Spotxel® 1.1 Microarray Data Analysis Software User's Guide
Contents
1. Figure 9 Processing Background Noise Since processing noise during quantification efficiently removes background signal it can be regarded as an implicit background correction method You can choose whether noise is processed during quantification with the Process noise option Figure 7 Please note that you can process noise during quantification with both Fixed Spot and Flex Spot methods Spotxel 1 1 User s Guide Page 12 4 Quantification of Microarray Data Imaging Quantified Data Mb Figure 10 Processing Foreground Noise Smallest Spot Size You can set the size limit of a valid spot by means of the Smallest spot size parameter Figure 7 Suppose that this value is 50 Imagine a virtual square whose side length being 50 or half of the spot diameter If the spot signal is smaller than or can be contained in that virtual square the Flex Spot method will reject this spot The software will then use the Fixed Spot method to compute the raw value for that spot The spots shown in Figure 8 are detected by the Flex Spot method with the Smallest spot size parameter being 50 It can be observed that small spots do not have a blue border Show Border After data quantification you can turn the detected spot border on and off using the Show border option Figure 7 The software supports saving the border information in the project file spotxelproj Therefore you can still observ2. The negative image of the current image can be created by selecting the Inversion check box 3 2 Image and Array Rotation Rotating images You can flip and or rotate images at angles of 90 180 or 270 These functions are located in the Image Rotation section of the Images control panel Figure 5 a Rotating Array If the array slightly deviates from the image at a small angle it is recommended that the array i e related blocks be rotated since image rotation may change the image data You can select blocks and rotate them at an arbitrary angle These functions are located in the Array Rotation section of the Arrays control panel Figure 5 b The degree change can be as small as 0 01 Selected blocks can be rotated in clockwise or counterclockwise direction with a rotation center defined as follows e Global the top left of the image e Local the top left of each block Spotxel 1 1 User s Guide Page 8 4 Quantification of Microarray Data Image Rotation Array Rotation Flip Center of rotation Vertically Horizontally Global Local Rotate at an angle Direction o age Clockwise Counterdockwise 180 gt 270 0 00 a Image Rotation b Array Rotation Figure 5 Rotation of Images and Arrays 4 Quantification of Microarray Data 4 1 Quantifying Microarray Data Click the Quantify button in the main toolbar to activate the Quantification control panel e Click the Quantify Array but
3. sicasyYs Spotxel 1 1 Microarray Data Analysis Software User s Guide 27 January 2015 Rev 4 Spotxel is only intended for research and not intended or approved for diagnosis of disease in humans or animals Copyright 2012 2015 SICASYS Software GmbH All Rights Reserved SICASYS Software GmbH Im Neuenheimer Feld 583 D 69120 Heidelberg Germany Phone 49 62 21 7 28 50 40 Fax 49 62 21 7 28 48 94 Email info sicasys de Web www sicasys de Table of contents A MAE OCMC CON Po APP O A O O 2 1 1 INS rada 2 1 2 Product Activation inn a E a E S 2 1 3 SOreWare User Internac E A a 3 1 4 Terms and EON CED S io ratico 4 1 5 Microarray DaI Ana VS Saura E N 5 2 Preparation for Microarray Data AnalySiS cccccccsssccccesseccceeseccccensceceeesecesseeceseenecessegeceesenseeeeas 5 2 1 GA ING O coats aie urecatetncimtncnaateceieds 5 2 2 AE A PP a 7 2 3 SPOe Pro eet FIE nea O d 7 3 image and Array PROCESSING ai a N 7 3 1 Mage ROCES nai ani 7 3 2 Mage ana Array Rotation a a ee aces 8 4 Quantification of Microarray Data iaa 9 4 1 Quantifying Microarray Dalal c sisceasusn cesasbtnceesteubsedersbesedaieoevenecadsacestontieewsbidadausanceoesabewessarins 9 4 2 Change OF INtersity Vales sy A 10 4 3 Spot Detection Methods iia da 11 4 4 Spot DETECHIOM ODIOS 12 4 5 Backs rouna Correcciones di 13 5 scatter Plot K Means Clusteri picas 15 G Balch PROCESSING ceesre a a a a a tall oat cd 17 7 Data Minna TOO Si
4. in the Block page first open the Block page by clicking on it and then hover the mouse over the block in the canvas Similarly you can open the Spot page and then points to a spot to view its properties Figure 3 b Spotxel 1 1 User s Guide Page 6 3 Image and Array Processing 2 2 Array Alignment As mentioned in Section 1 4 the array needs to be aligned with the image before quantifying the microarray data This can be done automatically You can also manually align the array with the image probably after processing the image or the array e g rotating a block or increasing the spot s visibility Please refer to Section 3 for image and array processing functions Aligning Array Automatically e Click the Align Array button in the Arrays control panel Aligning Array Manually e Click Ctr A to select all blocks in the array To select individual blocks click on them while pressing the Ctrl key e Click on the selection and drag the corresponding blocks to the intended position The aligned position of the blocks in the array can be saved with the Arrays gt Save Array menu In addition you can save the array to another GAL file using the Array gt Save Array As menu 2 3 Spotxel Project File It is recommended that the analysis of each microarray image be saved to a Spotxel project file spotxelproj using the Project gt Save Project menu The saved data includes the path to the image the aligned array
5. Arrays Plots Analysis Batch Configurations Help G Zoom In ZoomOut 70 v lt X gt Control Panel E X Imaging Quantified Data Array Object ee CICICIME T T Align Array AA TO TE E Quantify Block Blocki mM 11174 Scatter Plot Name ec_704 Spot position Local Column 9 HC Local Row 15 Horizontal um 7485 Green Channel Red Channel Combined TL Export to CSV File Vertical um 10010 Block Local Row Local Column ID Name Green Raw Mean Green Background Mean Green Foreground Mean Green Raw Median Green Background Me lg4 Blockl 7 41811 po101 40 235 73 1 931 48 38 304 24 65 535 00 1 8 los Blockl 7 5 11348 ec_348 12 891 67 1 931 48 10 960 19 13 118 00 18 96 Block 6 11596 ec_596 7 769 36 1 931 48 5 837 88 8 118 00 13 E JA eI 3850208 E 3657060 6553500 98 Block 8 11148 ec_148 11 951 19 1 931 48 10 019 71 14 499 00 18 4 m gt Ready Figure 6 Quantified Data Aggregating results of replicas When a spot is replicated the software also provides the intensity value of the spot calculated by aggregating pixels from its replicas Suppose that P1 and P2 are the replicas of peptide P The quantified results then include the intensity values for P in addition to those for P1 and P2 Taking the raw median value of P for example it is the median value of pixels from both spots P1 and P2 which cannot be calculated based on the median values of P1 and P2
6. OF THE USE OF OR INABILITY TO USE THE SOFTWARE EVEN IF SICASYS HAVE BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES FURTHER IN NO EVENT SHALL SICASYS BE LIABLE FOR ANY DIRECT DAMAGES ARISING OUT OF USER S USE OF THE SOFTWARE IN NO EVENT WILL SICASYS BE LIABLE TO USER FOR DAMAGES IN AN AMOUNT GREATER THAN THE FEES PAID FOR THE USE OF THE SOFTWARE Intellectual Property Right Infringement If a claim alleging infringement of an intellectual property right arises concerning the SOFTWARE including but not limited to patent trade secret copyright or trademark rights SICASYS in its sole discretion may elect to defend or settle such claim and or terminate this Agreement and all rights to use the SOFTWARE and require the return or destruction of the SOFTWARE with a refund of the fees paid for use of the SOFTWARE less a reasonable allowance for use and shipping Miscellaneous This Agreement is the entire agreement between USER and SICASYS with respect to the SOFTWARE and supersedes any previous oral or written communications or documents including if USER is obtaining an update any agreement that may have been included with the initial version of the Software This Agreement is governed by the laws of Germany If any provision or portion thereof of this Agreement is found to be invalid or unenforceable it will be enforced to the extent permissible and the remainder of this Agreement will remain in full force and effect Failure to prosecute
7. Plot No Yes ma Spot detection method a Hex soot raed ot ee i ee MEA A E A O A A A BT A ELA CPA NE HC y Show border 4 R y Y Process noise Green Channel Red Channel Combined a_i Smallest spot size 50 PCA Background correction 5 DDD O Local method Controls E Level Block v Batch Data Files PARTE Export to CSV File eplica Project a E Block Local Row Local Column ID Name Green Raw Mean Green Background Mean Green Foreground Mean Green Raw Median Green Background Medi Pte cado Blockl 7 5 11348 ec_348 18 588 26 142 55 18 445 71 17 573 00 o 95 wee abArray gal 19g Blockl 7 6 11596 ec 59 10 357 28 142 55 10 214 73 10 430 00 0 Image s 12_1 C1_111014131 F 11731 ec 731 52 0 E MO aci o Tae ewa T es asa wm 98 Block 7 8 11148 ec_148 15 299 55 142 55 15 157 00 15 826 50 0 l99 Blockl 7 91813 po103 1 289 29 142 55 1 146 74 433 00 0 p ETE ELERT er ee ee a a Figure 1 The Software User Interface O rere O remonta O resta O reinas O resonan O Pereo Table 1 Software Components The main toolbar enables quick access to a group of related functions They are described in Table 2 Clicking on a button on the main toolbar opens the control panel for the function group The software shows the data and the analysis results in the corresponding sheet on the right of the control panel E g Figure 1 shows the control panel with controls and settings for data quantific
8. Source Template array gt cessingltemplateArray gal Store generated files in The image s folder 9 Processing batches batch3 Tasks and Options Array alignment Data quantification 9 Process all images continuously Stop and review after each image Batch Entry 99 Image image_099 tif Status Quantifying Spotxel 1 1 User s Guide image_090 tif Done image_091 tif Done image_092 tif Done image_093 tif Done image_094 tif Done image_095 tif Done image_096 tif Done image_097 tif Done image_098 tif Done image_100 tif Waiting image _101 tif Waiting image_102 tif Waiting image_103 tif Waiting image_104 tif Waiting image_105 tif Waiting image_106 tif Waiting image_107 tif Waiting image_108 tif Waiting image_109 tif Waiting Array Quantified Data Analysis Results image_090 gal image_090 csv image_091 gal image_091 csv image_092 gal image_092 csv image_093 gal image_093 csv image_094 gal image_094 csv image_095 gal image_095 csv image_096 gal image_096 csv image_097 gal image_097 csv image_098 gal image_098 csv image_090 spotxelproj image_091 spotxelproj image_092 spotxelproj image_093 spotxelproj image_094 spotxelproj image_095 spotxelproj image_096 spotxelproj image_097 spotxelproj image_098 spotxelproj Figure 16 Batch Execution Page 18 7 Data Mining Tools Suppose that sample001 tifis an image in the batch The software creates th
9. a party s rights with respect to a default hereunder will not constitute a waiver of the right to enforce rights with respect to the same or any other breach Spotxel 1 1 User s Guide Page 26 10 Index A activate 2 23 24 analysis of microarray data 5 array 4 array alignment 7 automatically 7 manually 7 B background control 14 association 14 background correction 13 15 background control 14 for a block 14 global background value 14 Local method 12 15 block level 14 global level 14 spot level 14 background value 5 9 batch 4 5 17 create 17 execute 18 template array 17 block 4 properties 6 brightness 8 10 C canvas 3 6 canvas toolbar 3 cluster 22 contrast 8 10 control panel 3 D data mining 5 dataset 19 20 21 Hierarchical Clustering Analysis 21 K Means clustering 16 Principal Component Analysis 20 Spotxel 1 1 User s Guide 10 Index file CSV 19 GAL 4 7 19 GenePix Result gpr 19 microarray image 7 Spotxel project spotxelproj 7 19 TIFF tiff tif 5 flag 9 Flex Spot 12 detected spot border 11 13 smallest spot size 13 foreground value 5 9 H hardware requirement 2 HCA 21 clustering tree 22 heat map 22 Hierarchical Clustering Analysis 21 Installation 2 Mac OS X platforms 2 Windows Platforms 2 inversion 8 K K Means clustering 16 license b
10. oo oo oo oo o oo 0 oo oo o oo oo oo oo oo oo oo oo oo Data column 5500005000000 nmam 55555555555585555585858553535358 3 3 z E al J J al J d y ol J y ol ol E ol J ol J ol z 3 Analyze replicas Individually y gately Create clustering trees for V Features 4 Samples HCA Options Distance metric Pearson Correlation X Type of Linkage Complete Block Column Row Name ID 014_CS 010_073108_1224 Cyanine5 017_CS_030_073108_1243_Cyanine5S 017_CS_03u_073108_1248 Cyanine5S E020_CS 110_07 Single 1 1 4 1 ec 404 01 01 04 04 0 484 1 667 0 409 Weighted average 2 1 5 1 hs104 01_03_01_04 0 402 1 450 0 504 Unweighted average 3 1 6 1 ec_196 01_01_01_96 0 248 1 184 0 009 Dataset in Table 4 1 7 1 sp_102 01_04_01_02 0 120 0 829 0 031 Export To CSV File 5 1 8 1 ec_036 01_09_09_36 0 234 1 062 0 080 a 1 713 M Mm N7 12 NE 10723 nA 2 4 w p Figure 18 Hierarchical Clustering Analysis Spotxel 1 1 User s Guide Page 21 7 Data Mining Tools The clustering tree s are then constructed Figure 18 Two features considered being related are grouped into one cluster Their relationship is represented by a line connecting them A cluster might be related with a feature or another cluster The relationship between samples and clusters of samples are represented similarly The values in the dataset each representing the screening value of a feature with a sample are graphically represented by means of a
11. system uses a proxy server to connect specify it using the Proxy setting link Otherwise please contact the software provider for support 4 A completion message is shown when the product is successfully activated Click Next to use the software immediately or End to use it later Product Activation is completed Congratulation You have successfully activated Spotxel Figure 22 Completion of the Product Activation Please note that the license can be reviewed or renewed in the case of a time limited license by clicking on the Help menu and choosing License Information Spotxel 1 1 User s Guide Page 24 9 End User License Agreement 9 End User License Agreement SPOTXEL IS THE PROPERTY OF SICASYS SOFTWARE GMBH SICASYS THE USE OF THIS SOFTWARE IS GOVERNED BY THE TERMS AND CONDITIONS OF THE END USER LICENSE AGREEMENT AGREEMENT SET FORTH BELOW THE TERM SOFTWARE ALSO INCLUDES RELATED DOCUMENTATION WHETHER IN PRINT OR ELECTRONIC FORM AND ANY UPDATES OR UPGRADES OF THE SOFTWARE PROVIDED BY SICASYS BY INSTALLING THE SOFTWARE AND OR BY USING THE SOFTWARE YOU AGREE TO BE BOUND BY THE TERMS AND CONDITIONS OF THIS END USER LICENSE AGREEMENT License SICASYS grants to you USER a non exclusive non transferable license to use the SOFTWARE on the number of computers stated in the license contract A separate license is required for use on any additional computer Copy Restriction The SOFTWARE ma
12. you will be prompted to provide a system administrator s account and password Upon completion Spotxel is installed in the Applications Spotxel folder 1 2 Product Activation After installing Spotxel on Windows you need to activate the software with a trial serial number obtained from the software provider or its distributor This enables the use of Spotxel with full functionality for 14 days The trial use for Spotxel on Mac OS X platforms is handled automatically and does not require this step When the free trial time has expired you can buy a software license to continue using Spotxel Upon the purchase you receive a serial number and use it to activate the license Please refer to the product activation steps in Section 8 or at the online help here Spotxel 1 1 User s Guide Page 2 1 Introduction 1 3 Software User Interface Related software controls are grouped in labeled components as shown in Figure 1 We refer to a software component using the name listed in Table 1 gE T X Spotrel Grci Project Images Arrays Plots Analysis Batch Configurations Help E Zoom In Zoom Out 100 v AS S Control Panel X BatchProcessing Imaging Quantified Data i Data Quantification CON o EE eee a eee Ue OA Ad ee aani seeen eoe n e 1 1 eer oe namams aa e 0 Ue 0000 10 00 Qu contain Options eee Ree ee wee A ee aie a je Indude change of the image s intensity value Scatter
13. 1 00 32 Block 15 14 1621 ps_203 37 129 34 1 931 48 35 197 86 34 731 00 1 830 00 4 m Figure 13 Scatter Plot You can export the table s data to a CSV file It can be either the entire table or only main columns In the latter case only spots properties and the Foreground Mean values of the two channels are exported K Means Clustering The spots on the scatter plot can be classified into a number of groups according to their quantified values Figure 14 e Click the Analysis gt K Means Clustering menu e Enter the number of clusters i e groups that you would like On the plot spots close to each other will be grouped into one cluster and highlighted with the cluster s color At first the table shows the data of all clusters You can limit it to a cluster using the Clusters list box in the K Means Clustering section To obtain a different number of clusters click the Update Clusters button Like with the scatter plot the table s data can be exported to a CSV file Spotxel 1 1 User s Guide Page 16 6 Batch Processing X Spotxel Li Project Images Arrays Plots Analysis Batch Configurations Help Control Panel 8 X Imaging Quantified Data Scatter Plot K means Clustering Spot Details Scatter Plot ES Cluster 1 Block Block1 Cluster 2 r Local Local as Row 7 Column 7 Blocks All M X Axis Green F Mean x 1D 11731 Mi
14. This provides an additional view to the replicated data To control whether the aggregated data is calculated and shown use the Aggregate results of replicas check box Customizing the Quantified Data Click the Configurations menu and choose Imaging Result Values to show the setup dialog Here you can add or remove values to be calculated during the data quantification 4 2 Change of Intensity Values During the array alignment you may have adjusted the contrast and or the brightness of the images to make the spots visible This changes the image data and alters the analysis results It is recommended that the original image data be used for the quantification Therefore by default the Spotxel 1 1 User s Guide Page 10 4 Quantification of Microarray Data quantification procedure uses the original image s data i e it excludes changes made to the image by inversion noise filtering and adjustment of contrast and or brightness Quantification Options Indude change of the image s paced value No Yes Spot detection method Flex Spot Fixed Spot Show border Process noise air Smallest spot size 5 50 Background correction Local method Controls Level Block Figure 7 Quantification Options 4 3 Spot Detection Methods This option can be intentionally changed as follows In the Quantification Options section of the Quantification control panel select Yes for the nclude change of
15. and the quantified data When later opening the project file with the Project gt Open Project menu the software will load all the saved data This enables to manage all the analyzed data for one microarray image with a single project file In addition later you can use these project files directly with data mining tools The paths to the microarray image the GAL file and the project file are shown in the Data Files section of the Quantification control panel 3 Image and Array Processing 3 1 Image Processing You can change the image intensity Figure 4 for the convenience of array alignment Spotxel 1 1 User s Guide Page 7 3 Image and Array Processing Improving spot visibility Adjusting brightness and contrast can make the spots more Image Intensity visible and that eases the array alignment These functions Enhance contrast automatically are available in the Image Intensity section of the Images Change contrast manually o E control panel 5 Change brightness manually o E s J e Choose the Enhance contrast automatically option E Miter noise to maximize the spot visibility Invert the image e You can manually adjust the image s brightness and contrast by moving the slider Alternatively a value Figure 4 Image Processing between 99 and 99 can be entered directly Noise Filtering Noise in the loaded image s can be reduced by selecting the Noise Filtering check box Inversing
16. ation On the right side the Imaging sheet displays a microarray image in the canvas the signal of the currently selected spot in the Spot Image widget and the table of quantified data Spotxel 1 1 User s Guide Page 3 1 Introduction Images Select image channel change image s intensity rotate images Arrays View properties of blocks and spots rotate blocks align the array Quantify Quantify the microarray data and browse the quantified data Scatter Plot Display quantified data in a scatter plot and perform K Means clustering HC Hierarchical Clustering Analysis Principal Component Analysis Batch Setup and execute a batch Table 2 The Main Toolbar and Related Functions 1 4 Terms and Concepts In this manual the term array is used to refer to the spot layout and annotation of a microarray We assume that the array is saved as a GenePix Array List GAL file The term image or microarray image is used to denote a scanned image of the printed microarray An array consists of blocks Each block is a group of spots located next to each other In the canvas the spot s border is drawn as a white square or rectangle Figure 2 shows a block consisting of six spots arranged in two rows and three columns Within each spot the spotted region is defined as the area bounded by the dashed circle Its diameter is specified by the spot diameter parameter for Figure 2 A block with 6 spots each block The binding si
17. ca is 19 7 1 Dita aos 19 7 2 Principal Component Analysis PCA coccoonccncnnconanoccnncnnnacccononnanoccnnnonanoccnnoonanoccnnoonanincnnos 20 7 3 Hierarchical Clustering Analysis HCA scccsscssssssarscssossearconsoussarcenseusearsenseuseurcensousenreenes 21 Sr MPROCUCEACUIV aN ON O ais 23 9 End User license Arree ment cds 25 10 A O A D O II EE N E ace A E EE inane naa 27 Spotxel 1 1 User s Guide Page i 1 Introduction 1 Introduction Spotxel supports microarray image analysis and data quantification You can setup a batch to process a number of microarray images automatically Furthermore you can discover features and samples that influence the study and their relationship with data mining tools 1 1 Installation Spotxel is natively supported on Windows and Mac OS X platforms Installation of the software requires rights of a system administrator Hardware Requirement Minimum hardware 1 5 GHz Processor 1GB RAM Recommended hardware 2 0 GHz Dual Core or faster Processor 2 GB or more RAM Windows Platforms Spotxel works on Windows XP Windows 7 and Windows 8 Simply run the Spotxel setup If the current Windows account is not an administrator you will be asked to input an administrative account and its password Mac OS X platforms The software runs on Mac OS X 10 7 and 10 8 Unzip the package and double click on the pkg file to launch the installer During the installation
18. claims deviations in the program from the specification description USER has the right to request a fix patch work around or replacement of the SOFTWARE that does not meet such limited warranty If a rectification of the errors is not possible or if such rectification is not successful USER has the right to request a rescission of the contract where USER must destroy all copies of the SOFTWARE Spotxel 1 1 User s Guide Page 25 9 End User License Agreement EXCEPT AS EXPRESSLY SET FORTH ABOVE NO OTHER WARRANTIES OR CONDITIONS EITHER EXPRESS IMPLIED STATUTORY OR OTHERWISE ARE MADE BY SICASYS WITH RESPECT TO THE SOFTWARE AND THE ACCOMPANYING DOCUMENTATION AND SICASYS EXPRESSLY DISCLAIMS ALL WARRANTIES AND CONDITIONS NOT EXPRESSLY STATED HEREIN INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OR CONDITIONS OF MERCHANTABILITY NONINFRINGEMENT AND FITNESS FOR A PARTICULAR PURPOSE SICASYS DO NOT WARRANT THAT THE FUNCTIONS CONTAINED IN THE SOFTWARE WILL MEET USER S REQUIREMENTS BE UNINTERRUPTED OR ERROR FREE OR THAT ALL DEFECTS IN THE PROGRAM WILL BE CORRECTED USER ASSUMES THE ENTIRE RISK AS TO THE RESULTS AND PERFORMANCE OF THE SOFTWARE Limitation of Liability IN NO EVENT SHALL SICASYS BE LIABLE FOR ANY INDIRECT SPECIAL CONSEQUENTIAL OR INCIDENTAL DAMAGES WHATSOEVER INCLUDING WITHOUT LIMITATION DAMAGES FOR LOSS OF BUSINESS PROFITS BUSINESS INTERRUPTIONS LOSS OF BUSINESS INFORMATION OR OTHER PECUNIARY LOSS ARISING OUT
19. e the detected spot border when reopening the project file This is particularly useful for reviewing the quantified data generated by batch processing Undetectable by Flex Spot If the Flex Spot method cannot detect a spot due to e g being smaller than the size limit weak signal or noisy data the software employs the Fixed Spot method to compute the raw value No blue border is shown within the spot if the Fixed Spot method is used 4 5 Background Correction Background correction methods can be selected in the Quantification Options section of the Quantification control panel By default the Local method is employed Spotxel 1 1 User s Guide Page 13 4 Quantification of Microarray Data Local Method Spotxel supports different levels of local background correction Figure 11 The default one for GAL files is the block level ee eae At the block level all spots in a block will have the same Local method Controls background value It is computed as follows First a list Level of background pixels of all spots in the block is created Global Spot The mean and the median of the pixel intensity values in this list are then used as the background value Figure 11 Background Correction Options You can choose to have a global background value for the entire array by selecting the global level The calculation is based on the background pixels of all spots in the array At the spot level a spot s backg
20. gnals of a microarray tested with a sample are converted by a microarray scanner into a digital array image containing a matrix of pixels Each pixel has a gray value representing pixel intensity Array images are often saved in the TIFF format In 8 bit grayscale images the gray value ranges from O to 255 This value can be from O up to 65535 in 16 bit grayscale images Since they have a broader range of signal levels than the 8 bit format 16 bit grayscale images are recommended for array image analysis Quantification is the procedure that estimates the true binding signal for each spot and represents its signal value in terms of statistic measurement of pixel intensities within that spot Obviously the quantification quality depends on the spot finding or spot detection procedure which determines http partners adobe com public developer en tiff TIFF6 pdf Spotxel 1 1 User s Guide Page 4 2 Preparation for Microarray Data Analysis which pixels in the array image should belong to a spot in the array Background correction also contributes to the quantification quality It estimates signals caused by non specific binding and removes them from the spot s signal For each spot in the array the median and the mean of its raw background and foreground values are calculated Raw represents the intensity value of the spot s signal Background is the estimated value of the signal caused by non specific binding The value of inte
21. h with their spotted regions as specified in the GAL file Figure 8 illustrates such a case Here within a spot the white square the spotted region is depicted by the white dashed circle The spots signals shown in red have different shape and position from those of their spotted region Despite that fact the Flex Spot method can still precisely find the spots and highlight their border in blue Please note that the above described mechanism for background calculation uses only the background pixels within the spot Therefore it is called the Local method or local background correction By means of background controls you can have background values based on pixels from any region of the image 4 4 Spot Detection Options Process Noise The images may contain noises that mislead the spot detection procedure and result in wrong quantified data The noise can be background noise that span across the whole slide Figure 9 It can also be foreground noise like the two large red bands shown in Figure 10 In the case of background noise we want to remove the background layer so that only the meaningful signal remains Foreground noise like the two red bands in Figure 10 should not be part of a valid spot s signal The software can effectively handle this task The results can be seen in Figure 9 and Figure 10 only meaningful spots are highlighted with a blue border by the Flex Spot method Imaging Quantified Data
22. heat map You can save the clustering trees with the heat map to an image file using the Export to Image context menu Spotxel 1 1 User s Guide Page 22 8 Product Activation 8 Product Activation The product activation requires an internet connection You need to have a serial number obtained from the software provider or its distributors 1 In the Evaluation Time Has Expired dialog click the Next button Evaluation Time Has Expired The product needs to be activated with a valid Serial Number for further use Click here to purchase a Serial Number Figure 19 Starting the Product Activation 2 Enter the serial number and the licensee information in the Product Activation dialog Click Next Serial Number BPFPn E1t4e D6 8ck Ctg 7O ROT6 31n4 First Name Dale Last Name Jones Organization AZ Pharma Inc Email Address dale jones az pharma com Click here to purchase a Serial Number Figure 20 Entering the Licensee Information Spotxel 1 1 User s Guide Page 23 8 Product Activation 3 If the internet connection is ready click the Activate button and wait for the activation to finish Product Activation in Progress The product will be activated online Please prepare an internet connection and dick Activate Proxy setting Figure 21 Product Activation in Progress Please check the internet connection in the case the software could not reach the activation server If your
23. in the table Spotxel 1 1 User s Guide Page 15 5 Scatter Plot amp K Means Clustering X Spotxel e Project Images Arrays Plots Analysis Batch Configurations Help amp X imaging QuantifiedData Scatter Plot Spot Details Scatter Plot Block Block1 al Local 7 Local 7 Row Column Green F Mean y ID 11731 0 5 Name ec_731 2 45365 E 5 Green Channel Red F Mean X 2 u 0 E fag 46896 Spots between Thresholds Red Channel gt CS gt A r 2 A gt AS eas e Pm b gt ms Lo oe P Total number 71 y ba 8 eT eje e gt gt eje Select the largest 71 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 PCA Green Foreground Mean Table of Selected Spots Block Local Row Local Column ID Name Green Raw Mean Green Background Mean Green Foreground Mean Green Raw Median Green Background Median G Batch Export To CSV File 25 Block7 1 116711 ps7du 39 033 25 1 994 62 37 038 62 65 401 00 1 866 00 Eosti 26 Block7 1 31651 ps_505 38 670 73 1 994 62 36 676 10 65 535 00 1 866 00 9 Only Main Columns SS NTRA AAA AA AA aia 3 EEE 38 591 19 2 032 11 36 559 08 65 535 00 1 903 00 E 29 Block2 15 15 16211 ps 2du 38 403 63 1 916 44 36 487 19 58 550 00 1 838 00 30 Block8 1 116811 ps 8du 37 818 43 2 031 89 35 786 54 65 535 00 1 901 00 31 Block8 15 8 17931 mh 031 37 420 94 2 031 89 35 389 04 53 401 00 1 90
24. log xml One template array for all images y Template array rocessingltemplateArray gal The image s folder hProcessing batches batch3 Tasks and Options Array alignment Data quantification 9 Process all images continuously Stop and review after each image Image Status image_001 tif Waiting image_002 tif Waiting image_003 tif Waiting image_004 tif Waiting image_005 tif Waiting image_006 tif Waiting image_007 tif Waiting image_008 tif Waiting image_009 tif Waiting image_010 tif Waiting image_011 tif Waiting image_012 tif Waiting image_013 tif Waiting image_014 tif Waiting image_015 tif Waiting image_016 tif Waiting image_017 tif Waiting image_018 tif Waiting image_019 tif Waiting image_020 tif Waiting if Waiti Quantified Data Analysis Results Figure 15 Batch Setup After creating the batch click the Run button to execute it Figure 16 Please note the running mode e Process all images continuously The batch processes continuously without stopping e Stop and review after each image You can view the batch results for one image before proceeding to process the next one Scheduling Table Project Images Arrays Plots Analysis Batch Configurations Help E zoomi zomout 100 Batch Processing Order Image Status J Batch file datches batch3 batch3 xml Continue Log file res batch3 patch3_log xml Array
25. lysis Please note that compressed images are not supported Therefore please disable image compression when saving the scanned images with your microarray scanner software If you are using GenePix Pro software uncheck the Use TIFF LZW compression lossless option in the Save Images dialog Loading Input Data e Click the Images gt Open Image menu and select the microarray image file For grayscale images select to display each image with either the Red channel or the Green channel e Click the Arrays gt Open Array menu and select the GAL file After being loaded the images and the array are shown in the graphical canvas Figure 1 To obtain an appropriate view you can use the Zoom In and Zoom Out buttons on the canvas toolbar or select a predefined zoom level in the Zoom combo box Alternatively an arbitrary value can be entered directly into the Zoom combo box Block Block spot Name Block1 Number of columns 15 Block Block 1 Number of rows 15 ID 11731 spot spacing Name ec 731 Horizontal um 295 gt Spot position Vertical um 295 Local Column 7 Spot diameter um 250 Local Row 7 Top eft spot s position Horizontal lum 6895 Horizontal um 2618 Vertical um 7650 Spot a Block Properties b Spot Properties Figure 3 Properties of an Array Object Viewing Array Data In the Array Object section of the Arrays control panel you can view properties of a block or a spot To view a block s properties Figure 3 a
26. n 0 Name ec_731 Max 45365 Green Channel Red Channel Update Clusters Clusters Cluster 1 X CT Poo we pr gt Me ie 4 a ST P e L 5 a M ee lt A eo s E gt es a al a Al N 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 Green Foreground Mean Table of Selected Spots Block Local Row Local Column ID Name Green Raw Mean Green Background Mean Green Foreground Mean Green Raw Median Green Background Median G Y Axis RedF Mean z Min 0 Red Foreground Mean Max 46896 K Means Clustering Export To CSV File 21 Block3 9 15 17912 mh_012 39 834 79 1 949 33 37 885 46 62 689 00 1 872 00 22 Blockl 12 14 11731 ec_731 39 758 62 1 931 48 31 827 14 65 535 00 1 830 00 9 Only Main Columns 23 Block 1 116711 ps 7du 39 033 25 1 994 62 37 038 62 65 401 00 1 866 00 a 3 EA 38 591 19 2 032 11 36 559 08 65 535 00 1 903 00 26 Block2 15 15 16211 ps_2du 38 403 63 1 916 44 36 487 19 58 550 00 1 838 00 27 Block8 1 116811 ps 8du 37 818 43 2 031 89 35 786 54 65 535 00 1 901 00 28 Block8 15 8 17931 mh_031 37 420 94 2 031 89 35 389 04 53 401 00 1 901 00 _ 4 il b Figure 14 K Means Clustering 6 Batch Processing You can setup a batch to process a number of microarray images automatically Suppose that the experiment is to screen an antibody microarray with k samples The microarray design is a
27. n E Zoom Out 100 S A Control Panel X Principal Component Analysis eee Images Analysis Load Data Start Analysis Data column Analyze replicas Scatter Plot Simplify the dataset to three pal Features O Samples L HC Show the simplified dataset in gt e J gt 3D PCA o 2D o X Yy y z bbb E Batch Dataset in Table Block Column Row Name ID x y z 1 1 1 ps 1du 010601 11 6 56 2 94 0 26 Eigenvalues Original dataset Eigenvectors 0 Simplified dataset Export To CSV File 1 1 1 1 1 a in gt Lu N e Ready I Figure 17 Principal Component Analysis in 2D View Spotxel 1 1 User s Guide 2 3 4 5 6 1 ps103 01_06_01_10 1 ps 103 01_06_01_10 1 ec_404 01 01 04 04 1 hs 104 0103 0104 1 ec 196 0101 0196 25 59 10 67 2 09 26 92 10 92 1 99 3 70 1 93 1 61 1 75 0 37 1 54 1 62 1 54 0 95 Page 20 7 Data Mining Tools The PCA chart Figure 17 then depicts the features according to their values in the simplified dataset whose data is shown in the table below the chart You can select 2D or 3D view By observing the charts you can find the trends of the data For example features that have common characteristics locate near each other on the chart On the other hand those that are distinct are far from the others The simplified dataset can be exported to a CSV file for further analysis Similarl
28. nnotated by the so called template array From the screening result you have k scanned images and would like to quantify their data To this end for each scanned image the batch aligns the template array with the image creates the GAL file that contains the aligned layout and generates the quantified data Click the Batch button in the main toolbar to create a batch Figure 15 In the Batch control panel e Click the Add button and select the microarray images for processing They will be added to the scheduling table Use the Add Remove Up and Down buttons to modify the table e Double click on the Template array edit box to browse to the template array file e Specify the folder to store generated files and the running mode e Finally save the batch to a file using the Batch gt Save Batch menu The batch log is created automatically and named after the batch file We recommend using a separate folder for each batch to store the batch file and generated data Since the software uses the dot character for file extensions such as gal or csv please do not name folders or files used in a batch with dot characters except for the file extension to avoid errors Spotxel 1 1 User s Guide Page 17 6 Batch Processing Project Images Arrays Plots Analysis Batch Configurations Help E zonn E 200m out 100 ontro Order sep Continue patches batch3 batch3 xml ches batch3 patch3_
29. ow Column ID and Name of an individual feature For simplicity we only write Feature 1 instead of its five property values In addition to Spotxel project files you can create a dataset from a list of GenePix Result gpr files The third alternative is to manually prepare your dataset as a CSV file having the data format like Table 3 Spotxel 1 1 User s Guide Page 19 7 Data Mining Tools 7 2 Principal Component Analysis PCA PCA simplifies a complex microarray study to a simpler one with only three samples or features thus you can easily observe the study s data and its trends To start with click the PCA button in the main toolbar In the PCA control panel e Click the Load Data button and select the dataset Please refer to Section 7 1 for the preparation of the dataset e Select a quantified value in the Data Column list box e Choose to have the simplified dataset with three either Features or Samples e Click the Start Analysis button eno w Table 4 The Simplified Dataset Suppose that that you chose the Samples option Take the screening in the previous section again as example As shown in Table 3 the original dataset represents the features screening value against k samples where k is much larger than 3 PCA will simplify it to a dataset with only 3 samples as illustrated in Table 4 G X Spotxel Ll Project Images Arrays Plots Analysis Batch Configurations Help Zoom I
30. ree data files for it e sample001 gal the array file whose spot layout is aligned with the image samp e001 tif e sample001 csv a CSV file containing only the quantified data and e sample001 spotxelproj the Spotxel project containing the analysis data for this image 7 Data Mining Tools Data mining tools assist you to find useful information from the microarray study You can employ Principal Component Analysis to discover features and samples that influence the study and then Hierarchical Clustering Analysis to find their relationship The batch processing results e generated Spotxel project files can be used directly for data mining 7 1 Dataset A dataset can be compiled from a list of Spotxel projects Consider the example in Section 6 again in which the antibody microarray is screened with k samples After running the batch we obtained k Spotxel project files containing the quantified data If the dataset is created from these k projects it can be regarded as the table in Table 3 where Vi is a screening value of Feature 1 when the microarray is screened against Sample k and so on The screening value can be chosen among the list of quantified values e g log Green Foreground Mean Red Foreground Mean moa Row conn 10 rame Somes same rene w Table 3 A Sample Dataset Please note that the first five columns in Table 3 contain the spot s properties specified in the GAL file Block R
31. rest is foreground it is computed by subtracting the background value from the raw value Array alignment is the process of associating spots in the array with their signal in the image The spot s signal is presumably due to the binding of immobilized substance in the spotted region with the sample Therefore before quantification we will reallocate the array such that the spotted regions are as close to the spots signal as possible 1 5 Microarray Data Analysis From the software perspective typical tasks of microarray data analysis include 1 Quantification of microarray data e Load the scanned images and the array file e Align the array to the images e Quantify the microarray data 2 If necessary batch processing of multiple microarray images i e the automation of step 1 3 Discovery of parameters influencing the study and their relationship with data mining tools The following sections explain how to accomplish these tasks with Spotxel 2 Preparation for Microarray Data Analysis 2 1 Loading Data To analyze the microarray data two input data are required e Scanned images of the microarray in the TIFF format e The array file prepared in the GAL format gal Supported Image Format Spotxel supports 8 bit or 16 bit grayscale images or 24 bit color images For the best image quality 16 bit grayscale TIFFs are recommended Spotxel 1 1 User s Guide Page 5 2 Preparation for Microarray Data Ana
32. round value is based on its background pixels only Therefore it is likely that the background values are different between spots Background Controls Instead of having the background values locally calculated you can use a background control to explicitly specify the image region from which the background value is calculated You can define a global background value for the entire array using a background control as follows e Open the Block page in the Array Object section of the Arrays control panel e Right click at a point in the image where the pixels represent the background for the spots Choose Create Background Control Specify the shape and the number of spots e Perform data quantification All spots in the array now have the same background value which is obtained from the background control We call that an association between the blocks and the background control In addition to having a global background value for the entire array you can flexibly choose an individual background value for each block This is done by creating a background control just for the block and establishing the association between them e Right click on the block to popup the context menu Choose Background Control gt Remove Association to release the block from the first background control e Right click on the block again and choose Background Control gt Establish Association Then click on the background control with which you want
33. the images intensity value option Figure 7 To use the original image data for the data quantitation again select the No option Please note that when the No option is chosen the Spot Image widgets in the Imaging Quantified Data and Scatter Plot sheets show the spot s counterpart image using the original image s data even though the image s contrast or brightness has been changed In the Quantification Options section of the Quantification control panel the Spot detection method option defines how the raw value and the background value of a spot are calculated Figure 7 e Fixed Spot The software always uses the pixels in the spotted region to compute the raw value The background value is calculated based on the pixels in the remaining region within the spot e Flex Spot This method can flexibly detect the spot s signals in the image even though their shape and position are not in accordance with the spotted region The detected spot border is shown in blue Calculation of the raw value is based on pixels within the blue border Pixels in the remaining region within the spot are used to calculate the background value Imaging _ Quantified Data Data _ eeoooapaec i P Figure 8 The Flex Spot Method Spotxel 1 1 User s Guide Page 11 4 Quantification of Microarray Data The Flex Spot method is recommended because it does not require the spots in the image to rigidly matc
34. to associate this block You can check this association using the Highlight Associated Control context menu Figure 12 Spotxel 1 1 User s Guide Page 14 5 Scatter Plot amp K Means Clustering Figure 12 Associated Background Control Please note that the background correction method is automatically set to Controls after the creation of the first background control In the Quantification Options section of the Quantification control panel you can select Local method again for the Background correction option Figure 11 5 Scatter Plot amp K Means Clustering Scatter Plot After quantifying the data you can depict the microarray data on a two dimensional scatter plot Figure 13 This enables to visually examine and select spots according to their quantified values To start with click on the Scatter Plot button in the main toolbar Initially spots of all blocks in the array are shown You can limit the plot to a certain block using the Blocks list box The X and Y axes can be any quantified value By means of the two blue threshold bars on the plot you can select spots whose X values are between the two threshold values These spots are then populated into the table below the plot By clicking on or hovering over a spot in the plot you can view the signals of the spot and its neighbors in the Spot Image widget as well as its properties in the Spot Details widget This also highlights the spot s quantified data
35. ton to get the data quantified for the entire array e To quantify some blocks select them and then click on the Quantify Selection button For each spot in the array the median and the mean of its raw background and foreground values in each channel Red and or Green are calculated If the raw value of a spot is smaller than its background value the spot is flagged Error and its foreground value is set to zero Section 4 3 details the methods used to calculate the raw value and the background value In the Imaging sheet you can view the spots and their quantified data simultaneously Figure 6 The image part corresponding to the selected spot and its neighbors are displayed in the Spot Image widget In addition selecting a row in the table of quantified data highlights the corresponding spot in the canvas This also opens the Spot page in the Array Object section in the Arrays control panel and shows the spot s properties there Similarly when the Spot page is opened clicking on a spot in the canvas will highlight its quantified data in the table You can also browse the spots quantified data in the Quantified Data sheet which shows only the table of quantified data and the Spot Image widget In both Imaging and Quantified Data sheets you can export the quantified data to a CSV file for further analysis Spotxel 1 1 User s Guide Page 9 4 Quantification of Microarray Data X Spotxel o Project Images
36. uy 2 End User License Agreement 25 M main toolbar 3 4 menu 3 Page 27 N noise background 12 foreground 12 process noise at quantification 12 noise filtering 8 p PCA 20 original dataset 20 simplified dataset 21 Principal Component Analysis 20 Q quantification 4 5 9 11 13 15 change of intensity values 11 quantified data 9 export to CSV file 9 table 3 R raw value 5 9 replicas 10 Spotxel 1 1 User s Guide 10 Index aggregate results 10 rotate array blocks 8 9 image 8 9 S scatter plot 15 serial number 23 spot border 4 diameter 4 properties 6 9 15 smallest spot size 13 visibility 8 spot detection method 11 Fixed Spot 11 Flex Spot 11 Spot Image widget 3 9 15 spotted region 4 11 Z zoom 6 Page 28
37. y you can discover such information about the samples by choosing the Features option before starting the analysis By combining the results of these two analyses you may be able to discover features and samples that influence the variance of the study 7 3 Hierarchical Clustering Analysis HCA You can group features or samples that are related using HCA The relationship can be e g having similar effect in the study represented by close screening values Click the HC button in the main toolbar to setup the analysis e Click the Load Data button and select the dataset Please refer to Section 7 1 for the preparation of the dataset e Select a quantified value in the Data Column list box e Choose to construct the clustering tree for features or samples or both e Select the distance metric and the type of linkage You can keep the default options e Click the Start Analysis button a X Spotxel Project Images Arrays Plots Analysis Batch Configurations Help lt gt E 4 Zoom Out 100 v Control Panel X Hierarchical Clustering Analysis 9 97 0 31 16 61 MMMM IO HMMM HN NH NH KH KH KH KKH Nn KH Nn NHN Nn NM wuuvuvuvuvYeVvVHTVVYVVYVVVVVYVYVYYY ECECECECCECECLECEELECCCCECLEC la le de Le la le le de de de lu de de De de le Yu de de de le de du de OOOO LoadData Start Analysis 3555555555555993595959550585805 So A 0 DMNA A m OO OO JSGGREARRARRRSHSRASTAS TRS o oo 00 oo oo
38. y not be copied either in full or part by USER with the exception of making copy for security or backup purpose Copies must include all copyright and trademark notices Use Restrictions This SOFTWARE is licensed to USER for internal use only USER shall not and shall not allow any third party to i decompile disassemble reverse engineer or attempt to reconstruct identify or discover any source code underlying ideas underlying user interface techniques or algorithms of the SOFTWARE by any means whatever or disclose any of the foregoing ii modify incorporate into or with other software or create a derivative work of any part of the SOFTWARE iii attempt to circumvent any user limits or other license timing or use restrictions that are built into the SOFTWARE Ownership of the SOFTWARE SICASYS retains all titles of ownership all ownership rights and all intellectual property rights of the SOFTWARE SICASYS reserves all rights not expressly granted to USER Termination SICASYS may terminate this Agreement immediately if USER breaches any provision Upon notice of termination by SICASYS all rights granted to USER under this Agreement will immediately terminate and USER shall cease using the SOFTWARE and return or destroy all copies of the SOFTWARE Limited Warranty and Disclaimer USER is aware of the fact that technical errors in the program in accordance with the accompanying documentation cannot be excluded If USER
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