xi 514 X 15 Quantification 2 2655 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 67 Quality Parameter in Detail The overall quality value is defined as Q min q 1 where q q x 0 1 are the marginal scaled quality parameters defined on page for x CD DWS N D GS IS CVR UB AB S and w are the weights that control the input of the correspondent quality components into the overall quality value For the user provided overall quality threshold Q lt 0 1 one can establish a link between the weight w and the critic
101. n filtering Removal of the shift enhances the correlation between the two color channels thus making uncorrelated pixels easier detectable 11 Oct 06 3 MAIA27 D Images Wicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d He rco REIN 4 Hs O II x Bixp spcuizxe Characteristic Typical 0 1 Determination DurbinWatson Contamination Image Alignment Block Independent V Shift x 022 v Shift Y 012 2 Default w En PAANI 10010 1451 489 Bl Y 5 X Pap RES Ho ID Y 11 X 144 Localization 0 687 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 51 Image Alignment Settings P MAIA27 D lmages Microarray CGH First rawdata0 20805 020805FR1087_cy3 bin File Run Options Window Help i dfo Hm eco HHS e NE O INS x mnk seoizx9 Noname Ratio Charactfristic Typical 0 195 99 995 This Quality 1 4 3402 9402 3402 isymmetry 3 9402 CVRatios 3402 Several settings that may 77 wl influence the image EH EUM alignment procedure are Em i T er erT we Value Default available from the Menu pues BIB Block Independent Shift v iv mage Shift Range 2 0 2 0 Item OptionslAnaly
102. nation 025 Gum Quality Plot 00 0 1 02 03 04 05 06 07 08 09 10 11 12 13 14 15 18 17 18 Ratio CV Fit Limits 0 546 s 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 118 Institut Curie Select Bad Replicates Ctrl Left Clicks followed Global Analysis by Ctrl Right Click create the contour on the Z Normalized Quality plot This contour Characteristic Typical selects the replicates to be able to find them on the Contamination arrays from the globally analyzed selection of arrays 022 Quivimt Quality Plot selected 27 00 01 02 03 04 05 0 5 07 DH 0B 10 114 T2 13 174 15 48 t7 1H Ratio CV 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 119 Institut Curie Export Quality Limits To send the obtained quality limits and selected 2 Global Analysis replicates to each array a Normalized file from the given Characteristic selection press the Toolbar button Set Contamination Limits 00 0 1 02 03 04 05 06 07 DH UB TU TA 12 13 oa T5 T7 XH Ratio CV Ftimts05 s 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 120 Institut Curie The Selected Bad Replicates I The selected replicates are highlighted on both arrays The first
103. ndependent v JMllauaaonuonoonn o i SGEREEEBEBBEB Save _ Default v t K n ho Series ssl IDCDDE eee ie EB js aa sess eerste BBGBBEBOCCOOCODOCOEOBBB I OOOOOOOO 18 e 1432 524 Y 0 X 59 BI Y X Sp Y 4 Ho ID Localization 6 5625 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 25 Institut Curie Spot Localization Output Adjusted Grid Using the Toolbar buttons Show Hide Main grid or Show Hide Adjusted grid one can mask either of two spot localization grids Adjusted Grid is shown 11 Oct 06 File Run Options Window Help d amp Hm rco HES 0 WB O SU x B x Sp 5X NoNami Characteristic Typical 0 195 99 995 Thi ity Determination 0 1 DurbinWatson T 1 T Contamination 3402 Diametgy O 940 1 ry 0 3402 Symmetry lo aoz f CVRatios z o 3402 d u RBackground jo 3402 ABackground o 3402 Signal 0 940 Regression Plot 1 00 0 75 amp oso 0 25 0 00 00 0 25 0 50 0 75 1 00 Cy3 Image Alignment Block Independent V lt Bi Save Defaul w rx Ld 18 012 Bl Y 5 X Localization 6 5625 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 26 Manual Corre
104. o 85 cee GSymmetry o 7 o z IITTTTTTTTITITT cee Isymmetry 03 o sacese cas White crosses indicate Sa Eu em Ref m 3 bad spots i e spots ABackground 508 46 n f73 514 06 p 94 p LU pt Signal itgfo es 10 83 5 229 1 whose overall quality Tess 0762 value iS below the Q M jioececeneott LJ Limit as defined by the 3 HH EITHER Quality Limit spinner E 3 e See ee ais es aus Sus M ses Sogecsccoece 38 or equivalently if one of Quy jose sso 4 1 icti pani 18 990000909000 HP H the quality characteristics _Reset nena SSsssssscssssss S H r Image Alignment a Aeee tA Mte of a spot exceeds the Block Independent V M HMM Ht LI admissible limits defined Sx 028 shift Y 012 by the corresponding ESNA i 3 oe e 9 percentiles nn it 5 C 29 LLIZXILAIIIIJ 1437 540 Y 3 X 189 Bl Y X Sp Y X t Quantification 2 265 5 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 69 Institut Curie Used Quality Characteristics 2 MAIA27 D lmages Wicroarray CGH First rawdata0 2 020805FR1087_cy3 bin File Run Options Window Help b d Me eco BEL 4 6 Mi ORAM EX e Bizx2 sinxa Characteristic Typical 0 1 99 9 This Quality Determination 0 98 10 05 1 10 99 10 99 H e de e EXE E Contamination D o m b 0 58 Ss e Diameter pss b pos pss see EE Using the right button vH 29 4 Fixed pop
105. o si ia sa FL sa TOSS Dir Visible D Images Microarray 021007FR1077_cy3 tiF F D Images Microarray 021026FR1077_cy3 tif Dataset D Images Microarray CGH First Duplicates D1077 021007FR1077_cy3 tif 11 407 s Memory 184973360 Memory 184973360 Dataset D Images Microarray CGH First Duplicates D1077 021026FR1077_cy3 tiF 10 828 s Memory 185546320 z Ed Design 11 1 4 4 22 21 0 0 0 0 0 1 1 3 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie Select the Menu Item RunlGlobal Analysis to open the window for identification of the global Quality Limits Check the field Run to specify which arrays will be used for global quality analysis 113 Global Quality Analysis Main Window Global quality analysis panel shows up with the same set of quality characteristics as for each particular image 11 Oct 06 Global Analysis Replicated v Normalized Characteristic Typical Determination DurbinWatson 340282346638 340282346638 340282346638 340282346638 340282346638 340282346638 340282346638 340282346638 Contamination eojojojojojojojojo j o 1 Quality Lirnit hd Quality Plot 05 Ratio CV MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie Press the Toolbar button Get Experiments to copy quantification results from all selecte
106. ow Help d d Hm rco KEEN Bj OIN BX soso e Show Hide Main Grid Characteristic Typical 99 This Quality Determination DurbinWatson Contamination D 3402 9402 9402 5402 9402 3402 3402 Regression Plot Image Alignment Block Independent V J I A fs ir g Default w eoe PR 7l sjele les leise j r mM JOUODDODUI Bees 1452 483 FIRA Bl Y X X Ho ID Localization 6 562 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 34 Adjusted Grid Refinement Cuts Refinement When the adjusted grid is almost good further refinement procedure will try to place the separators cuts between neighboring spots more precisely use the Cuts Refinement button from the Toolbar or the Menu Item RunlCuts Refinement 11 Oct 06 File Run Options Window Help d d BE Plo BAS o e OR G x xJje l4 asx Spx We Show Hide Main Grid v 9 95 ji 97 15 Characteristic Typical 0 195 99 9945 Quality 7 Determination 0 1 DurbinWatson Contamination 3402 Diameter 3402 GSymmetr p 8402 ISypefftry o 9402 Ratios D 402 RBackground o 9402 ABackground 0 3402 Signal
107. s Dust Radius 2 5 Density Restore Defaults Noise to signal ratio for T EE St non specific hybridization for both color channels NoiseToSignal 0 1 NoiseType Proportional Seed 123455 Simulation finished 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 128 Institut Curie Dust Density of dust is defined with respect to the number of good spots on the array 0 no dust spots 1 the number of dust spots equals to the number of good spots Maximal dust radius The radius of the dust spot is randomly chosen from the interval from 0 to the given value Maximal intensity of dust in the Cy3 and Cy5 color channels Real intensity will be randomly chosen from the interval from 0 to the given value 11 Oct 06 Blocks x Spots Interval Blocks Interval x 151 Spot Radius Mean 3 Image 2 5 Density Restore Defaults Shift X Shift Y NoiseToSignal 0 1 NoiseType Proportional Seed 123455 Simulation finished MAIA 2 7 Manual Copyright C 2005 2006 129 Institut Curie Image Shift File Run Help gt a 0 amp Layout Margins Bottom Spots x Spots Interval x 151 Spot Radius Image shift in pixels in A horizontal direction 0 5 Back N S 10000 20000 Blocks Interval x 20 Y Position SD Spots 1 Blocks Dust Radius 2 5 Density Restore Defaults ust Shift X 1000 6
108. s of type TIFF Cy3 532 Images v 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 7 Institut Curie Directory Selection s All TIFF files extension MAIA27 Batch defaults MAIA tif tff from the Fie Model Run Options Help same directory can be Dow 9 b M SOSLIASQFL SA selected Visible Look in la CGH O Duplicates 7 GER CD rawdata021022 O rawdata021004 O rawdata021026 documents o rawdata021007 rawdata021029 recens rawdata021007 2 rawdata021030 3 rawdata021010 rawdata021102 rawdata021 010 2 ravedata021103 Bureau C rawdata021012 C rawdata02110j d D rawdata021017 rawdata021fl8 cJ O rawdata021017 2 resultatsCQ Mes documents rawdata021019 Spot Exefnples 48 File name rawdata020805 Poste de travail Files of type TIFF Cy3 532 Images 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 8 Institut Curie Batch of File Names The selected filenames g i appear in the table MAIA27 Batch defaults MAIA TBR File Model Run Options Help oi D Images Microarray D Images Microarray O20805FR326671_cy D Images Microarray Q20805FR9216 cy3 tif The whole batch a list of files and accompa nying options can be saved on the disk using the Menu Item FilelSave Group Ctrl S to be able to restore it using the Menu Item FilelLoad Group Ctrl O
109. sed over the image Main Grid is composed of the straight lines separating neighborhood spot rows or columns eAdjusted Grid is composed of the piecewise lines refined borders between the neighborhood spots E Novikov and E Barillot A noise resistant algorithm for grid finding in microarray image analysis Machine Vision and Applications 2006 17 337 345 11 Oct 06 2 MAIA27 D Images Wicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help E ED e e ONSE x No Hame This Quality Determination Burbin Watson T Diameter Symmetry ISymmetry CVRatios RBackground 3402 3402 ABackground Signal 3402 3402 Regression Plot Block Independent Image Alignment Ratio see DOCU BICI 1448 515 Y 10 X 275 Bl Y 5 X Ho ID Localization 6 5625 Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie 23 Spot Localization Output Spot Identification 2 MAIA27 D Images Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help db Wim Pleo MM 4 emm Ol x Bysx3 Sbqx No Name Ratio Characteristic Typical 0 1 This Quality Determination ponams Es DurbinWatson Contamination 3402
110. sis Options Alt O tab Alignment See next page for details Image Alignment Block Independent sitx 025 w Shifty 0 1 E lt gt Save Defaul w Y 5 Xi 13 i 1451 489 Y 11 X 144 Bl Y 5 X 125 xi Ho ID Localization 0 687 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 52 Institut Curie Image Alignment Settings in Detail Block Independent Shift defines whether the shift is the same for EON all blocks on the array on or it e is specific for each block off Localization Alignment Quantification Local Quality Title Value Default M d Block Independent Shift Image Shift Range 2 0 2 0 Image Shift Range establishes the boundaries in pixels for the relative shift between the two images 2 pixels by default Restore Defaults 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 53 Institut Curie Spot Quantification C MAIA27 D lmages Microarray CGH First rawdata020805 020805FR1087_cy3 bin File Run Options Window Help d d Hm rco REI 4 He O INS x BI Y 1 1 Sp 12 9 Mo Name Characteristic Typical 0 195 99 9945 Quality Determination DurbinWatson Contamination 3402 To start Spot oro b obe Quantification use the mooni e Spot Quantification TET button from the Toolbar
111. sults Global Analysis have been downloaded v Normalized Durbin Watson Contamination 2792 74 1 il Quality Limit Quality Plot 0 10 MEL 000i e n ze Rt oa os i 00 0 1 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 Ratio CV Fit Limits 0 531 s 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie The following quality analysis procedure is equivalent to the quality analysis performed for each particular image 116 Global Quality Plot A set of used quality awe Global Analysis characteristics can be eH 44 defined v Replicated v Normalized Characteristic 99 9 Determination Diameter v Used Fixed All Used C Ratios All Idle al Fixed All Free Ratio CV Limit 0 2 5 To identify the shape of the quality curve one can use the same tools as for each particular image 0 00 i s 0 0 0 1 02 03 04 05 06 07 O8 00 10 11 12 13 14 15 16 17 18 Ratio CV it Limits 0 531 s 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 117 Institut Curie Fit the Limits To initialize the Limits use To run fitting procedure use the Init Limits button the Fit Limits button from from the Toolbar 2 Normaized the Toolbar Global Analysis Characteristic Typical Contami
112. t Options The i visible toggle button SO is Fuer pressed D Images Microarray D20805FR9216 cy3 tif DE MAIA27 Batch defaults MAIA File Model Run Options Help Design 1 1 4 4 21 21 10 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie This is required only if the binary files have already been generated Otherwise new settings will be applied as defaults in the processing of each new image from the batch 111 Run Batch Batch processing can be stopped by pressing the Stop button on the Toolbar or selecting the Menu Item RunlStop Ctrl F5 Protocol of the Batch processing 11 Oct 06 Using the field Run one g MAIA27 Batch defaults MAIA may exclude include ie cu Mu 0 ch OOOO certain files from in the so SL ja sa JFL SA Batch processing Dir Name Visible DilImages Microarray Q20805FR1087 cy3 tif D Images Microarray D20805FR326671 cy Lm D Images Microarray 020805FR9216_cy3 tif Memory 192420200 Dataset D Images Microarray CGH Firstirawdata020805 020805FR1087_cy3 tif Design 1 1 4 4 22 21 10 0 0 0 0 1 1 3 MAIA 2 7 Manual Copyright C 2005 2006 112 Institut Curie Global Quality Analysis To start global quality g analysis two or more MAIA27 Batch defaults MAIA arrays have to be TEO selected and quantified DO b m s
113. t the obtained ratio is directly delivered from the regression analysis thus making the procedure of spot segmentation unnecessary Background pixels are concentrated at the initial part of the linear regression and do not influence the slope of the regression line However the linear regression approach suffers from the presence of the outlier or aberrant pixels within the spot cells These pixels occurring even in small quantities can distract the regression line and strongly bias the regression ratio With the aim to fully exploit the advantages of the linear regression approach we have reinforced this procedure by systematical filtering out aberrant pixels See page E Novikov and E Barillot A robust algorithm for ratio estimation in two color microarray experiments Journal of Bioinformatics and Computational Biology 2005 3 1411 1428 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 56 Institut Curie Spot Contours File Run Options Window Help d d Be pco HAs opi BI Y 1 x 1 Sp 12 9 NoName Ratio Characteristic Typical 0 1 99 9 This Quality Determination 0 98 Er p 99 0 99 DurbinWatson 1 35 0 51 2 36 1 29 Qag Contamination 0 o i p 1 Diameter lese 0 gw ess 0 94 GSymmetry 0 5 58 1 51 0 54 I5ymmetry ga Jo 2 65 029 0 77 Using the Toolbar button 27 ee Rgef round 0 14 03 8 87 lo12 0 93 Show Hide Spot ABackground
114. ter pss zs Be 4 0 57 eap 660686 4 ET T e e e pRpoo0o l 6 T TTERITIT Use the mouse pointer or 9 e 66 e eff cecaoe Fac TITT IT JGITEITIEFTITTTTT the spinners Ratio CV 00000 0900000090009 Po aa T e ITII Limit and Quality ceee TTTTTTTTTTTDS Limit to define the J coe E e og OGSDITIIETIIFIL Manual 0 85 gt E LI eseagaeace quality curve green line Reset Manual I AIIIIIIIIPIN Image Alignment 3 E G LJ E amp e L a EJ o Block Independent E E a s E E p Quality curve defines how fast 7 y shx 028 200 Se eteeococes i a CN g e o9 is the overall quality must o ee ds e dedere e e 0 22 Quali 0 252 v decrease with the decrease of the manually assigned quality The user defined quality curve is an exponent with the predefined decay constant cossett eecs SRCR Bon E OE Remeron XT ddedededer Ne 10 15 20 In Manual 1409 443 Y 226 X 4 Bl Y X 1353 Ho ID Quantification 2 265 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 80 Institut Curie Fit the Limits P MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help Fit the quality limits by clale Hals ame ete Y TEE TET
115. to reanalyze the batch Design 1 1 4 4 21 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 Institut Curie To remove filenames from the batch one may use the Toolbar button Remove Experiment or the Menu Item FilelRemove Experiment Ctrl E The toolbar button Remove All Experiments or the Menu Item FilelRemove All Experiments Ctrl Alt E will remove all filenames from the batch Ready for Analysis Check the Visible field to open download an image MAIA27 Batch defaults MAIA BEES D Im ages Microarray OSFR1087_cy3 tif D Images Microarray D20805FR326671 cy D Images Microarray D20805FR9216 cy3 tif 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 10 Institut Curie Main Processing Window Three panels are created Ratio image Cy3 and Cy5 channel images MAIA27 D images Microarray CGH First rawdata0 20805 C File Run Options Window Help E m Poo ME gt als eo d rel EET Sp Y X Another pair of images Cy3 Cy5 can be downloaded using the Ho Hame Ratio y 9 95 Jl 3 95 T Load Data button from the Toolbar or the m Menu Item gt en FilelLoad Data Ctrl 0 o Regression Plot For the new images image file format i e multi page TIFF versus single page TIFF can be changed using the Menu Item Options
116. trl Left ee o z JAIITZITTIPE TI i ecocesases eee Clicks followed by Iz amp s TIT Ctrl Right Click create the 2229 pis tos 6s Aaga contour of the selected Loi TTT cocee eee s80c ee eee 56 OR om ao EBEN spots l 425 aaa ni 440 E 5 e o e a bd L a a e v 9 J amp Quality 0 1 otea eee00 aa eee E LI Selected spots are marked roost 5 ee 2020 2 9 2 1222 by the dots in the left Ces MPMIN Soone ee THEE TIG LE RON os upper corner of the spot Bock Independent I MR TITT aa TTET Shftx 0 26 6000 EILE seo eee area v Shift 018 5 x _ save Default w 5 X 13 12 Quit 018 1443 464 Y 268 X 714 Bl Y 1 X 2 Sp Y 0 X 17 Ho ID Quantification 2 2655 Design 1 1 4 4 22 21 10 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 75 Institut Curie Manual Qualification of the Selected Group 9 MAIA27 D lmages Microarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help amp d d BE pch HAS e s Shift x 022 Bv1 x2 Sp 0 17 No Name Ratio Characteristic Typical 0 1 99 9 This Quality E TT ESPERE NI or camem TTT dii dide sid dme Contamination 0 11 1 bx b be vc dl LLET ooe eee Li TIITII b Bs r IIILIII LRESBEBINZIEI s oi i j sl ioca getn 27211 E
117. un Options Window Help d Nim ec MBS o B O SH x BI Y 1 1 Sp 12 9 Mo Name Characteristic Typical 0 1 99 9945 Quality Determination 1 DurbinWatson T 4 Contamination 3402 D 1 9402 8402 8402 T poo 8402 Using the Shift E spinners one can adjust if f I HE 3402 necessary the shift EUN The new values will be valid for all spots from the given block Block Independent is off or for all spots from the image Block Independent is on Tage Alignment BlockNind gendent v Shift x 1258 w Shift 0 7 lt gt ixl Save Default w 1 SEITE I Es 012 1396 424 4 452 X 4 BI Y X Sp Y Xt Ho ID mage Alignment 0 578 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 46 Institut Curie Compare Different Image Shifts 2 MAIA27 D Images Wicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin File Run Options Window Help d d Hm rco ie 4 omm BI Y 1 1 Sp 12 9 Mo Name Characteristic Typical 0 195 99 995 This Quality Determination 0 1 DurbinWatson T o 4 Contamination o 3402 Diameter 9402 Symmetry 3402 ISymmetry T B 3402 CVRatios 3402 The new values of the A E tp ul shi
118. up menu in the quality AI Used m Alide table select a set of quality ure EIE characteristics which can asao fune coda E a oo be relevant for this image 5 55 1 000 500 Idle characteristics are shown aoe in gray 102 Quality 0 58 Manual 1 Reset Manual Image Alignment eecoseeeos Block Independent Shift x 0 23 Shift Y 01 eeeeses000 Default trt tel eo ag lt 1986 1061 Y 199 X 34 Bl Y 0 X Q Sp Y 16 X 1 uantification 2 265 s Design 1 1 4 4 22 21 0 0 0 0 0 1 1 1 11 Oct 06 MAIA 2 7 Manual Copyright C 2005 2006 70 Institut Curie Manual Limits Adjustment P MAIA27 D lmagesWicroarray CGH First rawdata0 2080510 20805FR1087_cy3 bin DEAR Fie Run Options Window Help b ME pco ME 4 o ME OINEEHISX e 1 4 Bizx2 sxa Characteristic Typical 0 1 99 9 This Quality Ratio v 9 97 I i97 18 Determination 0 98 8 ji 0 99 0 91 M ns rbd E bsr TEN Jm Contamination 0 0 it 3 Qs8 8 HTHT t 2s L 00008 see H HHHEHIEETEH Ratios p 01 k Wz p oss nun 333 For each used quality po p MP sees T i FU characteristic the limits pare een can be adjusted RR 0765 RS 0762 The gray fields in the quality table are user modifiable Certain characteristics allow CEEE ET LICR 2 eceeeseceoeocececes

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