You can create a manual bin set to adjust the bin set created by Coffalyser.Net. This can be done as follows:
- Open the experiment.
- In the fragment analysis tab, right-click somewhere in the screen and select Edit Manual Bin Set. Select the appropriate channel with MLPA fragments (when an ABI instrument was used, this will be channel 1).
- Select a sample on the left to see a chart with the bin set at the top of the dialog. All identified signals are depicted as dots. A bin appear as a green bar when a signal was detected within the bin. When no signal was detected (or when the signal was too low), the bin will appear as a red bar.
- Right-click in the table below and select Manual Bin Set Bounds > Set Based On Current Experiment Results (All Rows) to save the current bin set as a starting point.
- Select a sample where all signals are present in the raw data (e.g. a reference sample or a binning SD). For each fragment that falls outside of the current bin settings, do the following:
- Find the fragment in the table with probes.
- In the column manual binset lower bound, change the current value to a value below that of the actual length of the fragment.
- In the column manual binset upper bound, change the current value to a value above that of the actual length of the fragment.
- Make sure that the new bins do not overlap with other bins.
- The chart will show the adapted bin.
- Click OK at the bottom of the open window and then Yes in the pop-up screen that appears. The manual bin set will be used the next time the fragment analysis is performed.
- Start (or repeat) the fragment analysis.
You can usually use the same manual bin set as long as your electrophoresis conditions do not change significantly. The manual bin set is stored in the analysis sheet, and can be re-used in another experiment by selecting manual as probe recognition method in the Fragment Analysis Settings that are displayed when you start the fragment analysis.
This procedure is explained in more detail in the video below.
In case you are unable to view the video on YouTube you can also download it here.
Specific electrophoresis conditions may sometimes result in fragment lengths that deviate too much from the expected lengths. These signals will not always be properly identified by the automatic binning procedure in Coffalyser.Net. As a result, peaks may be misidentified or missed completely, which can result in warnings for missing probes.
Experiments where none of the samples have a signal for a particular probe are also prone to binning issues. MRC-Holland supplies all probemixes with mutation-specific probes with a binning DNA that can aid in determination of bin boundaries.