Partek Flow Documentation

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The Chromosome view in Partek Flow is a visualization tool for next-generation sequencing (NGS) and microarray data. The viewer can display different types of information, including aligned reads, genomic databases (e.g. genes, transcripts, or variants), isoform proportions, and reference sequence. 

This chapter will illustrate how to:

Table of Contents
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Launching the Chromosome View

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SubtitleTextAccessing Chromosome view via the toolbox (the content of the Visualisation section depends on the selected data node)
AnchorNameToolbox

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The Chromosome view can be invoked from some data nodes on the Analysis tab, giving a global overview of the results; or from certain Task report or result pages, providing a focused view, i.e. pointing to a specific feature of interest. 

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A new Chromosome view task node will be added to the canvas (Figure 2) and in order to invoke the viewer <double-click> on the node (you can also select it and then go to Task report in the toolbox). When invoked in the aforementioned way, the default visualization in the Chromosome view is the first 100,000 bases of the first chromosome. 

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SubtitleTextSelecting Chromosome view from the toolbox adds a Chromosome view task node to the canvas. To open the view, <double click> on it
AnchorNameChromosome view task node

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Another way to get the Chromosome view is through a Task report; you can launch the viewer by selecting the chromosome icon Image Removed in the View column (Figure 3).

 

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SubtitleTextAccessing the Chromosome view from results table (mouse-over balloon is visible when hovering over the chromosome icon). The image is an example, based on gene expression pipeline
AnchorNameChromosome view access

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In that case, the Chromosome view will browse directly to the selected genomic location (i.e. a transcript or a variant, depending on the pipeline).

Navigating Through the View

A user can browse through the results by using one of the tools in the navigation bar (on the top of the view; Figure 4). Select tracks tool is the topic of a separate section, while the remaining tools are described below.

 

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SubtitleTextNavigation bar of the chromosome view (from left): Select tracks tool, Search box, Position box, mode selector (pointer, zoom, pan), zoom tool, bookmarks, save icon (the position in the figure is an example)
AnchorNameChromosome navigation bar

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You can use the Search box ito zoom to genomic features that are available in the annotation track. Start typing a search term and Partek Flow will show you the first 10 suggestions (Figure 5). To select one, use the arrow keys or mouse, or type the full feature name and hit enter.

 

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SubtitleTextSearch box of the Chromosome view. To zoom in on a feature, start typing the feature name; Partek Flow will show suggestions available in the corresponding annotation file (the current annotation is visible in the column on the right) (an example is shown)
AnchorNameChromosome Search Box

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The Position box enables the user to visualize a region in the genome. Coordinates are accepted in the following format: chromosome:start – end (zero-based). To show an entire chromosome, it is sufficient to enter just the chromosome number. The U-turn icon on the right Image Removed takes you back to the original view, i.e. resets the zoom level to the view that was shown when the viewer was first opened. 

Next, the mode selector (Figure 6) helps you to quickly navigate through the results.

 

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SubtitleTextMode selector (from left): pointer mode, zoom mode, pan mode
AnchorNameMode selector

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When panning mode is activated, the appearance of the cursor will change to an arrow (Figure 7). Pointer mode provides details on any item (e.g. short sequencing read, variant, microarray probe, annotation feature) selected on the canvas. The selected item is highlighted by a green box (Figure 7).

 

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SubtitleTextHighlighted item in chromosome view: the microarray probe highlighted by a green box was selected using the pointer mode (microarray probes are used just as an example)
AnchorNameMicroarray Highlight

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When zoom mode is activated, the appearance of the cursor will change to a plus (plus). With the zoom mode on, you can magnify a specific region by positioning the cursor (plus) to the left of the area of interest and then <left-click> & drag the mouse to the right of the area of interest (Figure 8). When the viewer refreshes, it will come "closer" to the region that was selected (by halving the number of basis displayed on the screen). 

 

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SubtitleTextUsing <left-click> & dragging mouse to zoom into a region of genome (start magnification shown on the left). After releasing the left mouse button, Partek Flow will zoom into the highlighted region (right panel; an example is shown)
AnchorNameChromosome view zoom

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Alternatively, <left-click> on the canvas and Partek Flow will zoom in one level, by halving the number of bases visible on the screen. To zoom out one level Ctrl & <left-click> should be used; as a result, the number of visible bases will be roughly doubled.

When panning mode is activated, the appearance of the cursor will change to four arrows (Figure 9). <Left-click> and drag the canvas to the left or to the right to move upstream or downstream in the genome, respectively.

 

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SubtitleTextAppearance of the mouse cursor when the panning mode is on
AnchorNamePanning mode

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Zooming out and in can also be achieved with the zoom tool (Figure 10) by moving the golden slider left or right, respectively, or by selecting the magnifying glass icons (– and +).

 

 

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SubtitleTextZoom tool
AnchorNameZoom

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The location of an interesting region can be bookmarked. Selecting the bookmark icon (i.e. the star) opens the dialog (Figure 11). To create a new bookmark, type the name of the region in the Create bookmark box and push Create.

 

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SubtitleTextBookmark dialog.The 'B2M' is shown as an example of an existing bookmark
AnchorNameBookmark

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The next time you want to go directly to the same location, select the name of the bookmark (example in the Figure 11 lists B2M - exon #4 as the bookmark name) and Partek Flow will plot the region as defined in the Location column. To remove a bookmark, select the delete icon Image Removed.

Once the plot has been modified, you can save the current appearance of the canvas by using the save icon Image Removed . The resulting dialog (shown in Figure 12) enables you to change the image Format (options include: .svg, .png, .pdf), Size, and Resolution. The image will be saved in your Downloads directory.

 

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SubtitleTextSave image dialog. Upon selecting Save, saves the current visualisation on the canvas
AnchorNameImage dialog

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Selecting Data Tracks for Visualization

Partek Flow plots genomic information on the canvas and is organized into horizontal sections called tracks. The exact number, type, and presentation of tracks depend on several factors, such as the underlying pipeline, available annotation, and the level of zoom. The tracks are added, removed, or customized via the Select tracks dialog (Figure 13).

 

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SubtitleTextSelect tracks button opens the Select tracks dialog
AnchorNameSelect tracks button

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The content of the Select tracks dialog depends on the data nodes present on the Analysis tab of the current project (an example is shown in Figure 14). Current pipeline is depicted in the center of the window, while data nodes that can be visualised are highlighted by the colour of their layer. Tracks can be turned on or off by selecting the check boxes in the list of possible tracks (and data nodes) on the right. To uncheck all, use the Clear selection button.

 

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SubtitleTextSelect tracks dialog (the pipeline is an example). Data nodes that can be visualised are highlighted by the colour of their layer (sky blue in this example). Tracks can be turned on or off by selecting the check boxes in the list of data nodes and respective tracks (right panel)
AnchorNameTrack selection dialog

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For the ease of use, the pipeline and the list of tracks are linked: hovering over the track list highlights the matching data node in the pipeline and vice versa, i.e. selecting a data node in the pipeline panel highlights the respective node in the track list (Figure 15). Once you decided on the tracks that should be plotted, push Display tracks to depict them on the canvas.

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Visualizing the Results Using Data Tracks

Data tracks section of the Select tracks dialog enables you to specify the tracks for visualization on the canvas. An overview of the available track types is provided in Figure 16. Note that not all tracks are visible at all times and that their presence depends on the zoom level. The tracks can be customised and their appearance changed by using the control panel on the right.

 

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SubtitleTextData tracks in Chromosome view (examples)
AnchorNameData tracks

Alignments track

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Isoform proportion track
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Variants track
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Amino acids track
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Reads pileup track
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Probe intensities track
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Alignments Track

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SubtitleTextAlignments track: different colouring options. When colouring reads by sample, the reads are stacked (on top of each other), i.e. in the example above there are more reads in the red sample than in the blue sample
AnchorNameAlignment track

Reads coloured by sample
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Reads coloured by base calls
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Isoform Proportion Track

The Isoform proportion track displays the reads mapped to transcripts and helps to visualize differential expression and alternative splicing, using standard symbols for exons (boxes) and introns (lines connecting the boxes). The size of each transcript is proportional to the number of reads that map to that transcript. The color indicates the samples to which the reads belong. Figure 18 shows a gene with two transcripts in RefSeq database; the top transcript is more abundant than the bottom transcript and is preferentially expressed in the "blue" condition (labeled as 0 uM). The bottom transcript, on the other hand, seems to be expressed at the same level across all three conditions (i.e. 0 uM, 5 uM, 10 uM). The number and structure of transcripts on the plot depend on the transcript model that was used for mapping.

 

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SubtitleTextIsoform proportion track: the transcripts are shown as present in the transcript model that was used for mapping. Exons are depicted as boxes. The size of each transcript is proportional to the number of reads mapping to it. Colors indicate samples to which the reads belong
AnchorNameIsoform proportion track

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Variants Track

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SubtitleTextVariants track at low power magnification: SNVs are symbolized by purple columns and an insertion is presented as a green bar (an example is shown). A deletion is presented as a red bar (none is visible on the figure)
AnchorNameVariants track at low magnification

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SubtitleTextVariants track at high power magnification: each SNV is presented as a pie chart and each slice symbolises the relative frequency of each base call (an example is shown). Base call colour codes are given by the track name
AnchorNameVariants track at high power magnification

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At higher modification, insertions are seen as green boxes, with individual inserted bases presented using a pie chart, while deletions look like red boxes and the affected bases are also presented by a pie (Figure 21). 

 

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SubtitleTextVariants track at high power magnification: insertion is presented as a green box, deletion is presented as a red box. An example is shown.
AnchorNameVariants track at high power magnification

Insertion
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Deletion
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Amino Acids

Amino acids track becomes available in the Select tracks dialog after completing the Annotate variants task. The actual appearance of the track depends on the zoom level. With low-power magnification, you will see a message View not available at this zoom level, Please zoom in to view amino acids.

When you zoom closer to the genome, all the amino acids become visible as colored boxes (Figure 22) and labeled using the single-letter amino acid code. Alternative amino acids are depicted as additional boxe on the top of the consensus sequence.

 

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SubtitleTextAmino acids track at high power magnification: consensus amino acid sequence is at the bottom of the track, while a variant is shown on the top (change from Threonine to Proline is shown)
AnchorNameAmino acids

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If an amino acid spans two exons, its box will be truncated and the line connecting the exons will be dashed. An example is in Figure 23.

 

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SubtitleTextAmino acids track: exon-spanning amino acids indicated by truncated boxes (i.e. Alanine on the left) (an example is shown)
AnchorNameexon-spanning amino acids track

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An empty gray box on the top of consensus sequence is used to indicate a STOP codon, which is a consequence of a mutation (Figure 24).

 

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SubtitleTextAmino acids track: A variant which is in fact a STOP codon is represented by an empty box, as seen on the top of the G (an example is shown)
AnchorNameSTOP codon

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Untranslated bases, such as ones downstream of a STOP codon are depicted by lighter shades. Figure 25 shows two transcripts in an amino acid track; the direction is from left to right, so amino acids downstream of a STOP codon (P > G > L) are lightly shaded.

 

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SubtitleTextAmino acids track: amino acids downstream of a STOP codon are depicted by lighter shades. STOP codon is represented by "." in the middle, direction is from right to left (an example is shown)
AnchorNameDownstream amino acids

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Reads Pileup Track

Reads pileup track plots the short sequencing reads, as present in the .bam file. The track is not on by default (go to Select tracks to turn it on) and its appearance depends on the magnification; if you are zoomed out a message - Zoom in to view individual reads - will be displayed.

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SubtitleTextReads pileup track: short sequencing reads are represented as bars. Paired-end reads are located within a gray box encompassing both pairs. Singletons, such as that on the top right, are depicted as thicker reads (an example is shown)
AnchorNameReads pileup track

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If you used a junction-aware aligner (such as TopHat or STAR), the junction reads will be depicted using dashed lines, which connect exon-spanning parts of the same read (Figure 27).

 

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SubtitleTextReads pileup track: junction reads are depicted using dashed lines. A RefSeq track is added at the top, to visualise the exons (an example is shown)
AnchorNamejunction reads

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Deleted bases can also be seen on a Reads pileup track, as fat black lines (Figure 28).

 

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SubtitleTextReads pileup track: deleted bases depicted using fat black lines (an example is shown)
AnchorNameDeleted bases

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Probe Intensities Track

Microarray probes are visualised by the Probe intensities track. The probes are shown as bars and their colour depends on the probe intensity, ranging from white (low) to admiral blue (high) (Figure 29).

 

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SubtitleTextProbe intensities track: probes are depicted as bars and their colour reflects the intensity (an example is shown)
AnchorNameProbe intensities track

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As with the Reads pileup track, probes may not be visible with low power magnification and you will see a message - Zoom in to view individual microarray probes.

Annotating the Results

Cytoband Track

By default, the Chromosome view shows a cytoband track at the top of the canvas. If a cytoband file for your genome has not been added to Partek Flow, a warning will appear (Figure 30). In that case, go to the Library File Management page and download or create a cytoband file.

 

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SubtitleTextWarning message indicating that Chromosome view can not be launched because of missing cytoband file
AnchorNameWarning

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The red box (Figure 31) indicates the part of the chromosome that is currently depicted on the canvas.

 

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SubtitleTextCytoband track: highlighted part is currently depicted on the canvas (an example is shown)
AnchorNameCytoband track

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Reference Genome

The sequence of the reference genome is added to the Chromosome view by default, as long as it has been added to the respective genome on the Library File Management page. However, its presence (or absence) in the viewer depends on the current magnification. At low power, the track is hidden and you will see the message - Track hidden (zoom to view). At high power, on the other hand, the Reference genome track becomes visible (Figure 32) and is supplemented by the genomic coordinates (below the sequence). A vertical guide helps you to align the bases between Aligned reads and Reference genome tracks. Depending on the reference genome file, some bases may be shown in lowercase letters, symbolizing repetitive sequences, or other sequences masked by a tool such as RepeatMasker.

 

 

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SubtitleTextReference genome track. Numbers beneath the sequence are coordinates
AnchorNameReference genome

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Variant Database
If a variant database file (such as dbSNP) for your genome is present on the Library File Management page, you will be able to include variant annotation track in your visualization (to add a variant database to the viewer, use the control panel on the right).

The variants will be shown adjacent to the Reference genome track (Figure 33). If the database contains no frequency information on alternative alleles, the alleles will be drawn as bars (an example is the SNP on the left in Figure 33). If the frequency information is available, the relative frequency of each variant will be represented by a column (the SNP on the right in Figure 33).

 

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SubtitleTextReference genome track with added variant annotation: single nucleotide variants present in the chosen database are depicted as bars (if no frequency information is available) or columns (columns reflect relative frequency of each alternative allele as stored in the database)
AnchorNameVariant annotation

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Note that the frequency information for each allele will be parsed out from the chosen database. That information can be retrieved by selecting a variant using the selection mode and will be shown in the Selection details section of the control panel. Using the example shown in Figure 33, the details of the left database variant can be seen in Figure 34. The most frequent allele at that locus is G (hence, yellow column is plotted above the Reference genome track), which matches the base call of the reference genome.

 

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SubtitleTextSelection details section of the control panel showing details of a SNV, as present in the selected database
AnchorNameSelection details

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If your variant database stores indels, they will be depicted using green (insertion) or red (deletion) symbols (Figure 35) pointing to deleted bases.

 

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SubtitleTextReference genome track with added variant annotation: insertions are shown in green, deletions in red. In this example, an insertion of a single base has described in the database, between G and T. An adjacent deletion of T and C bases has also been seen before
AnchorNameinsertions_deletions

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Other Annotation Tracks

Additional annotation tracks can be added to the viewer with the help of the Select tracks dialog (Figure 14) as long as they have been associated with the genome you are working on in the Library File Management.

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SubtitleTextTranscript database track: a gene with two transcripts is shown as an example. Exons are plotted as boxes and introns as lines connecting them. Untranslated regions (UTRs) are seen as narrow boxes. The arrows indicate directionality
AnchorNameTranscript database track

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Customizing the View

Controls
Chromosome view can be customized by using the control panel on the left (Figure 37). The Attribute and Order By controls show options depending on the current project, while the content of the Annotate amino acids control depends on the annotation files associated with the current genome build in the Library File Management. In order for any change to take place, push the Apply button.

 

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SubtitleTextControl panel (an example is shown)
AnchorNameControl panel

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The first option, Group data by, specifies the number of Alignments tracks (Figure 38). All will result in only one track, with all the samples on it. Sample creates one track per sample, while Attribute produces one Alignments track per level of the Attribute (i.e. one track per group). 

 

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SubtitleTextGroup data by: All creates one Alignments track for the entire project, Sample creates one Alignments track for each sample, Attribute creates one Alignments track for each group (an example is shown)
AnchorNameGroup options

All

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Sample

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Attribute

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Annotate amino acids by controls the appearance of the Amino acids track and allows you to pick the transcript database that will be used to plot codons (Figure 39). The drop down list shows the databases currently available for the selected genome (additional databases can be added via Library File Management). 

 

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SubtitleTextAnnotate amino acids by: transcript models currently associated with the chosen genome are displayed in the drop-down list and can be used to plot Amino acids track (an example is shown)
AnchorNameAmino annotation

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Color by option affects the colouring of the Alignments track and Isoform proportion track. When Sample is selected from the drop-down list, individual samples will be shown on the aforementioned tracks, each sample being given a different colour. If attributes were assigned to samples, they will also be visible in the Color by drop-down (Figure 40) and you will be able to highlight levels of the selected attribute (Figure 41).

 

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SubtitleTextColor by: the options control colouring of Alignments and Isoform proportion tracks. Sample, Base, and Match options are present by default. If attributes have been assigned to samples, they will appear in the drop-down list. In this example, that is the "Tissue" attribute
AnchorNameColor options

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SubtitleTextDifference between Color by Sample and Color by <Attribute>. Color by Sample uses different colours to depict individual samples; Color by <Attribute> uses different colours to depict levels of the selected sample attribute (as present in the Data tab). Alignments and Isoform proportion tracks are shown (an example)
AnchorNameColor difference

Color by Sample

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Color by <Attribute>

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The effect of the option to Color by Base can be seen with high power magnification (Figure 42). Individual base calls are highlighted by different colours. When that option is chosen at low power magnification, all the bases are shown in grey.

 

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SubtitleTextColor by Base highlights the base calls by colours. Different colours are visible with high power magnification; otherwise all the bases are shown in gray (an example)
AnchorNameColor by Base

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Finally, Color by Match can be used to quickly identify mismatches against the reference genome. A matching base is coloured in blue, while mismatch bases are shown in yellow. 

The maximum of the y-axis of Alignments tracks is set by Read histogram Y axis scales option (Figure 43). When using Independent, the y-axis for each track is set individually, based on the maximum within that sample. On the other hand, Linked uses the maximum across all the samples and uses that value as the maximum for all. 

 

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SubtitleTextRead histogram Y axis scales. When set to Linked, all the tracks have the same Y axis maximum, which depends on the sample with the highest coverage. Using Independent sets Y axis maximum independently for each sample.
AnchorNameHistogram Y axis

Independent
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Linked
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Read histogram type changes the presentation of the Alignments track and should be used in conjunction with the Group data by and Color by tracks to get the desired visualisation.

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SubtitleTextAlignments track: total coverage per locus is shown by using "Read histogram type" set to "Sum" and "Group data by" set to <Attribute>
AnchorNameTotal coverage

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To show average coverage per locus, switch Read histogram type to Average and leave Color by as is (i.e. by group) (Figure 45). With this setting, Chromosome view will calculate the average by dividing the total coverage per locus by the number of samples. Note that using Color by Sample would not make sense here. Although Figure 44 looks quite like Figure 43, the y-axis range is different.

 

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SubtitleTextAlignments track: average coverage per locus is shown by using "Read histogram type" set to "Average", "Group data by" set to "Attribute", and "Color by" set to <Attribute>
AnchorNameAverage coverage

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Finally, the option Overlay is useful if you want to directly compare base counts over several samples (or groups) as each will be represented by a line (i.e. no stacking). Example in Figure 46 is based on microarray data, showing three groups on the same Alignments track. The red group has the highest base counts, while the counts in the blue group are much lower.

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SubtitleTextranscript label: setting the control to Gene shows only gene label, while Transcript shows transcript labels. Both transcript database and Isoform proportion tracks are affected Short sequencing reads can be coloured by strand (Reads pileup color: Strand) or by base (Reads pileup color: Base). Both options are illustrated in Figure 48.
AnchorNameTranscript label

Transcript label: Gene
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Transcript label: Transcript
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SubtitleTextReads pileup color: colouring of the short sequencing reads by Strand or by Base
AnchorNameReads pileup color

Reads pileup color: Strand
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Reads pileup color: Base
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Probe color control customizes the appearance of Probe intensities track (Figure 49). When set to Intensity, colour of a probe reflects its intensity, using a colour gradient from white (low) to admiral (high). Alternatively, when Strand is turned on, probes on the reverse strand are in parakeet green, while probe on the forward strand are in sky blue.

 

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SubtitleTextProbe color: "intensities" colors probes proportionally to their intensity, "strand" uses colors to indicate probe positioning (an example is shown)
AnchorNameProbe color

Probe color: Intensity
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Probe color: Strand
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If a variant database is available for the current genome, the variants can be added to the Reference genome track (Figure 33). To show the variants, point the Variant database control to the database of your choice.

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SubtitleTextCustomize colors dialog: selecting a drop-down arrow opens the color-picker tool
AnchorNameCustom color dialog

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Track Order
The position of the tracks on canvas can be controlled by using the Track order tool. If you want a track to be visible all the time, i.e. while scrolling up or down, pin it to the top or to the bottom. Figure 51 shows Cytoband track pinnned to the top of the canvas and Reference genome track pinned to the bottom of the canvas. To unpin a track, click on the pin icon ( Image Removed ). The track will be unpinned and a message No tracks are pinnned to the top / bottom will appear. To pin a track, drag the track name to the No tracks… message. Alternatively, you can use the green arrows ( Image Removed ) to pin a track. When you mouse over an arrow, the new position of the track will be highlighted on the canvas; click on the arrow to accept.

A track can be hidden (meaning it will not be visible) by selecting the red minus, or unhidden by selecting the green plus icon.

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SubtitleTextTrack order tool: To change the position of a track drag and drop to the new position. To pin a track to the top / bottom of the canvas, use the up and down arrows. To unpin a track, select the pin icon. A track can be hidden by clicking on the red minus symbol and unhidden by selecting the green plus. Coloured dot by a track names indicates the layers to which the track belongs (an example is shown)
AnchorNameTrack order tool

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Selection Details
At the bottom of the control panel you will find the Selection details section (Figure 52). It is used to display information on the element selected on the canvas (using the Pointer mode).

 

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SubtitleTextSelection details showing information on the element selected on the canvas. The example shows details of a microarray probe. Note the two link-outs ("Browse on UCSC" and "BLAST this sequence")
AnchorNameSelection details

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Additional assistance

 

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