Subtraction Raw Flowgrams

3. GS Run Browser : 3.5 The Wells Tab : 3.5.5 Subtraction Raw Flowgrams

3.5.5

Subtraction Raw Flowgrams

In addition to single location raw flowgrams, the GS Run Browser can generate a Subtraction (raw) flowgram from any two locations on the main image (Figure 35). Subtraction flowgrams are produced by right-clicking on a subtraction pin and selecting the Subtraction flowgram action, as described in Section 3.5.1.4. This is an advanced troubleshooting function that can be used in two distinct ways:

•

to generate a raw flowgram that approximates a presumptive well’s (in the pin head, pentagon) processed flowgram by subtracting the local background: when the signal intensity at the pin tail (square) location is lower than a set threshold (450 counts for “A” flows and 150 counts for any other flow except PPi or ATP), the software will compute a simple local background subtraction.

•

to compare the flowgrams from two well locations (or any two locations): when the signal intensity at the pin tail (square) location is above the set threshold, the subtraction flowgram simply compares the flowgrams from the two well locations (or any two locations). In this case, since different wells have different light-generating efficiencies, the calculation first normalizes the raw flowgrams at both locations by the average pixel intensity of their respective “first PPI or ATP” signals, before doing the subtraction. This type of calculation is also always applied if the Subtraction flowgram is requested from a PPi or ATP image, irrespective of the pin tail signal.

Figure 35: The Subtraction flowgram of image pixels (region 2, position 2420, 838) minus (region 2, position 2452, 841). It is shown with a scale-to-fit y-axis, the lines style is chosen, and the wash steps are not displayed. The mouse pointer is over the data point of flow 154, a “T” nucleotide flow that had substantially more signal in the pin head pixel than in the pin tail, as shown in the mouse tracker.

3.5.5.1

Subtraction Raw Flowgrams Features and Functionalities

A Subtraction flowgram window is identical to a Location flowgram window (Section 3.5.4), except that:

•	The window title bar identifies it as containing a Subtraction flowgram, and references two pixel locations rather than one.

•	the y-axis may contain negative values, since the subtraction may yield “signals” below zero; this is akin to the “difference plot” of a tri-flowgram window (Section 3.5.3).