GMT Tutorial Exercise 22

The Automated Designers - How to perform marker screening using a custom set of markers

• This macro is designed to analyse up to eight DNA samples (e.g. the parental lines of a mapping population or complex cross, or the DNA pools for bulk segregant analysis) with up to 576 markers to select informative markers for the subsequent genetic analysis, e.g. of a segregating population.
• Below is a workflow showing how "GENica Marker Tools", the "Automated Designer for Marker Screening" and "GeneMapper" can be used in combination to pre-screen a custom selection of MRT^TM markers.
• Click on a step for details. Move between the steps forming a workflow with the [previous step] and [next step] links in the top right-hand corner of each step-wise instruction.
• Alternatively, this macro can be used to set up a genetic analysis of up to 8 samples with up to 576 markers without first pre-screening the markers. In that case, follow the first 17 steps of the workflow below, then switch to the instructions for scoring markers in GeneMapper, as described for the Automated Designer for Genetic Analysis .

Select Markers and create BINNER panels

1. Choose MRT^TMmarkers to be screened.
2. Use BINNER to develop marker panels for the selected markers

Design the experiment

3. Download the Automated Designer for Marker Screening macro from GMT.
4. Open the macro and enable the macro functions.
5. Check the macro's version number.
6. Using the macro - the "Data Entry" worksheet.
7. Using the macro - the "Primer Dilution Table" worksheet.
8. Using the macro - the "Primer Dilution Plate Layout" worksheet.
9. Using the macro - the "PCR Setup" worksheet.
10. Using the macro - the "PCR Master Mixes" worksheet.

Carry out marker assays

11. Prepare PCR products.
12. Using the macro - the "SAMPLE File Writer" worksheet.
13. Separate PCR products on the ABI3730.

Score marker data

14. Using the macro - the "PANEL File" worksheet.
15. Using the macro - the "Primer Screening Assistant" worksheet.
16. Import panel information into GeneMapper.
17. Import sample electrotraces into GeneMapper and determine peak sizes.
18. Pre-score marker data in GeneMapper.
19. Export the GeneMapper marker data table.

Identify polymorphic markers and their allele size ranges

20. Using the macro - the "Primer Screening Analyser" worksheet.
21. Using the macro - the "Polymorphism Summary" worksheet.