Mapdisto Version 2.0

MapDisto version 2.0

By Mathias Lorieux

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Manual: mapdisto.free.fr/Tutorials/files/MapDisto%202.0%20Tutorial.pdf

Installation:First, download the program at Go to the section Download, and follow the instructions. When you are done with the downloading, Unzip the downloaded file.Then, create a separate folder for your mapping project on your desktop, and copy the unzipped MapDistofilesin your mapping project folder that you created. This folder will contain all the files related to your mapping. Therefore, you should also copy and paste the genotyping file that you want to work with in this folder. (For our tutorial we have a mapmaker formatted file of Arabidopsis genotyping data).

Now Open MapDisto 2.0.b106.XL2013.Win.xlsb file. Click start icon which appears on the MapDisto screen. If “Enable Editing” and “Enable content” appeared on the toolbar of the excel file, don’t forget to click on them first and then click “Start” icon. The excel file that you see after clicking on “Start” icon is called Main menu

Basic Mapping steps

Step 1: In the “Linkage groups” sub-menu, click on “Clear linkage groups” and then click “yes”. Scroll up through the excel sheet to make sure no data is remaining.

Step 2: in the main menu click on “Data” icon. It will take you to the Data sheet. In the “Genotypes & Traits” menu In the Data sheet click on “Clear Data”. Click “yes” and then click “ok”.

Step 3: In the “Data” sheet, click on “Import data…” icon. Put number “1” in the dialog box (because your genotyping file is formatted for Mapmaker). Give the path of your mapping project and choose your genotyping file. Make sure to choose the correct population type in the “Population type” icon. For our tutorialtype “SSD” for population type. When the data are imported, click on “Read data” to check the validity of the data.

Step 4: click on “Main menu” in Data sheet. In the main menu click on “Find linkage groups” icon. In the Sequence for searching groups dialog box, click on Use all loci 3. In the Find groups dialog box, click on OK without altering the default values for LODmin and rmax. If you have missing data, a dialog box will ask you if you want to “Filter loci with too many missing data in 2-point matrices”. You can decide if you want to filter your missing data or not. For this project we want to filter missing data. So click on “yes” and enter number “50”in the dialog box. (It means you want to filter loci that have more than 50% missing data). Then click “ok”. The output of this command consists in sequences, which are series of linked molecular markers. For this tutorial you see 5 linkage group and oneunlinked group was created. The unlinked markers are the ones that you filtered them because of missing data. Validate the sequences with the “Add groups” command. Then click “Main menu” icon. Now the individual linkage groups are listed in your “Main menu”.

Step 5: In the “current working linkage group” icon put number “1”. It means you want to work on linkage group 1. Then click on “Order a linkage group“icon. This command implements SARF (Sum of Adjacent Recombination Frequencies) to find the best order of loci in the linkage group. The best order will have the lowest SARF. In the output, click on “Replace sequence” icon and then click on Main menu.

*Step 6 and 7 are used to locally improve your map for each linkage group.

Step 6:for the current linkage group use icon “Check inversions”. In the output it shows which block size between which loci can be inverted to give a better map. You can find the better map option in the “Best map from step ...” icon in the output. Click on “Replace sequence” icon and in the dialog box put number that you see in the “Best map from step“. Click “ok” and then click Main menu.

Step 7: for thecurrent linkage group use icon “Ripple order”. It uses the lowest SARF and SALOD/Log(L), or Sum of Adjacent LOD scores, to locally improve the map of your current linkage group. if in output it said “Ripple” did not find any better order”, just click on “Main menu” but if it indicated the “Best map from step…”, then Click on “Replace sequence” icon and in the dialog box put number that you see in the “Best map from step “. Click “ok” and then click Main menu.

Step 8. Repeat steps 5 to 7 for other linkage groups one by one.

Step 9. Once you done with all steps to improve your mapping, click on “Draw all linkage groups”. You can save your linkage map as PDF by clicking the “Save PDF” icon in the output.

If you are not satisfying with the font of markers names or distances on your linkage map, just go to the Main menu and click on the option. Here you can change your criteria or formats. And then run “Draw all linkage groups” again.

Step 10. To see some statistics of your linkage map, you can use different command by clicking on “detailed map” or “write a map”, “compute tables” or “show assignments”. Copy past your tables in another excel sheet for your report.

Alternative options for Mapping:

1-Your input map file can be imported with an excel format rather than mapmaker format. Arrange your data in a separate Excel worksheet, and then simply paste them into the Data window.

Here is how to proceed:

A)Prepare a matrix of data in a separate Excel worksheet. The data should look like as the ones showed in Figure below. This is an example of a genotyping data matrix of n = 8 individuals and m = 11 markers or loci.

marker / R31_01 / R31_02 / R31_03 / R31_04 / R31_05 / R31_06 / R31_07
ss715650814 / A / A / A / A / A / A / B
ss715642652 / A / A / A / A / A / A / B
ss715640195 / A / A / A / A / B / A / B
ss715641657 / A / H / A / B / A / B / A
ss715647488 / - / A / A / B / B / A / A
ss715649186 / - / A / A / B / B / A / A
ss715648335 / A / A / A / A / B / A / B
ss715649420 / A / A / A / A / A / B / A
ss715649854 / A / A / A / A / A / A / B
ss715645337 / A / A / B / B / B / A / A
ss715649616 / A / B / A / A / H / B / B

B)Open yourMapDisto. Do step 1 and 2 to clear the data. Now copy your data that you have it in the excel file and paste it in the “Data” window of MapDisto. Where to paste? select the B15 cell in “Data” sheet and paste your data there. If your data has no header, then select B16 cell and paste them there.

C)If everything went well, the loci names have to appear into the yellow cells, while the genotyping data will appear in the white cells. Click on “Read Data” icon.

D)fill the different fields of the “Data” window with the following parameters:

Population type:

“DH”, for a population made of doubled haploids derived from an F1 hybrid,

“BC1”, for a backcross population (similar to the f2 backcross code for Mapmaker/ EXP), “F2”, for a population derived from the selfing of an F1 hybrid,

“SSD”, for a population of recombinant inbred lines obtained from single-seed descent, “IRIL#”, for a population of intermated recombinant inbred lines, where “#” indicates the number of intermating generations.

Population size, i.e., the number of individuals in the population.

Total number of loci. Total number of traits, if you plan to perform a QTL analysis.

Number of trait. 0

Data encoding: the way the genotyping data were encoded. For example, you can follow the Mapmaker/EXP standard: “A”: Homozygote for Parent 1 allele, “B”: Homozygote for Parent 2 allele, “C”: Non-homozygote for Parent 1 allele, “D”: Non-homozygote for Parent 2 allele, “H”: Heterozygote, “-” : Missing data. Data format: this indicates the direction of the matrix. Enter “1” if the loci are arranged in columns, “2” otherwise.

2-If you didn’t reach the desired linkage groups,you may have to try different values for LODmin and rmax, until you reach the desired number of linkage groups. The quickest way to proceed is to start with less stringent values for LODmin and rmax, then “cut” the large linkage groups that seem to correspond to more than one chromosome using more stringent values. This is done by running again the Find groups command and indicating the number of the sequence to be “cut” in the Sequence for searching groups dialog box, then using more stringent LODmin and rmax values progressively until reaching the correct number of chromosomes.

3-You can always use AutoMap icon to successively run the "Find groups", "AutoOrder" and "Draw all sequences", to allow for a very easy and quick display of the map computed for all the loci declared in the Data window.

4-Click on “Bootstrap order” icon to evaluate the stability or robustness of a given order using resampling methods. Choose a sequence and click on Bootstrap order (with, for example, 100 trials).

5-Click on Drop locus to identify the problematic loci in the given linkage group. This icon drops one locus at a time for a specific sequence and precomputes the map. Output gives pairwise distances (cM) between the remaining markers after each one is dropped. Full map is the last column. A locus that causes an important negative difference in the map size is expected to contain erroneous data and should be removed of the analysis process this locus will appear with red color. Note that dropping the terminal markers can have a large effect on map order simply because they are loosely linked. Also, if a sequence of markers are loosely linked, then it is inevitable that dropping one will have a large effect on the map. Note: this command complements well Bootstrap order, it’s recommended to use both in order to identify a set of markers that will constitute a solid framework for the map.

6-To detect genotypes errors, after ordering and rippling your data you can click the “Data fit linkage groups” icon in the Main menu and then click “Yes”. Now click on “color”, then click on “Load data” and then click on “color” and finally click on “show double recombinant” icon. You can decide what you want to do with your double recombinant loci, using one of the three options given in this sheet for replacing this error and then click on “use this data”.

7-You can always use the icon Segregation X2sin Main menu to computes segregation chi-squared tests (which measure the deviation from a 1:1 segregation) and their associated probabilities for a particular sequence. It also computes deviations from 1:3 and 3:1 segregations. This feature can be useful for dominant markers such as AFLPs or RAPDs, when two bands have very similar sizes and cannot be separated on the gel. The apparent band segregates (1 absent : 3present), and has no unique location on the map.

8-To make sure if your linkage maps needed to be flipped or not, you can compare your map with other maps. To do this, put the genetic map from other study into your mapping project folder. This genetic map should be included the chromosome name, the marker name and the genetic distance. Now go back to your main menu where you just did your analysis. In the main window, click on Define as Map 1. Back to the main window, click on Extract map from DB…. Click on Load marker list, then Extract positions…. Locate the folder that contains the other study’s map file. Click on Open. Visualize loci position on the physical map using the Draw map command. Click on Back, then Define as Map 2. This will re-compute your genetic map based on the order of the defined sequences and will get you to the Compare Maps window. Click on Compare maps.