GMT Tutorial Exercise 22 - Workflow 1 - Step 18

Pre-score marker data in GeneMapper

• The Automatic Designer for Marker Screening macro can analyse MRT^TM marker data that has been pre-scored in GeneMapper for polymorphisms between a set of DNA samples. This is particularly useful when e.g. attempting to identify polymorphic markers for DNA samples representing the parental lines of a complex cross.

• Find instructions below on how to display and pre-score data in GeneMapper to create a report table that can be analysed by the macro.

• If the Automated Designer for Marker Screening macro is used to set up a genetic analysis of a few samples, rather than to pre-screen markers before a genetic analysis, follow the instructions for scoring markers in GeneMapper, described for the Automated Designer for Genetic Analysis macro, instead.

Displaying sample electrotraces

1. Click on the Genotypes tab in the main GeneMapper window.
   Select Edit » Select All to mark all the entries in the genotypes table.
   Then select Edit » Sort from the file menu. The Sort dialogue box opens.
   Choose to sort by "Panel", then by "Dye", then by "Marker", and click OK.
   This operation sorts the marker data into the same order as on the Primer Screening Assistant worksheet, so the type of PCR products expected for each marker can be easily looked up on the Primer Screening Assistant printout as data is pre-scored in GeneMapper.
   




2. Select Analysis » Display Plots from the main menu in the GeneMapper window.
   The Genotypes Plot window opens with electrotraces for all the entries that were selected on the Genotypes tab.




3. Adjust the number of electrotraces shown in the Genotypes Plot using the Panes drop-down box, so that all of the DNA samples to be compared for polymorphic markers are visible.
   For example, for a four-way cross, four electrotraces should be visible, one for each parental line.
   

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Editing allele calls on sample electrotraces

• To pre-score marker data in a format compatible with the macro, allele calls automatically made by GeneMapper may need to be edited.
  To use the allele-editing functions, ensure that the Single-click Editing option is turned off:
  In the Genotypes Plot window, select Alleles on the menu bar. Ensure no "tick" is displayed on the left-hand side of the Single-click Editing menu option.

Delete an allele call

• Left-click on the peak. The peak will become highlighted. Right-click on the peak to display the allele editing options list. Select Delete to remove the allele call.

Add an allele call

• Left-click on the peak. The peak will become highlighted. Right-click on the peak to display the allele editing options list. Select Add Allele Call.

Rename an allele

• Left-click on the peak. The peak will become highlighted. Right-click on the peak to display the allele editing options list. Select Rename Allele.

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Rules for Scoring Markers

The macro will automatically analyse marker data for polymorphisms between a set of DNA samples. To facilitate this operation, electrophoretic regions (or "zones") that you want the macro to analyse must be defined. There are two types of zones: monomorphic and polymorphic zones. The importance of defining both zone types is to prevent the BINNER software from building marker panels in subsequent work that contain markers with overlapping PCR fragment sizes. For example, if a marker amplifies two loci, one monomorphic and the other polymorphic then zones must be defined for the two electrophoretic regions within which the PCR fragments for each locus occur. If the monomorphic fragments were not described then BINNER could build a marker panel in subsequent work that contained another marker whose PCR fragments comigrated with the monomorphic amplicons of the first marker. In essence, zones describe the electrophoretic regions in which PCR fragments amplified by each marker occur for a set of DNA samples that are to be compared for polymorphism. The marker zones defined will be germplasm-specific, and allow for more efficient construction of marker panels in subsequent steps such as genetic mapping, compared to the default marker allele size range data contained in the Multiplex-Ready Marker Database. The rules used to define an electrophoretic zone in GeneMapper are described below using the parental lines of a four-way cross as an example.

Markers amplifying a single PCR fragment

When a single PCR fragment is detected for each of the DNA samples, the macro will automatically detect if the marker is polymorphic for these DNA samples:

• The marker is monomorphic, i.e. peak sizes are identical for the samples being compared:
  The macro will assign a monomorphic zone with an allele size range equal to that of the observed peak.

• The marker is polymorphic, i.e. peak sizes differ between samples:
  The macro will record the zone as polymorphic, with an allele size range (in base-pairs) defined by the minimum and maximum fragment sizes that are observed in the set of DNA samples being compared. See below for examples showing when allele editing can be required with this type of marker.

Note:
The macro allows for a one (1) base-pair drift in allele size to account for capillary to capillary variation, e.g. a 100 and 101-bp fragment are considered to be the same size and would not be rated as "polymorphic".

Example for a polymorphic marker amplifying a single PCR fragment: No allele editing required

Example for a polymorphic marker amplifying a single PCR fragment: No allele editing required.
In this case, GeneMapper has automatically detected one peak per lane. Peak sizes differ between the set of DNA samples to be compared, therefore, the marker will be recognised by the Automated Designer for Marker Screening macro as "polymorphic". A custom allele size range for this set of DNA samples will be defined, including the minimum and maximum allele sizes observed.
In the situation shown in this example, no allele editing is requried.

Example for a polymorphic marker amplifying a single PCR fragment: Allele editing required

When additional fragments are detected by GeneMapper that would not interfere with marker scoring if the amplicon(s) of another marker were to occupy the same electrophoretic region, then their allele calls should be deleted, as shown in this example:

Example for a polymorphic marker amplifying a single PCR fragment: Allele editing required.
In this example, the allele calls at 150 bp and 190 bp in the 2nd lane can be deleted, as they would not interfere with marker scoring if another marker was to occupy the same region. As a consequence, the macro will define a polymorphic zone with an allele size range of 150 bp to 158 bp for this marker.
If the 190 bp allele called by GeneMapper is not deleted, the macro would define a polymorphic zone with an allele size range of 150 bp to 190 bp.

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Markers amplifying multiple PCR fragments

• When multiple PCR fragments are detected for a marker, manual editing of the allele calls automatically made by GeneMapper may be required to accurately define the electrophoretic zone to be analysed by the macro. The rules used to define an electrophoretic zone are listed below.

• The application of these rules is flexible, and it will often be possible to define an electrophoretic zone in more than one way. The application of these rules is illustrated in the examples below, using the parental lines of a four-way cross.

Allele editing examples

"Noise" allele calls that extend the electrophoretic zone occupied by genuine marker peaks

In this example, the marker assay for the first DNA sample failed. An electrophoretic zone is defined according to rule (a) by removing the "noise" peaks detected in lane 1 by GeneMapper. Without this editing, the macro would assign an annecessarily wide polymorphic zone with an allele size range from 172 bp to 195 bp.

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"Noise" allele calls fall within the electrophoretic zone occupied by genuine marker peaks

In this example, the marker assay for the second DNA sample failed. However, it is not strictly necessary to delete these allele calls, as their presence will not expand the size of the electrophoretic zone that would be automatically assigned by the macro: The "noise" peaks fall within the allele size range of 351 bp to 371 bp, as defined by the peaks for the samples on the 3rd and 4th lane.

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Genuine marker peaks outside the marker area defined in the "panel file"

In this example, the marker allele amplified from the fourth DNA sample was outside the expected size range for this marker (as defined in the PANEL file). This peak was therefore not automatically detected by GeneMapper. To define an electrophoretic zone for this marker according to rule (a), the two peaks detected instead by GeneMapper are deleted, and the target peak for the fourth DNA sample is added. The macro will now assign a polymorphic zone with an allele size range from 128 to 157-bp.

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Multiple monomorphic marker peaks

In this example, multiple monomorphic fragments were amplified. By default, the Automated Designer for Marker Sreening macro would consider this marker "polymorphic", as the smallest and biggest peak for the DNA sample set differs by more than 1 bp. The marker therefore has to be manually marked as "monomorphic" by renaming alleles with "m", according to rule (b).
To define the correct electrophoretic zone occupied by peaks for this marker, the left- and right-most peaks in the first DNA sample were assigned the letter "m" .
Notice that the middle peak in the first DNA sample was also deleted to satisfy the requirement of a maximum of two allele calls per DNA sample (rule (a)).

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Multiple polymorphic marker peaks

In this example, two marker loci are amplified, one of which is polymorphic. An electrophoretic zone is defined automatically because the smallest and the biggest peak for the DNA sample set differs by more than 1 bp.
The correct allele size range would equally be recognised, as both the left-most and the right-most peaks are detected, and no lane has more than 2 peaks in it (according to rule (a), only the first two alleles, counting from the left, are considered. If the right-most peak appears as the 3rd or higher peak in a lane, it is ignored by the macro).)
The polymorphic "rating" can also be manually entered, following rule (b), by assigning the letter "p" to the left- and right-most peaks for this sample set (note the peaks in the first and second lane).

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A single peak from a dominant marker

In this example, a dominant marker is amplified. Following rule (d), the peak at 178 bp is marked "p" with the Allele Rename option. This allele edit is required to let the macro recognise the marker as "polymorphic", without it, the marker would be rated as "monomorphic".

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