GMT Tutorial Exercise 22 - Workflow 1 - Step 6

Using the macro - the "Data Entry" worksheet

• This screenshot shows the Data Entry worksheet of the "Automated Designer for Marker Screening".
• Enter experimental data following the instructions on this worksheet. See below for background explanations for each of these instructions.
• To start over, click the Reset Form button. This will reset the entire Automated Designer macro.

(1) Enter the name of this experiment.

• Entries in this field will be used by the Automated Designer when creating output files, helping to track different files used to set up and analyse a marker screening experiment.
• Because of their downstream use, entries in this field can only use a limited number of characters: Allowed characters: Regular letters (a-z); numbers (0-9); special characters: underscore (_) and dash (-).

Allowed, but undesirable character: full stop (.) (Use of full stops is undesirable, as they are usually interpreted as the separator preceding a file's extension. When presented with a file called e.g. "Sloop.Alexis.txt", the computer's operating system might have difficulty selecting the correct software application to open the file. Also, files with "double extensions" are usually considered high-risk by virus scanning software, making it potentially difficult to e-mail result files to collaborators.) Forbidden characters: All special characters, e.g. ü, é, æ, etc.; / \ & * # $ % , as well as spaces.

• A 3-letter "Project Abbreviation Code" is automatically created from the project name, by using the first, middle and last character of this entry, see cell "K4" on this worksheet. The "Project Abbreviation Code" is used to create unique panel names to meet down-stream requirements imposed by GeneMapper 4.0.

(2) Enter the names of the DNA samples to be screened in this experiment. Up to 8 samples can be screened at a time.

• Entries in this field will be used to track samples through the different analysis steps. Again, the only allowed characters are regular letters (a-z); numbers (0-9); full stop (.), underscore (_) and dash (-).
• Tip: When markers are scored for polymorphism, it is convenient to have the relevant DNA samples maintained as a group, e.g. so the peaks for the parents of a cross can be easily compared. As GeneMapper sorts DNA samples alphabetically, a simple way to maintain sample groupings is to put an integer at the beginning of the sample names, e.g.:

(3) Paste into the box below up to 96 marker panels from your BINNER output file. Each marker panel can contain a maximum of 6 markers. Only paste panel numbers and marker names.

• Open the BINNER output file (from the BINNER step of this workflow) in a spreadsheet viewer, e.g. Microsoft Excel. Select and copy the first two columns of data, and paste them into the appropriate macro box, as shown in the screenshots below:

(4) Select the level of marker scoring stringency required.

• Choose one of two available "marker scoring stringency" settings:
  • "Score All Marker Fragments" (default) will result in a GeneMapper panel file that includes all the zones where informative peaks have been observed when a marker was characterised on the ABI3730, ignoring only Noise Zones.
  • "Score Only Major SSR Fragments" generates a GeneMapper panel file including only zones where Major Peaks have been observed when a marker was characterised. This option is useful to speed up the marker scoring process, but can result in a significant loss of polymorphism information.
    

Background:

• When multiplex-ready markers are characterised on the ABI3730, "zones" are defined to indicate where PCR products were observed. BINNER uses this information to ensure that markers with overlapping fragment sizes are not included in the same marker panel.
• Several types of "zones" are distinguished and can be included / excluded in the GeneMapper panel file, depending on the chosen "marker scoring stringency" setting:
  • Noise Zone - indicates non-specific, unscorable fragment(s)
  • Low Peak Height - indicates a scorable peak(s) with a peak fluorescence less than half that of other major PCR fragment(s)
  • Rare Peaks - indicates the presence of null alleles
  • Major Peaks - indicates the presence of scorable, target fragment(s)

(5) Enter the PADDING (in base-pairs) used to construct the BINNER marker panels.

• This refers to the Padding setting that was used in the BINNER step of this workflow.
• If unsure, open the downloaded BINNER results file. The Padding setting that was used to generate the output is reported above the binning results.

(6) Press the Format Panel Data button.

• The macro will re-format the marker panel information entered following instruction 3 above. This can take some time.

(7) Click on the Next Sheet button to move to the next spread sheet.

• Congratulations - time to move on to the [next step] in the workflow...