CLONETRACKER DB™ CLONETRACKER DB™
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1. 95 695 29 006 56 06 ALLELE EE 86600800 505 6 585 LEO ENS EEI LLIT LEA Ea 626 920 TI LE a eee 2090200020009 500 2921 8 22252 85 9 29 9 222 8887 7 222092999 ore 55898 00888568 oe 00 000 09090 1 EE CI EE TILILLE ISLE ITTE 30 Microarray Design Using CloneTracker DB Version 2 0 USER MANUAL APPENDIXES APPENDIXES Appendix 1 Frequent Asked Questions APPENDIX FAQ Frequently Asked Questions Q Why does CloneTracker DB use microns instead of millimeters A Using microns allows CloneTracker DB to be highly accurate within it internal calculations Also as spot size and spacing is typically determined in microns CloneTracker DB maintains unit consistency Q Why does CloneTracker DB offer so many possible combination of arraying styles A CloneTracker DB 2 has been designed from the start as a comprehensive clone management sys tem capable of performing virtually any task required of researchers As a result CloneTracker DB pro vides greater flexibility than any proprietary solution available 0 15 CloneTracker DB cross platform compatible A Currently CloneTracker DB is only available for the windows platform However BioDiscovery is actively porting to Unix systems and the Mac OS X Please contact BioDiscovery Support for the latest listing of supported OS o8 Microarray Desig2. Ci Merge plates choose plate ID Enter additional info about plate Fig 4 3 3 30 Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL Importing Merged Plate In the Array Design Panel of CloneTracker click on the Field tab Under the Plates tab select the type of plate you wish to import In this project we want to use the 384 well plate we had just creat ed from the four 96 well plates so we select 16x24 384 well plates as the Plate Type Click on the Edit Plate List icon to bring up the Edit Plate List window You will see that the newly created Mouse Scale Up plate is there Choose your choice of plate s from the Available Plates list click on the right arrow icon to bring your chosen plate s to the Selected Plates list Hit the OK button B SPLITTING ONE 384 WELL PLATE INTO FOUR 96 WELL PLATES Some of your projects may require performing PC in a 394 well PCR plate and subsequently split ting the materials into 96 well plates Figure 4 3 4 Under the View pull down menu select Plates Click the Split One Plate into Four icon on the right vertical toolbar of Plates Panel to display the Split plates dialog box Select the 384 button for plates to be split Select the Mouse Scale Up plate Click the right pointing arrow on the right The 384 well plate is automatically split into four 96 well plates which have the fo
3. gt BioDiscovery Inc 4640 Admiralty Way Suite 710 Marina del Rey CA 92092 Phone 310 306 9310 Fax 310 306 9101 E mail support biodiscovery com CONTENTS CHAPTER 1 Introduction to CloneTracker DB and its role in Array Design 5 Microarray Experimental Design to Assist Normalization of Data 6 CHAPTER 2 CloneTracker DB terminology 8 Experimental Terms 9 Array 005005 aa ia ao RG ebook ee PEED 000 04 tans ED REED Ro de nci 9 10 LL a CONES 0 43 029 0 9 mis 10 Plates PCT 10 Array Design Terms 10 Field osse rr as 10 Layout 11 00 20 02 mes 9 9 0 9 99 eee eae eae te 11 11 SUGCING ca 00 5 eu ORO Pe 0 993 11 Spot Staggering 444 4 12 CHAPTER 3 Moving from CloneTracker DB v1 X 0 V2 uuaa Qu Qu LL LL La 13 Database Converter 14 User Interface Changes 15 Naming Convention Changes 10 CHAPTER 4 Design 19 Example 1 Designing a cDNA macro Array working with a nylon membrane 20 Example 2 Designing a a High
4. 3 Click the drop down triangle under Spot Ordering to change the ordering to something different than the original field 22 Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL Change Sector Ordering 1 Select the duplicated field tab typically Field 2 2 Select the Sector tab 3 Click the drop down triangle under Sector Ordering to change the ordering to something different than the original field The other way to duplicate spots is to make duplicates within the same sector as the original spots This is called editing the Replicates Pattern This tool is a little complicated but it is very powerful Edit Replicates Pattern 1 Under the Field tab click on the Spots sub tab 2 Click the Edit Replicates Pattern button to display the Replicates Editor dialog box In the left panel is an empty grid and on the right panel is a grid full of spots The left grid rep resents the layout of every sector in this field A blank grid indicates a default layout Fig 4 4 2 A blank grid means a default layout Indicate Spot Layout 1 On the right grid click on the spot that you wish to replicate 2 On the left grid click on the location s that you wish to have the selected spot in 3 Repeat steps 1 and 2 for all of your desired spot s to be replicated In the example below we have indicated that we want the first clones to be duplicated three more times marked in red Any left grid boxes that y
5. Step 3 Spots For simplicity purposes we will not change the default values under the Spots tab Researchers usually change the spot spacing and diameter to achieve special printing patterns Depending on the type of arrayer machine one is able to decrease the vertical and horizontal spot spacing to fit more spots into each sector area Decreasing the spot diameter allows one to further decrease the spacing between each spot Step 4 Sectors A sector is most easily defined as the total number of spots printed by one pin from one plate in Regular mode please see the following examples for further discussion The sector size specifies the layout of all those spots In our case we have 96 wells that are print ed by 96 pins There is only one spot in one sector Therefore the only sector size possible is 1x1 in Regular Mode 96 wells 96 pins 1 In fact in this case one spot is equivalent to a sector The numbers of Sector Rows and Sector Columns define in what pattern the sector as a geometri cal entity will be repeated Enter the number of Sector Rows and Sector Columns and then change to another pair of numbers to see how the printing pattern alters Let us choose 4x4 29 CHAPTER 4 Design Examples Select a specific order for Sector Ordering from the pull down menu The Sector Ordering refers to the order of clones that are spotted by one pin you choose Left to right Top down and
6. BioDiscovery for data normalization procedures Replicate the array on the same slide array replication This is a blocking strategy performing an experiment on a homogenous material i e the same slide which can be achieved by the click of the mouse with CloneTracker DB Use control spots CloneTracker DB keeps track and can color code control spots Replicate the experiment on different slides experiment replication CloneTracker allows you to use an array design that is automatically applied to many slides Randomize the spot replicates on the array You may use CloneTracker Replicates Editor to design your array CHAPTER 2 CloneTracker DB Terminology CHAPTER Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL Due to the flexibly associated with various parts of the arraying processes CloneTracker DB has developed a unique terminology covering various design aspects The terms listed below reoccur through out the usage of CloneTracker DB As a result users are encouraged to have a working knowledge of their meaning Most terms covered below are found throughout microarray design and clone management For additional information please consult the CloneTracker DB v 2 User s Manual EXPERIMENTAL TERMS Array Designs Array Designs are templates of spotted arrays describing how a particular array is spotted Figure 2 1 and from which plates the clones are taken Figure 2 2 Array Designs
7. are used to create multiple Arrays of the same template or design Some of the parameters used to describe an Array Design include the array dimensions whether for a slide or a membrane pin and spot configurations pin and spot spac ing and sectors Fig 2 1 Regular 5 Fig 2 2 Example f a particular set of genes we denote any length of DNA whether an EST or cdna as a gene for simplicity needs to be printed on hundreds of slides by a core facility then the set up applying to each slide is the array design This blue print contains all the information about the dimen sion of the slide how many pins are used for printing what plates with DNA probes were utilized etc CHAPTER 2 CloneTracher DB Terminology Arrays Arrays are particular instances of Array Designs corresponding to physical arrays The plates used in the Array Design are the plates used in the Array Additional information such as barcode information can be applied to each Array Clones Clones are the biological materials contained in Plate wells Therefore a clone may mean a PCR product cdna or other material to be printed Clones may be imported into the database along with Plate Importation or they may be added to existing plates that are not populated with Clone data Plates Plates are small plastic containers consisting of 96 384 and 1536 wells These wells contain the material to be spotted onto the microarray slide With
8. sector The Compact layout mode will be explained in another project example 24 Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL Step 6 Saving the Design Clone Tracker There are two ways to save the array design that you have created By using either method the Array Design can be easily recalled for future analysis or modification Saving to the CloneTracker Database Click on File gt Save on the menu bar Figure 4 1 10 This will save the current Array Design to the CloneTracker database Saving to a Specific Location Click on File gt Export on the menu bar The Save dialog box is dis played where you can browse to the location of where you would like your array design exported to Click the Save button and your slide set is saved in xml format in the desired location Fig 4 1 10 2 CHAPTER 4 Design Examples EXAMPLE 2 Designing a High Density Array working with glass slides OBJECTIVE Design a high density array which will be printed on a glass slide This example will require 2x2 pins and 96 well plates Array Design Specify the array design name and the name of the user under the Array Design tab Let us call this array design Slide Mouse The dimensions of the slide by default are width 5 000 x height 25 000u 7 5 cm x 2 5 cm The frosted part of the slide is 25 000u With these dimensions the slide width is horizontal to the viewer and the frosted side is to yo
9. v2 Fig 3 11 The two versions differ because v2 allows users to create multiple or different fields for additional designs within the same slide Fig 3 12 Fig 3 10 Fig 3 11 Fig 3 12 Microarray Design Using CloneTracker DB Version 2 0 USER MANUAL CHAPTER FOUR CHAPTER 4 Design Examples EXAMPLE 1 Designing a cDNA Macro Array working with a nylon membrane OBJECTIVE Print a cDNA array on a nylon membrane with a hand held device which has 96 pins Array Design Specify the array design name and the name of the user under the Array Design tab Let us call this array design Membrane Mouse The dimensions of this membrane are 20 cm x 20 cm Therefore you may enter the corresponding dimensions in microns i e 200 000 see frequently asked questions You many choose margins depending on your experiment For this example enter 10 000 for all margins After all the information is specified press the Apply button Fig 4 1 1 Cens Tomek 0 Spred Telal of sabia D FIELD DESIGN Click on the Field Tab Specify the name of the field under the Field tab For example type in Device 96 pins Step 1 Plates Initially only the Plates tab is enabled Figure 4 1 2 Depending on your microarray experiment choose either 96 8x12 384 16x24 or 1536 32x48 well plates to be used for the final stage of print ing by selecting from the Plate Type drop down menu For this experiment we
10. you wish to import Figure 4 1 6 In this case select all columns The selected columns will be colored in red Click on the first row of plate 501 and select it Scroll down to the last row of plate 503 Hold the SHIFT key on your keyboard to select all rows including the last one row h column 12 of plate 503 All the columns become highlighted Click the Down Arrow button at the bottom of the Plates Importer win dow The selected plates appear in the lower half of the Importer window Figure 4 1 7 You may enter some remarks about each plate in the column termed Info Click OK Now you may go to the Field Tab and click on the Add Plates icon Select plate 501 502 and 503 Click the right arrow Now plate 501 502 and 503 are added to the Selected Plates panel And then click on the OK button 109888 110844 414775 Fig 4 1 6 22 Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL ITI Tracker Fig 4 1 8 Step 2 Pins The Pins tab allows you to customize the desired pin configuration You could modify the pin spac ing by selecting either Consecutive wells or Every other well Choose the 8x12 pin configuration for this experiment as shown if Figure 4 1 8 Several well dipping order patterns could be selected by clicking on the Well Dipping Order drop down menu The most common dipping pattern used by most arraying machines is Left to Right Top Down
11. Solution Fabrication limage Analysis Data Rin CloneTracke DE gt TmaGene GeneSighi CeeDuuiectorTM Svnergistic Anolydis Manezement of Data EM De cul 28255 Clone Expression house Data Data Data fu E M Slide Data Data Chitsecke ata Fig 1 1 The software tool CloneTracker DB from BioDiscovery Inc Marina Del Rey CA www biodiscovery com has been designed to make management of array fabrication data easy and rewarding The CloneTracker DB software offers Laboratory information management A management system that provides access interface to the underlying fabrication database It helps the user monitor the overall fabrication process by allowing queries of fabrication data updating data step by step according to the progress or tracing a particular spot on a particular array back to the originating plate and well position Array design An easy to use visual interface that allows the user to design or adjust the array layout based on the type of arrayer used It can also export the settings for the arrayer or even generate complete programs to control the arrayer robot directly Utilities for complex data manipulation The information flow in the fabrication process involves several transformations that cannot be handled using simple database operations Examples i transformation of clone data from a set of plates plate data into spot data
12. ah wii d UsiNG 121 Tg 127 145 3 N 154 147 ERSION 2 0 USER MANUAL L 5 a MICROARRAY DESIGN USING CLONETRACKER DB A Supplement to the CloneTracker DB Version 2 0 User nual 19 LE T Va 222 uy 1 tT FEM oot Hoe TILI uim 5 nee Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL Every effort has been made to ensure the accuracy of this document However BioDiscovery makes no warranties with respect to this documentation and disclaims any implied warranties or merchantability and fitness for a particular purpose BioDiscovery shall not be liable for any errors or for any accidental or consequential damages in connection with the furnishing performance or use of this document or the examples herein The information in this document is subject to change Trademarks CloneTracker DB GeneSight and GeneDirector are trademarks of BioDiscovery Inc Other product names mentioned in this document may be trademarks or registered trade marks of their respective companies and the sole property of their respective manufacturers Copyright 2002 BioDiscovery Inc All Rights Reserved Written at BioDiscovery Inc Marina Del Rey CA 90292 Printed in the United States of America
13. density Array working with glass slides 26 Example 3 Plate to plate to slide mapping 29 Example 4 Designing an array with replicated Duplicated spots 32 Example 5 Exercise for 36 APPENDIX 1 FAQ Frequently Asked Questions 00000000 38 APPENDIX 2 Solution to Exercise in Chapter 5 39 References 40 New Features 41 Microarray Design Using CloneTracker DB Version 2 0 USER MANUAL CHAPTER ONE n TE TT il CHAPTER 1 CloneTracker DB and its role in Array Design Introduction Microarray projects involve acquisition and validation of large data sets Each study typically requires iterations of series of processes which start with experimental design and array fabrication pro ceed with array scanning image analysis and finally gene expression data analysis The initial data resources required to design and fabricate gene expression microarrays that need to be documented include cDNA sequence data cDNA clones information experimental procedures phys ical location of materials and others This information needs to be integrated within the data flow dur ing array design analysis and interpretation of results Figure 1 1 Integrated Informatics
14. dow The data is now available within CloneTracker DB 2 Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL USER INTERFACE CHANGES CloneTracker DB 2 has included the best aspects of version 1 and applied several new options for the main user interface which were required by a world wide group of scientists employing CloneTracker DB in their research The user interface in version 2 as it was in 1 x is primarily geared to virtual array design Within this virtual design the key elements slide array geometry clones and mapping infor mation are placed predominantly before the user Please see diagram below for similarities Virtual Design m umo um um ah ah bh a a bou mh mb o uno Nh c pu 5 Experiment Information jue HEE 6 om a NU NS i 1 EON WW WE M 8 amp JM 85 o 5 NNUS GE de ook d CHAPTER 3 Moving from CloneTracker DB version 1 x to version 2 CloneTracker DB 2 also adds several new features as outlined below Fig 3 5 When zoomed
15. e IDs from a 96 well plate to a 384 plate and then to the glass slide array 96 well PCR plates 384 well PCR plates Fig 4 3 1 Alternatively some protocols call for transfer of biological material from 384 well plates to 96 well plates which are subsequently used as a source for printing the arrays CloneTracker has a module which assists in mapping clones in such protocols A MERGING FOUR PLATES INTO ONE Under the View pull down menu select Plates Click the Merge Four Plates into One icon on the right vertical toolbar of the Plates Panel to display the Plate Merger dialog box Select the four plates that you want to merge from the All Available Plates list Click the left arrow button to move these plates to the Four Plates list For example if you have the plates 501 502 and 503 then transfer them to the left Now select plate 501 again and transfer it to the left Figure 4 3 2 CHAPTER 4 Design Examples 2 za m En MEI 2 2 DENM Ed EE MUI RR Fig 4 3 2 Click the Merge button to merge the plate tables Figure 4 3 3 Specify a name for the merged plate under Plate ID as an example Mouse Scale Up and click the OK button to save your changes You should always enter notes about the created plate in the Description field These notes prove always useful in future data analysis Return to the CloneTracker Plates Panel Em mii UM 26
16. e parameters under the Spot tab users can design arrays for machines capable of high density printing Exportation of a gene ID file After generating an array design you can export the gene ID file to your image analysis software maGene from BioDiscovery automatically imports gene ID files generated by CloneTracker Select Export Mapping under the File menu in CloneTracker Choose maGene format Figure 4 2 3 You have the option to assign the name blank to all empty spots Those are the spots where nothing is printed This is particularly useful if you want to carry out your background corrections based on such spots for the data analysis Alternatively you can choose not to export any empty spots With the Browse button select the directory and folder where you would like to save the file Assign a name to the file and click the save button 5 sport Happy Fig 4 2 3 28 Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL 29 EXAMPLE 3 Plate to plate to slide mapping OBJECTIVE t is a common practice to perform a PCR reaction in 96 well plates and then transfer some volume of the PCR reaction from each well to a well in a 394 well plate Subsequently the printing of the microarray will be carried out by using the 384 well plate Figure 4 3 1 This protocol allows for printing of high density arrays and storing some of the initial PCR material for future use Therefore we need to map the clon
17. forming more complicated arraying this is not true however such arraying is beyond the scope of this document In v1 all the spot layouts generated by a single pin are grouped into a subgrid Fig 3 8 In v2 the sectors are grouped into a subgrid Fig 3 9 but are loosely called simply Rows and Columns of Sectors The subgrid term is not used much in v2 except to indicate to which direction printing should continue upon filling the allocated sector rows and columns 99999999999 6606064649 0600066 006066616 ssa s 08589 6 E s s eae 959 5 B 9525 E ea pos s O Fig 3 9 The top level of design in v1 is the supergrid The supergrid was simply all the subgrids printed within the slide However the problem of a supergrid is that it only allows for a single supergrid design per slide For example this prevents users from generating a slide where extra marker genes are placed outside of the supergrid as is displayed below It also prevents different arraying designs or shapes to occur on a single slide V2 addresses these issues by introducing the concept of a field CHAPTER 3 Moving from CloneTracker DB version 1 x to version 2 Typically a single field represents a the entire supergrid structure on the slide For example if the slide has a supergrid structure of 1x3 Fig 3 10 then this design would be contained within a single field in
18. in spotting orders become visible All Arraying Parameters conveniently displayed on main screen New Toolbar for quick access to important features de d da do de de 8 97 105 113 121 129 137 145 153 88 106 4 422 430 438 436 101 8 Fig 3 5 Ability to edit individual Replicate Patterns Ability to control the mapping information that is displayed NAMING CONVENTION CHANGES CloneTracker DB 2 uses a unique naming convention which allows for the description of highly complicated arraying design Note although there have been terminology changes between previous ver sions of CloneTracker DB and version 2 the same designs can still be created CloneTracker 1 used the notions of spot layout subgrid and super grid to describe all the design elements of the slide CloneTracker DB 2 also has a set of terms sector sub grid and field which per form the same task The following information explains the relationship between naming conventions Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL 5808055559 000060666 8 5 BRERE ER 000006600 EEES 8 4 d e Fig 3 7 The smallest level of design in v1 is the spot layout Fig 3 6 In v2 this is the Sector Fig 3 7 When arraying in Regular Mode spot layout and sector represent the same thing Note When per
19. in CloneTracker DB the Plate Type indicates whether the plate is 96 384 or 1536 well plate ARRAY DESIGN TERMS Field A field is the largest design element within a slide Figure 2 3 A field typically consists of the arraying done by a single print head on the slide For example if the arrayer has a print head with 8 pins in a 2 x 4 configuration the region of the slide containing the resulting printing is a field For most experiments and arrayers users will only have a single field However CloneTracker DB 2 fully supports the creation of multiple Eia fields if necessary 1 mm 16 CES CES CAS Common uses for multiple fields include Fig 2 3 Generating slides which contain arrays of different geometries for example some microarray core facilities procedures include a set of designated genes on each slide produced in the facility for calibra tion purposes These genes are not within the field with the genes under investigation Such sets of genes will be located in a separate field CloneTracker DB 2 offers the flexibility and ease to use to cre ate this field Generating slides with duplicated arrays next to each other This will be needed if you decide to replicate the whole array on the same slide as discussed in Lee et al 2000 Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL Layout Mode Regular In Regular Mode Fig
20. licate yields 24 Let us choose sector size 3x8 3 rows x 8 columns Enter the number of Sector rows 3 and Sector columns 2 Watch the array Flip these numbers and see the design In this way you will understand the architecture of the array and the flexibility of CloneTracker DB to create your designs To understand the design of the finished array and to conceptualize what will be printed by one pin we can compute a subgrid size The subgrid is simply the total numbers of rows and columns of spots printed by a pin from several plates The actual calculation is Number of sector rows x number of rows in each sector total number of rows of each subgrid Number of sector columns x number of columns in each sector total number of columns of each subgrid So in the above example we have 3x3 9 rows and 2x82 16 columns in each subgrid Step 4 Spot Information To create a high density array we will need to change the information regarding the size and posi tion of spots Click on the Spots tab to access this information First let us change the spot stagger ing to Staggered Right Upon selection the array design to the right will be updated to reflect the new 2 CHAPTER 4 Design Examples design To further increase the number of possible spots within the array let us decrease the Spot Space Horizontal and Spot Space Vertical The default values of 250 should be changed to 225 As a result by manipulation th
21. lide Design after editing Replicates Pattern After creating this customized Replicates Pattern we may want to save our design and load it back in at a later time Export Replicates Pattern 1 Click the Export Replicates Pattern button to display the Save dialog box 2 Select a destination folder for the replicates file 3 Click the Save button to save the file to the designation location Import Replicates Pattern 1 Click the Import Replicates Pattern button to display the Open dialog box 2 Locate and select a replicates file to import 3 Click the Open button to display the selected file 35 CHAPTER 4 Design Examples EXAMPLE 5 Exercise for practice Using the examples outlined in the previous sections try to perform the following exercise by self The proposed design configurations are given in an Appendix of this tutorial Let us try to design the following experiment You will print your arrays with an arrayer equipped with 4x4 pins You will use 384 well plates the distance between spots is 240 um The printing of the array sectors will be performed Right to Left Top to Bottom Genes will be replicated on this array by creating a duplicate meta grid The slide should be aligned vertically and there is no frosted area You need to print the spots in sectors of 18x18 Figure 4 5 1 Additionally the objective is to have 12x4 such sectors gt gt gt 9999 9
22. llowing names Mouse Scale Up 0 Mouse Scale Up 1 Mouse Scale Up 2 and Mouse Scale Up 3 If you decide to design an array with 96 well plates and repeat the plate import procedure as out lined in example 1 you will see that the newly created 96 well plates are available for mapping to the glass slide array Transfer from 384 well plate to 96 well plates o6 well plates PCE performed m 384 well plates Fig 4 3 4 CHAPTER 4 Design Examples EXAMPLE 4 Designing an Array with Replicated Duplicated Spots As discussed in Chapter 1 replication is one of the basic principles of experimental design Many researchers prefer to have replicates of their genes on the same array There are a two ways to achieve this in CloneTracker The easiest way to duplicate spots is to copy the entire field to a different location on the slide In the Array Design Panel start off by loading some plate files See Example 1 Step 1 Plates Copy Field 1 Select Copy Field from the Fields menu 2 Click the Field Mode button on the toolbar 3 Click on the new field and drag it into the desired position Fig 4 4 1 Duplicated Fields Some researchers prefer to have their duplicate clones positioned differently in each field Changing the Spot Ordering or Sector Ordering in the duplicated field easily does this Change Spot Ordering 1 Select the duplicated field tab typically Field 2 2 Select the Spot tab
23. mats with virtually any number of columns including using the use of XML Enhanced Visual Manipulations Zoom move and align arrays with a mouse click Advanced Spot Duplication Tools CloneTracker DB includes the replicates editor allowing for almost any form of spot replication pattern New Online Help System ava based help system Enhanced Query Capability New built in query tools allow researchers to retrieve information on a variety of microarray information Java Core allows CloneTracker DB to be a robust cross platform application with enhanced memory management Staggered Spotting Users whose machine s support spot staggering are now supported within CloneTracker DB 2 MySQL Database CloneTracker DB includes a powerful cross platform relational database system 4640 Admiralty Way Suite 710 Marina del Rey 2 2002 All rights resrved
24. mns After we have created this array field with 4x4 sectors and each sector with 18 rows and 10 columns we need to print to more arrays on the same slide with the same configuration 22 Click on the Apply button to apply these changes to the current field 23 Select Copy Field from the Fields menu at the top of the screen CloneTracker DB will now iden tify the first Field as Field 1 and the new one as Field 2 24 Click on the Field 2 tab 25 Select the general sub tab 26 Type 4 000 for Field location X and 53 000 for Field location Y 21 Click on the Apply button to apply these changes to the current field 39 REFERENCES REFERENCES Lee M L Kuo F C Whitemore G A and Sklar 3 2000 Importance of replication in microarray gene expression studies statistical methods and evi dence from repetitive cDNA hybridizations PNAS USA 29 9834 9839 Montgomery D C 1991 Design and analysis of experimental design John Wiley amp Sons New York NY 40 Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL NEW FEATURES Dynamically Structured Database Unlike common databases CloneTracker DB s has been designed to grow with new data and information For example virtually any type of additional clone data whether assesion number or chromosomal info can be added to the database during import Extremely Flexible File Formats supporting XML Researchers are now free to import files in var ious for
25. n Using CloneTracker DB Version 2 0 e USER MANUAL APPENDIX Il Solution to Exercise in Chapter 5 Exercise 1 Solution 1 Open CloneTracker 2 Click on the Array Design Tab 3 Enter Exercise 1 as the name for this design 4 Set slide height to 75 000 and the width to 25 000U because we would like to have the slide positioned vertically 5 Assign 300u to all margins This will remove the frosted area 6 Click on the Field Tab 7 Select the Plates Tab 8 Select 384 as the plate type 9 Click the Add Plates button 10 Pick any plate from the Available Plate list 11 Click the Right Arrow button to add this plate to the Selected Plates list 12 Click OK This will activate all other Field sub tabs 13 Select the Pins Tab 14 Pick 4x4 from the Pin Configuration list 15 Click on the sub tab General 16 Choose Compact layout mode Because we need to print spots in a structure 18x18 324 we cannot use regular mode as explained earlier in one of the examples If we use regular mode then we will have to choose a sector size equal to a combination of two numbers which have been multiplied and their result is 24 384 4x4 17 Enter 4 000 for Field Location X and 15 000 for Field location Y to position it 18 Click on the Sectors sub tab 19 Type 18x18 in sector size this size will NOT appear on the list 20 Select Left to Right top down sector ordering 21 Type in 1 for sector rows and for sector colu
26. neTracker DB version 1 x to version 2 DATABASE CONVERTER CloneTracker DB 2 includes a handy database conversion utility designed to transition data from CloneTracker version 1 4 4 and higher to CloneTracker DB version 2 User with older versions of CloneTracker DB will need to export plates from version 1 then recreate the slides within CloneTracker DB 2 Through use of the DB Converter all information within the database is automatically transferred to the new MySQL database To convert the database perform the following 1 From within the Start Menu click DB Converter within the CloneTracker DB 2 menu 2 Read the initial message click OK to continue Fig 3 1 un adbr 11 jdbe odbe Clone Tracker get database Sectings successful yet coemection For seucce database succeed get comection for target database succeed setup data store Using oracle dome Fig 3 2 3 The next window is the Database Transfer Window For most users the correct parameters will be provided However the user must enter the source password By default this password is sql There is no need to enter a Target password 4 Click OK to continue 5 The information is transferred and Log window is then displayed If the same data exists with in the new database the user is prompted what to do with the data Fig 3 2 6 Upon completion Logout Succeeded is displayed in the Log win
27. need to choose 96 8x12 20 Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL Fig 4 1 2 Click on the Add Plate icon to bring up the Edit Plate List window figure 4 1 3 Choose your choice of plate s from the Available Plates panel For this membrane printing project select 8x12 96 well plates and choose Plates 501 502 and 503 Click on the right arrow icon to bring your chosen plate s to the Selected Plates panel Hit the OK button lf hin Columns Lenrriptan CHAPTER 4 Design Examples If initially there are no plates listed or you would like to import plates then click on the Plates icon in the toolbar Alternatively select Plates under View menu Figure 4 1 4 Click on the I mport Plates From Text icon on the right icon bar selection Figure 4 1 5 A Plates Importer window shows up Click on Browse and find the folder Samples located in your CloneTracker folder Choose 96 plates txt file which contains clone information for four 96 well plates called 501 502 and 503 i e they are concatenated A file can also contain information for one plate only You may want to view or edit this file by using Excel Once editing within Excel is complete remember to save the file as a tab delimited text file for compatibility with CloneTracker Fig 4 1 4 Fig 4 1 5 In the Plates Importer click on the selection buttons squares for the columns
28. on the array array data ii generation of fabrication data in the format recognized by the image analysis system maGene and the data mining tool GeneSight iii tracking clone transfer processes from 96 well to 384 well plates to a DNA array and back Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL The primary goal of this document is to illustrate uses of CloneTracker DB with examples while explaining to existing users new features and terminology Readers should review the CloneTracker DB User s Manual when using this software CloneTracker DB is useful not only as a virtual design tool to facilitate visualization before print ing of the array and organizing data but also to help the actual experimental design Microarray Experimental Design to Assist Normalization of Data A designed experiment is a test or series of tests in which purposeful changes are made to the input variables of a process or system so that we may observe and identify reasons for changes in the output response Montgomery 1991 The basics principles of experimental design are replication randomization and blocking Approaches for experimental design in microarray experiments are as follows Use more than one cDNA spot for normalization purposes spot replication CloneTracker DB offers an easy approach for creating duplicates triplicates and more spot replications This information is submitted to the data analysis tool GeneSight
29. ou do not explicitly select are assumed to be empty 22 CHAPTER 4 Design Examples 1 2 3 4 5 CEC ees eed doesn t equal HEEE Fig 4 4 3 Blank areas on the grid does NOT mean a default layout Fig 4 4 3 Blank areas on the grid does NOT mean a default layout Looking at the right grid above you may wonder what happened to clones 1a2 1a3 and 1a4 Since they are not on the grid they will not be printed In order to have all the clones in the sector AND have duplicated spots you must increase the size of the sector and add the missing clones back in marked in green Change Sector Size 1 Enter the new number of rows to use in the Number of Rows field 2 Enter the new number of columns to use in the Number of Columns field 3 Press ENTER on your keyboard to apply the changes Fig 4 4 4 Expanded sector with missing clones added back in 34 Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL Now that we have our customized Replicates Pattern we can apply the changes by clicking OR and exiting the Replicate Pattern Editor Our new slide design now shows up on the Spotted Array window box 4 2 2 402 434 435 424 422 482 de m 2 28 42435 420 425 425 492 e de 29 41485421 426 Go 454 m 422 427 431 des amp n7 Ti g JP mh pol 4 1 32 37 Fig 4 4 5 New S
30. r of Sector Rows and Number of Sector Columns 8B 5 6 E 8 5 ee 9 5 8 5 8 5 8 8 8 B g amp W M OG 8 mw a si m uw a m 55 m HE m em e 2 d s 888225 8 n m m 79 m TIE 3 gt ze Pin Configuration The arrangement and number of individual pins on the print head CloneTracker allows you to use either hand held devices with 96 pins or any arrayer with any combination of pins Pin Spacing Pin Spacing determines whether the pins on the print head dip into wells next to one another or whether the pins skip a well in the middle Typically users should select Consecutive wells CHAPTER 2 CloneTracher DB Terminology Spot Staggering Spot Staggering allows researchers to fit an increased num ber of spots within an array Spot Staggering prints spots in a 9 7 5 3 4 hexagonal structure minimizing the space between spots Left staggered means the initial row is spotted to the left of the row 2 10 18 E 34 42 below it Right staggered means the opposite where the top row Is position slightly to the right of the row below it Fig 2 6 3 11 19 2 35 43 Fig 2 6 Microarray Design Using CloneTracker DB Version 2 0 USER MANUAL CHAPTER THREE TT 4 y LILIS Im eun CHAPTER 3 Moving from Clo
31. the well dipping order is Left to Right Top Down the then the first spot row one column one in the first sec tor will come from well al in plate 501 the second spot left to right row one column two will come from well al in plate 502 and so on Let us consider the following design Now your objective is to replicate the same clone material from the same well on the same array twice Several research teams print the spots resulting from the same well adjacent to each other For example the material from well al plate 501 will be printed in row 1 column 1 and row 1 column 2 To achieve this we need to enter the plates in a different order In the Plate importer enter the plate in this case 501 twice as shown in Figure 4 1 9 Then do the same with plate 502 503 and so on Additionally some researchers like to leave blank spots for either background corrections or other reasons Let us imagine that in row 4 column 4 for each pin you would like to leave a BLANK spot The easiest way is to create a fake plate with all gene IDs BLANK Then import this plate in such an order after all the others so that the spots coming from this plate appear in row 4 column 4 96 Edit Plate List El Destrinton Fig 4 1 9 Step 5 General For this project we will stay with the Regular layout and keep the default settings under the General tab Select the Regular layout mode ensures that the spots from one plate are spotted In a rec tangular
32. ur right Figure 4 2 1 If you wish to rotate the slide then you can enter 75 000u for the height and 25 000u for the slide bottom margin Let us leave the slide with the default settings Fig 4 2 1 Field Design Click on the Field Tab Specify the name of the field under the Field tab Enter 2x2 for Pin Configuration 26 Microarray Design Using CloneTracker DB Version 2 0 e USER MANUAL Step 1 Selecting Plates To select the plates you wish to use in CloneTracker click on the Add Plates icon on the right ver tical toolbar in the Plates Panel The Edit Plate List window is displayed there are no plates listed see Example 1 for details on importing plates Select plates 501 502 and 503 and click on the right arrow to the right to import these plates Select plate 501 again and import it so we can work with 4 plates in this experiment Step 2 Pins Select 2x2 pin configuration pe Clone Tracks Step 3 Sector Click on the Sector Size tab Your Sector Size pull down Menu gives you options which are all combinations of 24 i e 1x24 2x12 3x8 4x6 etc Figure 4 2 2 The reason for this is that 24 is the number of times each pin will touch the slide from one plate during the printing If this number is denoted by N then x SR where W is the number of wells in the plate P is the num ber of pins and SR is the number of duplicate spots In this case 96 wells 4 pins only one gene rep
33. ure 2 4 the printing is done in such a way that each pin prints the required number of spots from a single plate as to use all samples from all wells See the examples in following chapters Compact n Compact Mode users are free to design the rows and columns to be printed irre gardless of the relationship to the plate This allows for more complicated array design Sector A sector is a region on the array containing spots printed by one pin The source for a sector could be one or several plates depending on the Layout Mode Fig 2 5 Example when printing in Regular Mode a sector is the printing done by a single pin from a single plate In this case each sector is displayed as a different color within the software Please note that when the Layout Mode is switched to Compact the notion of a sector increases in complex ity Under Compact Mode a sector defines the rows and columns of printing however this may or may not be equal to the printing done from a single plate Sector Size refers to the number of rows and columns of clones spots placed on the slide by a single pin In Regular Mode this is FIXED to a size which allows the print head to dip to every well of the plate In Compact Mode the sector size can be modified to virtually any val ues by the user Due to the gap between pins on the pin head researchers can spot several Sectors between each pin CloneTracker DB allows this to be done by specify ing the Numbe
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