Skip to content

User Interface

Jump to bottom
ufcyg edited this page Sep 13, 2016 · 7 revisions

Features

  1. Settings
    • "[M]" - For Marker, changes display of data to the marker dye channel
    • "[D]" - For Dye, changes display of data to the by the pulldown menu defined dye channel
    • "Selected dye channel" - Pulldown menu, names are extracted from read data
    • "Minimum threshold" - Here the user can define the threshold that shall be applied to dye channels
    • "Ignore approximately first X bases" - Here the user can define how many bp shall be ignored in analysis
  2. Figure Options
    • Allows the user to open and close *.fig files to view their contents
  3. Dataset Options
    • Allows to save or load a previously saved dataset, dataintegrity is checked by MD5 hash
  4. Status Box
    • Shows all status messages the FSAnalyzer outputs
  5. Progress Bar
    • Shows the status of the current calculation
  6. Data Management In- and Output
    • "Select Data" opens a dialogue where the user is asked to select a folder, if this folder contains a minimum of 2 .fsa files, a SizeStandard.txt and SampleIDs*.txt file, these will be automatically loaded as experiment, if none of these are found the user is asked to locate them, optionally if no or more than one save file (*.mat) is detected the user can define a save to be immediately loaded without preanalysis of data. While only one save file is present, this will be automatically loaded (also dataintegrity is checked by MD5 hash)
    • "Graphics" starts the plotting and saving of all loaded files data to *.fig (editable) and *.jpg (static) files as pictures
    • "Analysis" starts the Analysis defined in the data selection process
    • TBP
    • SSR
    • "Graphics and Analysis" Starts a combined execution of previously mentioned "Graphics" and "Analysis"
  7. Switch files
    • Arrowbuttons allow to switch between data of all loaded files
  8. Manual correction of data
    • "Correct" - TOGGLE - Allows do disable or enable detected peaks. (Detected peaks are marked with a red circle)
    • "Select" - TOGGLE - Allows to manually define peaks in loaded data
  9. Current File indicator
    • Shows the name and index of file shown at the moment as the number of files
  10. Graphic manipulation tools
    • "Move" - TOGGLE - Rightclick into plotwindow, while holding move the mouse to move data
    • "Zoom in" - TOGGLE - Rightclick to zoom in, hold and drag rectangle to zoom in selection, doubleclick to zoom to default
    • "Zoom out" - TOGGLE - Rightclick to zoom out, doubleclick to zoom to default
    • "Select Point" - TOGGLE - Rightclick on datacurve (blue) to show coordinates of selected point

Guide

Troubleshooting

Marker channel Data

There have been more or less peaks detected than required How can one decide what peak is the right one?
In preanalysis after detection of peaks the (perfect) distance relation is calculated depending on selected SizeStandard.
for n=SizeStandardFragmentCount:-1:1
relativeDist(n) = ( SizeStandardFragment(n) - SizeStandardFragment(n-1) ) / biggestSizeStandardFragment
The distance relation between every detected peak, starting from the left (biggest fragment, and assumed to be of high quality over a high amount of experiments) and the peaks which are closer to the perfect distance are tagged with the correspondingly label
In some rare occasions more peaks been detected, but haven't been tagged, the user must deactivate them.
In this case two peaks are close to each other and only one has a tag. In this case the user must decide which of the peaks is the right one. The black bars are the same length and the distance between the 3 fragments (420 bp, 440 bp, 460 bp) must be the same, 20 bp, too. So it is clearly the tagging algorithm has choosen the wrong peak, and the user has to correct the data.

Dye channel Data


Above, many peaks are found in the noisy low RFU zone of data, this can be fixed by adjusting threshold to a higher value as can be seen below.<br<
The magnification shows 2 peaks at the base of a much higher peak. These are false detected peaks and must be removed before running an analysis.

General



Dye Blobs


Disassociated primer dyes, more commonly referred to as dye blobs, are fairly common in STR analysis. While it isn’t entirely understood why dye blobs occur, evidence suggests that the fluorescent dye tags attached to the primers begin to break down over time. Disassociated primer dyes can show up in the sample analysis range and can mask true data. Dye blobs are usually wider than real peaks and are typically only seen in one color. By following the manufacturer’s specifications for storage of amplification kits, problems with disassociated primer dyes can be avoided.


Spikes


Spikes are narrow peaks usually attributed to fluctuation in voltage or the presence of minute air bubbles in the capillary. Spikes can also be caused by crystals in the polymer and/or fluorescent material in the polymer or formamide. Spikes, unlike other artifacts, are generally seen in the same position in all colors. However, it is possible to detect spikes in a single color. If detected, they must be removed by the user.


Noise

Analysts may confuse actual alleles with noise and vice versa; they should be familiar with the signal-to-noise ratio of the instrument and/or the specific data. Noise is not reproducible; one way to differentiate alleles from noise is to rerun the sample.

Clone this wiki locally