Step-by-Step Guide to Bi-Parental Linkage Mapping WHITE PAPER

Transcription

1 Step-by-Step Guide to Bi-Parental Linkage Mapping WHITE PAPER

2 JMP Genomics

3 Step-by-Step Guide to Bi-Parental Linkage Mapping Introduction JMP Genomics offers several tools for the creation of linkage maps using marker data from bi-parental mapping populations. These tools can accommodate framework maps, fitting new markers in and around framework markers that have fixed linkage group membership (and optionally a fixed order). The main steps in generating linkage maps include: 1) dividing the markers into distinct linkage groups based on their recombination frequencies, 2) determining the order of markers within linkage groups, 3) calculating genetic distances between them, and 4) assembling the results into a map. This guide will present detailed examples of linkage mapping in JMP Genomics, both with and without framework maps, using the Basic Linkage Mapping Workflow. This workflow performs the grouping, ordering and display steps from a single dialog. Advanced processes, not available from the workflow dialog, will be accessed from individual process dialogs (found under Genetics > Linkage Maps and QTL in the Genomics Starter). Objectives In this document we will cover the following tasks: Learn how to run the Basic Linkage Mapping Workflow with or without a framework map. Learn to interpret output, and to examine and adjust the linkage group membership. Explore advanced options. 1

4 SAS JMP White Genomics Paper Input Data To build linkage maps in JMP Genomics, you must start with a marker genotype data file containing the data from an experimental cross. The data file must be in standard wide format, with lines (plants) in rows and markers in columns. Our example data comes from a cross of oat recombinant inbred lines derived by selfing. We start with the file oat_markers.sas7bdat, which has 90 lines and 466 markers. The example data set (shown below) is available for download with this document. The genotypes are in numeric format: homozygous genotypes are represented as 0 and 2, while heterozygous genotypes are represented as 1 (Note: This data set contains no heterozygous genotypes.) There is a framework map for this example, found in the oat_framework.sas7bdat file. There are 172 rows in the framework map file these are a subset of the original 466 markers for which we have a known map position. When this framework map is included in the analysis, the rest of the markers will be fit in and around this subset. 2

5 Step-by-Step Guide to Bi-Parental Linkage Mapping Note that the Marker column in the framework file matches the marker names found in the data file. The framework map may consist of grouping information only, or both groups and positions or orders of the framework markers, as in this example. Annotation for the markers may or may not be present. When included, this information is generally presented in a separate annotation file. Annotation files may contain any arbitrary information about the markers. We are not using an annotation file for this example. However, if we had included one, the format of that file would be very similar to the framework map file, with one row for every marker in the data set. For all analyses in JMP Genomics, the order of the rows in an annotation file must be the same as the order of the marker columns in the genotype data file. If you need to adjust the order, use the Subset and Reorder Genetic Data tool found under Genetics > Genetics Utilities in the Genomics Starter menu. 3

6 SAS JMP White Genomics Paper Setting up the Basic Linkage Mapping Workflow 1. Launch the Basic Linkage Mapping Workflow process from Workflows > Basic from the Genomics Starter menu. 2. Select oat_markers.sas7bdat as the Input Genotype SAS Data Set. 3. Highlight all the marker variables in the Available Variables list (all variables except Line) and move them to the Marker Variables box. Another option is to make a list of all markers between the first and the last. To use this method, type GMI_ES_CC12738_ GMI_ES01_c7940_496 into the List-Style Specification of Marker Variables box Choose an Output Folder to hold the result files. 5. Specify Oat Map as the Workflow Output Name. 6. The completed General tab should appear as shown below: 7. Select the Options tab. 8. Select Numeric Genotypes as the Format of Marker Variables. 1 You can access the context-specific Help for any parameter by clicking on the question mark (?) link next to the parameter. For example, click the List-Style Specification Help link for more details about the syntax used for specifying lists of variables. 4

7 Step-by-Step Guide to Bi-Parental Linkage Mapping 9. Leave the Annotation and Framework Map tabs blank. We will adjust this analysis to include the framework map later. 10. Select the Linkage Groups tab and complete the tab as shown below: Select the Cross Type corresponding to your experiment. The cross used in this example was RILself. If your experimental cross has a different set of expected segregation ratios than the standard ratios, you can specify custom ratios in the box labeled Expected Segregation Ratios for AA AB BB. See the question mark icon help for this item for more details. Linkage groups are formed on the basis of a hierarchical clustering analysis, and three clustering methods are available here: Average, Complete, and Single. The default Average method is robust and performs well in many situations; try one of the others if results are unsatisfactory. The number of linkage groups can be determined as a specified number (based on your intuition or knowledge of the organism), or by setting a recombination fraction cutoff. 5

8 SAS JMP White Genomics Paper 11. Select the Linkage Map Order tab and complete the tab as shown below: Linkage Groups with large gaps in genetic distance or large recombination fractions can be automatically subdivided here. The Run Subset and Reorder Genetic Data process to order marker data for QTL analysis checkbox will create a new data file and annotation file in the order of the newly computed linkage map, which can be used for subsequent QTL analysis. 12. Leave the Linkage Map Viewer tab options at their default values 13. Click Run in the lower left to launch the analysis. 6

9 Step-by-Step Guide to Bi-Parental Linkage Mapping Basic Linkage Mapping Workflow Output When the computations are finished, a workflow journal will appear: Four processes were run as part of the workflow: 1. Recombination and Linkage Groups, 2. Linkage Map Order, 3. Linkage Map Viewer, and 4. Subset and Reorder. Results can be viewed by clicking the blue Results links; the individual process dialogs with advanced options can be viewed by clicking the blue Reopen Dialog links. To review the dialog that launched this analysis, click the Reopen BasicLinkageMappingWorkflow Dialog button. 7

10 SAS JMP White Genomics Paper Recombination and Linkage Groups 1. Click the Results link for Linkage Groups (the first process in the list above). 2. Inspect the Linkage Group Results tab 2. The graphic shows the recombination frequencies for the first one of the 22 linkage groups. To examine different linkage groups, click the LGroup rows in the data filter on the right, or click the play ( ) button in the Animation Controls panel. The number in parentheses to the right of the linkage group label is the number of markers in the group. For example, there are 33 markers in linkage group 1. The heatmap is a visual representation of the matrix of recombination frequencies. Hot colors are regions of low recombination, and cool colors are regions of high recombination. To view the recombination matrix, click the Linkage Group Results button (in the Tabs panel in the upper left); choose View Data from the drop-down menu. Genotype frequencies for AA and BB are displayed in the dotplot on the left side of the heatmap. Each marker is represented by a pair of dots. When the dots are far apart, BB is more frequent than AA, and when the dots are close together, AA is more frequent than BB. If you wish to remove one or more ill-fitting markers from the analysis, select the corresponding rows in the heatmap or in the underlying data table and click the Exclude Markers and Rerun Analysis button. 2 You can access information on the organization of the Results dashboard by clicking on the Output Description link in the upper left portion of the window. 8

11 Step-by-Step Guide to Bi-Parental Linkage Mapping 3. Select the Segregation and Linkage Groups tab. 4. Examine the Bivariate Fit plot. The frequency of the A allele less the frequency of the B allele is plotted on the horizontal axis. Markers with higher A frequency are to the right; markers with higher B frequencies are to the left. The statistical significance of the segregation distortion test is plotted on the vertical axis. Values lying above the red dashed line are statistically significant (i.e., distorted from expected segregation ratios) at p < The different colors of the points represent the 22 different linkage groups, and counts for the number of markers in each linkage group are displayed in the histogram. To examine each linkage group separately, select individual histogram bars. 5. Click the Close All button to close the dashboard. 9

12 SAS JMP White Genomics Paper Linkage Map Order 6. Click the Results link for Linkage Map Order in the Journal. 7. Inspect the first tab, Linkage Order Results. A schematic overview of the linkage map is displayed. The vertical axis depicts genetic distance in centimorgans. Hover over individual markers in the plot to view the names. Note that there are now 24 linkage groups, rather than the original 22; this is due to automatic splitting of linkage groups with gaps greater than 40cM, as specified in the workflow dialog. The Reverse Linkage Group Marker Orders allows you to select individual linkage groups and reverse the display order of the markers, in order to match the order of established maps. 8. In the Tabs section, click Linkage Order Results > View Data. The data file oat_markers_lmo.sas7bdat is displayed. It contains all the markers and their linkage groups and computed genetic distances. 9. Double-click the thumbnail image in the lower-left of the data table window to return to the results dashboard. 10. Click on the Genotype Color Plots tab. 10

13 Step-by-Step Guide to Bi-Parental Linkage Mapping The plot shows marker genotypes, with ordered markers in columns and individuals in rows. Missing genotypes are denoted with X. 11. In the Action Buttons section, click the Linkage Group 12 button to view a recombination plot: 11

14 SAS JMP White Genomics Paper This interactive plot shows recombination rates between pairs of markers within the linkage group. The markers are labeled at the top of the plot, with a line pointing to their relative positions along the horizontal axis, representing genetic distance. The red triangular regions represent groups of markers with low recombination, and thus the genetic distances among them are small. Click inside the plot to highlight different blocks. The buttons along the bottom allow you to zoom in on a selected block, retain a block, and edit block labels. 12. Close the triangular recombination plot and the Linkage Map Order Results dashboard, and return to the workflow results Journal. Linkage Map Viewer 13. Click the Results link for the Linkage Map Viewer in the Journal. Two views are produced, a 3D view on the first tab, and a 2D view on the second tab. A section of the 3D view is shown below: You can adjust the sliders along the top of the display to optimize the visualization. There are many options in the Linkage Map Viewer dialog, such as coloring the markers according to different values of a variable, and filters to view subsets of markers. To modify the dialog, click the Reopen Dialog button from the Results dashboard, or click the Reopen Dialog link in section 3 of the workflow journal. 14. Close the Linkage Map Viewer dashboard and return to the workflow Journal. 12

15 Step-by-Step Guide to Bi-Parental Linkage Mapping Subset and Reorder Genetic Data 15. Click the Results link for Subset and Reorder Genetic Data in the Journal. This window contains links to output files that were created: oat_markers.sas7bdat contains the marker genotypes, and oat_markers_lmo_sr.sas7bdat contains the map information. These files have been reordered to match the newly computed linkage map order. They are now ready for QTL analysis (not discussed here). Adding a Framework Map This section describes a modification of the previous analysis, adding a framework map to fix the grouping and ordering for a subset of the markers. 1. Click the Reopen BasicLinkageMappingWorkflow Dialog button on the Journal dialog. 2. On the General tab, assign a new Output Folder so as to avoid overwriting previous results. 3. Type WithFramework in the Workflow Output Name box. 4. Select the Framework Map tab. 5. Choose the supplied oat_framework.sas7bdat file as the Framework Map Data Set. 6. Complete the rest of the tab as shown below: 13

16 SAS JMP White Genomics Paper Groups specified in the Framework Linkage Group variable will be honored in the grouping process, and the order of the elements in the Framework Order Variable will be used in the ordering process. 7. Select the Linkage Groups tab. Note that the Automated Hierarchical Clustering Options that were used previously are now grayed out, and instead you must use Minimum Recombination Grouping options. 8. Leave the Minimum Recombination Grouping option set to the default threshold value of Click Run to start the analysis. 10. Click on the Results button for the Recombination and Linkage Groups on the resulting Journal. 11. Select the Segregation and Linkage Groups tab on the Results dashboard. 14

17 Step-by-Step Guide to Bi-Parental Linkage Mapping 12. Click on the histogram bars in the original framework group s histogram (labeled Framework_Groups) to visualize the relationships between framework markers and new markers. In the segregation plot and elsewhere, markers from the framework map are shown as asterisks. 13. Click Close All to close the dashboard and return to the workflow results Journal. Manual Regrouping and Reordering, and Other Advanced Options Up to this point our analysis has used the Basic Linkage Mapping Workflow. This workflow simplifies the input, but it offers less flexibility than is available when running the processes separately. Certain advanced options can only be accessed using the AP dialogs. It is, thus, a good idea to examine the AP dialogs and documentation, to learn all the different options that are available. As an example, we show how to make manual changes to the grouping and ordering of markers. To manually specify linkage groups and marker order, you will need to create a file that contains markers in the desired grouping and ordering. One easy way to create this file is to run the Linkage Map Order process and then manually edit the resulting map file. In the interest of brevity, we will use the ManualOrder.sas7bdat edited file (shown below) that is included with the data described above. 15

18 SAS JMP White Genomics Paper The ManualOrder.sas7bdat edited file contains three columns: The Marker_Name column contains all the markers in the SNP data set. The LGroup column contains the desired groups. The Map_Order contains the orders of the markers. This file was edited to create a new linkage group (number 27) from five markers that were originally assigned to linkage group 1. Order of the five markers was also adjusted. Note: For the purpose of manual reordering, only the sorted order of values in the order column is important. The actual values have no inherent meaning or restrictions. 1. From the workflow results Journal, click the Reopen Dialog link for the Linkage Map Order process. The dialog for the Linkage Map Order AP is now open, preloaded with the values that produced our previous output. 2. Specify a new output folder on the General tab, so as to avoid overwriting existing results. 3. Click within the Framework Order Variable box and then click the leftwardpointing arrow button ( ) to remove the specified variable. This process can either run manual ordering or fit markers around a framework map, but not both at the same time. Because we are performing manual ordering in this step, we must remove the framework map information. 4. Select the Order Data Set tab, and choose the ManualOrder.sas7bdat file as the Order Data Set. 5. Complete this tab as shown below: 16

19 Step-by-Step Guide to Bi-Parental Linkage Mapping When a Marker Order Variable is specified here, the software does not attempt to compute an order for the markers; instead it takes the order as given by the variable and computes the genetic distances between adjacent markers. 6. Select the Analysis tab and uncheck the Break linkage groups between markers with large ordered distances checkbox. 7. Click Run. 8. Inspect the new Results dashboard for Linkage Map Order. 17

20 SAS JMP White Genomics Paper 9. Examine the newly formed linkage group number 27 on the far right, with the five chosen markers arranged in the desired order. Conclusion We have learned about the tools in JMP Genomics 6.0 for creating, modifying, and displaying linkage maps. The resulting maps can be used for QTL analysis. 18

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