Wells Tab Features and Functionalities

3.5.1

The Wells tab contains many features with the functionalities described in the sub-sections below.

3.5.1.1

This is a drop down menu used to select the well display category (Figure 24). Each category has its own color chart to identify the specific attribute of each well, within the category (see Section 3.5.1.2). If the data from one or more regions of the PicoTiterPlate Device is missing, “(Data unavailable)” will appear in the Well Categories drop down menu.

Figure 24: Wells Tab, Well Categories Menu

•	Status category

◦	No Key: Identified as a well (generates signal), but not one with recognizable data

◦	Library Passed/Failed Filter: Library read (key is TCAG or GACT) that either passed or failed quality filters. Note that the GS Run Browser does not make pass/fail decisions; it only reads results provided by the quality filtering algorithms of the GS Run Processor application.

◦	Control DNA Passed/Failed Filter: Control DNA read (key is CATG or ATGC; see Section 7.1 for details) that either passed or failed quality filters.

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Control DNA category: This is similar to the Status category except that it uses separate colors for the reads matching each specific Control DNA sequence, or wells where an appropriate Control DNA key was detected but the sequence of the read does not match any of the known Control DNA fragments (see Section 7.1 for details on Control DNA keys). Wells where the library key or no key at all was detected are also displayed, using the same color scheme as for the Status category.

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Raw Density and Key Pass Density: Measures of local well crowding, calculated individually for all identified wells in which a signal is detected (Raw Density) and for all identified wells in which the initial signals match a known key, i.e. excluding the wells with a No Key status. For data sets processed with the software version 2.0.00 or later, this option will appear only if the signal processing step of Data Processing was carried out. For versions 1.1.03 or earlier, this will normally show (requires the region.wells files in the data set). Color gradient bins of +2.5% can be selected.

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Carry Forward and Incomplete Extension: Level of CAFIE correction applied to each well due to the detection of carry forward and incomplete extension events (described in Section 1.4.1). This option will appear only if the signal processing step of Data Processing was carried out. Individual color gradient bins can be selected.

3.5.1.2

Color chart

Located just below the Well categories drop down menu (Figure 24), this area allows the user to select the range of well values to display in the main image area, for the category selected. These controls have the following general features and behaviors:

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Checking the box for an attribute (or numerical range) in the list causes wells matching that attribute (or range) to be displayed on the image. If an item in that list is not checked, the corresponding wells are not displayed. Shift-clicking deselects all attributes; or, if all are already deselected, it selects them all. Control-clicking deselects all attributes except the one clicked; or, if the user control-clicks on an attribute that is currently the only one selected, it selects all attributes except the one clicked.

•	For the categories whose attributes are continuous variables, the color scheme uses the “color wheel” order for the gradation of value ranges.

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For the attributes that denote “goodness” or “badness” of the wells, the “good” values are indicated by green hues and the “bad” values by red hues, when possible. For example, the most desirable values (wells marked green) for well density in the mid-range values (40-45%) but for CAFIE correction the best wells have minimum values (<0.1%).

•	The GS Run Browser “remembers” attribute selection for each category when a user navigates to another tab and then returns to the Wells tab.

Figure 25: Examples of color charts for well category display

3.5.1.3

Flows

Located below the color chart, the Flow selector allows the user to choose an image to underlay the well circles, in the main image area. If images are available in the Run data set, this selector is displayed; it lists the steps of the sequencing Run where an image was taken by the instrument camera.

If any image is unavailable in the data set (e.g. it was removed), its appearance in the Flows list will depend on whether the current data set was opened from a raw instrument data set (R_ directory) or a data processing data set (D_ directory). If an R_ directory was opened, the image is grayed out in the Flows list. If a D_ directory was opened, the image will be enabled, but there will be a

icon next to it. This indicates that the raw image is unavailable, but a low-resolution image, stored in the .cwf file, is available. If you hover the mouse over the image, the tool tip will indicate Low Resolution, as shown in Figure 26.

Figure 26: Partial view of Flows, showing an image that is missing, but available in low-resolution format

•	The Flow tags have the following meanings:

◦	SUB: Substrate buffer (background)

◦	WASH: Buffer wash flow (GS 20 and GS FLX standard chemistries only)

◦	Apyrase##: flow used for Apyrase pulse calibration (GS Junior Titanium and GS FLX Titanium chemistry only)

◦	ATP: Flow of ATP for the GS Junior Titanium and GS FLX Titanium chemistry (all wells)

◦	PPI: Flow of PPi for the GS 20 and GS FLX standard chemistries (all wells)

◦	T, A, C, G: Nucleotide flow

•	Clicking on a step displays the corresponding image in the main image area (and overview image area). After clicking in the Flows selector list, a user can use the keyboard arrow keys to navigate quickly up and down the list of images.

•	When the Show All box is checked, all the images captured during the sequencing Run are listed. If the box is not checked, only the images captured during nucleotide or PPi / ATP flows (i.e. excluding SUB and Apyrase flows) are listed.

•	The wells themselves exist during all flows, so this selector has no effect on the display or coloring of the well circles. If the background image is a distraction, the user can disable the images by deselecting “Show image” in the contextual menu that appears if a user right-clicks on the image.

3.5.1.4

Image area

This area provides a zoomable, scrollable view of the entire PicoTiterPlate Device area, with well data overlaid on the camera image, if available. Overall, the image is comprised of four layers:

•	The camera image selected; the displayed image is corrected to emphasize the well intensities and deemphasize the background intensities, for better visualization.

•	Green rectangles delimit the bead loading regions of the sequencing Run; the top of each region is labeled with the region name.

•	Wells, per the options selected.

•	Display control buttons and overview image

Settings chosen in the Options section are reflected in the image area, such that colored circles displayed at the x,y location of the wells identified during data processing reflect the Well Categories option, and the selected range of attribute values.

Wells remain in the same locations across all flows of a sequencing Run, irrespective of what image is selected for display in the Flow selector (or of whether images are available at all in the data set).

Pausing or moving the mouse slowly over the main image area has the following effects (Figure 27):

•	The well closest to the pointer is highlighted.

•	The pointer position is marked by a blue “+” on the overview image area

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The Mouse Tracker box appears near the pointer, providing information about the image pixel under the pointer and the closest well. This includes the location under the cursor (region number and x,y coordinates), the corresponding pixel signal intensity value (for the flow selected, if images are available), and the attribute value for the closest (highlighted) well, in the category currently selected in the options. Note that setting the image brightness using the White Threshold slider does not change the intensity value of the pixel, only the brightness at which it is drawn on the screen.

Figure 27: Wells Tab image area mouse tracker information and overview image

Clicking on the image can have the following effects:

•	A simple left-click selects the closest well for further action. A selected well is marked by an additional light blue circle around it.

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A double-click also applies to the closest well. This constructs a Well Flowgram (for wells that contain a library bead; TCAG or GACT key) or a Tri-Flowgram (for wells that contain a Control DNA bead, CATG or ATGC; see Section 7.1 for details), and opens the flowgram viewer to display it (see Sections 3.5.2 and 3.5.3 for a full description of well flowgrams and well tri-flowgrams, respectively).

•	A right-click opens the contextual menu shown in Figure 28 and described below.

•	A left-click-and-drag slides the image in the direction of the drag, revealing the part of the image that was just out of view.

•	A right-click and drag zooms the image to the area circumscribed by the dragging action.

If a mouse wheel is available, rolling forward zooms in and rolling backward zooms out the image. The Magnification Slider control (see below) moves along with the mouse wheeling action.

Figure 28: Contextual menu evoked by right-clicking the image area of the Wells tab

The contextual menu of the image area offers the following actions:

•	Reset view: resets the image to its default size and location, in the top-left corner position. The accelerator key is Home.

•	Go to location: opens a data entry field just above the image area allowing a user to specify a location on the image (Figure 29), for immediate navigation when a user presses the “Enter” key on the keyboard. Valid entries and their meanings are as follows:

◦	a single positive integer scrolls the image to the top of the region number specified (e.g. 1-16).

◦	two positive integers equal to of less than the number of the image pixels scroll the image to that x and y location. The two values can be separated with characters such as a space, comma, period or a slash.

◦	a well ID or accession number scrolls the image to that location and selects the well

If the entry is not valid, an icon showing an “x” in a red circle appears at the lower left corner of the field, and the image location does not change. The accelerator key is Ctrl-G.

Figure 29: The 'Go to location' field, for the Image area of the Wells tab.

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Well flowgram: opens the “Well flowgram” for the closest well. This will be a regular flowgram if the status of the well is “Passed filter” (or “No key”, or “Failed”), and a tri-flowgram for “Control DNA” wells. See sections 3.5.2 and 3.5.3 for a full description of Well flowgrams and Control DNA Tri-flowgrams, respectively. The accelerator key is Ctrl-F.

•	Expand images: pre-expands camera images to decrease image loading times. The accelerator key is Ctrl-E.

•	Location flowgram: opens the “Location flowgram” for the image pixel on which the user clicked. See Section 3.5.4 for a full description of Location flowgrams. The accelerator key is Ctrl-L.

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Subtraction flowgram: this action requires that a pair of subtraction pins (see below) be first added on the image. Then, selecting this menu item opens the “Subtraction flowgram” (Section 3.5.5) for the pair of image pixels selected by the pin, that is, for each flow of the sequencing Run, the difference between the signals at the two ends of the pin. The accelerator key is Ctrl-S.

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Add subtraction pin: this action deposits a subtraction pin on the image (Figure 30). A subtraction pin is a visual indicator showing two image locations that can used for calculating a subtraction flowgram. A small circle circumscribes the area that will be used for the calculation; a pentagon and a square around each of the location serve as “handles” that can be dragged to position the circles at the locations of interest; a bar between the two squares shows which two locations are associated with other. The direction of the subtraction is: “circle in the pentagon” (pin head) minus “circle in the square” (pin tail). A user can add multiple subtraction pins on an image; the “active” one, i.e. on which an eventual “Subtraction flowgram” command would apply, is shown in blue; all the others are yellow. The accelerator key is Ctrl-P.

•	Clear pins: removes all the subtraction pins currently in view. The accelerator key is Ctrl-M.

•	Show image, Show regions, and Show wells check boxes: control whether the corresponding layers of the image area are displayed (checked) or not (unchecked).

•	Show comments if enabled, will show all the Run and region comments entered by the operator when the run was performed.

Figure 30: The image area of the Wells tab, with three subtraction pins. The one on which an eventual “Subtraction flowgram” command would apply is in blue. The direction of the subtraction is: “circle in the pentagon” (pin head) minus “circle in the square” (pin tail).

The image area of the Wells tab has the following special navigation controls:

Button	Name – Description
	Hide controls – Hide the icons for the image area controls
	Show controls – Show the icons for the image area controls
	Reset view – Reset the view of the image to the default top left location and default magnification
	Adjust white threshold – Adjust the upper threshold for white values in the image. Clicking this button elicits a slider that can be used to set the white threshold (Figure 31). For the GS FLX+ Instrument, the range is from 0 to 2500 and the default setting is 1000. For the GS Junior Instrument, the range is from 0 to 8000, with a default setting of 2000. Clicking the blue button to the right of the slider resets the threshold to its default value. The white threshold value selected applies to all images. Figure 31: The Adjust White Threshold slider These default values can be changed by editing the GS Run Browser startup script, grRunBrowser.sh.
	Magnification Slider – Set the magnification of the image. Higher values reflect greater magnification (zoom in).
	Overview image: shows a small representation of the entire PicoTiterPlate Device, with the image, if available, but without the wells, as an inset near the upper right corner of the image area. A blue “+” shows the current location of the mouse pointer, in the main image. Clicking (left or right) on the overview image scrolls the main image directly to the pixel on which was clicked.

3.5.1.5

Average well density summary

This area provides the statistical averages for raw well density and key pass well density, calculated for each region and for the entire PicoTiterPlate Device.

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For data sets processed with the GS Run Processor version 2.0.00 or later, these values are taken from the [region].cwf files, if available. For older processed data sets, they are calculated by the GS Run Browser as the wells are loaded. For each well, the application counts the number of other wells in a local area around the well’s center (~50 pixels); the raw well density calculation includes all wells, whereas the key pass density calculation omits wells that did not key pass (had a No Key status value). Note that the GS Run Processor and the GS Run Browser use different algorithms to perform these calculations, and will not yield identical well density values, but regions of fairly uniform density will have close results.

•	An invisible divider is present between the image area and the well density summary table that can be dragged to adjust the part of the window allotted to these two areas.

•	Image and Data Capture buttons are located to the left of the well density summary tables. The functions of these buttons are described in Section 3.3.4.