The Flowgrams Tab

1. GS Amplicon Variant Analyzer Application : 1.8 The Flowgrams Tab

1.8	The Flowgrams Tab

1.8.1 Populating the Flowgrams Tab

1.8.2 The Triflowgram Plot

1.8.3 Navigation on the Flowgrams Tab

The Flowgrams tab allows you to view the flow-by-flow signals of any individual read included in the Project, highlighting any departure from the signal intensities that would be expected of the Reference Sequence to which that read (Amplicon) is associated. The display is designed to help you evaluate the significance of differences between an individual read and a Reference Sequence. To this end, the tab does not simply display the raw flowgram of the read, but rather a computationally processed version of it. In particular, flow cycle-shifts may be introduced into one or both flowgrams in order to optimize their alignment; and the flowgram of the read may be computationally reverse-complemented in order that it is always shown in the 5’-->3’ orientation of the Reference Sequence. Finally, the flowgram only displays the subset of flows relevant to the read’s sequence alignment as displayed in the Global Align or Consensus Align tabs.

The Flowgrams tab’s main feature is a “tri-flowgram” plot showing (Figure 1‑66):

•	an aligned, idealized flowgram for the Reference Sequence.

•	an aligned, (possibly reverse complemented) flowgram of the read.

•	a difference flowgram (read minus reference), where any variation from the Reference Sequence is seen as a non-zero value (whereby extra signals in the read, relative to the Reference Sequence, show up as positive differences in this panel).

In addition to the tri-flowgram plot, the Flowgrams tab contains a small set of display option and navigation tools in the upper-left corner, and the usual “Mouse Tracker” and color-code legend, in the lower-left corner.

Examining flowgrams can be useful when trying to assess whether a variation may be genuine or due to an artifact. For example, the flowgram of a mononucleotide from the Reference Sequence called as a dinucleotide repeat in a given read may show that the signal was barely over the threshold for calling a two-nucleotide incorporation, casting doubt on the second base of the call. Conversely, variations that induce a cycle shift in the flow alignment are particularly compelling since such shifts would not be expected as a result of simple overcalling or undercalling during signal processing, nor would they result from sequencing artifacts such as incomplete extension or carry forward. If the flowgram indicates that a variation appears genuine, the user should still consider whether it also occurs in other, overlapping reads, especially in the opposite orientation, or in a replicate experimental Sample; or could the variation simply be due to a PCR artifact introduced early in the sample preparation process? Anticipating these types of questions should play a large role in the experimental design of an Amplicon sequencing experiment.

A brief explanation of the information contained in a flowgram is provided along with a full description of the tri-flowgram display, in section 1.8.2. For more details on flowgrams and on the processing of data that generates them, see in the description of the Wells tab of the GS Run Browser in Part B, Section 3 of this manual.

Figure 1‑66: The Flowgrams tab

The flowgram alignment algorithm works only by introducing cycle shifts, with the goal of minimizing the sum of the absolute value of the signals in the difference plot while simultaneously attempting to minimize the number of cycle shifts introduced. The alignment algorithm does not attempt to split larger individual flow values into multiple flows of lesser magnitude, which could allow it to produce results that more closely mimic the alignments one would obtain by working in nucleotide-space (for an example where this situation arises, see section 2.3.2, below Figure 2‑30).