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1. GS Amplicon Variant Analyzer Application : 1.8 The Flowgrams Tab : 1.8.2 The Triflowgram Plot
1.8.2
The Triflowgram Plot
A flowgram is a graphic representation of the number of nucleotides added to the nascent DNA strands present in a given well of a PicoTiterPlate Device, during each nucleotide flow of a sequencing Run. Simply put, it shows the succession of nucleotide flows of the sequencing Run on the horizontal axis, and the number of nucleotides incorporated during each flow on the vertical axis (as histogram bars, with the nucleotides color-coded per the legend at the lower-left of the tab).
In Amplicon sequencing, because the software not only knows the data from the reads but also has a Reference Sequence to which each read is to be compared, the AVA application can calculate an “ideal flowgram” of the Reference Sequence corresponding to the read (i.e. the Amplicon, in AVA software language), and display the difference. The AVA software presents this to the user in the form of a “tri-flowgram”. The three plots of a tri-flowgram show the following:
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The top plot shows the calculated flowgram of the segment of the Reference Sequence corresponding to the Amplicon that produced the read being displayed. This is simply the even number of bases that would result from the perfect incorporation of all nucleotides along a sequencing template of that sequence, during a sequencing Run on the GS Junior or GS FLX+ Instrument.
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The middle plot shows the exact signals recorded at each flow of the sequencing Run for the read being displayed, converted into “nucleotide” units. If the read was from the DNA strand opposite the Reference Sequence, the plot will display the signals after calculating the reverse complement of the read sequence, so the read flowgram will align with that of its Reference Sequence.
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The bottom plot is not truly a flowgram; it shows the difference between the two plots above (read minus Reference), such that if the read matches the Reference Sequence at a given flow, the histogram bar height is zero; if the read has a stronger signal than expected from the known Reference, the bar height is greater than zero; and if the read has a weaker signal than expected from the known Reference, the bar height is less than zero. This “difference flowgram” thus conveniently shows all flow-by-flow variations between the read and its Reference Sequence as departures from zero.
The tri-flowgram plot has all the standard navigation features (scrollbars, zoom buttons, mouse tracker, etc.) described in section 1.1.3.3. A separate set of zoom buttons is available for the difference flowgram because it is sometimes convenient to examine it at a different scale than the Reference and read flowgrams. Also, the “Save plot snapshot to image file” and “Save plot data to spreadsheet file” buttons will save all three plots together, in a single file. The histogram bars can be displayed in your choice of three styles (Bars, Lines, and Lollipop), by selecting the corresponding radio button near the upper-left corner of the tab. The three plots of the Flowgrams tab can also be resized and collapsed, as described in section 1.1.3.2.
The tri-flowgram plots have another feature that is important to maintain the alignment between the two upper plots: due to the interplay between the nature of a variation and the flow order of the nucleotide cycle during the sequencing Run, the situation sometimes occurs whereby the Reference and read flowgrams fall out of phase by one or more cycles of four flows. If this were allowed to happen, all the flows beyond the cycle shift would be misaligned, and all the values in the difference flowgram would be wrong. Cycle shifts are therefore inserted in either the Reference or the read flowgram, as appropriate to maintain their synchronicity; these “empty” inserted cycles are marked in gray on the flowgrams (Figure 1‑67).
Figure 1‑67: Example of a flowgram with inserted nucleotide cycles