Well Tri-Flowgrams (for Wells Generating Control DNA Reads)

3.5.3

When the selected well produces a Control DNA read (starting with the ‘CATG’ or ATGC; see Section 7.1 for details), a “tri-flowgram” plot appears (Figure 33). Tri-flowgrams allow the well’s flowgram to be compared to an idealized flowgram of this Control DNA reference sequence, or to be compared to the consensus flowgram generated from all the wells in the Run that contain this Control DNA sequence.

Figure 33: The tri-flowgram view of a Control DNA well (sequence AVTF90), at (region 2, position 2459, 877)

3.5.3.1

Well Tri-Flowgram Features and Functionalities

The tri-flowgram window contains two flowgram plots, plus a “difference” flowgram showing the flow-by-flow difference between the top two flowgrams. This flowgram viewer has functionalities similar to the standard flowgram viewer, but with the following differences:

•	Three flowgram plots are calculated per viewer window:

◦

An “ideal” flowgram constructed from theoretical values for the (known) sequence of the Control DNA fragment identified in the selected well. This is generated by taking the known nucleotide reference sequence and converting each homopolymer stretch into a corresponding signal (so, for example, “AAA” becomes a signal of 3.0 in an A flow).

◦	A “consensus” flowgram calculated as a flow-by-flow average of all the wells that contained the same Control DNA sequence as the selected well.

◦	The flowgram of the selected well.

•	The Options area offers the following choices (specific to the tri-flowgram viewer):

◦	Compare flowgrams:

▪	Ideal versus Well – Display the idealized flowgram in the top plot, the selected well’s flowgram in the middle plot, and the flow-by-flow difference of the two in the bottom plot.

▪	Consensus versus Well – Display the consensus flowgram for the selected well’s Control DNA sequence in the top plot, the selected well’s flowgram in the middle plot, and the flow-by-flow difference of the two in the bottom plot.

▪	Ideal versus Consensus – Display the ideal flowgram in the top plot, the consensus flowgram in the middle plot, and the flow-by-flow difference of the two in the bottom plot.

◦	Consensus from:

▪	Well Region – Average the signals of the reads (for this Control DNA sequence) only from the same region as the selected well.

▪	All Regions – Average the signals of all the reads (for this Control DNA sequence) across the whole PicoTiterPlate Device.

Only the N-mers flowgram signals are shown, because the Corrected Intensity signals are not defined for ideal or consensus flowgrams or relevant in this type of comparison.

•	The Navigation and data capture buttons and flowgram display have the following special functionalities (compared to the standard well flowgram view):

◦	The scrolling and zooming of the three plots are correlated. All three plots scroll and zoom together along the x-axis, and the top two plots scroll and zoom together along the y-axis (the bottom “difference” plot scrolls and zooms separately along the y-axis).

◦	Since the bottom plot zooms separately along the y-axis, it has its own zoom buttons.

◦	The horizontal bars between the plots allow for resizing the heights of the three plots.

◦	The text file and snapshot image buttons will save a file containing the data or view for all three plots.