The  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:

Launching the Chromosome View

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. 

On the Analysis tab, selecting a data node containing aligned reads, variants, gene or transcript counts, or feature lists, shows Chromosome view in the Visualization section of the toolbox (Figure 1). 

If Partek Flow has no information on the genome build, you will need to provide the species and genome build in a subsequent dialog (not shown). Otherwise, chromosome view will come up directly. 

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. 

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

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.

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.

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  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.

When pointer 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).

When zoom mode is activated, the appearance of the cursor will change to a plus . With the zoom mode on, you can magnify a specific region by positioning the cursor  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). 

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.

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 +).

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 .

Once the plot has been modified, you can save the current appearance of the canvas by using the save icon . 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.

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).

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.

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.

Figure 15: Selecting data tracks for the visualisation using the Select tracks dialog (an example). 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 (e.g. Isoform proportion track) highlights the respective node in the track list (Normalised counts and Isoform proportion).

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.

Alignments Track

Alignments track displays a view of alignments present in .bam files in a stacked histogram fashion (similar to Partek® Genomics Suite®). The y-axis shows number of (raw) base calls per position. By default, reads are coloured by sample; the exception is invocation of the chromosome view on a variant table, when the reads are coloured by base calls. The difference is shown in Figure 17.

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.

Variants Track

Variant tracks show single nucleotide variants (SNVs) and indels, and appear in the Select track dialog if Detect variants task has been performed. Presentation of variants depends on the level of zoom. With low power magnification, SNVs are seen as purple columns and indels are bars (insertions: green bars; deletions: red bars) (Figure 19).

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). 

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.

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.

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).

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.

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.

Forward strand reads are in sky blue, while reverse strand reads are in parakeet green. If paired-end chemistry was used, the paired reads will be depicted as half reads within a gray rectangle encompassing the pair (Figure 26). Singletons will be depicted as thicker reads.

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).

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

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).

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.

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

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.

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).

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.

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

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.

A common choice of an additional track is a transcript database, such as RefSeq (Figure 36). All the database entries are displayed, using common depiction of exons as boxes and introns as lines connecting them. Untranslated regions (UTRs) are seen as narrow boxes. The arrows indicate directionality.

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.

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). 

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). 

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).

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.

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. 

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.

When set to Sum, the Read histogram type shows the sum of base calls at each position, i.e. total coverage per position. Figure 44 shows an Alignments track with three samples. With the Sum option, number of reads at each base in each sample is added and displayed. Contribution of individual samples is not visible, since the track is Colored by Group (but that would make sense in this example).

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.

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.

Figure 46: Alignments track: coverage per locus is shown by using "Read histogram type" set to "Overlay". Each plot is a single experimental condition ("Group data by" set to "Attribute", "Color by" set to <Attribute>). Lines are rectangular since microarray data is used (an example)

Next, you can use the Transcript label selector to specify labels on the reference transcript track and Isoform proportion track (Figure 47).

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.

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.

To change any of the colours on the canvas, use the Customize track colors tool. A resulting dialog will help you to pick another color (drop-down button opens the colour-picker) (Figure 50).

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 ( ). 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 ( ) 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.

The tracks can be reordered by drag and drop.

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).