GMT Tutorial Exercise 22 - Workflow 1 - Step 12

Using the macro - the "SAMPLE File Writer" worksheet

• This screenshot shows the Sample File Writer worksheet of the "Automated Designer for Marker Screening".
• Update details for your experiment, if required, and create a "sample submission file" with the Write Sample Files button, following the instructions on the worksheet. See below for further explanations.
• Ensure that the "panel names" in the "sample submission file" correspond to the names used in the automatically created "panel file", see details below.
• The "sample submission file" helps to track sample and marker details when DNA fragment analysis is done on the ABI3730, see the next step of this workflow.

How to get here

1. If required, open the Automated Designer for Marker Screening macro for your experiment and enable macro functions as described previously in this workflow.
2. Use the Save As... option and re-save the macro without changing its name or location. Click yes at the "file overwrite" warning. This step is required to ensure that macro-generated files are saved in the same directory as the macro.
3. Use the Next Sheet / Previous Sheet buttons on each worksheet to find the Sample File Writer worksheet shown above.

(1) If the layout of the samples for ABI analysis shown above is not correct, then press the Edit button and manually edit the plate layout.

Background explanations:

• A table at the top of the worksheet (rows 4-29) shows which markers belong to which Capillary Code.
• Beneath, a second table with a grey background (rows 32-34) shows which DNA Sample Codes refer to which samples.
• Underneath, two 96-well ABI Sample Plate(s) are drawn. For each well of the plate, a combination of a Capillary Code and a DNA Sample Code shows which pooled PCR products, from which DNA sample, are found in that particular well (note the areas highlighted in orange on the screenshot above).
• The well positions in this layout of the ABI Sample Plate(s) are automatically derived from:
  1. The pooling protocol followed in our lab and
  2. The entries made on the PCR Setup worksheet.

1. Using the information from the two tables at the top of this worksheet and a record of how your pooled PCR products were distributed onto the ABI sample plate, compare the automatically derived plate layout with the layout of your own ABI sample plate.
2. If the information on the worksheet is correct, proceed to worksheet instruction (2). If you find the ABI Sample Plate on this worksheet needs to be updated, click the Edit button. This overrides the macro's functions that automatically enter this information.
3. Enter the Capillary Code and DNA Sample Code combinations in the wells where the corresponding DNA samples' pooled PCR products are really found on your ABI sample plate. Please do this by actually typing the capillary/sample-identifyers into the cells, rather than by using the mouse to "grab and drag" entries. If doing the latter, the changes are not recorded correctly, which will lead to incorrect sample file entries and problems with the Primer Screening Analyser worksheet.

(2) Enter the results folder to which the ABI instrument should save your data.

• Check with your DNA fragment separation service provider if they would like you to enter a particular "results folder" entry on your Sample Submission Sheet. If unsure, leave this field blank.

(3) Press the Write Sample Files button.

1. Click on the Write Sample Files button. This will automatically create two Sample Submission Files (for two ABI sample plates), based on the information entered into the macro.
2. Click "yes" at all prompts. The file will be saved in the same directory where the Automated Designer for Marker Screening macro is saved. The macro creates file names by appending .plate1_sampleFILE.txt and .plate2_sampleFILE.txt to the experiment name (as entered on the Data Entry worksheet).
3. Find the file(s) and open them with a spreadsheet viewer, e.g. Microsoft Excel.
4. Compare entries under the "Panel" column heading of the "sample submission file" with the panel names in the "panel file" (generated by the PANEL File worksheet of the macro), as highlighted in the screenshot below:
   
5. If the panel names used in the "sample submission file" and in the "panel file" differ, use the Find / Replace option of the spreadsheet viewer to update the entries in the "sample submission file" with those in the "panel file". In the example shown in the screenshot above, panel name "A" would have to be replaced with "Eae.A", "B" with "Eae.B", etc.
   Background explanations:
   • The panel file and the panel names in it are imported into GeneMapper, which requires unique panel names are used. The macro assigns a 3-letter prefix, derived from the experiment name, to each panel name, to reduce the likelihood of two identical panel names being used.
   • If the panel names in the sample submission file are not identical to these panel names, GeneMapper will not be able to automatically correlate samples with their panels when samples are imported into GeneMapper, requiring that the correct panels are manually assigned for the samples.

• Forward this file to your DNA fragment separation service provider together with your DNA samples, see the next step of this workflow.

The Print Preview button

• If desired, e.g. as a reference when pooling PCR products in the lab, print a copy of the information on this worksheet by clicking on the Print Preview button.