Display Option Tools

1.6.4

The upper-left corner of the Global Align tab contains various navigation and filtering tools that modify the information displayed on the Variation Frequency Plot and the multiple alignment panels of the tab (Figure 1‑63).

Figure 1‑63: The display option tools of the Global Align tab

1.6.4.1

Alignment Data

There are two navigation controls located at the top of the display option tools box, that allow you to select new sets of data to display in the Global Align tab. The first is a drop down menu that contains a list of all the Samples defined in the Project that are associated with at least one of the Amplicons you are viewing in the multiple alignment currently displayed. Selecting a new Sample from the drop down menu will update the ‘Global Align’ tab with the alignment data for the new Sample, replacing the current data. This allows you to quickly compare various Samples over a single or a given set of Amplicon(s).

The second Alignment data control is the Amplicon selection button, located just below the ‘Alignment Data’ drop down menu (Figure 1‑63). Clicking this button opens the “Choose Alignment Data” window (Figure 1‑64).

Figure 1‑64: The Choose Alignment Data Window

This window allows you to browse over the entire Project and select data for display in the Global Align tab. It is used in three steps:

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Step 1: choose a Reference Sequence for which you want to display the data. This will update the list of available Amplicons, in the second column, to those that are associated with that Reference Sequence and for which there is an alignment computed for at least one Sample (excluding, however, Amplicons associated with the Reference Sequence selected but for which no Read Data sets have supplied any reads).

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Step 2: select one or more of the available Amplicon(s) of interest. The Global Align tab can display the reads from multiple Amplicons, merged into a single multi-alignment, as long as they all belong to the same Reference Sequence. This selection will update the list of eligible Samples, in the third column, to those that are associated with at least one of the Amplicons selected and for which there are currently computed results.

•	Step 3: select one of the eligible Samples, and click “OK” to load the selected alignment in the Global Align tab. The number of Amplicons included in the displayed multi-alignment is indicated to the right of the Amplicon selection button.

If you activate the “Choose Alignment Data” window and click “OK” without changing any of the currently selected choices, the AVA software will freshly reload the currently selected alignment. This can be useful to reset the view of the alignment after using the “Remove reads, reset selections” action (see section 1.6.3.3)

1.6.4.2

Read Type

The ‘Read Type’ radio buttons (see Figure 1‑63) give you the choice to display the reads in one of two ways:

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Consensus: This is the default read type. When this option is selected, reads that are substantially similar are grouped together into consensi, which results in fewer sequence entries in the multi-alignment table. The consensi are listed in the multi-alignment in decreasing order of the number of reads they represent, so the ones near the top have the more “weight”. This option is intended to reduce noise and speed up navigation.

•	Individual: This option displays every read as a separate sequence line in the multi-alignment, even if they are identical to other reads. This can greatly increase the volume of alignment lines and slow navigation, but hides no noise.

It is usually easiest to perform an initial analysis with the default Consensus view, with its lower volume and decreased noise. Delving into the individual reads can be useful if you need to search for a particular variation that may have been erroneously spread amongst several consensi, treated as noise in basecalling, rather than being exposed as a separate variation.

1.6.4.3

Reported Frequency

The next set of radio buttons controls the type of ‘Reported Frequency’ (see Figure 1‑63). This applies to:

•	the Variation Frequency Plot left axis (which relates to the histogram bars)

•	the information reported in the mouse tracker panel

•	the frequency information for a nucleotide, in the screen tips that appear when you pause the mouse over a nucleotide in the multi-alignment panel

•	the frequency information for the nucleotide selection options, in the contextual menu that appears when you right-click on a nucleotide in the multi-alignment panel

•	the reported read depth in all of the above

The two options are as follows:

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The default ‘Global’ frequency option uses the coverage from the full data set as the read depth denominator when calculating the frequency of occurrence of a given nucleotide at a given alignment position, regardless of any positional ‘Select’ filters that may have been applied to hide reads (or consensi) from the multi-alignment display. (This also applies to the display of the reads from a single consensus on the Consensus Align tab, which is another form of read selection; see section 1.7.)

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The ‘Relative’ option recalculates frequencies using only the visible data, i.e. ignoring reads (or consensi) hidden from the multi-alignment display after you applied any selection(s). This can be useful when you have selected reads (or consensi) for a variation at a given coordinate and you want to examine other variations relative to the first selection(s) (now set at 100%); for example, variations linked as a haplotype should show near 100% relative frequency in this situation. (This is also useful when examining the reads from a single consensus on the Consensus Align tab, which is another form of read selection; see section 1.7.)

If you did not make any ‘Select’ filter choices on alignment positions, the ‘Global’ and ‘Relative’ frequencies will be the same (but not so on the Consensus Align tab, where all results displayed are inherently selected for a single consensus; see section 1.7). Once you make selections to focus on a subset of the data, you will notice the difference between the reported frequency types. Note that the reported read depth is that used in the frequency calculations. So, if you want to know how many reads are present amongst the selected reads of the multiple alignment, you must switch to ‘Relative’ frequencies.

1.6.4.4

Read Orientation

The final set of radio buttons controls the display of the reads or consensi by ‘Read Orientation’ (see Figure 1‑63).

•	The default is ‘Any’ which means both forward and reverse reads are presented in the multi-alignment.

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Choosing ‘Forward’ or ‘Reverse’ will restrict the multi-alignment view to display only the reads of the selected orientation. This can be useful when you have coverage of a variation in reads from both orientations; the presence of the variation at a similar frequency in both orientations would be a strong argument that it is “real”, whereas its presence in only one orientation (or a large discrepancy in its frequency between the two orientations) would be an indication that the variation might be due to an artifact. (See section 2.5 for guidelines and factors to consider when trying to determine whether a Variant is genuine).

In a restricted orientation view, the behavior of the ‘Global’ reported frequency option is slightly modified so that the coverage in the denominator comes from the full data set, but restricted by orientation. This prevents the global frequencies from deceptively dropping by about 50% when an orientation is chosen. As mentioned in the description of the Variants Tab (section 1.5.2.2), failure of a variation to show in one orientation for which a good number of reads are available is an indication of a possible artifact; however, you may want to not “penalize” a variation on this account if it is covered in only one orientation, or if too few reads cover it in one orientation to provide for statistically valid data.