GMT Tutorial Exercise 23 - Workflow 2 - Step 2

Use BINNER to develop marker panels for the selected markers

• BINNER creates panels of markers that have correct spatial separation to avoid allele overlap when separated on an automated DNA fragment analyser.
• BINNER can use two different types of input: Marker names can either be uploaded as a list file or "tagged" for binning within GENica Marker Tools, see below.

With custom binning information from a "pre-screening" experiment

* This section applies if markers have been pre-screened with the Automated Designer for Marker Screening or with the Automated Designer for Whole Genome Scan. If this was not the case, please refer to the section on doing binning with default marker information below.

1. Prepare an import file for BINNER»MRT UPLOAD:
1. Using a spreadsheet viewer such as Excel, open the the BINNER file created with the "Polymorphism Summary" worksheet of the "Automated Designer for Marker Screening" or of the "Automated Designer for Whole Genome Scan".
   The name of this file ends with *_BINNER.txt, and it should have been saved in the folder created for setting up the pre-screening experiment.
2. Copy the first column containing marker names only into a text file (e.g. using "Notepad"). The file should look as described here.
3. Save this BINNER»MRT UPLOAD file in the same directory as the BINNER file. Choose a suitable file name, e.g. one ending in *_BINNER_UPLOAD.txt.
2. Login to GENica Marker Tools (https://www.genica.net.au/gmt/).
3. Import the custom binning information from the pre-screening experiment:
1. Go to MARKERS»IMPORT and click on [Add Marker Set].
2. Choose the User Group where the marker set is to be stored, if more than one is available.
3. Enter a Marker Set Name, e.g. the name of the population for which the binning information applies (e.g. "Sunco x Tasman") or an experiment name.
4. Choose Access level "private", "group" or "public", as desired. Usually, "private" is a good choice.
5. Click Save.
6. Back in MARKERS»IMPORT, Browse for the BINNER file created with the "Polymorphism Summary" worksheet of the "Automated Designer for Marker Screening" or of the "Automated Designer for Whole Genome Scan".
   This is the same file referred to in step 1.1 above: The name of this file ends with *_BINNER.txt, and it should have been saved in the folder created for setting up the pre-screening experiment.
7. Click on Import.
4. Go to BINNER»MRT UPLOAD.
5. Browse for the BINNER»MRT UPLOAD file created in step 1 above (with a name e.g. ending on *_BINNER_UPLOAD.txt).
6. From the Marker Set drop-down box, select the marker set where the *_BINNER.txt was imported (step 3 above, e.g. "Sunco x Tasman").
7. From the Padding drop-down box, select the desired "padding", i.e. the minimum distance between adjacent allele size ranges belonging to different markers. As the binning information is based on a pre-screening experiment, a value as small as "5 bp" can be chosen.
8. Leave the Sort order setting unchanged ("Sort by longest loci range before binning").
9. Click Bin Markers.
10. When binning results have been prepared, click [Download results file] and save binned markers to your computer, e.g. to a new folder created for the "Genetic Analysis" experiment.
11. Repeat the binning process, but this time change the Sort order input option ("Sort by highest loci midpoint before binning").
12. Compare the two different outputs (downloaded files can be opened with Microsoft Excel or similar).
    The BINNER results interface displays a table with the panel number in the first column, the marker name in the second column, followed by up to 8 columns, indicating the minimum and maximum allele sizes of up to 4 allele size ranges for the marker. Markers that have the same panel number (i.e. the same number in the first column) can be analysed together in one capillary of the ABI3730. An example of the BINNER results table is shown below:

1	ta0094	214	252	
1	ta0121	256	280	
1	ta0134	146	164	
2	ta0011	232	241	
2	ta0063	153	162	
3	ta0015	148	154

13. Select the binning result with:
    • Fewer bins (the highest number in the first column gives the total number of bins) and / or
    • More even distribution of markers per bin.

With default binning information (if no "pre-screening" experiment was carried out)

1. Login to GENica Marker Tools (https://www.genica.net.au/gmt/).
2. Go to BINNER and submit markers to be binned:
   • If marker names have been saved in a list file... (The preparation of the marker list file is described here)
     1. Select BINNER, then UPLOAD.
     2. Browse for the file with markers to be binned, then click Open.
   • If markers have been "tagged" with GENica Marker Tools... (Tagging markers in GMT is explained in the [previous step] of this workflow.)
     1. Select BINNER, then TAGS.
     2. Choose the tag with markers to be binned from the Tags drop-down box.
3. Select the applicable marker set from the Marker Set drop-down box:
   • "Wheat (MPBCRC:public)" for MRT^® wheat markers (names beginning with "ta")
   • "Barley (MPBCRC:public)" for MRT^® barley markers (names beginning with "hv")
4. Select a minimum padding of "10 bp" from the Padding drop-down box, unless the allele size range for each marker is known for the germplasm of interest.
5. Leave the Sort order setting unchanged ("Sort by longest loci range before binning").
6. Click Bin Markers.
7. When binning results have been prepared, click [Download results file] and save binned markers to your computer, e.g. to a new folder created for the "Genetic Analysis" experiment.
8. Repeat the binning process, but this time change the Sort order input option ("Sort by highest loci midpoint before binning").
9. Compare the two different outputs (downloaded files can be opened with Microsoft Excel or similar).
   The BINNER results interface displays a table with the panel number in the first column, the marker name in the second column, followed by up to 8 columns, indicating the minimum and maximum allele sizes of up to 4 allele size ranges for the marker. Markers that have the same panel number (i.e. the same number in the first column) can be analysed together in one capillary of the ABI3730. An example of the BINNER results table is shown below:

1	ta0094	214	252	
1	ta0121	256	280	
1	ta0134	146	164	
2	ta0011	232	241	
2	ta0063	153	162	
3	ta0015	148	154

10. Select the binning result with:
    • Fewer bins (the highest number in the first column gives the total number of bins) and / or
    • More even distribution of markers per bin.