Visualizing reads and enriched regions

We can visualize the reads and enriched regions using the Partek Genome Viewer.

Select ChIP-Seq from the spreadsheet tree
Select Chromosome View from the Visualization section of the ChIP-Seq workflow

The Chromosome View tab will open. The default tracks are the transcript tracks, the sequence visualization tracks, and the cytoband track (Figure 1).

Figure 1. Viewing ChIP-Seq data in Chromosome View

We can add additional tracks to view the results of our analysis.

Select New Track from the left-hand side of the Tracks tab
Select Add tracks from a list of spreadsheets
Select Next >
Select the p-value_filtered.txt and Motifs_instances.txt tracks on the Tracks panel
De-select Aligned Reads
Select Create (Figure 2)

Figure 2. Adding spreadsheet tracks to Genome Viewer

This will display the enriched regions found in the samples and the locations of the motif instances from the de novo motif discovery (Figure 3).

Figure 3. Adding p-value and motif binding tracks

The two track display the detected regions at each location on the chromosome for the NRSF-enriched sample, chip, and aligned them to the de novo discovered motif binding sites. If you have not gone through the steps for peak detection and motif discovery, these tracks will not be available.

Select RefSeq Transcripts - 2016-10-18 (hg18) (+) on the Tracks panel
Set Strand to Both using the drop-down menu in the Track section of the track properties panel
Select Apply

The RefSeq Transcripts track now shows transcripts from both strands. We can remove the dedicated (-) strand track.

Select RefSeq Transcripts - 2016-10-18 (hg18) (-) on the Tracks panel
Select Remove Track

Several other tracks will not be used in our analysis; we can remove them as well.

Select Legend: Base Colors, Genome Sequences (hg18), and Cytoband (hg18) on the Tracks panel by left clicking on each while holding the Ctrl key on your keyboard
Select Remove Track

The height of many tracks can be adjusted by changing the Track height setting in the Profile tab.

Select Regions (1/p-value_filtered (p-value filtered.txt)) on the Tracks panel
Set Track height to 25 using the slider
Select Apply
Repeat for Regions (1/p-value_filtered/motifs/instances (Motifs_instances.txt))

This view clearly shows the Bam Profile tracks for chip and mock samples alongside smaller track indicating motif binding sites, significant peaks, and RefSeq transcripts (Figure 4).

Figure 4. Genome Viewer tracks can be modified to facilitate data exploration and analysis

We can also see strand-specific information by color-coding forward and reverse strand read information on the Bam Profile (chip) track.

Select Bam Profile (chip) on the Tracks panel
Select Alignments in the Display Color Options section of the Style tab
Select Strands under Color by
Select Apply

A legend track will be added at the bottom of the viewer showing that forward strand reads are colored green and reverse strand reads are colored red. We can move this track to below Bam Profile (chip).

Left-click Legend: strands in the Tracks panel and drag it up the list to below Bam Profile (chip) (Figure 5)

Figure 5. Move tracks by left-clicking and dragging them in the Tracks panel

Chromosome View opens to a whole-chromosome view of chromosome 1. To analyze the data we can zoom in on the data (Figure 6).

Figure 6. Viewing the zoomed-in view of an enriched region showing two possible binding sites at the location

We can practice this using a gene highlighted in the paper describing this data set, NEUROD1.

Type NEUROD1 in the navigation bar
Select Enter on your keyboard to navigate to NEUROD1 (Figure 7)

Figure 7. Zooming to the NEUROD1 gene

NEUROD1 contains a binding site for the NRSF motif. Notice that the enriched region for the NRSF transcription factor is within the NEUROD1 gene. As discussed in the Johnson et al. paper, NRSF is implicated in the repression of NEUROD1, but it was unknown exactly where the NRSF binding occurred. This data indicates that the binding site is within the NEUROD1 gene itself, as shown by the orange box in the Regions track. We can zoom in further to view the forward and reverse strand read histogram.