Using the Variants Tab

2.10	Using the Variants Tab

2.10.1 The HC Diffs Sub-Tab

2.10.2 The HC Structural Rearrangements Sub-Tab

2.10.3 The HC Structural Variants Sub-Tab

There are three sub-tabs on the Variants tab that contain information about differences (relative to the reference) that are indicated by alignments of reads mapped to the reference: one reports on nucleotide differences and the others on structural variations. The sub-tabs contain tables of variations showing statistics, loci, and (optionally) annotations. Using the mouse, you can right-click any of the entries on the HC Diffs and HC Structural Variants sub-tabs and choose to view the alignment for the selected variation.

The sub-tabs are described in more detail in the sections below; they share the following common features:

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Sorting:

◦	All columns are sortable by clicking on the column header. A small triangle appears to indicate the direction of the sort (see Figure 63 and Figure 65, below). Clicking on a column header of a column that is already sorted will reverse the order of the sort.

◦	If multiple rows have the same value for the sorted column, these rows stay in the order in which they were before the sort, relative to one another. This allows for “nested sorting”. For example, if you:

▪	sort first based on “Start Position in Ref”,

▪	then sort based on “Reference Accession Number”,

▪	the result is a table where the high confidence differences are grouped by reference sequence, and the ones that belong to the same reference are displayed in order of their position on the reference sequence.

•	Summary tooltip (mouse-over information):

◦	For the HC Structural Rearrangement sub-tab, hovering the mouse cursor over any row provides a tooltip that indicates the type of rearrangement and position or length, depending on the type of variation.

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Hovering the mouse cursor over any row in either of the other two sub-tabs provides a tooltip that summarizes the basic statistics of the high confidence difference described in that row (see Figure 63). In particular, the bases involved in the variation coupled with the total variation percentage, the depth of sequencing, and the separate statistics for both forward and reverse reads are presented.

•	Links to read alignment data:

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Right-clicking in the row for a particular HCDiff or HC Structural Variant opens a contextual menu with a single item: “Show Alignment”. Selecting this loads the Alignment results tab with the alignment that supports the variation in question. The first base of the variation appears as the leftmost base in the view.

◦	You can also select multiple variants, then right-click to load these variations into a list. The Alignment results tab will display the first variation in the list and the list itself will comprise the dropdown menu contents of the Positions of interest (Figure 65).

◦	You can then scroll the alignment to examine the context of the variation.

•	Export of Variations Tables:

◦	You can export variations data, in its current sort order, in png, txt or csv format. The txt and csv export includes the complete table. The png format only includes the currently visible portion of the table.

◦	The export dialog box is launched by clicking the button, for txt or csv format, or the button for png format. (See Figure 66)

Note on duplicate reads handling when computing Variants:

Duplicate reads of a single DNA input are a known potential artifact of the emPCR Amplification process which may occur, for example, when two or more beads are amplified in a single emulsion microreactor. In an attempt to compute more accurate variation percentages, the GS Reference Mapper, by default, groups individual reads into groups of duplicates (reads that start at the same base of the reference sequence). Groups of duplicate reads count as only one read in the computation of variants (HCDiffs and HC Structural Variants). However, the corresponding alignments on the Alignment results tab display all the reads whether or not they were considered duplicates. When grouping occurs, the variant summary tooltip provides the statistics for both grouped and for individual reads (see Figure 63).

On the command line, the “-ud” option (see Table 4, in Appendix) instructs the GS Reference Mapper to treat all reads individually and to not perform this grouping operation. This option is available in the GUI, on the Parameters/Computation sub-tab. This should be used, for example, when using the GS Reference Mapper to analyze data from an Amplicon sequencing experiment, where every read is a distinct data point.