The intention of this video is to answer some frequently asked questions. This segment of the video discusses how to manually perform size calibration.
Why are there yellow (or red) size quality sheets next to the sample names in the main screen? Is there a way to manually perform size calibration?
The screenshot below shows the size standard color channel for three samples.
-Sample (A) is spread out across a larger region compared to the other two samples.
-Sample (B) has a green sizing score and is the least problematic compared to the other two samples.
-Sample (C) has a loss of resolution at the higher molecular weight region and cannot be resolved with manual calibration in that region, but manual calibration is possible in the lower molecular weight region.
Below shows the same samples zoomed in on a higher quality low size region 0-120 where manually calibration can be successfully used to correct inaccurate and inconsistently sizing shown in these samples.
To perform manual calibration:
- Go to the Calibration Charts using the linear line icon
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- Select a sample from left hand panel to manually calibrate.
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Hold the left mouse button to drag a box from top left to bottom right around region that needs editing to zoom in.
Note: On the image below for sample 03_3_.fsa, question marks on peaks with green triangles indicate uncertainty in peaks and some peaks are without green triangles because they did not match the expected pattern during automatic size calling. Peaks with green triangles and no questions marks matched the expected size pattern with high certainty.
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To add peaks, hold the shift button and the left mouse button to drag a box from top left to bottom right around multiple peaks or single peaks that have missing green triangles.
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When yellow peaks appear on selected peaks, select Yes to Add the peaks to calibration. Repeat process until all true peaks are added.
- Once all peaks are added, right mouse click on empty space somewhere in sample electropherogram and select Update Calibration.
The below screenshots show before (top) and after (bottom) manual calibration. (A) are the scores and (B) shows the gap and then gap correction in the ratio plots once the correct size peaks are added and the calibration is updated.
-Below shows loss of resolution in example 03_3_.fsa at the higher molecular weight; a region that cannot undergo manual calibration, so a low score of 66 from 56 is the highest achievable score.
Moving onto sample 01_4_.fsa below, the actual size electropherogram displays a series of baseline peaks identified as matching the expected pattern, instead of the actual taller size peaks shown.
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Right mouse click each baseline peak with a triangle and select Delete Peak until all baseline peaks are removed.
- Now hold the shift button and the left mouse button to drag a box from top left to bottom right around multiple true peaks or single true peaks that have missing green triangles.
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Then select Yes when prompted to Add the peaks to calibration.
- Once all true size peaks are added back in (marked with green triangle), right mouse click on empty space somewhere in sample electropherogram and select Update Calibration.
-Take note of score (A) prior to Update Calibration shown below. Also note the ratio plot (B) for sample 01_4_.fsa shows poor linearity and missing points prior to Update Calibration.
-Following Update Calibration, the score (A) is now 96 instead of 54, and the ratio plot (B) for sample 01_4_.fsa shows improved linearity and completeness.
-Below are three example samples shown before (A) and after (B) manual calibration. The size calling in (B) is improved and samples are consistently and accurately sized in the region shown, following manual calibration.