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Manual - NCSU Bioinformatics Research Center

1. moi _ wo Fill in the dialog based on your source data Map function A map function is a mathematical relationship between recombination probabilities and map distances Select the function you want based on the interference to be assumed e Haldane The default option Assumes no crossover interference e Kosambi Assumes some interference e Fixed The Morgan mapping function Assumes complete interference Position type 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 47 e Position Indicates that the numbers indicate positions from the left telomere of the current chromosome This means the numbers should be in increasing order e Interval Indicates that the numbers are for the interval distance after a marker This means that the last number and the last number only should be zero Position units e centiMorgans cM Very small 100 centiMorgans 1 Morgan e Morgan The distance over which on average one crossover occurs per meiosis e Recombination Percentage of crossover events that occur between two markers Data window All the numbers you see here are place holders waiting for you to input the real data Edit window Data from your raw data file or in Windows clipboard is copied to this window first You can edit data in this window Browse Displays Windows Open dialog so you can select the data file Selecting the file copies its contents to the Edit window
2. 2010 N C State University Bioinformatics Research Center 4 Windows QTL Cartographer 2 5 Marker genotype table Edit or add tokens you want to use for these genotype markers you can enter any alphanumeric character s as a token These translations apply to the Crossing type you selected from the drop down You must ensure that the chosen symbols in this translation table match those in your marker genotype data file Note WinQTLCart assumes that the allele is diagnostic for the High parental 1 line and the a allele is diagnostic for the Low parental 2 line A minus sign means the allele is unknown WinQTLCart uses the numbers 2 1 0 12 10 1 to determine how to encode the output of the genotypes The alphanumeric tokens you enter here indicate how you have coded the markers in your source data Source data file stem name Enter a name for your source data file As WinQTLCart creates new source data files WinQTLCart will append extensions to this stem 2 Click Next button to accept the values The Create New Source File Step 2 of 6 appears Create New Source File Map Infomation 1 Step 2 of 6 Information Map function Haldane D Position types Position D Position units cent Morgan D Chromosome Chromosome label Marker number m Input chromosome lable and marker number for each chromosome k 13 i Browse C2 2 Clipboard C3 13 I Include label Send Data NotePad
3. Clipboard Paste contents of Windows clipboard to Edit window Send Data Check Include label if necessary then click Send Data to update the data window Notepad Open a blank Notepad file for use as a holding area for data provide an editing scratchpad and so on 3 Click Next button to accept the values The Create New Source File Step 3 of 6 appears 2010 N C State University Bioinformatics Research Center 4 Windows QTL Cartographer 2 5 Create New Source File Map Infomation 2 Step 3 of 6 Marker labels Marker positions AXR 1 HH 335C COL EC 480C EC 65C 24 76 6 93 2 97 0 115 5 116 5 HH 3423L EG 12L COL GH 53L COLC 49 6 50 6 60 5 91 0 92 0 97 0 PGK 12 NIAA C121 PRD 675 COL DD 2 93 3 106 1 108 3 125 1 142 0 Labels Positions Click radio button Labels to input and send marker label information and click radio button Positions to input and send marker positions information Other operation is similar to above step 2 4 Click Next button to accept the values The Create New Source File Step 4 of 6 appears 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 49 Create New Source File Cross Infomation 1 Step 4 of 6 m Specify raw data file s number All data in one file Arranged in individula order Data in three files Marker_genotypes Trait_values Other_Trait_values m Select data format of three raw files situation
4. 6 Select one or all chromosomes to include in the analysis 7 Select one or all traits to include in the analysis The Traits you select may change the value of the Threshold controls on the right side of the form 8 Set the threshold level via either manual input or permutations For more information see Setting threshold levels IM amp CIM f56 9 Click Start to begin QTL mapping analysis WinQTLCart will open a Save As dialog for you to save the result file that will be created 10 If you selected Set control markers manually in step 4b then WinQTLCart will display the Select CIM Control Markers dialog box Enter or edit the marker numbers you want to using the text box separate each number with a space Click on the marker row s cells to toggle their display in the text box When the analysis is complete WinQTLCart will e Create a QTL mapping result file QRT and open it in the Graph window e Create a QTL summary information file using the EQTL function e Display a confirmation box asking if you want to display QTL summary information in the Main window s Data pane Multiple Interval Mapping What it is Multiple interval mapping MIM uses multiple marker intervals simultaneously to fit multiple putative QTL directly in the model for mapping QTL The MIM model is based on Cockerham s model for interpreting genetic parameters and the method of maximum likelihood for estimating genetic parameters MIM is well suited to t
5. Dominant as well as co dominant markers can be encoded The middle column is how the output of these genotypes will be encoded The third column is how you will code the marker genotype data in this source data file Just about any set of tokens can be used for the third column corresponding to your dataset but DO NOT change the first two columns The above TranslationTable maps 2 to 2 1 to 1 0 to 0 etc Just about any set of tokens can be used for the third column but DO NOT change the first two columns If you encoded your P1 homozygotes as BB heterozygotes as Bb etc your translation table might appear as TranslationTable AA 2 BB Aa 1 Bb aa 0 bb A 12 B a 10 b 1 e Anything in the data file that is not recognized doesn t match something in column 3 will become unknown 1 in the output Important You need all 18 tokens following the TranslationTable token and the first two columns can t be altered You should only alter the last column Token start markers and stop markers Line 70 and Line 109 Use these tokens to start and end the marker genotype data of all chromosomes Please keep the same chromosome and marker order as the data between token start and token end above You can organize the data by marker or individual e Order by marker For each marker you provide the genotype data for all individuals The order of the 2010 N C State University Bioinformatics Research Center WinQTLCart P
6. MCD file successful dialog click OK button to the dialog and display the Main window with the MCD file you just created loaded into the Source Data form Where to go from here Select an analysis methodl 2 Creating simulation data To use WinQTLCart s results graphing abilities you need data for it to work on If you haven t collected any data though you can still simulate a dataset that WinQTLCart can work with To create a data simulation file select File gt Simulation This displays the Create Simulation Data series of dialogs 2010 N C State University Bioinformatics Research Center 52 Windows QTL Cartographer 2 5 Simulation Data Control Dialog Basic Information Ran Seed Replications 1301 126861 10 300 Map Function Haldane e Sample Size Step1 Translation Table AA 2 Aa 1 Trait Information Total Trait 2 f 36 85 Current Trait aa 0 A 12 a 10 1 Trait Mean Cross Information e Bi C B2 WE Chromosome and Marker Information The Total Chrom Numbers E m From File EES Gem Gem The Current Chrom Number f Average Space between Markers cM PZ Marker Numbers for Chr 1 12 Variations of the Marker Positons Cancel From OSI File Fill in the blanks for yourself or accept the defaults Random seed Click the button to generate a new random seed Replications Produce a file with many replications of simulation data wi
7. Save the data as the EQTL format that is used on Command line version of QTL Cartographer Print Graph Print the graph to a selected printer PR Closes the Graph window If you have unsaved data you ll be prompted to save it Graph window Menus Chrom Save As Excel File Save the data as an Excel file Use Excel s charting capabilities to draw 2010 N C State University Bioinformatics Research Center Windows QTL Cartographer 2 5 Next Chrom gt gt Display the next chromosome in the file Display the previous chromosome in the file the order of the chromosome display See Selecting chromosomes for graph display Show All Chroms Shows all chromosomes in the file in a single graph Graph window Menus Traits Select Chroms Choose the chromosomes you want to graph You can also change Jemen Jee SE Next Trait gt gt Display the next trait in the file Display the previous trait in the file W Select Trait s Choose the traits you want to graph See Selecting traits for graph display 20 Show All Traits Shows all traits in the file in a single graph Graph window Menus Effects Jemen ame A Show Additive Effect Display additive effect graph at bottom of window This is on by default for new Graph windows D Show Dominant Effect Display dominant effect graph at bottom of window if cross type is SFn or RFn a Show Values of R2 Display R2 value graph at bottom of window
8. Select Menu Item File gt Open or click OPEN to open source data file mouse mcd Select Menu Item Method gt Interval Mapping or click to open the Form of IM analysis Select By Permutation and click button OK to start threshold value calculation Click button Start to start IM analysis Click Y to show QTL summary in Text Window aaron Composite interval mapping 1 Select Menu Item File gt Open or click OPEM to open source data file wqcart samp0 mcd You can just click the tree item if the file has already been opened wes 2 Select Menu Item Method gt Composite Interval Mapping or click ON to open the Form of CIM 3 Click button Control to set the parameter Set Control marker number as 8 Window size as 15 0 Other trait as 1 2 and click button OK to back Select All traits in trait selection pull down menu 5 Click button Start to begin CIM analysis A Multiple trait analysis Select Menu Item File gt Open or click OPEN to open source data file wqcart samp0 mcd Select Menu Item Method gt Multiple Traits Analysis Select IM Method and input 1 3 in edit box for Multiple Traits Setting Dialog Click button Start to begin multiple traits analysis Pons Multiple Interval Mapping 1 Select Menu Item File gt Open or click OPEN to open source data file wqcart samp0 mcd 4 Select Menu Item Method gt Multiple Interval Mapping or click MM and select trait MECIAIA to enter the Form of MIM Click button New Mod
9. Y Coordinate o Font size 8 F Show trace hairs Color and Style for Trait 001 s00_M_GY X Color Penwidth P Threshold value for Trait 001 v Maximum LR value in graph 3 3 Set all traits with this value F Minimum LR value in graph Marker label font size z Number of scale lines for X axis j 1 Number of scale lines for Y LOD axis E Number of scale lines for Y effect axis 3 Space between two chromosomes 5 EE LE REER Show LOD profile as block graph view Check to show color block for LOD LR profile instead of line curve Use continuously or block radio button to set the total colors in color block display Ratio between effect window size and LOD window size Use the spin dials to affect the display ratio You can choose for example to make the LOD window the same size as the effect window by selecting 1 1 By default the LOD window is 3 times the effects window size Title Enter a title for the graph display Use the X and Y coordinate boxes and the Font size box to precisely place the title so it looks as you want Show QTL info Check to toggle display of QTL information See Showing QTL information 24 for more information No LOD window Check to suppress display of the LOD LR graph window upper window and only show the effect window No LOD line Check to suppress display of the LOD LR line curve in upper window 2010 N C State University Bioinformatics Research Cente
10. You may see the WinQTLCart Main window s status bar underneath the dialog begin filling in as the search progresses When the search is complete the model is presented in the cells 3 To manually edit the model e Click Add QTL or Del QTL e Click on a cell and enter a new value in the Cell editing box Click the Cell Update button to enter the new value 4 Click OK to accept the parameters close the dialog and return to the MIM form The model you created is now displayed in the form Where to go from here From here you can refine the MIM modell zt manually edit the model by clicking the Add QTL and Del QTL buttons or click in the model field to change the value of Position Chromosome Additive or epistatic values Click Save Model to save the model as a MDS file Related topics About the MIM form 63 Refining the MIM modell 70 2010 N C State University Bioinformatics Research Center ER Windows QTL Cartographer 2 5 CIM search option In the Create New MIM Model dialog If enabled click the Criterion button to display the Set Composite Interval Mapping Control Parameters dialog box 1 For the CIM Model field specify the markers to be used as cofactors in the CIM analysis Model 1 All Marker Control Use all the markers to control for the genetic background Model 2 Unlinked Marker Control Use all unlinked markers to control for the genetic background Model 6 Standard Model The default model that selec
11. partition of the variance explained by QTL main and interaction effects and estimates of genotypic value of individuals based on the model Parameters for current model QTLs Number of QTLs in model 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 65 Epistasis Number of epistatic genes in model L k Likelihood of the mode k is the QTL number BIC Bayesian Information Criteria BIC value of the mode QTL Effects Click to test additive dominant and epistatic effects The data pane under the form will show the test results You can select the text and then Edit gt Copy to copy it to the clipboard Refine Model Select an option and click OK to refine the modell70s parameters Add QTL Adds a QTL to the model Del QTL Select a QTL column and click Del QTL to delete that QTL from the model Cell Edit Cell Update Click on a cell in the model to select it and then update its value in this field Click the Cell Update button to write the value to the cell Close Close the MIM form and return to the Source Data form If you have not saved your work you can save your work at this time The Model Occupies the right half of the form Click the blue lt lt QTL cell to expand the model so it fills the form pane click the QTL gt gt cell to re display the MIM form Creating MIM initial model Click the New Model or Add Model button on the MIM form The following Create New MIM Model di
12. 2 5 e Create a QTL mapping result file QRT and open it in the Graph window e Create a QTL summary information file using the EQTL function e Display a confirmation box asking if you want to display QTL summary information in the Main window s Data pane Composite Interval Mapping What it is Composite interval mapping CIM adds background loci to simple interval mappingl 58 IM CIM fits parameters for a target QTL in one interval while simultaneously fitting partial regression coefficients for background markers to account for variance caused by non target QTL In theory CIM gives more power and precision than simple IM because the effects of other QTL are not present as residual variance Furthermore CIM can remove the bias that would normally be caused by QTL that are linked to the position being tested Background markers are usually 20 40cM apart High level workflow Here s a quick overview of how to use WinQTLCart s CIM implementation The first few times you run this analysis go with the WinQTLCart default values for the form s parameters The defaults provide the best all around parameter settings especially for initial analysis sessions 1 Select the CIM analysis method 2 Select the chromosome s and trait s you want to analyze 3 Select a threshold level to apply to the selected trait s Select either By manual input 57 the WinQTLCart default or By permutations 57 to have WinQTLCart determine an optim
13. E Show Values of TR2 Display TR2 value graph at bottom of window Show Values of S Display S value graph at bottom of window One Standard Deviation Normalize the additive and dominant effect in one standard deviation 2010 N C State University Bioinformatics Research Center WinQTLCart Windows amp Menus 15 Graph window Menus Tools Jesse Ire Display One Page Format Show the graph information in a smaller one page format for publication purposes See One page display window Menus 4 for more information Show QTLs information Display QTL information from a simulation parameter file or summary QTL peaks See Showing QTL information 24 Allows you to customize the graph display See Setting display parameters 21 Set Test Hypothesis Display result of different tests such as H1 H3 See Setting a test hypothesis Ja Show Graph in LR LOD Toggles between LR and LOD scale displays Look for LR or LOD at Scale the top of the y axis line Show Black and White Toggle between color and black and white display Use black and Graph white graphs for publication Show Colorful Background Activate a color or white background might be useful for printing or to provide better contrast for color graph lines Show Horizontal Grids Toggle display Show Vertical Grids Toggle display Show Trait Names or Toggle display If traits are present WinQTLCart defaults to showing legends on the right side of t
14. QTL in centiMorgans 5 Click OK WinQTLCart places a band over the chromosome graphic indicating the QTL location 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 81 Chr 3 0 0 2 EBmec0521 3 5 1 2 EBmsc0727 A chromosome graphic before adding QTL positions Chr 3 0 0 2 EBmac0521 2 5 1 2 EBmsac0737 and after adding QTL positions Tutorials Import data files Create source data file mcd by using different formats of data file s Import data INP format i Start WinQTLCart and select menu item File gt Import or click Import Select QTL cartographer INP format and click Next button Click button Map File to select file wqcart map inp Click button Cross Data to select file wqcart cro inp Enter file name wqcart_inp_In in edit box and click button Finish arFwns Using Emap function i 1 Start WinQTLCart and select menu item File gt Import or click Import 2 Select QTL cartographer INP format and click Next button 3 Check Infer map information from cross data file 4 Click button Cross Data to select file Csamp0 inp 5 Enter file name Csamp0_inp_In in edit box and click button Finish Import data OUT format be 1 Start WinQTLCart and select menu item File gt Import or click Import 2 Select QTL cartographer OUT format and click Next button 3 Click button Map File to select file wqcart map 4 Click button Cross Data to select file w
15. Select Effects gt Show QTL Information or click di on the toolbar to display the Show QTL Information dialog From here you can show QTLs from a simulation parameter file or show summary QTL information from the likelihood ratio graph peaks Select the Open QTL information file option and then the Browse button to select a file that has the QTL positions and effects settings you want to use for the display Select the Show one or two LOD interval options as desired to show empiric QTL confidence intervals 95 percent is one LOD and 99 percent is two LOD Select the Automatically locate QTLs option to specify parameters WinQTLCart will use to find QTLs in the results Use the spin dials to specify the minimum acceptable cM range that defines a QTL peak if the peak s distance is less than this value then the highest peak will be considered a QTL The minimum acceptable LOD scale as measured by the highest and lowest points of a QTL peak on the graph Both of these requirements must be met for a peak to be considered a QTL 2010 N C State University Bioinformatics Research Center WinQTLCart Windows amp Menus 25 Related topics Creating simulation datal 51 WinQTLCart Procedures Setting the working directory By default WinQTLCart looks for data files and other working files in its home directory typically C NCSU WinQTLCart or directory of last opened source data mcd file WinQTLCart saves all files it creates to the
16. Setting Graph window Procedures ss EE RRER EE ERER EE ERER EE RRER mennun nnmnnn nnmnnn Tracing coordinates Om the graphes nn nero rene detre ren eee ENEE 19 Selecting traits for graph dis play si ininrnrrrrearesrennennenennenrenesnnsnesnnsnnsneeneaneaneanrnrnnnne 20 Selecting chromosomes for graph display nn iiirrrnnnrnrrennesnesneeneeneanenneanrnnnennnne 21 Setting display parameters nn iiirrrnreeeneaneaneannncnncnnsnennenneeneennean ere snasnasnnennsneene ane aseisiin 21 GIE dt TEE 23 Showing QTL info rim ation sgp EES Eaa aa aeaa dae Aaaa aas 24 2010 N C State University Bioinformatics Research Center Contents I WinQTLCart Procedures 25 Setting the working directory cccceeeeeeceeeeeeeeeeeeeeeeeeeeeseeeeeeeseeeeeeeseeeeeeeseeeeeeeseeeaeenseeseneets 25 Importing and exporting WE 25 Importing AO 25 Exporting source data and results ccccsssssssssssessseesesseeseesesesseseveevsevsnesneseeneennasaesaesaesaeeseesoeseesaesaesaesanvanvanse 29 Exporting source data to QTL Cartographer A 30 Exporting source data to an MCD file iii 30 Exporting results fromthe Graph window iii 31 Working with source data files cceeeeseceeeeeeeeeeeeeeeeeeeeeseeeeeeeseeeeeeeseeeeeeseeeeeeeseeeeeeeseeeeneess 31 Opening SOUFCC ata TINGS eege ssh nn eaa aa aAa Aar Aa AA een 32 Working with a source file s Marker genotype data csecssecssecseeesseeseeeseesseesenessesseneseeesensseeeseesseeseeesenesees 32 Wor
17. State University Bioinformatics Research Center Windows QTL Cartographer 2 5 Index analysis methods Bayesian interval mapping 76 composite interval mapping 60 interval mapping 58 multiple interval mapping 63 multiple trait 78 single marker 55 58 bayesian 85 Bayesian interval mapping 76 77 R Project for Statistical Computing 76 BIM 85 chromosome graphics display 78 adding QTL positions 80 CM 84 composite interval mapping 60 78 84 setting threshold levels 57 threshold levels 56 current working directory 29 30 32 datapane 9 copying data from 12 source files color codedin 12 Emap 25 Excel 82 exporting 29 results 31 source data 30 file formats import and export 1 form pane 9 31 disabled commands 12 leaving analysis modes 12 Graph window display parameters 21 exporting results 29 31 menus 12 one page display of graphics 16 selecting chromosomes for display 21 selecting traits for display 20 showing QTL Information 24 test hypothesis 23 tracing coordinates 19 IM 84 importing 25 INP 81 interval mapping 58 78 84 setting threshold levels 57 threshold levels 56 likelihood ratio 24 LOD 12 19 21 24 LR 12 21 Main window components of 9 datapane 12 form pane 12 status bar 9 tree pane 9 10 MapMaker 82 MapMaker QTL 1 importing 25 Microsoft Excel 1 expected worksheet names for importing 25 exporting results to 29 31 MM 84 mulitple trait 84 multiple interval mapping 63 84 f
18. active source data and click Delete radio button to delete current chromosome or marker s in current chromosome from active source data Select chromosome Select one chromosome from the pull down menu as the current chromosome 1 Insert one marker in current chromosome WinQTLCart can read the marker information through a properly formatted text file and click Filename button to indicate the filename To check From Clipboard button if the marker information is already in Windows clipboard 2 Insert one chromosome WinQTLCart can read the chromosome information through a properly formatted text file and click Filename button to indicate the filename To check From Clipboard button if the chromosome information is already in Windows clipboard 2010 N C State University Bioinformatics Research Center ER Windows QTL Cartographer 2 5 3 Delete current chromosome Click Delete current chromosome radio button and click OK button 4 Delete marker s in current chromosome Click Delete markers radio button and in the Marker Number text edit box enter the marker s you want to delete separated by commas or hyphen Working with source file s trait information At the Source data manipulations pane on the Main window click the Trait button to open the Add or Delete Traits dialog Add or Delete Traits Append Delete Enter trait numbers or number ranges that WinQtiCart should process Enter the numbers separated by commas and the
19. phenotyping on a cross B1 derived from that intercross Enter an integer indicating the generation T B1 RF Test cross with genotyping done on an intercross RF and phenotyping on a cross B1 derived from that intercross Enter an integer indicating the generation T B2 SF Test cross with genotyping done on an intercross SF and phenotyping on a cross B2 derived from that intercross Enter an integer indicating the generation T B2 RF Test cross with genotyping done on an intercross RF and phenotyping on a cross B2 derived from that intercross Enter an integer indicating the generation Map Function A map function is a mathematical relationship between recombination probabilities and map distances Select the function you want based on the interference to be assumed e Haldane The default option Assumes no crossover interference e Kosambi Assumes some interference e Fixed The Morgan mapping function Assumes complete interference Note These mapping functions among others are discussed at length in Ben Lui s book Statistical Genomics Linkage Mapping and QTL Analysis 1998 Distance type e Position Indicates that the numbers indicate positions from the left telomere of the current chromosome This means the numbers should be in increasing order e Interval Indicates that the numbers are for the interval distance after a marker This means that the last number and the last number only should be zero Distance u
20. please try put your MCD file in a simple folder such NCSU Work1 and only letter and digital are in file name 2010 N C State University Bioinformatics Research Center s Windows QTL Cartographer 2 5 2 Why my trait values are truncated into integers Correct Windows setting by Control Panel Regional and Language Regional Options Make sure that the Number format is 123 456 789 00 3 WinQTLCart cannot import Map information from selected file The Map file you ve selected doesn t have a format that WinQTLCart recognizes Open your file in Notepad and compare its format to one of the sample MAP or CRO files in the WinQTLCar directory Problems also occur when a map file is selected as the cross file or a cross file is selected as the map file Related topics Compatible programs and formats 1 4 Invalid file or wrong format messages You may see WinQTLCart error messages complaining that a file you re opening or importing is invalid is in the wrong format cannot be recognized and so on The file s data may not be in a format WinQTLCart recognizes or the file may have been formatted incorrectly or its extension might have changed Open up the file in Notepad and compare it to a sample file of the same extension in the WinQTLCart directory Related topics Compatible programs and formats 1 5 Failures when I try to creat MCD file from text files The easiest way to create a MCD file is by using method of Import C
21. ranges separated by hyphens Trait For example 2 5 8 10 12 are 2 5 8 10 11 and 12 Number 3 oO Cancel a i e A Add and Append Click Append radio button to append trait s into active source data and click Delete radio button to delete trait s from active source data 1 Append trait s WinQTLCart can read the trait information through a properly formatted text file and click Filename button to indicate the filename To check From Clipboard button if the trait information is already in Windows clipboard Please check the Data include trait label s button if the trait data include trait label Click OK button to append the trait s after set the correct Total trait number 2 Delete trait s In the Trait Number text edit box enter the Trait s you want to delete separated by commas or hyphen 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 39 Working with source file s other trait information At the Source data manipulations pane on the Main window click the OTrait button to open the Add or Delete Other Traits dialog Add or Delete Other Traits Filename null tt D From Clipboard e Append Total number i H F Data include otrait label s f Enter other trait numbers or number ranges that WinQtlCart should process Enter the numbers separated by commas and the ranges separated by OTrait hyphens For example 2 5 8 10 12 are 2 5 8 10 11 and 12
22. rat Trait2 Joint Traits Click anywhere in a column to toggle display of the trait in the Graph window In the screen shot above the Trait 1 Deletion cell is cleared meaning this trait will be displayed An asterisk in the Deletion cell a trait means it will not be displayed e Click Select All to show all the traits e Click Select First to show only the first trait e Click Help to display help text for this dialog Note You cannot change the display order of traits However you can change the display order of chromosomes 2010 N C State University Bioinformatics Research Center WinQTLCart Windows amp Menus 21 Selecting chromosomes for graph display Select Chrom gt Select Chroms or click c when you want to focus the graph on only a few chromosomes rather than all of the chromosomes in the data Selecting the command displays the Select Chromosomes dialog Use this dialog both to select chromosomes for display and to juggle their display order Chromosomes Selection Dialog The lower text field shows the chromosomes and the order in which they will be displayed To delete a chromosome from the graph display click the cell in the Delete row below that chromosome An asterisk in a field means the chromosome will not be displayed a clear field means the chromosome will be displayed Click the Deletion cell to toggle display on and off In the screen shot above the Deletion cells for 1 2 3 a
23. the bottom and the detailed result is in the result file You can compare the likelihood values between two QTL models likelihood test to do certain decision such as the new QTL is significant or not The two QTL is the same or not etc 2 Test all QTL effects To test the value is zero or not for each of the QTL effects additive dominant and epistasis 3 Optimize QTL positions To go through whole genome you can set the walk speed such 1 0 cM find best position for each QTL 4 Search main QTL s If you do not know the new QTL s chromosome and position then to assign value 1 to the chromosome string and WinQTLCart will search the best chromosome number and position for you automatically 5 Search epistasis interaction s If you want to find the best QTL interaction then assign value 1 to QTL 1 and QTL 2 and WinQTLCart will find out the best QTL number 1 and QTL number 2 for you automatically 6 Graphic and summary result To obtain the graph result file and text summary file 7 Test Pleiotropy in current population 2010 N C State University Bioinformatics Research Center Windows QTL Cartographer 2 5 To do the pleiotropy vs close linkage test according to above parameter setting 8 Genome wide test of QTL effects To do the QTL effect s equality test according to above parameter setting 9 Trait pairwise co relation in current population To calculate QTL co relation between pairs of traits according to a c
24. the form after the analysis completes 1 From the Trait Selection pull down do one of the following e Select one trait to find the threshold value for that trait e Select All Traits to find threshold values for all traits 2 Click the By Permutations option 3 Enter a value for Permutation Times to set the permutation repeating times Note If you want a quick run use the default value of 300 But if you re going to publish use the 1000 value this wil take several hours to run but will yield very precise results We recommend starting this analysis before you leave for the day and letting it run overnight e Enter a value for Significance Level 5 Click OK to begin threshold analysis 2010 N C State University Bioinformatics Research Center s Windows QTL Cartographer 2 5 Note You cannot update the All Traits checkbox WinQTLCart checks or clears this box based on whether you select one or all traits from the drop down list After you click OK WinQTLCart displays a countdown showing its progress through the file When the threshold analysis is complete WinQTLCart will display the results in the Data pane and display the results file s name WinQTLCart saves the file to the current working directory 25 After setting the threshold value move on to finish the IMI5 or CMleg analysis procedure Interval Mapping What itis Interval mapping IM is an extension of single marker analysis 55 In single marker
25. 0 N C State University Bioinformatics Research Center WinQTLCart Procedures 71 Refine MIM Model Fuction selection Optimizing QTL positions Test both main and epistatic effects Searching for new QTLs C Testing for existing QTLs Criteria of MIM model selection Score 0 05 significant level e MIM Walk Speed in cM 1 Window Size 10 et At the refine MIM model dialog select a model selection criteria from the drop down list Choose the first 6 options WinQTLCart will do search test or optimizing in the principle of LR test and use BIC as criteria By select the last 4 options WinQTLCart will use score statistics test and certain significant level as search test and optimizing criteria 1 Optimizing QTL positions Move main QTLs one by one along the chromosome to maximize LR or Score statistics choose the first 6 options is LR and otherwise is score statistics Check box Test both main and epistatic effects is only worked in score statistics testing By check this check box both main QTL and its interaction with other QTL s are considered in score statistics calculation 2 Searching for new QTLs Main QTLs Search for new main QTL s using LR or Score statistics test QTL interactions Interaction between Identified QTLs Try to find more interaction among existing main QTLs 1D Scan of 1 new QTL and Interactions Search one new main QTL plus interaction between the new QTL and QTL in the model b
26. 1 211211112222222222222222 39 8114 22 4848 7 0315 222122222221122222222222 37 9753 23 1155 11 2001 ATA Bete 22 222 2 22 201 395619 21093435133 111122222222222211111112 35 9214 24 4051 6 5268 11121111111112112111122 35 7827 22 6201 6 6770 fie Re Re Be ie ee Be ee ee ee ee Be ee el le aa Raw data filename s and action s Marker genotype filename 521 mkgenindiab bd Browse Send Data Trait value filename 5224tindeb bt Browse Send Data Other trait value filename 523 otindiab b Browse een _ ww Please notice that the buttons in this dialog is depend on the selections of previous dialog For marker genotype information use Browse button to indicate the filename and Send Data button to update the Data Window It is same for the trait value also The Browse and Send Data button will be disabled if there is no other trait Otherwise you can do the same as trait value Only one set of Browse and Send data button will be enabled if you choose one data file format in the previous dialog Note The Data file s format should be comparable with the setting of previous dialog For example if there is no individual label in the genotype data file and you check the include individual labels button in previous dialog Then a warning message box will pop out when you click the Send Data button in Marker genotype filename group WinQTLCart 4 Number of data in file is not enough 1901 it should be 1952 6 Cl
27. 19 Related topics Graph Window Menus 1 Tracing coordinates on the graphlis Selecting traits for graph display 20 Selecting chromosomes for graph display 21 Setting display parameters 21 Setting a test hypothesis 23 Showing QTL information 2 Tracing coordinates on the graph Select Settings gt Trace Coordinate in Graphic or click E As you mowe the cursor around the screen note that the graph coordinates are displayed in the graph s upper right corner 1 104 8 0 70 Double click a point on the graph WinQTLCart marks that point with a dot and displays the coordinates Take the following coordinates as an example 1 46 7 3 0 1 the chromosome number 2010 N C State University Bioinformatics Research Center Windows QTL Cartographer 2 5 46 7 the cM location along the chromosome 3 0 the LOD score After marking one or more points copy the graphic to the clipboard File gt Copy to Clipboard for use in another application or for publication Re select the command to toggle the coordinate display WinQTLCart also clears from the display the coordinate points you selected Related topics Setting display parameters 21 Selecting traits for graph display Select Traits gt Select Trait s or click Y when you want to focus the graph on only a few traits rather than all of the traits in the data Selecting the command displays the Select Traits dialog ons j Traits Selection Dialog
28. 64 1 30 4 1 8 1 1 1 6028 1 6934 0 8715 0 3281 0 1610 1 4650 0 3580 0 1410 0 2736 Chromosome Position and Effects Window T 44 F ap T DA PF DD Pos cM Chromosome Additive Dominant Update Save Load First Trait Adjustment After inputting the parameters in the previous two dialogs the final dialog presents the data simulation model you have built Click in any cell to see that cell s values represented in the fields below Next Trait or First Trait Click this button to reach each trait Use the fields to edit a cell s contents Click the Update button to write the change to the model e Click Save to save your model to a QSI QTL Simulation Information file The current working 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 55 directory is selected as the default Click the Xin the dialog s upper right corner to close without saving e Click Load to load a QSI file to this window The file s values replace the values you see in the current window e Click Adjustment to adjust values for the entire genome Click OK to produce the simulation data file You can then use that file for analysis Related topics Showing QTL information 24 Single marker analysis When to use For quick scanning of the entire genome all chromosomes to find best possible QTLs and identify missing or incorrectly formatted data Use single marker analysis first to ensu
29. Center WinQTLCart Windows amp Menus 17 Row Number lt lt Decrease graph number in a row by 1 Column Number gt gt Increase graph number in a column by1 Column Number lt lt Decrease graph number in a column by 1 One Page window Menus Setting Command Function HSpace Between gt gt Increase horizontal space between chromosomes HSpace Between lt lt Decrease horizontal space between chromosomes Graph window Procedures To see the Graph window open a WinQTLCart results file with a QRT extension by click the Result button on the Main window s toolbar The Graph window opens presenting a graphical overview of the results file data From this window you can e Spot the location of QTLs A graph peak that extends past the threshold line is the site of a QTL e Load multiple results files at one time and compare them This could be useful when comparing the results of the same dataset pushed through different analysis methods and parameters 2010 N C State University Bioinformatics Research Center Windows QTL Cartographer 2 5 Ph1 0 Graph for QTL Mapping Result C NCSU WinQTLCart2 5 Examples Ph1 0 qrt DER Fie Chrom Traits Effects Tools Setting Ta TV A I S 16 di PE Toolbar 1 sh1 2 sh2 3 sh3 Graph Showing of LOD Score Profile Graph Showing of Effects From top to bottom here s what you see 1 2 3 Title bar Shows the name of the selected results data file You can ha
30. Composite Interval Mappingl ef use threshold values to determine QTLs If you don t know the threshold value for a trait you can have WinQTLCart find them out and use them as inputs for the QTL search The threshold levels control the rate of Type 1 errors false positives A lower threshold value means more false positives but a higher value means you may miss more QTLs When you look at a result graphl17 the horizontal line you see running across the graph is the threshold level When the graph peaks over the threshold level that is good evidence for a QTL So setting the threshold level in this form controls how high or low that line will sit on the graph Too high and you ll see no QTLs too low and you ll see too many The threshold value can be changed later in the Graph window Getting a very accurate threshold level may take a long time Recommendations are included in the threshold procedures 1 Follow the instructions for the form you re working with either IM or CIM Be sure to select a trait or all traits for which you want to set a threshold level 2 The Threshold group box on the right of the form controls inputting or obtaining mapping threshold 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 57 values for each trait Select a different trait number to see the threshold value of that trait 3 Select whether you want to set the threshold levels manually or have WinQTLCart d
31. Increasing the number of control markers will allow better resolution for mapping linked QTLs 4e Regression method selection Select a method e 1 Forward Regression e 2 Backward Regression e 3 Forward amp Backward 4f Probability for into Probability for out 4g Ifthe OTrait number field is enabled enter other trait numbers and their ranges to be included in the model Note OTraits is another term for categorical traits Use QTraits for background control as nuisance factors we want to account for 4h Click OK to close the dialog and return to the CIM analysis form 5 The Walk speed cM default is 2 The walk speed is the genome scan interval Click the spin dial beside the Walk speed value to increase or decrease the walk speed by 5 increments e Increasing the walk speed greater than 2 means less precision but the analysis takes less time e Decreasing the walk speed less than 2 yields a more precise result but will take more time You should set the walk speed value once for the entire dataset A single walk speed establishes a consistent norm against which the data can be graphed If you change the walk speed between runs the graph displays will be skewed If you want to check your data against a different walk speed create a separate directory for your data files and then run the new walk speed against those files 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures e
32. Marker genotypes Trait values Other trait values Te Arangedin individual order Aranged in individual order Arranged in individual order Je Include individual labels Iw Include individual labels M Include individual labels Aranged in marker order Aranged in trait order Arranged in other trait order bk Inelud bh Inclui IV includ ee First to specify raw data file s number by click one of the radio button 1 All data in one file Use one file that includes marker genotype trait and other trait information Select Data format 1 in Select data format of one raw file situation group box if the data include individual label Otherwise select Data format 2 All data must arrange according to individual order 2 Data in three files Use three files for marker genotype data trait data and other trait data For each file you can choose arrange according to individual or according to marker trait or other trait in Select data format of three raw files situation group box To check corresponding box if there are labels in file 5 Click Next button to accept the values The Create New Source File Step 5 of 6 appears 2010 N C State University Bioinformatics Research Center so Windows QTL Cartographer 2 5 Create New Source File Cross Infomation 2 Step 5 of 6 Marker genotypes BC231_1 BC231_2 MECIA Celia Mecinia 122222222221122222222222 40 1609 25 078911 1917 112122222222222222222222 42 1206 24 6962 14 235
33. Number Cancel Add and Append Click Append radio button to append other trait s into active source data and click Delete radio button to delete other trait s from active source data 1 Append other trait s WinQTLCart can read the other trait information through a properly formatted text file and click Filename button to indicate the filename To check From Clipboard button if the other trait information is already in Windows clipboard Please check the Data include otrait label s button if the other trait data include label Click OK button to append the other trait s after set the correct Total other trait number 2 Delete other trait s In the OTrait Number text edit box enter the Other Trait s you want to delete separated by commas or hyphen MCD file format WinQTLCart MCD source data files use tokens to indicate the meaning of data This topic describes valid tokens used in MCD source data files The line numbers in this topic refer to the sample MCD file NewMcd mcd which is included as part of the WinQTLCart distribution Token FilelD line 1 2010 N C State University Bioinformatics Research Center 40 Windows QTL Cartographer 2 5 File s ID number usually a 10 digit number Token and Lines 2 5 Insert multiple line comments between these tokens Token bychromosome Line 6 Indicates start of chromosome information Token Lines 6 10 Insert one line comments afte
34. Population Group Total population refers to the total population number in the source data Selected number indicates how many populations will be in the QTL mapping model To use Index edit box to indicate the index of selected populations comparing to all populations in original source data Parameter setting for current population Group Click the spin dial beside the Current population value to set current population number first Then to fill other parameters in the rest of group Note The current population number depends on selected population number only For example if the selected population number is 3 then the current population will be 1 2 and 3 despite where these population is located in source data file For each current population Total traits in current population The number refers to the total traits or envronments in the source data Selected traits in current population The number indicates that how many traits will be in the QTL mapping model index The number string indicates the index of selected traits Selected QTLs in current population Click the spin dial to set the QTL number for current population Chromosome String Chromosome number for QTL1 QTL2 Position cM String Positions for QTL1 QTL42 Selected Epistasis in current population Click the spin dial to set the QTL interaction terms for current population QTL 1 QTL number list They are the first QTL number between two QTL interaction
35. QTL 2 QTL number list They are the second QTL number between two QTL interaction Pleiotropy vs Close Linkage Test Group The purpose of the test is to see that two or more QTL is the same or not statistically The QTL number list is in QTL Number String edit box Please notice that you can do the test for one or more population all together Genome wide test of QTL effects The purpose of the test is to see that two group of QTL effects QTL main effect or epistatic effect are the same or not statistically Effect number is the QTL effect numbers in a effect group Effect1 and Effect2 indicate group 1 effects and group 2 effect respectively Each QTL effect includes three number that are population number trait number and QTL effect number Trait Pair wise Test Click Trait Pair wise Test button to open the control file and click button to open the control file with Windows notepad Refine Model Group Click the pull down menu to select a function please to see Mt MIM Functions 75 and click Go button to start calculation Modifying Step Edit Box to indicate the walk step in cM while searching through chromosomes Update Click the button after changing parameters setting Finish Click the button to finish Mt MIM analysis and the software will remind you to save the parameters setting into a new control file 2010 N C State University Bioinformatics Research Center Windows QTL Cartographer 2 5 Mt MIM Control File The foll
36. SV format You can use the sample file NSimuB1 01 OnePop csv as a model to create your own CSV data file Technical Support Ensure you have the latest release 1 Select Help gt About WinQTLCart and look at the software release date Click on the Update Site link to go to http statgen ncsu edu aticart WQTLCart htm the WinQTLCart release page 2 If the date on the page is more recent then scroll down to the Downloads section and get the latest copy 3 Upgrading to a new version of WinQTLCart does not overwrite your working files However the upgrade will replace the sample files that are part of the WinQTLCart distribution If you have questions Send an email to WinQTLCart Tech Support at lt mailto shchwang statgen ncsu edu subject WatlcartUser gt Please include the following details in your mail e WinQTLCart version you re using e Describe the task you re trying to accomplish what you expected to see and what WinQTLCart actually did e Please describe the error message you received if any 2010 N C State University Bioinformatics Research Center Technical notes 87 Credits amp acknowledgements Windows QTL Cartographer was created by Shengchu Wang C J Basten and Z B Zeng Thanks to P Gaffney Sara Via Ann Stapleton Heike Kross and Janice Cuthbert This file was originally written by Michael E Brown and was produced using EC Software s Help amp Manual Site http www helpandmanual com 2010 N C
37. Sum IM 3 Messages r Summary information gt r Source data view and modify fe Analysis S EE Ge Population 1 E Marker values b MCI D File name envl jun3 mcd Chromosome 1 Chr 1 Markers L Er File ID number 1148497108 l H nul H r Trait values Go ait Viet Single Marker Analysis Result Files Cot O art Gg BI Trait View Dr a Sample size 100 Seren a Dt Chromosome numbers 3 LA Z Mouse qrt Trait numbers 3 5 Kl Files EE 0 Basic Info Individual Chromosome Trait OTrait NewModel txt 5 Form Pane Source data manipulations FileID 1148497108 bychrormosore type interval function 1 units cH seer named y 6 Data Pane nared yes start Chromosome Chr 1 cimi clm2 cin3 clm4 clins cin cin cing cing cimi0 cimii cini2 cini3 cimi4 EI gt For Help press F1 7 Status bar Men Feb 19 2007 7 57 PM From top to bottom here s what you see 1 Title bar Shows the name of the selected source data file 2 Menu barf AN 3 Toolbar with one click access to the program s major functions Hover the pointer over a button to see a brief description of that command The button s function is also described in the Status bar at the bottom of the window 4 Tree pane 10Yor file management Lists open files and organizes files under various category names Source Files Text Files Results Fil
38. Traits Note OTraits is another term for categorical traits Use OTraits for background control as nuisance factors we want to account for 5 The Walk speed cM is the genome scan interval and the default is 2 Click the spin dial beside the Walk speed value to increase or decrease the walk speed by 5 increments e Increasing the walk speed greater than 2 means less precision but the analysis takes less time e Decreasing the walk speed less than 2 yields a more precise result but will take more time You should set the walk speed value once for the entire dataset A single walk speed establishes a consistent norm against which the data can be graphed If you change the walk speed between mapping runs the graph displays will be skewed If you want to check your data against a different walk speed create a separate directory for your data files and then run the new walk speed against those files 6 Select one or all chromosomes to include in the analysis 7 Select one or all traits to include in the analysis The Traits you select may change the value of the Threshold controls on the right side of the form 8 Set the threshold level via either manual input or permutations For more information see Setting threshold levels IM amp CIM f56 9 Click Start to begin QTL mapping analysis Following the analysis WinQTLCart will 2010 N C State University Bioinformatics Research Center ER Windows QTL Cartographer
39. Ve By Permutations 7 Click lt Control gt to Change Parameters Setting Chromosome Selection Ja Chromosomes X Or Click lt Start gt to Begin Mapping Analysis Trait Selection Tran Ks Cancel Start Aal 3 Click Result File to select the QRT file you want to create 4 Click the Control button to display the Set CIM Control Parameters dialog Set Composite Inteval Ma Control Parameters _ ol x CIM Model Model 6 Standard Model Set control markers manually Background Controls Control marker numbers F Window size cM 10 0 Regression method D Forward Regression Method Probability for into Probability for out l Enter other trait numbers separated by commas or number ranges separated by hyphens Example 1 2 5 6 6 are 1 2 5 6 7 8 No other trait will be selected if the field is blank 4a For the CIM Model field specify the markers to be used as cofactors in the CIM analysis e Model 1 All Marker Control Use all the markers to control for the genetic background e Model 2 Unlinked Marker Control Use all unlinked markers to control for the genetic background e Model 6 Standard Model The default model that selects certain markers as control markers by using additional parameters control marker number and window size Selecting this model requires you to fill in extra fields on the dialog Control marker numbers Window size cM and Regression method selection
40. View Proportion of Marker Number Show length of chromosome graph in proportion of marker number Proportion of Chromosome Show length of chromosome graph in proportion of chromosome length Len in cM Next Page gt gt Show next page of the graph if there are multiple pages First Page Show First page of the graph Add QTL Positions Display QTL positions in the graph 2010 N C State University Bioinformatics Research Center CS Windows QTL Cartographer 2 5 Chromosome graph Menus Setting Select Chromosomes Select chromosomes to be showed in graph Show Chromosome Name Toggle display between chromosome names or chromosome labels produced by WinQTLCart Chromosome Name lt lt Decrease font size of chromosome names Column Number gt gt Increase the number of chromosome displayed horizontally Column Number lt lt Decrease the number of chromosome displayed horizontally Chromosome Name gt gt Increase font size of chromosome names 2010 N C State University Bioinformatics Research Center WinQTLCart Windows amp Menus 9 Main window tour When you start up Windows QTL cartographer the program s Main window displays WinQTLCart C NCSU WinQTLCart2 5 Examples env1 jun3 Source Data mcd Title Bar Sac File Edit View Method Tools Help 2 Menu D se amp SIM Slt we mw F EE OQ 2 R 3Toolbar CIM MIM DrawChr Graph DR Note About Help New Open Import SData SaveAs Print D
41. Windows QTL Cartographer 2 5 User Manual 2010 N C State University Bioinformatics Research Center Windows QTL Cartographer 2 5 Table of Contents About Windows QTL Cartographer 1 WINQTL Cart features ENEE EEN ege ENEE etecadeccuceuads EE NEEN 1 Compatible programs and formats 0 ccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeseeeeeeeeesseeeeeeseeeeeeesseneeeee 1 System ME UE LCE 2 Installing uninstalling upgrading ss sssssrennnernennnneenennnnes 2 Using WinQTL a high level overview 2 When to use WinQTLCart ii ee ee KEE ee eeee ee eeeeeeeeeeeeaeeeseeeeeseeeseeeaeseseeneeeseeeeeeesees 4 WinQTLCart Windows amp Menus 4 Main window Menus ccceeececceeeeeeeeeeeeeeeeeeeeseeeeeeeseeeeeeeseeeeeeeseeeeeeeseeeeeeeseeeaeeeseeeeeesseeeaneess 4 Main window Menus File eecht 4 Main window Men s UE 5 Main window Menus View we 5 Main window Menus Method An D Main window Menus Tools we 6 Main window Menus Help ccccccesssesseseeseesenssnsseeseesseseessesseeseeseeseesaesaesaesaesaevaevaeesneseeseesnesaesaesaesoeeseesnesneeanoas 7 Chromosome graph display Menus ss KEEN KEEN ERRR KEEN KEEN 7 Chromosome graph Menus File scssccssesssesseesesesseesereneeesensneessnsenneseneesnessnesseeseeeseneseesseeeseesseeseessneeseesene 7 Chromosome graph Menus View cesscsseceesseessesseee
42. al data include individual label Click OK button to append the individual s after set the correct Total individual number There are two ways to read data If your data is Then click In a text file Filename to displaythe In the Open dialog select the Open dialog box text file that has the values you want to add and click OK On the Windows clipboard Data from clipboard check You should select and copy itto transfer the data to the the data in other text editor edit window such as Windows notepad 2 Delete individual s In the Individual Number text edit box enter the individual s you want to delete separated by commas or hyphen Click OK button to finish 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 37 Working with source file s chromosome information At the Source data manipulations pane on the Main window click the Chromosome button to open the Chromosome and Markers Manipulation dialog Chromosome and Markers Manipulation Select chromosome Chromosome 1 Chr 1 ct C Add Ce Cc e Delete Delete markers Delete current chromosome Enter marker numbers or number ranges that Wirt at should process Enter the numbers separated by commas and the ranges separated by hyphens For Marker example 2 5 8 10 12 are 2 5 8 10 11 and 12 Number 20 1 Cancel Add and Delete Click Add radio button to add a chromosome or a marker into
43. all explained below 4b Click Set control markers manually if you do not want WinQTLCart to automatically select the control markers This will display a dialog box after you start the analysis so that you can 2010 N C State University Bioinformatics Research Center e Windows QTL Cartographer 2 5 manually select the control markers Skip to step 10 for a description of this dialog box The Background Controls group box specifies the number of background controls and regression type WinQTLCart should use in applying the selected CIM model 4c Control marker numbers Enter the number of markers to control for the genetic background WinQTLCart will use up to the number of markers entered here 4d Window size cM Enter the window size in centiMorgans The window size will block out a region of the genome on either side of the markers flanking the test site Since these flanking regions are tightly linked to the testing site if we were to use them as background markers we would then be eliminating the signal from the test site itself If the control marker number is And if the window size is This is the result The total number of markers loos Model 6 reduces to Model 1 The total number of markers Large such as the size of the Model 2 largest chromosome Zero N a Model 3 Recommendations e Model 6 is good for starting an analysis Start with the default values of 5 for control markers and 10 for window size e
44. alog appears At the Create New MIM Model dialog select an enabled option from the Initial MIM model selection method group box Create New MIM Model Initial MIM model selection method Commands C Regression forward selection on markers C Regression backward selection on markers C Forward and backward selection on markers C Scan through QTL mapping result file e MIM forward search method Parameters of the initial MIM model Main QTLs only QTL Position cM Chromosome Add OTL Del OTL Click OK to start model creation Cancel 2010 N C State University Bioinformatics Research Center s Windows QTL Cartographer 2 5 Regression options 66 e Regression forward selection on markers Enables the Criterion button e Regression backward selection on markers Enables the Criterion button e Forward and backward selection on markers Enables the Criterion button CIM search option s e Scan through composite interval mapping Enables the Control and From File buttons From File displays only after you select this option MIM search option ec e MIM forward search method Enables the OK button After finishing the initial model creation the MIM form redisplays with the buttons enabled the Parameters group fields populated the new model available in the drop down list and the model values on the right The Parameters fields are now populated Note To see the entire m
45. an 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 41 e r Recombination frequency If you choose this parameter then token function should be 3 Morgan Token chromosome Line 10 Indicates total number of chromosomes for source data Token maximum Line 11 Indicates the maximum number of markers for a chromosome for all of the source data Token named Line 12 Either yes or no e Yes means markers have names e No means markers will not have names Token start and end Line 13 and Line 55 Use these tokens to start and end the marker position data of all chromosomes Token Chromosome Line 14 28 and 41 Indicates chromosome name The marker position data for this chromosome will start with the next line Token bycross Line 56 Indicates that cross information is to begin Token SampleSize Line 57 Indicates sample size or individual number Token Cross Line 58 Indicates codes for crosstype mating design see the following table Code Design Examples Bi Backcross to Pi B1 B2 Bij Backcross j times to Pi B13 B25 SFi Selfed generation i intercross SF2 SF6 RFi Randomly mated generation i intercross RF2 RF3 RIO Doubled haploid RIO HI Recombinant inbred via selfing RH RI2 Recombinant inbred via sib mating RI2 T Bi SFj Testcross of SFi to Pj T B1 SF3 T SFi j SFi Testcross of SFi for j generations T SF4 SF3 T Bj RFi Testcross of RFi to Pj T B1 RF3 T D3 SFi D
46. analysis only one marker is used in QTL mapping but effects are underestimated and the QTL position cannot be determined Interval mapping provides a systematic way to scan the whole genome for evidence of QTL IM uses two observable flanking markers to construct an interval within which to search for QTL A map function either Haldane or Kosambi is used to translate from recombination frequency to distance or vice visa Then aLOD score is calculated at each increment walking step in the interval Finally the LOD score profile is calculated for the whole genome When a peak has exceeded the threshold value we declare that a QTL have been found at that location When to use it IM is a good general standard to use for all datasets Use it in combination with or as part of a process including You may wish to start with a single marker analysis 5 and then run IM to further refine the analysis High level process Here s a quick overview of how to use WinQTLCart s IM implementation The first few times you run this analysis go with the WinQTLCart default values for the form s parameters The defaults provide the best all around parameter settings especially for initial analysis sessions 1 Select the IM analysis method 2 Select the chromosome s and trait s you want to analyze 3 Select a threshold level to apply to the selected trait s Select either By manual input 57 the WinQTLCart default or By permutations 57 to have WinQTLC
47. andomize each trait independently Otherwise to keep the co relationship among traits when doing randomization Refer to the IM 58 and CIM 60 procedures in this manual for more information on using the form Drawing a chromosome tree You may need to use chromosome graphics when it s time to write an article on your research WinQTLCart displays all the source data file s chromosomes including each chromosome s markers and intervals in a single display window that you can copy to the Windows clipboard From the Main window with a source data file loaded select Tools gt DrawChrom to show the Chromosome Graphic Display window 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 79 Chr 1 0 0 1 Bmag0206 0 0 3 2 1 HVMO4 0 5 36 5 56 5 75 5 Chr 5 82 5 0 0 2 Bmag0140 93 5 2 8 2 GMS002 122 3 2 1 2 EBma00558 1 3 1 2 Bmsc0082 4 7 2 EBmsc0640 5 2 2 EBms00521 1 6 2 2 EBmsc0521 2 0 0 8 7 2 EBmec5214 1 5 37 2 2 EBmsc 415 1 2 0 38 7 2 HVM54 115 15 9 5e 8 Chr 9 61 2 0 0 4 5 Bmag0579 71 5 EBms00782 88 Chr 3 Chr4 1 Bmsc2731 0 0 2 EBms00521 3 0 0 2 Bmag0125 14 Bmag0110 1 51 2 EBmac727 32 1 2 EBmact415 1 Bmao0273 4 12 Bmag0010 2 1 Bmag0221d 6 Bms00218 2 Chr 7 Chr 8 1 Bmag0120 4 HVMO3 5 Bmac 213 d o o in w 1 Bmao 15 6d 4 Bmeg0253 16 2 5 Bmag0872 129 4 Bmsac210 228 5 Bmsg0718 40 9 4 EBmso0679 27 1 5 Bmsg0347 65 5 34 Bmsg0128 2 29 8 5 Bmsg0345 Ch
48. art determine an optimum threshold See Setting the threshold ewell sei for more information on the impact of each of these choices 4 Click OK to start the calculations for the threshold level 5 Following threshold calculation set IM form parameters 58 Select a walk speed in cM It s recommended you use the same walk speed for your entire dataset Don t reset the walk speed between runs or your results will not be comparable 6 Click Start to begin the analysis Running interval mapping analysis WinQTLCart provides default values for the parameters in this form The defaults provide the best all around parameter settings especially for initial analysis sessions 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 59 Interval mapping analysis uses WinQTLCart mapping source data files MCD files Use WinQTLCart s import commands to move your source data files from text to MCD format 1 Open a source data file into the WinQTLCart main window 2 Select Method gt Interval Mapping WinQTLCart displays the interval mapping analysis controls in the form pane PENMUETONNNIMEES SIGNNBENNLEVE 3 Click Result File to select the location of and to name the QRT file that will be created when the analysis is complete 4 Click the OTraits button to enter other trait numbers or number ranges WinQTLCart will use these as a co factor in the analysis WinQTLCart s default is no O
49. ata panel12 to the Windows clipboard Select All Ctrl A Click in the Data panel and then choose this command to select all text in the data pane Enables you to easily select and copy the text to a separate file Main window Menus View st Data Summary Summarizes statistical information of active source DSum data file and displays in Text window yG Result Graph Ctrl G View result file in the Graph window See Graph Window Result tour t m Status bar a Select to toggle the Status bar display Main window Menus Method ee Single Marker Analysis Displays the Single Marker Analysis 5 form 2010 N C State University Bioinformatics Research Center Windows QTL Cartographer 2 5 a Interval Mapping Displays the Interval MappingI 58 form Composite Interval Mapping Displays the Composite Interval Mapping form let ea Multiple Interval Mapping Displays the Multiple Interval Mapping 6 63Yform Click OK button to start MIM analysis and click MtMIM button to choose multiple trait MIM analysis el Multiple Trait MIM Analysis Display the Multiple Trait MIM Analysis form a Multiple Traits IM CIM Analysis Displays the Multiple Trait Analysis 7 Yorm Category Trait Analysis Displays the Category Trait Analysis form Bayesian Interval Mapping Displays the Bayesian Interval Mapping 76 form Ed eQTL MIM Analysis Displays the eQTL MIM Analysis form Main win
50. ber of other traits in the source data Other traits are also known as categorical traits Individual number Enter the number of individuals in the source data Symbol for missing trait value Enter a symbol to use for missing traits based on your trait data Crossing type Select the correct cross type for your data B1 Backcross with 1 parental line to which the F1 line was crossed B2 Backcross with 2 parental line to which the F1 line was crossed Ri0 Recombinant inbred line derived by doubled haploid lines Ri1 Recombinant inbred line derived by selfing Ri2 Recombinant inbred line derived by sib mating SF Selfed intercross line Enter an integer indicating the generation Limit of 2 RF Randomly mated intercross line Enter an integer indicating the generation T B1 SF Test cross with genotyping done on an intercross SF and phenotyping on a cross B1 derived from that intercross Enter an integer indicating the generation T B1 RF Test cross with genotyping done on an intercross RF and phenotyping on a cross B1 derived from that intercross Enter an integer indicating the generation T B2 SF Test cross with genotyping done on an intercross SF and phenotyping on a cross B2 derived from that intercross Enter an integer indicating the generation T B2 RF Test cross with genotyping done on an intercross RF and phenotyping on a cross B2 derived from that intercross Enter an integer indicating the generation
51. cts Distribution C Equal C Normal C Equal C Normal Gamma e Gamma Parameter 3 Parameter 0 3 Filename Directory C NCSUSWInQTLCart2 5 lt lt Prey Next Trait WS Filename The file name of the MOD file that will be created Directory The directory that the simulated MOD file will be created 2010 N C State University Bioinformatics Research Center s Windows QTL Cartographer 2 5 Next Trait Click this button to reach each trait and you can set following parameters QTL numbers Set the number of QTLs to appear in the file Heritability Requires a value in the range 0 0 to 1 0 Default is 0 8 Vd Va Dominant variance Additive variance Vi Va Epistatic variance Additive variance For the Additive Dominant and Epistatic effects you want to see in the simulation you can select the following Effects direction e Same All values are positive e Both Values are positive and negative e Sign s Ratio Ratio of negative positive Effects distribution e Equal All effects have the same value e Normal Values derive from normal distribution e Gamma Values derive from gamma distribution e Parameter Value for gamma distribution Note For more information on effects distribution and the additive dominant and epistatic effects please refer to the QTL Cartographer s manual Simulation Data Control Dialog Step3 Trait 2 QTL Positions and Effects Va 3 636 Vi 0 364 Ve 1 000 Vi a 0 100 h 2 0 800
52. current working directory But if your data files reside in another directory or on a network drive you can tell WinQTLCart to look for and save its files there 1 Select Tools gt Set Working Directory to display the Set Working Directory dialog Set Working Directory 2 To change the directory click Modify navigate to the directory you want and click OK The new directory appears in the Set Working Directory dialog 3 Click Set Importing and exporting Importing files Unless the files you re working on have already been saved as WinQTLCart mapping source data files files with a MCD extension or are already in the MCD format 3 then you need to import them into WinQTLCart WinQTLCart will read in the files verify the data formatting and save the files in MCD format automatically WinQTLCart can import files from the following applications Formats supported MapMaker QTL MAP Map file MPS Map file RAW Cross data file QTL Cartographer INP Map and Cross data files 2010 N C State University Bioinformatics Research Center Windows QTL Cartographer 2 5 MAP Map file CRO Cross data file Microsoft Excel XLS CSV Note f WinQTLCart displays an error message saying the file format is invalid or can t be recognized then the extension may be wrong or the file s formatting renders it unusable in WinQTLCart Open the file in Notepad and compare it to one of the sam
53. dow Menus Tools Set Working directory Set the default working directory See Setting the working directoryl25 Draw chromosome graph SE Show and print graphic displays of chromosomes from the Current active MCD file See Drawing a chromosome tree 78 Delete markers of same position Notepad Shift Opens Notepad Use Notepad as a convenient text dee editor to help format source data files WW Calculator SC Shift Open the Windows Calculator accessory Delete markers that have the same position in a chromosome and only keep one marker Copy between trait and Copy normal trait to Other trait category trait or vise otrait verse 2010 N C State University Bioinformatics Research Center WinQTLCart Windows amp Menus 7 Main window Menus Help SN EEN About WinQTLCart Display About dialog of WinQTLCart You can open WinQTLCart upgrade site in this dialog Chromosome graph display Menus Open a mcd source data file From the Main window select Tools gt DrawChrom or click BrawChr to draw the trees of chromosome graphI78 and markers in a single large window that is suitable for copying to an image program for later editing or printing and publication Menus in Chromosome graph display window include File 7 Viewl7 Setting 8 and Copy_ Graph that will Copy content in window to the clipboard Chromosome graph Menus File aE a Chromosome graph Menus
54. e select File gt Export In the Export Source Data dialog select MCD file only selected traits are included Edit the source file name as needed The file will be saved to the current working directory 25 From the Trait Number Edit Box type in trait numbers separated by comma or hyphen Click OK WinQTLCart exports the file to the current working directory 25 Exporting results from the Graph window I amp D The Graph window doesn t export results per se there s no Export menu but you can save the results in these formats es WinQTLCart mapping result file QRT e Excel file XLS LR LOD values to a worksheet labeled LR QTL information to a worksheet labeled QTLs and points obtained through graph trace function to a worksheet labeled Points e Text file TXT in a tab delimited format Simply select the appropriate command from the Graph window s File menu specify the directory and filename in the Save As dialog and click OK WinQTLCart will display a confirmation dialog that the file has been created Working with source data files WinQTLCart s source data files have a MCD extension A MCD file is a text file that adheres to a specific format 3 that includes all the information WinQTLCart needs for QTL mapping analysis When you open a source data file 321 WinQTLCart verifies the file s formatting and displays its basic information in the Main window s Form pane Summary informatio
55. eeee cece ee ee ee ence ee ee seen ee ee ence eee eeee ee eeeeae ee nunnu nenn nnmnnn nnmnnn nnmnnn nnmnnn 84 Multiple Interval Mapping ccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeseeeeeeaseseeeeeseseeeseeeseeeeeeeseaneeeesees 84 Bayesian interval mapping ccccceeeeeeeeeeeeeeeeeeeeeeeeee ee eeeeeeeeeeeeaeeeeeseeeeeseseaeeeeeeauseeeseaneeeesees 85 Result manipulation ege cece NEESS EEN EENENEEEECEENEEEE KEE EENEG 85 Technical notes 85 Trouble Sh OOting WE 85 1 Errors even to run Single Marker Analysis ccccccsessessessesseseevsesseeseeneesnenesessesseeseeseeseesaesaesesaesanvanse 85 2 Why my trait values are truncated into integers mm iinnnnnnnnnnenrenrennennnnte 86 3 WinQTLCart cannot import Map information from selected file e cseceseseeesseeseeeseeeseeseneeseteneneees 86 4 Invalid file or wrong format messages cecescseseeesseeseeenseeseeeseeeseeeseeesneeneesseeseneseessenesenesenesenesesneessenseeetenee 86 5 Failures when I try to creat MCD file from text files iirrrrnneeneaeenranrnnnennenne 86 Technical Support EE 86 Credits amp acknowledgements ececeeeeeee cece ee ee ee eeee ener eeee ee eeeeee se ERER EE nnn EE ERER EE ERER EE EREE EEN 87 Index 88 2010 N C State University Bioinformatics Research Center About Windows QTL Cartographer About Windows QTL Cartographer WinQTLCart features Windows QTL Cartographer maps quantitative trait loci QTL in cross populations fro
56. efresh View ud Maize l grt weng bers 3 Mouse l grt Single Marker Analysis 3 Ki Text Files Interval Mapping s 0 Composite Interval Mapping Multiple Interval Mapping S5 NewModel txt bb Multiple Trait Analysis CHU s 3 maximum 20 Source file selected with right click options displayed Tree ltem Action Function Message window Left click Displays WinQTLCart startup message in the data pane Tree Item Action Function Source files root Double click Open a source data file MCD Tree Item Result files root Tree Item Text files root Right click gt Open a File Action Double click Right click gt Open File Action Double click Right click gt Open File Open a source data file MCD Function Open a result file QRT Open a result file Function Open a text file TXT Open a text file 2010 N C State University Bioinformatics Research Center WinQTLCart Windows amp Menus 11 Action Left click Tree Item MCD file Double click Right click options gt Open File gt Open with Notepad gt Refresh gt Single Marker gt Interval Mapping gt Composite IM gt Multiple Traits gt Multiple IM gt Bayesian IM Action Left click Double click Tree Item QRT file Right click options gt Open File gt Open with Notepad gt Refresh gt Close File gt Graphic Dialog Action Left click Double click Tree Item TXT file Right click options g
57. el click Y and click button OK to obtain an initial MIM model Click button Refine Model Select Optimizing position and click button OK Click button Start to start QTL position optimization procedure Click button Save Model to save the QTL model Click button Refine Model Select Searching for new QTLs and click button OK Select Search for QTLs and click button Start for searching for more QTL Click button Refine Model and optimize QTL position again Click button Test All to see LR values for each QTL in Text window Click button Save Model to save the QTL model again You may change filename if you want to keep the previous QTL model SSB eC eCONoauro ND 2010 N C State University Bioinformatics Research Center Tutorials 85 12 Click button Refine Model Select Searching for new QTLs and click button OK Select Search for Epistasis and click button Start for searching for QTL interact epistasis Answer Y to add the epistatic effect 13 click button Refine Model Select MIM Model Summary and click button OK 14 Select Graphic Result File and click Start to produce result file and show it in graph dialog 15 click button Refine Model Select MIM Model Summary and click button OK Select Model Summary File and click Start to produce MIM summary information 16 In tree pane you can click to show information of breed value QTL information R2 partition and variance and co variance table 17 Click button Fin
58. el 6 e Increasing the number of control markers will allow better resolution for mapping linked QTLs 5 Regression method selection Select a method 1 Forward Regression 2 Backward Regression 3 Forward amp Backward 6 Probability for into Probability for out 7 If the trait number field is enabled enter trait numbers and their ranges to be included in the model 8 Click OK to close the dialog and return to the Create New MIM dialog 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 69 9 Click the Criterion button The Set Threshold dialog appears Enter a threshold value or click Default to accept the default value recommended Click OK to accept the value and close the dialog and return to the Create New MIM dialog 10 Click the Start button to begin the search The CIM form may display during the calculations When the search is complete the model is presented in the cells 11 To manually edit the model e Click Add QTL or Del QTL e Click on a cell and enter a new value in the Cell editing box Click the Cell Update button to enter the new value 12 Click OK to accept the parameters close the dialog and return to the MIM form If you selected Set control markers manually in step 2 then WinQTLCart will display the Select CIM Control Markers dialog box Enter or edit the marker numbers you want to using the text box separate each number with a space Click on the marker ro
59. er Labels are the column headings Individuals are the rows You can enlarge the dialog by dragging its border toward or away from the center of the screen The pointer is a two headed arrow when it is in the correct position 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 33 W Marker Information Dialog Marker Genotypic Information for Chromosome Chr 1 Marker number 20 length 190 0000 cM Edit selected cell value 1 Update Cell Cancel Markers cim1 cim2 cim3 clm4 clm5 cim6 clm cim8 cim9 Distance 0 00 10 00 20 00 30 00 40 00 50 00 60 00 70 00 80 Ind 1 Ind 2 Ind 3 Ind 4 Ind 5 Ind 6 Ind 7 Ind 8 Ind 9 Ind 10 Ind 11 Ind 12 Ind 13 Ind 14 Ind 15 Ind 16 Ind 17 Ind 18 Ind 19 4 Sa 2 1 1 2 2 1 2 1 1 1 2 1 2 1 1 2 2 1 d N9 ech sch NO sch Ni ech sch Ni bi sch b i 1 bi sch ech Ni ech No sch No sch sch N9 sch Ni ech sch NO NO sch bi bi sch eh Ni sch N sch Ow sch PN sch PN sch sch NN sch ND ON sch sch PN sch NN sch NY sch NY sch bb bM PM PM bM sch NO sch NID NY sch IN sch NY NIN NNN sch ROR N sch eh sch ech NN sch NUNN sch bb bM PM S NSS sch Changing a cell s contents For modifying a cell s value click in the cell and type in a new value Or click in a cell and enter the value in the Edit selected cell value field You can change as many cel
60. es 5 Form pane 12Yor displaying and controlling analysis of source data files The form pane contents change based on the analysis method you select 6 Data panel12 for displaying data of currently selected file 7 The Status bar displays a variety of system messages select View gt Status bar to toggle its display Click on each node in the tree pane or hover the pointer over a toolbar button or menu command to see the displayed message The right area of the status bar also displays the current date and time and also displays CAP NUM or SCRL if the Caps Lock Num Lock or Scroll Lock keys have been pressed e Double click on a category name in the Tree pane to open files of that type e Left click on a MCD filename to make that the active source data file on which to run an 2010 N C State University Bioinformatics Research Center 10 Windows QTL Cartographer 2 5 analysis e Right click on a filename to see appropriate commands for that file Main window Tree Pane The Main Window s Tree pane allows you to manage open files The following table describes the many different options available via left click right click and double click operations Messages m Summary information Population 1 v File name Open with Notepad EN Source Files EN Phl mcd ty env1 jun3 meq Result Files envi un3 mcd 148497108 Open a File B1 A Cot O qrt Close the File SE R
61. esign Ill T D3 SF5 Token traits Line 59 Indicates trait number of source data Token otraits Line 60 Indicates other trait number of source data Other trait also called a categorical trait is the trait with qualitative or categorical values such as sex color and so on Other traits can be used as factors that can be regressed out in regression analysis This means a regression of the quantitative trait of interest on the categorical trait will have been performed and the residuals used as the phenotypes in the analysis 2010 N C State University Bioinformatics Research Center e Windows QTL Cartographer 2 5 Token missingtrait Line 61 Indicates the symbol for missing trait value Token case Line 62 Either yes or no e Yes means all comparisons are case dependent e No means all names of individuals markers and traits are converted to lower case to make comparisons Token TranslationTable Line 62 The token with the table next 6 lines of data will define how marker genotype data are translated There are six rows and three columns 18 positions in the table Here is the default translation table TranslationTable AA 2 2 Aa T 1 aa 0 0 A 12 12 a 10 10 Ste Ss SE The first column is the genotype The program assumes that the A allele is diagnostic for the High parental 1 line and the a allele is diagnostic for the Low parental 2 line A minus sign means the allele is unknown missing
62. etermine the threshold via permutations Ke em Jemen Know the threshold values you Input them into the Manual Input want to use F5 box Don t know the threshold values Select By Permutations 5A to have Permutations take a long time to you want to use WinQTLCart calculate them run If 1 permutation runs for 10 seconds than 1000 permutations will take 2 3 hours Setting threshold levels manually Use this procedure to set the threshold for each trait manually This is useful if you know the value for each trait Alternatively select All Traits and apply this threshold setting to all the traits 1 From the Trait Selection pull down select a trait to find the threshold value for that trait The All Traits selection is available for permutations only 2 Click the By Manual Input option 3 Input a threshold value into the box 4 Optional Click Set as Default if you want to assign the threshold value you entered to all traits Setting threshold levels via permutations Setting values via permutations allows WinQTLCart to empirically estimate the genome wide significance threshold Use this procedure if you don t know the value and you want WinQTLCart to find the optimal threshold Threshold values will be filled in automatically for every trait according to significance level Following threshold analysis WinQTLCart will display the results in the Data pane and save them ina text file The text file s name is displayed in
63. fferent parameters and controls that help you control the analysis When you first open WinQTLCart you see the standard Source Data File Information form Most of the options are disabled because no source data file has been loaded Select an analysis method from the drop down list in the Analysis box on the right to begin working with the data When you have opened a file WinQTLCart enables the buttons and controls on the Source Data File Information form These enable you to perform some basic manipulations to the source data Such as add traits map information etc Forms and disabled commands When you select an analysis method WinQTLCart assumes you want to keep working with that method until you save your data or cancel the analysis If you select the Interval Mapping IM method WinQTLCart disables several toolbar and menu commands such as the other analysis methods setting the working directory and so on You need to either save your data or press the Cancel button on the IM form to leave the IM analysis mode Leaving an analysis method re displays the Source Data File Information pane See the Source data file information 3 topic and the topics for each analysis method for the appropriate screen shot relevant to that method Main window Data Pane The large pane under the Form pane displays the content of the active data or results file in text format You cannot edit the displayed information from this pane However you can
64. he graph Use Set Display Parameters 2 to switch the legends to above the graph Turn the trait name display on when you re loading more than one result file into a graph Show Marker Names Toggle display Show Chromosome Names Toggle display 2010 N C State University Bioinformatics Research Center a Hide Show Threshold Lines Toggle display al 16 Windows QTL Cartographer 2 5 Provides coordinates for a specific point on the graph See Tracing coordinates on the graph FE One page display window Menus From the Graph window select Tools gt Display One Page Format to display the graphs in a single large scrolling window that is suitable for copying to an image program for later editing or printing for publication The chromosomes and traits shown in the one page display depends on the Select Chromsl zi and Select Traits 20 ettings in the Graph window Menus in One page display window include Filete Viewl16 Setting 17 and Copy_Graph that will Copy content in window to the clipboard One Page window Menus File a Copy to Clipboard Copies content in window to the clipboard Print Graph Print the graph Close the window and return to the Graph window One Page window Menus View Show Threshold Line Toggle display of threshold line Show Marker Number Toggle display of marker numbers Increase graph number in a row by 1 2010 N C State University Bioinformatics Research
65. he identification and estimation of genetic architecture parameters including the number genomic positions effects and interactions of significant QTL and their contribution to the genetic variance High level process Here s a quick overview of how to use WinQTLCart s MIM implementation 1 Select the MIM analysis method 2 Pick a trait you want to work with MIM works with only one trait at a time 3 Decide if you want to create a model using WinQTLCart s default search procedures or an alternative such as Forward Backward or CIM 4 Run the analysis to generate the model 5 Refine the model as needed by editing individual cells in the model adding or deleting QTL searching and testing QTLs or epistatics and re estimating This part of the analysis can iterate for as long as you want to search for QTLs 6 Save the model as a MDS file or as a result file using the Refine Model function About the MIM form From the Source Data form open a source data file and select Multiple Interval Mapping as the analysis method If there is more than one trait in the dataset the Select Trait for MIM Analysis dialog appears Select a trait from the drop down list and Click MIM button to open MIM form 2010 N C State University Bioinformatics Research Center ER Windows QTL Cartographer 2 5 Select a Quantitative Trait for MIM Analysis Select one trait net e MT MIM eQTL MIM The MIM form appears Bef
66. he left of the information pane 5 In the Single Marker Analysis group box click Result to view the analysis result for the selected trait You can change the font used by the display window to make the results easier to read Click the Save button in this group to save the marker analysis results to a text file 6 In the Statistical Summary group box click Result to vew the summary in a larger display window Click the Save button to save the statistical results to a text file The statistical summary includes e Basic summary of the data e A histogram for the quantitative trait e WinQTLCart s summary of missing individuals that should be present as indicated by the data If markers show 0 data there was likely an import problem e Summary of marker segregation Combines LR map QTL and Q stats 7 Click the Graphic File button to save the results to a QTL mapping result file QRT You can open this QRT file later to view the results as a graph 8 Click Close to end the single marker analysis session and return to the Form View of Source Data Qstats show some important results e Tells you that the data were imported correctly If a marker has 0 data that indicates a problem likely an import problem For example the original marker may be Marker_1 but QTL sees that as Marker 1 and the values won t match e Tests for segregation distortion Setting threshold levels IM amp CIM Both Interval Mapping sand
67. ialog appears Click Mt MIM button to open MT MIM Parameters and Analysis Dialog MT MIM Parameters Selection Dialog Result File C working O MtMIM 01 B15enyv1 jun3 mtmim rlt txt Control File M TMIM CONTROL FILE txt Total population 4 Selected number 3 Index 134 Parameter Setting for Current Population Current population Fi Total traits in current population 3 Selected traits in current population 3 S Index 123 Selected QTLs in current population 4 Selected Epistasis in current population F2 3 Chromosome String f1 23 3 QTL 1 H 2 Position cM Sting 46 09 155 23 60 09 172 53 QTL 2 2 3 Pleiotropy vs Close Linkage Test QTL Number String Genome wide test of OTL effects test if the effect s lt p t e gt 1 and 2 are equal or not Effect DK Effect2 11 Effect number E Trait Pairwise Control _ TPControl txt Refine Model 1 Model parameter estimation e Update i Finish Result File Click Result File button change the result filename and click button to open the result 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 73 file with Windows notepad Control File Click Control File button to change the control filename and click button to open the control file with Windows notepad The control file includes all initial parameters setting in the dialog detail please to see Mt MIM Control File 74
68. ick Next button to accept the values The Create New Source File Step 6 of 6 appears 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 51 Create New Source File View and Finish Step 6 of 6 ChrNum Chat zb MkNum Marker label Marker position 13 AXR 1 HH 335C COL EC 480C EC 6 8 72 4 76 6 93 2 97 01155116 5 12 HH 3423L EG 12L COL GH 53L COL 13 PGK 12 NIAA C121 PRD 675 COL D Marker genotypes 122222222221122222222222 401609 25 0769 11 1517 112122222222222222222222 42 1206 24 6962 14 2351 211211112222222222222222 39 8114 22 4848 7 0315 222122222221122222222222 37 9753 23 1155 11 2001 121111111112222222222211 39 5613 21 0934 3 5133 111122222222222211111112 gt 35 9214 24 4051 6 5268 1 1121411411 1117021121111221 35 7827 22 6201 6 67 70 222222222211122222222222 40 3456 25 6763 13 9114 2222222101 2210414141 14140114 4 9150 18 4293 9 6165 222222222221111122221111 38 2035 21 4365 12 5332 lt Back Cancel Help This is the data summary dialog The up window is map information and lower window is cross information Click Back button to do the modification if the information is not right After you check all the necessary data click Finish button WinQTLCart will e Verify the data and display error messages if the data file is incomplete or formatted incorrectly e Create the MCD file using the filename stem you entered at the first Create New Source File dialog e Show a create
69. include Filel 4 Editf 5 Viewf 5 Methodf 5 Tools ef and Help 7 Main window Menus File New Ctrl N Create a new source data file from raw text files See Creating a new source data file 44 Ctrl O Open a data file result file or text file Source data files you can open in WinQTLCart have the MCD extension Results files have the QRT extension Files with any other extension are opened as text files Close Close the currently selected file The currently selected MCD file s name is in the title bar and highlighted in the Tree pane Save As Save the currently active source data to a file with mcd Save As Import Ctrl Import files in a variety of formats See the importing files Import F2S topic Se Simulation Opens the Simulate Data dialog See creating a SS simulation data file 5 Export Export the selected file to a different format See Exporting source data and results 20 EN format or a file with Microsoft Excel format D ew LG i 2010 N C State University Bioinformatics Research Center WinQTLCart Windows amp Menus 5 amp Print Ctrl P Print the selected file in data pane Print Opens Windows Print Setup dialog box Print Setup Closes WinQTLCart If you have unsaved data you ll be prompted to save it D Recently used WE WinQTLCart displays the last 6 data files you ve worked files with Main window Menus Edit Ctrl C Copy selected text in D
70. ish and button Save to finish the MIM session Bayesian interval mapping Select Menu Item File gt Open or click OPEM to open source data file wqcart samp0 mcd Select Menu Item Method gt Bayesian Interval Mapping to enter the Form of BIM You can click button Parameter to set the BIM parameters Click button Start to begin CIM analysis Fkrobh z Result manipulation vG 1 Start WinQTLCart and select menu item View gt Visualize Result or click Result to open result file wqcart samp0 C qrt 2 Use menu item Chrom to select and show one chromosome all chromosomes and any number of chromosomes with different order 3 Use menu item Traits to select and show any number of traits 4 Select menu item Tools gt Display one page format or click 1G to show graph in different format 5 Select menu item Setting gt set display parameters or click PE to adjust parameter of graph 6 Select menu item Tools gt Show QTL information or click El obtain the QTL summary information from the graph or open a QTL summary file qtl 7 you can trace on graph by selecting menu item Setting gt Trace coordinate in graph or click a Double click to show the coordinate numbers in graph 8 You can combine two graphs into one by selecting menu item File gt Add QTL result file Technical notes Troubleshooting 1 Errors even to run Single Marker Analysis WinQTLCart has problem to deal with complex folder and file name To avoid this problem
71. issing trait value Enter a symbol to use for missing traits value based on your trait data Marker translation table Edit or add symbols you want to use for these genotype markers you can enter any alphanumeric character s as a symbol These translations apply to the experimental design you selected at the left of the dialog Note WinQTLCart assumes that the A allele is diagnostic for the High parental 1 line and the a allele is diagnostic for the Low parental 2 line A minus sign means the allele is unknown WinQTLCart uses the numbers 2 1 0 12 10 1 to determine how to encode the output of the genotypes The alphanumeric tokens you enter here indicate how you have coded the markers in the source data Cross Information Select the experimental design option based on your data You can select only one option B1 Backcross with 1 parental line to which the F1 line was crossed B2 Backcross with 2 parental line to which the F1 line was crossed Ri0O Recombinant inbred line derived by doubled haploid lines Ri1 Recombinant inbred line derived by selfing Ri2 Recombinant inbred line derived by sib mating SF Selfed intercross line Enter an integer indicating the generation Limit of 2 RF Randomly mated intercross line Enter an integer indicating the generation 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 35 T B1 SF Test cross with genotyping done on an intercross SF and
72. ity Bioinformatics Research Center 2 Windows QTL Cartographer 2 5 Related topics Creating a new source data filea Troubleshooting import errors 86 Importing files 25 Exporting source data and results 29 System requirements WinQTLCart can run on the following operating systems Windows 95 98 ME NT 2000 XP and Windows 7 Because some WinQTLCart windows are quite large the suggested minimum monitor resolution is 1024x768 20MB free disk space for program files 512MB RAM Any mouse or pointing device supported by Windows Installing uninstalling upgrading Installing To install double click the WQTLSetup exe file and follow the prompts The default install directory is C NCSU WinQTLCart2 5 though you can specify a different directory The installer places a shortcut to the Windows QTL Cartographer program on your PC s Desktop labeled WinQTLCart Double click the icon to run the program Uninstalling To uninstall run the Add Remove Programs control panel and select Windows QTL Cartographer from the installed programs list Upgrading If you have a prior version of WinQTLCart already on your PC simply run the installer program Upgrading to a new version of WinQTLCart does not overwrite your working files However the upgrade will replace the sample files that are part of the WinQTLCart distribution Note You should close current running version of WinQTLCart first before installing the upgrading versi
73. king with a Source file s traits values inner 33 Working with a source file s basic information inner 34 Working with source file s individual information seen 35 Working with source file s chromosome information inner 37 Working with source file s trait information ceseeseeesseeseeeseeeseeeneeeseeeneeeseseenesseesenesenesensseeeeeesneeseenennerenee 38 Working with source file s other trait information seen 39 MCD file format ind dcescees sues rise E E T scdsnaedagerzecesaectaerieuenacence 39 Creating a new source data file from raw data RRE EEERREREERRER EE RRER EE ERER EE ERER EE EREE EEN 44 Creating simulation data ss EE ERER EE ERER EE RRER nennu mennene 51 Single marker analysis ss nnssssrennsnrennnnnneennnnnnennennnennnnnnneenennnnne 55 Setting threshold levels IM amp CIM REENEN ENEE is eerneensss 56 Setting threshold levels manually ccccsesetessesseseeeseeseeseeseeseesseseseeseeeseesoeseesaesoesaesaevaevanvaneaneaeeneesnesaesansaes 57 Setting threshold levels via permutations nn inirrarnnenrrsresnesnesnasneenesneeneaneenranrannnnnne 57 Interval iMa pping DEE 58 Running interval mapping analysis cesses ese eeeeeeeeeeeeseeeseveevenvseeseeseesoeseesaesausausaeeseesoeseesaesousaesaevaevanse 58 Composite Interval Mapping ececeeeeeee erence ee eeeeee ee eeeeee ee eeeeee se eeaeeeseeeeeeeseeeaeeeseeeseeeseeeaeeees 60 Running composite interval
74. ls as you like Click Update Cell to save your changes as you go you can click OK to save all changes and close the dialog Click Cancel to close the dialog without saving any of your changes including any changes saved with the Update Cell button Working with a source file s traits values Traits At the Trait values pane on the Main window click the Trait View button to open the Trait Information dialog Other Traits If the source data file contains other traits then click the OTrait View button to open the Other Trait Information Dialog For more information on working with this dialog and on modifying values see the following topics Working with a source file s marker genotype datal 32 2010 N C State University Bioinformatics Research Center 34 Windows QTL Cartographer 2 5 Working with a source file s basic information At the Source data manipulations pane on the Main window click the Basic Info button to open the Manipulation of Basic Information dialog Minipulation of Basic Information Basic information Marker Genotype Table Chromosome linkage group number 3 A Trait such as yield or weight number Aa Other trait binary value such as sex number z Individual sample size number ho Symbol for missing trait value Eo 1 Cross type B1 v Marker Position Information Map function Haldane D Position types Position e Position units cent Morgan D Cancel Symbol for m
75. m inbred lines WinQTLCart includes a powerful graphic tool for presenting mapping results and can import and export data in a variety of formats WinQTLCart incorporates many of the modules found in its command line sibling QTL Cartographer and provides a graphical interface to many of QTL Cartographer s features WinQTLCart implements the following statistical methods e Single marker analysis 55 e Interval mappingl 58 e Bayesian interval mappingl76 e Multiple trait analysis 78 e Multiple trait MIM analysis 7A Features e Supports various QTL mapping methods e View copy and print graphs e Includes an interface to help you build a source data file that WinQTLCart can use for analysis e Import 25 data from Mapmaker QTL and Microsoft Excel and CSV formats e Export 29 graph data to Windows Excel format e View copy and print chromosome information 78 graphically e Produce a simulation 51 data file Compatible programs and formats WinQTLCart can import and export data files in a variety of formats Import success depends on the data file s format Some data may need to be formatted manually before WinQTLCart can import it Applications Formats Supported Import Export MapMaker QTL MAP Map file MPS Map file RAW Cross data file EE WE QTL Cartographer INP Map and Cross data files MAP Map file CRO Cross data file WinQTLCart MCD Source data file ee 2010 N C State Univers
76. m selected filel s Invalid file or wrong format messages se Your source data may not have come from another program but may instead exist as raw source files In that case using WinQTLCart s Create a New Source File command steps you through all of the steps needed to translate the raw data into a readable form The new source file will conform to WinQTLCart s MCD file format See these topics for more information Creating a new source data file from raw data 44 MCD file format 39 Step 3 Analyzing data using QTL Mapping Methods With WinQTLCart able to view the data you can then select any of seven different analysis methods The end result for some of these methods is another MCD file but in most cases the process will create a QRT result file that WinQTLCart can use to graph QTL information See these topics for more information Single marker analysis 55 Interval Mapping 58 Composite Interval Mappingl60 Multiple Interval Mapping 63 Bayesian Interval Mapping z i Multiple trait Analysis 6s Multiple trait MIM Step 4 Viewing results and graphs WinQTLCart can present your data in graphics suitable for publication You can show all chromosomes and their intervals in one display while the Graph window display offers many parameters to help you fine tune the visualization See these topics for more information Drawing a chromosome treel 78 Graph Window jour Step 5 Saving and exporting results You can sa
77. mapping ANALYSIS cece eseveeveevseeeeeseeneeseeseesaessessesseeseeseesaesaesesaevanvanye 60 Multiple Interval Mapping cccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeseeeeeeeseaesaeeeeeeseeeseaeneeesees 63 ADOUt the MIM forint tee Seege EES 63 Creating MIM initial model EENEG 65 Regression Options eenegen aa a aae Eae i Ea a E conve age du dr dan R Re diese g n stoke CES 66 CIM search option MM search option Refining the MIM model eege ei ne 70 Multiple trait MIM eegne eEeg Ee telnet seen marne EES SES EE ns EAA 72 ADOUt the MM IM Tor ea a aan aare raaa deet cgente ceancuaeqesnseseeretncsaevendevderyueyeuaectcecaepceesnasucedestvnuernense 72 Mt MIM Control File 0 0 cccsssssssessessesseeseeneeneesessesesseeseeseeseesaesaesaesaevaesaevaeeseeseennesnasaesausaesaeesessaeseesaesaesaesaeuanvanye 74 Mt MIM FUN CtIONS cece eteesensesseveevsesseeseeneeseesaesaessesseeseeseeseesaesausaesauvaesaevaeeaneaeenneanenaesaesaesaesseeseeseesaesaesansaevanvante 75 Bayesian Interval Mapping cccceeeeceeeeeeeeeeeeeeeeeeeee ee eeeeeeeeeeeeaeeeeeseeeeeeeseaeaaeeseeeseeeseaeeeeesees 76 Running Bayesian interval mapping ANALYSIS cece eteeseeseeeeveeveeeeeeseeneeseeneesaessesseeseesoeseesaesaesesenanvanse 7 Multiple trait analysis ee ee ee ee ee ee ee eeee eee eeeeeeeeeeeeeeeeseaeeeseseeeaaeesseeseeessenseeesees 78 Drawing a chromosome tree ccccceeeeceeeee ence ee ee e
78. mosome and marker information Total chromosome number The total number of chromosomes Click the spin dial to the right of this field to select more or fewer Current chromosome Shows the active chromosome you re working on Click the spin dial to the right of this field to select a new chromosome You ll see the new chromosome s information in the Distance and Label boxes to the right Markers for chromosome Total number of markers for this chromosome Average space between markers cM Produces marker positions randomly Variations of the marker positions How many dewations are allowed on average Check Read marker positions from a text file and click Browse to select a text file containing that information Also select a marker distance type Position Indicates that the numbers indicate positions from the left telomere of the current chromosome This means the numbers should be in increasing order Interval Indicates that the numbers are for the interval distance after a marker This means that the last number and the last number only should be zero From QSI file To produce the simulation data by loading a QSI file alone In Step 3 you can save all simulation parameters into a QSI file Simulation Data Control Dialog Step2 Trait 1 OTL Numbers Heritability 0 8 viva 01 7 Additive Effect LE M Epistatic Effect Effects Direction Effects Direction e Same Same C Both Both Effects Distribution Effe
79. mosomes and traits Minimum LR value in graph Check and input a value to set the minimum LR not LOD value into the value at Y axis default value is 8 0 Marker label font size To adjust marker label s font size after selecting showing marker label in LOD LR window Setting a test hypothesis Select Setting gt Test Hypothesis to play with the results further by trying out different LOD LR and effects additive dominant R2 TR2 S settings on the displayed results Note This option is only for crosses with three kinds of genotype such as SF2 2010 N C State University Bioinformatics Research Center 2 Windows QTL Cartographer 2 5 Set Hypothesis Hypothesis definitions LR setting HO a O d 0 HO H3 0 C HO H1 3 a lt gt 0 d 0 e H3 e uO a 0 d lt gt 0 H1 H3 1 HO H2 4 a lt gt 0 d lt gt 0 C H2 H3 2 C H4 GxE Additive effect setting Dominant effect setting Additive under H1 a Ze Dominant under H2 d Additive under H3 al Dominant under H3 di 5 H1 R2 H0 H1 TR2 H0 H1 CC S H3 R2 H0 H2 TR2 HO H2 C 5 H3 C R2 H0 H3 C TR2 HO H3 Cancel This dialog is fairly self explanatory The Hypothesis definitions box describes the conditions for each hypothesis with a additive and d dominant The LR setting box describes pre set likelinood ratio hypotheses Click OK to apply the selected hypotheses options to your display Showing QTL information
80. n Source data view and modify Analysis Population 1 e Marker values Single Marker Analysis File name envl jun3 mcd Chromosome 1 Chr1 E Markers File ID number 1148497108 Trait values Go Cross type B1 E ES Trait View Sample size 100 Chromosome numbers 3 Source data manipulations Trait numbers 3 Other Eat nurbers 0 Basic Info Individual Chromosome Trait OTrait Note Although you can open other text and result files in WinQTLCart only MCD file information is displayed in the Form pane WinQTLCart will continue showing the last MCD file you viewed in the Form pane even if you open or swtch to text or result files The Summary Information box tells you the basics on the selected source data file From here you can go ahead and select an analysis method if you wish However the form s buttons and pull down lists let you look at the source data file in more detail and also perform the following actions e View information of markers traits and other traits also called categorical traits in a formatted way e Add edit delete marker genotype data 32for each chromosome in the file including adding or 2010 N C State University Bioinformatics Research Center 32 Windows QTL Cartographer 2 5 deleting individuals Add edit delete trait values 33 and other traits Edit map sand cross 3s information Add new experimental data individuals Try out different data configurations such as
81. nd 6 are cleared meaning those chromosomes will be displayed When a chromosome is removed from the display its number disappears from the lower text field also In the box above clicking the empty cell under 3 would remove chromosome chromo3 from the graph display To reorder chromosomes click on a number in the Chromosomes row It swaps places with the next displayed cell to its right Clicking the last displayed cell swaps it with the first displayed cell Example In the screen shot above clicking 1 will swap 1 and 2 and the display order will be 2 1 3 6 Clicking 3 will swap 3 and 6 so the display order would be 1 2 6 3 Clicking 6 will swap 6 and 1 so the display order would be 6 2 3 1 Click Select All to show all the chromosomes Click Select First to show only the first chromosome Click Help to display help text for this dialog Setting display parameters Select Settings gt Display Parameters or click SI from the toolbar to display the Set Graph Display Parameters dialog Use this dialog to refine the display change font sizes and colors used and so on 2010 N C State University Bioinformatics Research Center 22 Windows QTL Cartographer 2 5 Set Graph Display Parameters Parameter Setting Show LOD profile as block graph view Continuously Block D Show QTL info z F NoLOD window Ratio between effect window size and LOD window size f E F NoLOD Line Title M Legend on right x Coordinate fo
82. ner seen eeeeeeeeeeeeaeeeeeseeeeeseseeeseeesseeseeeseaneneesees 78 Adding QTL positions to the chromosome graphics iirrannnnnnnrnrnnennre 80 Tutorials 81 Import data UU 81 2010 N C State University Bioinformatics Research Center m Windows QTL Cartographer 2 5 Import data INP Gd ELE 81 Using Em ap fUnCtION ccc ssescessesseeseeneeneeneessessessesseeseesaeseesaesaesaesaevaesaevaneaeeseennesnesaesaesaesaeeseeseeseesaesaesaesaevaevanve 81 Import data OUT format oo ccc eseeseseneneeseesseseeeseeseeseesoesaesaesaevaesaevanesneseeneesnesaesaesaesaeuseeseeseesaesaesansaeuanvanye 81 Import data MapMaker format 0 cc ccssscsssssessessseeseeseeseesaesoesesaevsevonvaneseeseeneeseesaesaesaesaeeseeseeseesaesaesansaevanvanse 82 Import data Excel format cscs ssseeseestestesseseessesseeseeseeseesaesaesaesaevaevaevenssneseennesnasaesausaesaeuseeseeseesaesaesansaevaevanse 82 OICHT ENEE 82 Simulation source data file cceeecceceeeeeeeeeeeeeeeeeeeeeeeeeeeeseeeeeeeseeeeeeeseeeeeeeseeeeeeeseeeaeeeseesseeess 82 Create new source data file ccceeeeceeeeeeeeeeeeeeeeeeeeeeeeeeeeeseeeeeeeseeeeeeeseeeaeeeseeeeeaeseeeeeeeseeeseeess 83 Single marker analysis ceccceeeeeeeeeeeeeeeeeeeeeseeeeeeeseeeeeeeseeeeeeeseeeeeeeseeeeeeeseeeeeeeseeeeeeeseeeseeees 83 interval mappi E 84 Composite interval Mapping ssssneneeneensneeneennneennennneenennnneenennnnee 84 Multiple trait analysis ceceee
83. nits e centiMorgan cM Very small 100 centiMorgans 1 Morgan e Morgan The distance over which on average one crossover occurs per meiosis e Recombination Percentage of crossover events that occur between two markers Related Topics MCD file formatio Working with source file s individual information At the Source data manipulations pane on the Main window click the Individual button to open the Add or Delete Individuals dialog 2010 N C State University Bioinformatics Research Center s Windows QTL Cartographer 2 5 Add or Delete Individuals fe Delete Enter individual numbers or number ranges that Wd at should process Enter the numbers separated by commas and the ranges separated by hyphens For example 2 5 8 10 12 are 2 5 8 10 11 and 12 Individual 1 Number 10 D d Cancel Append and Delete Click Append radio button to add individual s into active source data and click Delete radio button to delete one or more individual from active source data 1 Append individual s WinQTLCart can read the individual information through a properly formatted text file data separated by spaces not commas and click Filename button to indicate the filename Use the Notepad button to call up an empty text file if you need to do impromptu editing To check From Clipboard button if the individual information is already in Windows clipboard Please check the Data include individual label s button if the individu
84. nteracted Token pvsitest line 14 Pleiotropy vs close linkage test The number after the token is how many populations that are in the test Token p line 15 16 One line started with p token for each population of pleiotropy vs close linkage test The first number indicates which population Then the total QTL number to be test and followed by the QTL indexes For example p 1 2 3 4 Population 1 to test QTL 3 and QTL 4 are actually one QTL or are two different QTLs 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 75 Token gqtltest line 17 To test two sets of QTL effects see token str1 and str2 have the same effects or not The number followed by the token is the QTL effect number of each set Token str1 and str2 line 18 19 Every three numbers population trait and QTL indicate a QTL effect For example gqtltest 2 str1 111 112 str2 211 212 This is to test the following assumption population 1 trait 1 QTL effect 1 equal to population 2 trait 1 QTL effect 1 as well as population 1 trait 1 QTL effect 2 equal to population 2 trait 1 QTL effect 2 or not Token end line 20 End of Mt MIM Control File Mt MIM Functions In the Refine Model Group you can select one of following functions from the pull down menu 1 Model parameter estimation To do the parameters estimation for current model The likelihood value is displayed in information window on
85. o look deeper at the properties of those estimates such as how effects estimates are related to loci estimates It also provides handy tools to explore research questions such as the posterior chance of multiple QTL in an interval How to use The defaults are quite robust and it is recommended you use them during your first BIM runs For more information on the BIM parameters Please refer to the R implementation particularly the function bmapatl options The best way to get library bim is to already have R 1 9 0 installed on your PC 1 If you don t have R 1 9 0 installed on your computer go to http cran r project org 2 Download the precompiled binary distribution of R Linux or Windows is preferred Follow 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures instructions for installation and run R 3 While connected to the Internet select R s Packages menu and select Install packages from Bioconductor 4 Scroll down to the bim package and click on it 5 Use the R Help to get HTML Help 6 Select Packages and click BIM from the list There is an overview document but the options are explained in detail in bmapatl options Note Pre 1 9 0 releases of R can get the bmapatl package from the CRAN website cited above Bioconductor is a companion project to CRAN focused on biological applications Brian A quick summary of BIM implementation in a previous version of QTL Cartographer can be f
86. odel wthout scrolling click the blue lt lt QTL cell To return to the MIM form click the blue QTL gt gt button Where to go from here From here you can refine the MIM modell zt manually edit the model by clicking the Add QTL and Del QTL buttons or click in the model field to change the value of Position Chromosome Additive or epistatic values Click Save Model to save the model as a MDS file Related topics About the MIM form 63 Refining the MIM modell zt Regression options In the Create New MIM Model dialog If enabled click the Criterion button to display the Set Criterion dialog box 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 67 Set criterion of model selection Criteria of BIC Significance level BIC related criteria Probability of partial R 2 Significance level 0 0100 D Help KVat DH Specify QTL number range From f To e ECHT ET Cancel Select the BIC related criteria option and select a criterion from the drop down list Use the spin dial to select an appropriate X Value The Help button displays a message box explaining the meanings of the BIC related criteria Select the Probability of partial R 2 and select a significance level from the drop down list 1 Specify a QTL number range using the spin dials and click OK The dialog closes and you re returned to the Create New MIM Model dialog 2 Click Start to begin the search
87. ome as 1 Markers for chromosome 1 as 15 Average space between marker as 9 Variations of the marker position as 10 Click button Set as default for all chromosomes to set parameter for other chromosome Click button Next to step 2 3 dialog window 4 In step 2 3 set parameters as QTL numbers 7 Heritability 0 82 Additive effect Effects direction gt Same Effects distribution gt Normal Epistatic effect None by uncheck the check box Filename Nsimu 01 Click button OK to step 3 3 dialog window 5 In step 3 3 Click button Adjustment to adjust all parameters Click button Save to save the parameter setting to file Nsimu 01 qpe Click button OK to finish 2010 N C State University Bioinformatics Research Center Tutorials 83 Create new source data file D 1 Start WinQTLCart and select menu item File gt New or click S Set parameters in step 1 of 2 as Chromosome number 2 Trait number 2 Other trait number 1 Individual number 125 Symbol of missing trait Crossing type B1 File stem name Test AA 2 Aa 1 Aa S Click OK to step 2 of 2 Click button Notepad to open file Text txt in default directory C NCSU WinQTLCart In Notepad select chromosome label CHROM 1 CHROM 2 and copy to Windows clipboard Minimize Notepad 6 Back to Create New Source File Step 2 Of 2 Click button Paste to show chromosome labels in Edit Window Select Chromosome Labels in Data Type Selection Click button Send Da
88. on Using WinQTL a high level overview Your goals in using WinQTLCart may include preparing data for publication or continued research into possible QTL sites Step 1 Preparing your source data Your data files may come from another program or they may exist as raw data files For WinQTLCart to work with your files they need to conform to the program s MCD file formatis Review that file as well as the other files included in the WinQTLCart distribution such as the QRT QPE and other files 2010 N C State University Bioinformatics Research Center Using WinQTL a high level overview 3 These are all text files that you can view in any text editor Or you may not have any data files or any data ready for import You may instead want to use WinQTLCart to create simulation data to try out some hypotheses to view potential results See these topics for more information MCD file formats Creating a new source data file from raw datal44 Creating simulation datals1 Step 2 Bringing data into WinQTLCart WinQTLCart can import map and cross data files from MapMaker QTL QTL Cartographer and Microsoft Excel As part of the import WinQTLCart runs verification checks against the data If the data does not conform to the accepted format WinQTLCart displays an error message that should indicate the source of the problem See these topics for more information Importing files 25 WinQTLCart cannot import Map information fro
89. ontrol file TPControl txt Example of TPControl file begin 1 gene 563 gene 2694 3 3 54 6 15 52 5 13 288 1 2 gene 563 gene 4898 2 3 54 6 15 52 5 3 gene 563 gene 5323 2 3 54 6 15 52 5 end The control file start with line of begin and end with line of end For each line between is the information for a pair of traits The first number is the numbering and second and third are the marker labels for the first and second trait After that is the total QTL number follow by chromosome number and position for each QTL Bayesian Interval Mapping WinQTLCart s Bayesian interval mapping BIM module is an implementation of the command line BIM library provided courtesy of the R Project for Statistical Computing What it is Bayesian interval mapping library R bim provides Bayesian analysis of multiple quantitative trait loci models This includes posterior estimates of the number and location of QTL and of their effects Bayesian interval mapping for controlled experiment provides a nice complement to the classical analysis for mapping QTLs It is recommended that the standard IM CIM MIM etc analyses be run first The Bmapqtl implements Bayesian analysis for either a fixed or random number of QTLs WinQTLCart s default is random The program generates a random sample from the joint posterior of QTL and effects Note that the BIM estimates should generally agree with those of MIM and should be similar to those from CIM BIM allows one t
90. ore you can use this form you need to load or create a MIM analysis model You can open existing files containing MIM model parameters or you can use WinQTLCart to create a model The following screen shot shows the MIM form with a model loaded and analyzed MIM Model 1 of Trait 1 bi D lt lt OTL aie i i MT ai O7L Position cM 76 01 Leo gess ss E Summary of current model JE ee 0 3810 aen 0 3810 0 3677 ans P La FREE wes JE 0 1408 01006 Epistais nat 425 Reine Mode fonz p SEN QTL 3 a Delete QTL Close Cara 976 CelUpdate Model drop down list Contains the list of MIM models to be used for the analysis You can create or load several different models for selection New Model Add Model Have WinQTLCart create a new inital MIM model or create additional MIM model for analysis Save Model Save the model you ve created or modified to an MDS file Load Model Load an existing MIM model parameters file MDS Click the button to display an Open dialog navigate to the MDS file containing the parameters and click OK Summary Click to create a text summary file and a graph result file QRT At the prompts confirm you want to create the files WinQTLCart by default saves them to the current working directory 25 but you can specify a different location and filename Note The summary file information includes position likelihood ratio and effect of each QTL epistatic effects of QTL
91. orm pane 63 refining MIM model 70 summary files in tree pane 10 multiple trait analysis 78 new source data 83 one marker 83 other traits 33 deleting an OTrait during export 29 deleting during export 30 OUT 81 QRT 85 Qstats 55 QTL Cartographer 1 4 2010 N C State University Bioinformatics Research Center QTL Cartographer 1 4 whentouse 4 export formats 30 working directory exporting to 29 setting 25 importing 25 refine the model s parameters 63 result 85 results files 32 setting the working directory 25 setting threshold levels manually 57 permutations 57 simulation 82 simulation data file 24 creating 51 single marker 83 single marker analysis 55 58 source data files 31 checklist for creating 44 creating from raw data 44 cross information 31 35 format of 39 map information 31 34 marker genotype data 31 32 opening 32 trait values 31 33 status bar 9 threshold levels 56 traits 33 tree pane 9 32 mouse click options in 10 troubleshooting 86 troubleshooting import errors 1 WinQTLCart compatible programs 1 exporting 29 features 1 high level overview 2 installing 2 source data files 31 system requirements 2 technical support 86 uninstalling 2 upgrading 2 2010 N C State University Bioinformatics Research Center
92. ormats 1 Exporting source data and results The following table summarizes WinQTLCart s export options Lane _ to in these formats Ea QTL Cartographer INP Map and cross data files BA Source data Main window QTL Cartographer MAD Map file a CRO Cross data file 2010 N C State University Bioinformatics Research Center so Windows QTL Cartographer 2 5 Source data minus Main window WinQTLCart 30 MCD with option to delete individuals with certain individuals with a certain OTrait OTrait values value Source data one or more Main window WinQTLCart 30 MCD with reverse marker chromosome s with position in some chromosomes reverse marker position Source data with certain Main window WinQTLCart 30i MCD with some selected traits selected traits Microsoft Excel Graph window __ Microsoft Excel Graph window TXT tab delimited Notes Files are exported to the current working director 25 Exporting source data to QTL Cartographer 1 With the source data displayed in the Main window s Data pane select File gt Export 2 In the Export Source Data dialog select one of the following options QTL Cartographer INP format INP files for both the map and cross data QTL Cartographer OUT format MAP for the map file CRO for the dross data file 3 Edit the Map or Cross filenames as needed 4 Click OK WinQTLCart exports the files to the current working directory Exporting
93. ound at http mmw cs wisc edu yandell atl software Bmapatl Bmapatl pdf High level workflow The first few times you run this analysis go with the WinQTLCart default values for the form s parameters The defaults provide the best all around parameter settings especially for initial analysis sessions 1 Select the BIM analysis method 2 Select the chromosome s and trait s you want to analyze 3 Click New Seed to select a new random seed 4 Click Parameters and set the BIM parameters of interest to you 5 Click Start to begin the analysis Running Bayesian interval mapping analysis 1 Open a source data file into the WinQTLCart main window 2 Select Method gt Bayesian Interval Mapping WinQTLCart displays the BIM analysis controls in the form pane iene Fans 1058995109 All Chromosomes D 3 Click Result File to name the QRT file you want to create and to specify its location 4 Click New Seed to generate a new random seed number 5 Select one or all chromosomes from the Chromosome Selection drop down to include in the analysis 6 Select one or all traits from the Trait Selection drop down to include in the analysis 7 Click the Parameters button to display the Set BIM Parameters dialog For more information on each of these parameters please refer to the R bmapatl documentation cited in the topic Bayesian Interval Mapping 7e 8 Click Start to begin QTL mapping analysis 2010 N C S
94. owing is a example of control file used for source data file envi jun3 mcd env2 jun3 mcd env3 jun3 mcd and env4 jun3 mcd There are totally 4 populations in the source data populations 3 1 3 4 line 1 population 1 line 2 traits 3123 line 3 qtls 4 1 46 09 2 155 23 3 60 09 3 172 53 line 4 epis 21223 line 5 population 2 line 6 traits 223 line 7 qtls 3 1 46 09 2 155 23 3 60 09 line 8 epis 113 line 9 population 3 line 10 traits 213 line 11 qtls 3 1 46 09 3 60 09 3 172 53 line 12 epis 0 line 13 pvsitest 2 line 14 p1i234 line 15 p3223 line 16 gatitest 2 line 17 stri 111 112 line 18 str2 211 212 line 19 end line 20 Token populations line 1 The first number indicates how many populations will be in the QTL mapping model The rest number s is are selected population s using index of selected populations comparing to all populations in original source data Token population line 2 6 10 This is the star line of parameters setting for each selected population Token trait line 3 7 11 The first number is the total trait number for current population and the rest is are selected trait index Token qtls line 4 8 12 The first number is the total QTL number and the rest are chromosome number and position in cM for each QTL Token epis line 5 9 13 The first number is the total QTL interaction number Then for each QTL epistasis there are two numbers that indicate which two QTLs that are i
95. ple files in the WinQTLCart directory Importing source data files 1 Select File gt Import The Source Data Import dialog appears Source Data Import Step 1 of 2 Select format of source data file QTL cartographer INP format inp mp map info This option can infer linkage map from cross data file cross info inp format c e 7 excel form QTL cartographer OUT format map cro emap C MapMaker QTL format map or mps raw C Microsoft Excel format D xls C Microsoft CS format D csv c Cancel Help 2 Select the import option you want The first extension listed for each option is for the Map file the second extension for the Cross Data file After clicking Next the second import dialog appears 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 27 Source Data Import Step 2 of 2 Data files for import Map File Infer map information from cross data file Cross Data Enter name of source data file to be created Directory c Home ScoreT est01 SC100116 5 ent mm 3 For the MapMaker QTL and QTL Cartographer options you need to locate and select the Map and Cross Data files by clicking the buttons For Excel spreadsheets you only need to specify one spreadsheet WinQTLCart disables the Cross Data button for Excel imports 4 By check Infer map information from cross data file WinQTLCa
96. qcart cro 5 Enter file name wqceart_out_In in edit box and click button Finish 2010 N C State University Bioinformatics Research Center ER Windows QTL Cartographer 2 5 Import data MapMaker format i Start WinQTLCart and select menu item File gt Import or click Import Select Mapmaker QTL format and click Next button Click button Map File to select file mapmaker samp map Click button Cross Data to select file mapmaker samp Enter file name mapmaker samp_In in edit box and click button Finish Import data Excel format i Start WinQTLCart and select menu item File gt Import or click Import Select Microsoft Excel format and click Next button Click button Excel File to select file wqcart samp xls Enter file name wqcart samp_xls In in edit box and click button Finish Import data CSV format 1 2 3 4 ii Start WinQTLCart and select menu item File gt Import or click Import Select Microsoft CSV format and click Next button Click button CSV File to select file NSimuB1 01 OnePop csv Enter file name NSimuB1 01 OnePop_in in edit box and click button Finish Simulation source data file _ Start WinQTLCart and select menu item File gt Simulation or click Simu 2 In step 1 3 set parameters as Sample size 200 Trait mean 15 85 Random seed any Map function Haldane Cross type B1 Translation table AA gt 2 Aa gt 1 A gt Total chromosome number 3 3 Set The current chromos
97. r WinQTLCart Windows amp Menus 23 Legend on right Check to show the legends to the right of the graphs Uncheck to show the legends above the graphs Show trace hairs By default WinQTLCart will not show X and Y cross hairs when you select use the Trace Position command Check this box if you do want to see the cross hairs Number of scale lines for X axis Specify the number of hash marks spread across the cM scale of the graph Number of scale lines for Y LOD axis Specify the number of hash marks spread along the LOD scale Number of scale lines for Y effect axis Specify the number of hash marks spread along the effect scale of the graph Space between two chromosomes Specify a distance as a percentage of the graph scale to separate the chromosomes in the graph Put in about 5 or 10 to see the effect Threshold value for traits Select a trait from the drop down list and enter a number to set as that trait s threshold value Click the Set all traits with this value button to impose a consistent threshold on all displayed traits Trait color and line styles Select a trait from the drop down list Press the Color button to select its color select a Line Style from the drop down list to further differentiate it from other traits in the display Maximum LR value in graph Check and input a value to limit the max LR not LOD value into the value for the LOD LR curve line default value is max LR value in the selected chro
98. r the more precise the model but the longer the analysis will take We recommend accepting the default value Click OK WinQTLCart builds the model based on the trait and the model parameters you selected Under the Cancel button in the form WinQTLCart displays its progress as it works through the file The process may take several minutes or several hours depending on your data You can safely minimize the WinQTLCart window and work on other apps in the foreground The MIM form redisplays with the buttons enabled the Parameters group fields populated the new model available in the drop down list and the model values on the right The Parameters fields are now populated To see the entire model wthout scrolling click the blue lt lt QTL cell To return to the MIM form click the blue QTL gt gt button Where to go from here From here you can refine the MIM modell zc manually edit the model by clicking the Add QTL and Del QTL buttons or click in the model field to change the value of Position Chromosome Additive or epistatic values Click Save Model to save the model as a MDS file Related topics About the MIM form e3 Refining the MIM modell zt Refining the MIM model After you ve specified the model and criteria for the search the model and its values are loaded into the MIM form At this point you can further refine the model From the MIM form click Refine Model to display the Refine MIM Model dialog 201
99. r 6 20 9 5 Bmac0090 1 13 Bmag0010 3 31 9 5 Bmsc0090 2 3 2 Bmsg0006 d 2 Bms290 02 Chr 11 3 Bmag0112 N 0 0 7 Bmag02223 2 Bmsag0225 49 7 Bmag0337 13 Bmsag0010 1 6 5 7 Bmagd294 3 Bmag0013 d 9 1 7 HVM20 2 Bmag0877 d 262 7 Bmsg0812 2 EBmso0541 3 EBmsG0705 d Chr 10 6 Bmacd316 6 EBmso0602 6 Bms00218 1 6 HVM31 Bmsg0496 6 Bmsg0009 6 Bmsg0867 6 Bmso0040 This sample graphic shows you the chromosome names the markers on the chromosomes and their distances Chromosome Graphic Display menus File menu Command Function Copy to Clipboard Print Graphic Exit View menu Command Proportion of Marker Number Proportion of Chromosome Len Next Page gt gt First Page Add QTL positions Copies the graphs to the Windows clipboard Print the graphic to a selected printer Closes the window If you have unsaved data you ll be prompted to save it Function Show length of chromosome graph in proportion of marker number Show length of chromosome graph in proportion of chromosome length in cM If there are lots of chromosomes displays the next group of graphs If there are more than one screen of chromosomes return to the first page Mark a QTL position on the chromosome See Adding QTL positions to the chromosome graphics ot for more information 2010 N C State University Bioinformatics Research Center so Windows QTL Cartographer 2 5 Setting menu Command Select Chromosomes Show Ch
100. r the double slash Token type Line 7 Indicates how marker positions are numbered along the chromosome It takes one parameter that can be either position or interval e Position indicates that the numbers are positions from the left telomere of the current chromosome So numbers should be in increasing order e Interval indicates that the numbers are the interval distance after a marker So the last number and only the last number in the series should be zero type position 0 0 9 3 17 2 29 9 38 7 52 8 57 8 TZA 76 6 93 2 97 0 115 5 116 5 interval 9 3 7 9 12 7 8 8 14 1 5 0 14 6 4 2 16 6 3 8 18 5 1 0 0 0 Token function Line 8 Indicates which map function is used to transfer recombination frequency r between markers to distance in Morgan M The parameter can be an integer from 1 8 Haldane and Kosambi are the two most useful map functions Code Reference Note 1 Haldane 1919 Default 2 Kosambi 1944 3 Morgan 1994 Fixed 4 Carter and Falconer 1951 5 Rao etal 1979 O p i 6 Sturt 1976 L 7 Felsenstein 1979 lt K lt K 2 8 Karlin 1984 Binomial N gt 0 The following Haldane and Kosambi formula can be used to convert marker distance from r to M or vice versa Haldane dM 0 5in 1 2r r 0 5 1 exp 2dM Kosambi dM 0 25In 1 2r 1 2r r 1 exp 4dM 2 1 exp 4dM Token Units Line 9 Indicates unit of marker positions There are three choices e cM centiMorgan e M Morg
101. re your data file is clean then move on more sophisticated analysis methods such as Interval Mapping 5 and Composite Interval Mapping 60 How it works Single marker analysis is based on the idea that if there is an association between a marker genotype and trait value it is likely that a QTL is close to that marker locus Comments Single marker analysis can be somewhat useful for a quick look at data but it has been superceded by Interval Mapping and Composite Interval Mapping IM and CIM are more thorough and accurate indicators of QTL The prime value of WinQTLCart s single marker analysis is its identification of missing data that could affect later analysis Running a single marker analysis 1 Open a mapping source data file an MCD file into the WinQTLCart main window 2 Select Method gt Single Marker Analysis WinQTLCart analyzes the data and displays the single marker analysis controls in the form pane The information pane on the right includes the analysis results 3 Select a trait for display from the Trait Selection pull down list All the traits present in the file will be on the list 4 For each trait the information pane on the right displays WinQTLCart s statistical summary of the 2010 N C State University Bioinformatics Research Center s Windows QTL Cartographer 2 5 file You can view this summary in a larger window by clicking the Result button in the Statistical Summary group box just to t
102. retaining certain individuals chromosomes You may find that you need to add edit or delete data due to import errors Some errors can be caused by mistaking cross data for map data and vice versa WinQTLCart s tools for editing marker trait and other information offer a safer and more organized approach than if you were to do the same thing by hand in a text editor If you need to alter the source data s information in any way it is highly recommended you use WinQTLCart Opening source data files There are several ways to open a prepared source data file from the Main window e Select File gt Open e Click the Open button on the toolbar e Use the keyboard command Ctrl O e Double click or right click on the Source Files node in the Tree panel 10 Any of these methods opens the standard Windows Open dialog box The dialog defaults to the current working directory 25 If you open an MCD file from a different directory WinQTLCart will save the mapping result files to that location Also you can double click a MCD file in Windows File Explorer to open the source data file automatically Working with a source file s marker genotype data At the Source data view and modify pane on the Main window select the chromosome you want to work with from the pull down list and click Markers button to open the Marker Information dialog Marker values Chromosomel C1 Markers Chromosomel C1 At the Marker Information dialog Mark
103. rocedures 29 Anchor marker parameters set Current anchor marker f H Maker label Chromosome Number Position on chromosome AXA f H On Left Cancel Note e f the import was successful you ll see a dialog saying the files were successfully imported and the source data file has been saved e f the import didn t work the reason is likely that the files formatting does not conform to a standard WinQTLCart expects To troubleshoot 8e this problem open one of WinQTLCart s sample files in Notepad and compare it to the file you specified The topic MCD file Tomat adi in this manual also describes the file format Correct any formatting problems in your specified meand try importing again If you re still unsuccessful please contact WinQTLCart tech support 86 e More detail help for Emap function see QTL Cartograhpers manul Notes e WinQTLCart includes sample source data files for import Run some tests using these files or open them up to see the kind of data formatting WinQTLCart expects to see e For Excel worksheets WinQTLCart expects to see the following worksheet names in the file BasDat ChrDat and CroDat WinQTLCart includes a sample file NewMcd xls that demonstrates the formatting it expects to see If you want you can make a copy of NewMcd xls and modify it for your data e For INP format one cross data file is needed if you use Emap to infer map information Related topics Compatible programs and f
104. rocedures 43 individuals must be the same for each marker Order by individuals For each individual you provide the genotype data for all markers all chromosomes The order of chromosomes and markers has to be the same start individuals markers TAAL 42 2 An he oA AAA 2 End 2 2 12 TL 22 2 2 Ends 2 52 2 2 2 21 ae 3 Ind4 21 2 2 2 2 4 Inds 2 Te 25 2h Ar stop individuals markers Note that the tokens are different and the first column is the individual s label Token start traits and stop traits Line 110 and Line 114 Use these tokens to start and end the trait values The data should be organized by the trait s order for the trait value That is for each trait you give the trait value of all individuals If organized by the individual then for each individual you provide the trait value of all traits start individuals traits 2 Trait_1l Trait_2 named Indl 5 0 15 0 Ind2 5 3 1543 Ind3 6 2 16 2 Ind4 4 1 24 1 Ind5 54 9 25 5 stop individuals traits Note that the tokens are different and there are trait number and trait labels after the token start individuals traits The label named means data has trait names Token start otraits and stop otraits Line 115 and Line 118 Use these tokens to start and end the other trait values The data must be organized by the other trait s order that is for each other trait you give the other trait value of all individuals If you order by indi
105. romosome Name Font Size gt gt Font Size lt lt Space Between gt gt Space Between lt lt Chromosome Name gt gt Chromosome Name lt lt Column Number gt gt Column Number lt lt Function Select the chromosomes you want displayed in the graphic Toggle between showing the chromosome name or its label Increase font size for graph Decrease font size for graph Increase the space between markers graph gets longer Decrease the space between markers graph gets shorter Go to the next chromosome in the series Go to the previous chromosome in the series Increase number of graphics displayed in a column Decrease number of graphics displayed in a column Adding QTL positions to the chromosome graphics At the Chromosome Graphic Display window select View gt Add QTL Positions to display the Add QTL Positions dialog box Add QTL Positions EZ Enter QTL chromosome and position The current QTL number fo e Total QTL number fo el In which chromosome fo QTL position cM fO Cancel 1 Click the spin dial in the Total QTL number dialog to select the number of QTLs you want to add to a graphic 2 If you re placing more than one QTL in the Current QTL number box use the spin dial to select which QTL you want to place 3 For the In which chromosome field use the spin dial to select the chromosome that will hold the QTL In the QTL position cM field enter the position of the current
106. rt will infer map information from cross data file by using Emap function 5 Enter a file name for the source data file that WinQTLCart will create You don t need to specify an extension WinQTLCart will take care of that 6 Click the Finish button 2010 N C State University Bioinformatics Research Center 2 Windows QTL Cartographer 2 5 EMap Parameters Setting Anchor Marker Setting Total anchor markers Random seed 1038932410 Parameter a Si Linkage map method 10 D Permutations 0 Notectve Ii Segregation test size 0 D Ei Map function Haldane Linkage test size 0 26 m Objective function SAL Cancel 7 Emap parameters setting dialog will pop out if you use Emap function to infer map information Click the button to change random seed for Emap function Linkage map method can take value 10 11 12 or 13 Segregation test size can be 0 01 to 0 20 Linkage test size can be 0 15 to 0 49 Now permutaion function is not active Map function can be Haldane or Kosambi Objuective function can be SAL or SAR 8 Set anchor markers by using control group of Anchor marker setting Click to select anchor marker number Click button parameter to open window of Anchor marker parameters set Select marker label chromosome and position on chromosome for each marker Current anchor marker Click button OK to finish parameters set 2010 N C State University Bioinformatics Research Center WinQTLCart P
107. select the text with your cursor and copy the selected text to the clipboard For MCD source data files WinQTLCart color codes the data so you can easily determine what are comments labels headers and so on Graph window Menus From the Main window select View gt Visualize Result to display the result file qrt in result graph window Menus in Graph window include File Chroma Traits MA Effects Tools 15 and Setting 15 In addition to the toolbar and menu commands some functions are available by right clicking on the graph 2010 N C State University Bioinformatics Research Center WinQTLCart Windows amp Menus 13 Graph window Menus File SE Open QTL Result File Open a result file Files with the QRT extension are considered result files Add QTL Result Graph Adds a new graph to the current display Files with the QRT extension are considered result files Note The added result file should have same chromosome number and marker number as original one You could add more than one new graph Sa Copy Graphic to Copies the graph to the Windows clipboard Clipboard Save As New Name Save the file under a different name in QRT format You may want to do this if you plan to work with the results in a later WinQTLCart session the text file in another program E Save As Text File Save the results as a text file You may want to do this if you plan to use the graph Save As EQTL File
108. source data to an MCD file Although WinQTLCart saves source data to its own MCD format you can use the export dialog to strip an MCD file of individuals with OTrait values You would do this when you want to analyze the data separate from the traits A MCD file delete individuals with a certain OTrait value With the source data displayed in the Main window s Data pane select File gt Export In the Export Source Data dialog select MCD file delete individuals with a certain OTrait value Edit the source file name as needed The file will be saved to the current working directory 25 From the OTrait Value pull down menu select the trait to be stripped from the MCD file Click OK WinQTLCart exports the file to the current working directory 25 IRON w MOD file reverse one or several chromosomes With the source data displayed in the Main window s Data pane select File gt Export In the Export Source Data dialog select MOD file reverse one or several chromosomes Edit the source file name as needed The file will be saved to the current working directory 25 From the Chrom Number Edit Box type in chromosome numbers separated by comma Click OK WinQTLCart exports the file to the current working directory 25 IRON 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 31 C MCD file only selected traits are included With the source data displayed in the Main window s Data pan
109. sseesensseneseneneeeeaeeneeseeseneeseneseeeseeeseeeseneseeeseesseaeseeteeeseatens 7 Chromosome graph Menus Setting nnnenennnnnennnnnennnnnennenennennnns 8 Main WINdOW TOUr 2 c idee en deeee SEENEN SEENEN bonus 9 Main window Tree Pame Eeer 10 Mai Window Form EE 12 Main Window Data Pane 1 028 500 aa aeaaaee n iaaea aae aa naiai aaaeeeaa 12 Graph window Monu S erisin rnense rana ARANAN PAON AE VENA TAARA 12 Graph window Menus File uuu cc eeeetesteesesseesessesseveevseeseeseenoeseesaesaesseseeseesoeseesaesaesaesaeusevanvaneaneaneneesnesaesaneaes Graph window Menus Chrom ou cc ceetstessseesessesseseesseeseeseeseeseesaeseesaeseesesseeseesaesaesaesaevaevenvaneaneaeeneesnesaesansans Graph window Menus TraitS ni srrnrereneaneaneaeannnnnesnesnennesneenreanannenasnasnnsnnsneeneaneaneneananene Graph window Menus Effects ui nrnrirenranranrennennennennesnenneeanenenasnasnnsnneneeneanenrnrnranene Graph window Menus Tools ini rrrnreneeneaneaneaneanrannenrennenneeneenneanennesrasnasnnsnnsneeneaneanenrnranente Graph window Menus Setting iinnnnrnrannnneneneenennesnesnesneeneeneeneeneeneannnnnnenee One page display window Menus ssssnsennneeneennneenennnneenennnnse One Page window Menus File ssccssssssesseeseesscessneneeesensneesseeennessneceneseneeseeseeesneneesseeeseessnecseessnenseesenensness One Page window Menus View One Page window Menus
110. t Open File gt Open with Notepad gt Refresh gt Close File 2010 N C State University Bioinformatics Research Center Function Show file contents in the Data pane and set as current working MCD file Open the MCD file in Notepad Open a new source data file Open the MCD file in Notepad Re load the file after modification Start single marker analysis Start interval mapping Start composite interval mapping Start multiple traits analysis Start multiple interval mapping Start Bayesian interval mapping Function Show this result file in text format via the Data panel Open the QRT file in the Graph window 17 Open a new result file Open the QRT file with Notepad Re load the file after modification Close the result file Open the QRT file in the Graph window Function Show this text file in the Data pane 1 Open the text file with Notepad Open a new text file Open the TXT file with Notepad Re load the file after modification Close the text file 12 Windows QTL Cartographer 2 5 Main window Form Pane The Form pane is the control panel you use to analyze your source data It serves as a dashboard that presents a lot of information about your source data file at a glance The Form pane is keyed to the MCD source data file only it does not show information for any other file format This control panel changes based on the analysis method you select For each analysis method WinQTLCart displays di
111. t nolab txt e Other trait values and labels One file for all other traits Case 1 Individual order with other trait label See 523 ot ind lab txt Case 2 Individual order without other trait label See 523 ot ind nolab txt Case 3 other trait order with other trait label See 543 ot ot lab txt Case 4 other trait order without other trait label See 543 ot ot nolab txt An alternative to use separate files is to create a single file holding marker genotype trait and other trait information But the data must be in individual order Case 1 has individual label See 511 mkttot lab txt Case 2 has not individual label See 51 1 mkttot nolab txt 1 Select File gt New to open the Create New Source File Step 1 of 6 dialog box 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 45 Create New Source File Basic Information Step 1 of 6 Basic information P Chromosome linkage group number 8 Trait such as yield or weight number 3 AA Other trait binary value such as sex number 2 H Aa Individual sample size number 50 Symbol for missing trait value Cross type BI RP H Filename C ANCSU WinQT LCart2 5 Add New NewMcd01 mcd Fill in the dialog based on your source data Basic information group box Chromosome number Enter the number of chromosomes in the source data Trait number Enter the number of traits in the source data Other trait number Enter the num
112. ta to input chromosome labels 7 Select and copy Marker Number in Notepad Click button Paste click Marker numbers in Data Type Selection and click button Send Data to input marker numbers 8 Select and copy Marker Label and Position in Notepad Click button Paste Select Marker positions in Data Type Selection and click button Send Data Answer Y in the dialog to input marker labels and positions 9 Select and copy Marker Genotype and paste Click Marker genotypes and click button Send Data Answer Y to input marker genotype data 10 Select copy and Paste Trait Value Select Trait Values and click button Send Data Answer Y to input trait values 11 Select copy and Paste Other Trait Value Select OTrait Values and click button Send Data Answer Y to input other trait values 12 Click button OK to create source data file Test mcd DF w Single marker analysis E 1 Select Menu Item File gt Open or click OPEN to open source data file wqcart samp0 mcd Select Menu Item Method gt Single Marker Analysis to open the Form of single analysis 3 Use the poll down menu to select a trait Click button Result to show information of single marker analysis or statistical summary Click button Graph File to produce result file and show in graph dialog 5 Click button OK to finish single marker analysis D gt 2010 N C State University Bioinformatics Research Center Windows QTL Cartographer 2 5 Interval mapping Ge 1
113. tate University Bioinformatics Research Center Windows QTL Cartographer 2 5 Multiple trait analysis Most of the analysis methods in WinQTLCart work on traits one by one The multiple trait analysis lets you pick and choose the traits you want to work with jointly This allows you to perhaps derive and correlate structures from among many separate traits 1 2 Open a source data file in WinQTLCart If the data contains multiple traits WinQTLCart enables the Method menu s Multiple Traits Analysis command Select Method gt Multiple Traits Analysis The Multiple Traits Analysis dialog appears WinQTLCart disables portions of the dialog box that cannot support analysis of the selected data file e BI crosstype has additive effects and enables only method selection e F2 crosstype has dominant effects and enables hypothesis test selection open the WinQTLCart sample file cod mcd for an example Select the analysis method you want to use IM or CIM Select the traits you want to use in the analysis Click the Trait List button to see available traits for selection In the Trait selection text box enter the traits you want to use separated by commas So if you want to use traits 1 4 and 5 you would enter 1 4 5 without the quote marks Click OK The form pane takes on the standard IM CIM look and function but the countdown pane prefixes the method with Multiple Traits For permutation procedure check Independent check box to r
114. th the same parameter setting The filename look like SimuRep 1 dat and the mcd is the simulation MCD file Sample size Individual number Map function Haldane assumes no crossover interference or Kosambi assumes some crossover interference Total Trait Indicate how many traits in the simulation data Current Trait You can change the current trait and set parameter of Trait mean Cross Information Select an experimental design option You can select only one option to modify at a time At this time only the three options below are available for selection e B1 Backcross with 1 parental line to which the F1 line was crossed e B2 Backcross with 2 parental line to which the F1 line was crossed e SF2 Selfed intercross line generation 2 Translation table Edit or add tokens you want to use for these genotype markers you can enter any alphanumeric character s as a token Note WinQTLCart assumes that the A allele is diagnostic for the High parental 1 line and the a allele is diagnostic for the Low parental 2 line A minus sign means the allele is unknown 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 53 WinQTLCart uses the numbers 2 1 0 12 10 1 to determine how to encode the output of the genotypes The alphanumeric tokens you enter here indicate how you have coded the markers in the source data See the topic MCD file ona z i for more information Chro
115. topic Although WinQTLCart can t automate this process for you it does provide you with tools to make a clean transition from raw source data to rigorously formatted data file The easiest way to work with this function is to have individual properly formatted text files for each data type marker genotype individuals etc Individual files are quicker to select and import Otherwise you do have to do a lot of copy and paste Here s a checklist of files you should have on hand along with sample files in the WinQTLCart2 5 Add New directory you can use to create your own files e Chromosome labels and number See 201 chlab mknum txt e Marker labels See 301 mklab txt that shows one file for all chromosomes e Marker positions See 302 mkpos txt that shows one file for all chromosomes e Marker genotypes One file for all chromosomes and there are 4 different cases Case 1 Individual order with individual label See 521 mkgen ind lab txt Case 2 Individual order without individual label See 521 mkgen ind nolab txt Case 3 Marker order with marker label See 541 mkgen mk lab txt Case 4 Marker order without marker label See 541 mkgen mk nolab txt e Trait values and labels One file for all traits Case 1 Individual order with trait label See 522 tt ind lab txt Case 2 Individual order without trait label See 522 tt ind nolab txt Case 3 Trait order with trait label See 542 tt tt lab txt Case 4 Trait order without trait label See 542 tt t
116. ts certain markers as control markers by using additional parameters control marker number and window size Selecting this model requires you to fill in extra fields on the dialog Control marker numbers Window size cM and Regression method selection all explained below 2 Click Set control markers manually if you do not want WinQTLCart to calculate the number of control markers This will display a dialog box after you start the analysis so that you can manually select the control markers Skip to the end of this topic for a description of this dialog box 3 Control marker numbers Enter the number of markers to control for the genetic background WinQTLCart will use up to the number of markers entered here 4 Window size cM Enter the window size in centiMorgans The window size will block out a region of the genome on either side of the markers flanking the test site Since these flanking regions are tightly linked to the testing site if we were to use them as background markers we would then be eliminating the signal from the test site itself If the control marker number And if the window size is This is the result is The total number of markers on Model 6 reduces to Model 1 The total number of markers Large such as the size of the Model 2 es Recommendations e Model 6 is good for starting an analysis e The default values of 5 for control markers and 10 for window size should be good starting points for Mod
117. um threshold See the Setting the threshold levell5 topic for more information on the impact of each of these choices 4 Click OK to start the calculations for the threshold level This may take from several minutes to several hours to run 5 Following threshold calculation set CIM form parameters 60 Select a walk speed in cM It s recommended you use the same walk speed for your entire dataset Don t reset the walk speed between runs or your results will not be comparable 6 Click Start to begin the analysis The analysis may take from 20 minutes to several hours to run Running composite interval mapping analysis WinQTLCart provides default values for the parameters in this form The defaults provide the best all around parameter settings especially for initial analysis sessions Composite interval mapping analysis uses WinQTLCart source data mapping files MCD files Use WinQTLCart s import commands to move your source data files from text to MCD format 1 Open a source data file into the WinQTLCart main window 2 Select Method gt Composite Interval Mapping WinQTLCart displays the CIM analysis controls in the form pane 2010 N C State University Bioinformatics Research Center WinQTLCart Procedures 61 S m Threshold of Trat Result File 54salinity C qrt Control Gem etas Default Composite Interval Mapping Analysis By Manual Input Precision Selection Walk speed cM 2 el
118. ve multiple results files loaded and multiple Graph windows open at a time Menu bar MA Toolbar with one click access to the program s major functions Hover the pointer over a button to see a brief description of that command The large graph charts the data as a LOD or LR score profile The higher the LOD the greater the evidence for a QTL The smaller graph at the bottom is the QTL effects window showing additive or dominant effects or R2 or TR2 or S values Graph window tips You can add several result files so they display at the same time on the current graph You might want to run your data through the IM CIM and MIM analysis methods for example and then pull them all into the same graph to see how they compare e Peaks above the threshold line indicate a QTL A high LOD value on the graph indicates a good QTL candidate Right click on the graph to see appropriate commands Commands described in the Graph window Menus 12 topic You can minimize the Graph window to the bottom of the Main window a small bit of the title bar is visible You can have the same result file open in several windows at the same time This might be useful if you re testing various viewing parameters To do this minimize the current Graph window go back to the Main window ensure the result file is still active and click the Graph toolbar button 2010 N C State University Bioinformatics Research Center WinQTLCart Windows amp Menus
119. ve your source data in MCD format and your results files in QRT format so you can work with them later in WinQTLCart You can also export your results to other selected formats See these topics for more information Exporting source data and results 2 Exporting results from the Graph windowls1 Exporting source data to an MCD filefso Exporting source data to QTL Cartographer 30 2010 N C State University Bioinformatics Research Center Windows QTL Cartographer 2 5 When to use WinQTLCart You can use WinQTLCart for any kind of data that is cross populations from inbred lines WinQTLCart is a particularly powerful tool when you want to explore your results graphically Prior to doing experiments you could use WinQTLCart to explore some what if scenarios in planning your experimental design In WinQTLCart you can create simulation data and then vary parameters setting to explore various QTL models However if you re working on a repetitive task that would be better off scripted you may want to turn to QTL Cartographer WinQTLCart s command line sibling For example if you have expression data with thousands of features you might want to run interval mapping on each feature which would take a long time This task can be automated via a shell script and made to run overnight The results can then be imported into WinQTLCart and its graphs charted WinQTLCart Windows amp Menus Main window Menus Menus in Main window
120. viduals then for each individual you give the other trait value of all other traits Example start individuals otraits 2 sex brood named Indl M 1 Ind2 F 1 Ind3 M 0 Ind4 M 1 Ind5 M 1 stop individuals otraits Please notice that the tokens are different and there are other trait numbers and other trait labels after the token start individuals otraits Label named means data has other trait names Token quit and end Line 119 and Line 120 Indicate the end of the source data file Token population Indicate the population number and the rest of MCD filename Please notice that you should build one MCD file for each population Example 2010 N C State University Bioinformatics Research Center Windows QTL Cartographer 2 5 population 4 env2 jun3 mcd en8 jun8 mcd env4 jun3 mcd There are totally 4 populations and the 4 MOD files include current file and file env2 jun3 mca file envB jun3 mcd and file env4 jun3 mcd All MCD files should be in same directory Token xchromosome Indicate the X chromosome number and cross type that will not be the same as rest of chromosomes Creating a new source data file from raw data WinQTLCart can help you convert your raw source data including map and cross information into formatted MCD files WinQTLCart can run analysis methods only on MCD files The goal of this process is to create a file that looks like the MCD data file described in the MCD data file format 3
121. w s cells to toggle their display in the text box Where to go from here From here you can refine the MIM modell zc manually edit the model by clicking the Add QTL and Del QTL buttons or click in the model field to change the value of Position Chromosome Additive or epistatic values Click Save Model to save the model as a MDS file Related topics About the MIM form e3 Refining the MIM modell zt MIM search option 1 At the Select Parameters dialog select a model selection criteria from the drop down list Select Parameters Criteria of MIM model selection Score 0 05 significant level MIM search walk speed in cM j H BIC MO gt c n In n 2010 N C State University Bioinformatics Research Center Windows QTL Cartographer 2 5 AIC gt C n 2 BIC M1 gt c n 2ln In n BIC M2 gt c n 2ln n BIC M3 gt c n 3in n BIC X gt c n 10 X In n Score 0 05 significant level Score 0 10 significant level Score 0 20 significant level Score X significant level Note The first 6 options are BIC search criteria BIC n In Q Q p c n n sample size Q Q residula varance of model p regressor marker number Choose last 4 options Score WinQTLCart will use score statistics not LR to do the forward search for both main and epistatic QTLs as initial MIM model 2 Note Click the spin dial beside MIM walk speed in cM to select the walk speed The smaller the numbe
122. y test the interaction effect only Aavailable in Score statistics test situation 2D Scan of 2 new QTL and Interaction Search two new main QTLs plus interaction between them by test the interaction effect only Aavailable in Score statistics test situation 3 Testing existing QTLs Main QTLs Test each main QTL to see it is significant or not The QTL will be deleted from the MIM model if it s not significant In Score statistics test to check Only QTLs without interaction check box will do test 2010 N C State University Bioinformatics Research Center Windows QTL Cartographer 2 5 only on those main QTL s that have no interaction with other main QTL s The reason is that WinQTLCart is allow existing of main QTL that has no very little effect but has strong interaction effect in score statistics test situation QTL Interactions Test each QTL interaction to see it s significant or not Clicking Start returns you to a slightly modified MIM model where the operation will continue until the result is obtained Note To create a MIM results file in QRT format select the MIM model summary option Multiple trait MIM Multiple QTL Mapping MIM for multiple trait or multiple environment in multiple population About the Mt MIM form From the Source Data form open a source data file and select Multiple Interval Mapping as the analysis method If there is more than one trait in the dataset the Select Trait for MIM Analysis d

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