GMT Tutorial Exercise 23 - Workflow 2 - Step 10
Using the macro - the SAMPLE file worksheet
- Below is a screenshot of the SAMPLES worksheet of the "Automated Designer for Genetic Analysis, University of Adelaide Special Edition".
- Enter details for your experiment following the instructions on the worksheet, as applicable. See also further explanations below.
- Click on the Create Sample File button to save a Sample File to be submitted with the actual samples for the electrophoretic separation step on the ABI3730. The information in this file is required to track sample and marker information, when samples are first electrophoretically separated and then analysed in GeneMapper.
(1) What type of sample plate will be submitted for ABI3730 analysis
- From the drop-down box, choose from "96-well" plate or "384-well" plate, as applicable.
- This will update the details in the sample submission file for the ABI3730 accordingly, see also instruction (7) below.
(2) Enter a filename for the ABI3730 sample submission file, if it should be different to the name of project
- The Automated Designer automatically uses the experiment name, entered on the Project Details worksheet, to create file names for output files (i.e. the PANEL file and the SAMPLE file). The experiment name is also used to create a "Run Name", which will later be used to identify all the results from a given ABI3730 sample submission plate in GeneMapper.
- It is possible to choose another name for the SAMPLE file and "Run Name" by entering a new name next to instuction (2) on this worksheet. Occasionally, this can be useful, e.g. when choosing a name for a sample plate containing more than one kit, e.g. "Sunco_x_Tasman_Kits1-4"
- Generally, it is a good idea to leave this field blank to take advantage of the consistency in naming that the automatically generated names offer. This greatly facilitates data tracking in the future.
(3) Enter the ABI3730 sample plate number for this Sample Sheet if the DNA samples in this project are distributed across more than one (1) ABI3730 sample submission plate
- This is feature allows distinguishing between different runs, containing different samples, belonging to one experiment.
- Any number entered in this field is appended as "_plate<number>" to the "Plate ID" entry of the ABI3730 sample submission file, e.g. "Tutorial_Example_plate3".
- The "Plate ID" entry will be used to automatically create a "Run Name", identifying all the results from a given ABI3730 sample submission plate in GeneMapper.
(4) Enter the results folder to which the ABI instrument should save your data
- The entry in this field tells the capillary separation machine (e.g. the ABI3730) where to save results.
- If in doubt, leave this field blank, the entry will be made by the ABI instrument operator.
(5) If the ABI3730 sample plate will contain more than one marker kit, enter the names of the additional marker kits
- The macro's default assumption is that all the samples on an ABI3730 sample plate have been amplified with the markers from the same kit.
- For smaller sample sets, however, samples analysed with more than one kit will often be submitted on the same ABI3730 plate. For example, a set of 96 DNA samples might be assayed with four different marker kits and submitted on the same ABI3730 sample plate for electrophoretic analysis.
- In this case, details where samples assayed with "additional" marker kits (i.e. the names of marker kits whose details were not entered on the Marker Kit worksheet) are found, can be entered with the macro:
- Enter the name of the "additional" marker kits in this table.
- After doing this, the names of these additional marker kits will become available when assigning marker kits to DNA samples (see instruction (7) below).
(6) Enter the name of the Analysis Method file that GeneMapper should use to analysis your data
- If a custom analysis method has been created in GeneMapper and its exact name is known, it can be entered here.
- Leave this field blank to use GeneMapper's default analysis method. Alternatively, other analysis methods can be easily assigned later when electrotrace results are analysed in GeneMapper.
(7) Paste into the box below the list of DNA samples as they appear on the ABI3730 sample plate, and assign a marker kit to each DNA sample
- Fill in the DNA sample names in the column entitled DNA Sample, ensuring that well positions on the ABI3730 sample plate are correctly assigned.
- If both the ABI sample submission plate and the DNA sample plate are 96-well plates (see entry made for instruction (1) above), well positions in the columns entitled Location on ABI plate and Sample location on 96-well DNA plate are identical. Sort by either column and copy / paste the list of DNA sample names into the DNA sample name column.
- If the ABI sample submission file was identified as a 384-well plate under instruction (1), well positions of one ABI plate and four DNA "extract" plates are aligned in the table under instruction (7), according to the pipetting pattern used in our lab, as described in the brown "background" box below. If the same 96-well to 384-well pipetting schema was followed, carry out the following steps:
- Click on the Sort by DNA well button in the box under instruction (7).
- Click on the grey Generate a table for DNA sample names and identifiers that can be sorted button to generate the
DNA Sample Sorter worksheet for the first time. The DNA Sample Sorter page automatically opens.
(To access this worksheet at a later time, use the Go to DNA sample sort worksheet button.)
- On the DNA Sample Sorter worksheet, sort the DNA sample names by plate, then by well position, as described in the next step of the workflow.
- Select and copy the DNA sample names for the relevant plate(s).
- Click on the Previous Page button to return to the SAMPLE file worksheet.
- Paste the DNA sample names into the DNA sample names column of the box under instruction (7).
Background: Pipetting DNA samples from 96-well extraction to 384-well ABI3730 plates
- DNA samples are transferred "column-wise" with an 8-channel multipipette from the 96-well "extract" plates to the 384-well ABI3730 plates.
- The first 6 columns of the first 96-well DNA "extract" plate (columns 1-6) are pipetted into the "top-most" rows of the first 6 columns on the 384-well ABI3730 plate (i.e. rows A, C, E, G, I, K, M, O are filled, see wells filled in "light blue" in the sketch below).
- The next 6 columns of the first 96-well "extract" plate (columns 7-12) are then pipetted into the "lowermost" rows of the first 6 columns on the 384-well ABI3730 plate (i.e. rows B, D, F, H, J, L, N, P are filled, see the "dark blue" wells in the sketch below).
- Samples from the second 96-well DNA "extract" plate are then pipetted in the same pattern into the next set of 6 columns on the 384-well ABI3730 plate (columns 7-12, "red" wells in the sketch below), and so on:
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For each well position, assign the Marker kit with which the DNA sample in that well was assayed. The available kit names are displayed in a "drop-down" box when clicking into cells under the column header Marker Kit. Refer to instruction (5) if samples have been assayed with "additional" marker kits other than the marker kit whose details were entered on the Marker Kit worksheet.
The Generate a table for DNA sample names and identifiers that can be sorted button
The Go to DNA sample sort worksheet button
The Create Sample File button
- Click on the Create Sample File button and answer yes to all following prompts to save the Sample File.
- Include the Sample File when submitting samples for electrophoretic separation on the ABI3730.
- The macro creates a name for this file by appending _sampleFILE.txt to the experiment name, or to the entry made following instruction (2) above, as applicable.
- The file is automatically saved in the same directory where the "Automated Designer for Genetic Analysis" is saved.
The Print Preview button
- Click on this button if you desire a hard copy for your records, see an example below.
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