Heatmap Analysis

Jump to Section:

• View Settings & Filters
• Setting Initial Filters
• Viewing Details
• Removing Taxa
• Analysis by Category

Overview

You can customize the heatmap visualization by applying multiple filters and adjusting various view settings. For example, you can reduce the initial complexity and noise shown in the heatmap by applying a set of threshold filters (see Setting Initial Filters). 

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View & Filter Settings

You can adjust the heatmap View and Filter settings to customize the heatmap. Note that every time you adjust the settings, the heatmap is recalculated to meet the specified criteria. Therefore, you may notice new taxa after changing settings. Below we describe the heatmap View and Filter settings.

 

View Settings

The View settings will enable you to adjust the level of taxonomic resolution used for taxa (i.e., genus vs species), the way taxa and samples are sorted, the metric used to calculate values displayed in the heatmap, and the scale.

• Taxon Level: Changes the granularity of the taxon on the heatmap. You can choose either the species level or genus level.
• Metric: The chosen metric dictates the metric used to calculate the value displayed in each heatmap square using a color scale. You can select to show values based on reads per million (rPM), total reads (r), or Z scores.
• Sort Samples: Changes the way that samples are sorted. By default, we cluster taxa using average-linkage hierarchical clustering. You can also sort samples alphabetically.
• Sort Taxa: Changes the way the taxa are sorted. By default, we cluster taxa using average-linkage hierarchical clustering based on Euclidean distances between rows. When you have selected species as your taxon level you can also sort taxa by genus.
• Scale: View the heatmap using a log or linear scale.

Filter Settings

The Filter settings will help you adjust the heatmap visualization to focus on relevant taxa (e.g., by filtering out low abundance taxa).

• Categories (Taxon group): Lets you focus on a particular group of taxa (e.g., bacteria, viruses, eukaryotes).
• Thresholds: These filters work just like the filters on the single sample report page. You can add as many threshold filters as you want. This will help reduce the complexity of the heatmap. 
• Background: If you chose Z-score for Metric, the heatmap will use your background model selection here to calculate that value.
• Taxa Per Sample: Choose how many taxa per sample to include in the heatmap. Increasing this value will increase heatmap load time.
• Read Specificity: Select “Species Only” to hide reads that were not defined at the species level.
• Pathogen Tag (Known Pathogens Only): Select this filter to only show known pathogenic taxa in the heatmap. Taxa are tagged as "Known Pathogens" based on the CZ ID pathogen list. Note that this list is not fully comprehensive and does not distinguish pathogenic strains or subtypes from non-pathogenic ones in many cases. Therefore, the pathogen list should be viewed as a starting point to quickly identify potential pathogenic organisms in samples.

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Setting Initial Filters

To reduce the initial complexity of the heatmap, we suggest using the following View and Filter settings:

• Metric: NT rPM
  • • This default setting displays reads per million (rPM) values in the heatmap, such that higher rPM values appear darker.
• Taxon Level: Genus
  • • Exploring detected taxa across samples at the genus level helps you zoom out and see high level trends before diving into species.
• Threshold Filters: NT rPM >= 10, NR rPM >= 1, and NT L >= 50
  • • We recommend applying these threshold filters to reduce noise and get a high level view of the data.

If you apply the suggested View and Filter settings to the heatmap created from the Feline Gingivostomatitis project, it would look like the one below. 

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Viewing Details

The heatmap gives a broad picture of the landscape of detected taxa across samples. To view more information about a taxon detected in a given sample hover over the heatmap grid of interest. Clicking the highlighted grid will open the Sample Report page for that sample in a new tab.

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Removing Taxa

To remove specific taxa from the heatmap, hover over the taxon name. Click the small blue "X" that  appears to the left of the taxon name.

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Analysis by Category

Sometimes it is useful to explore the heatmap based on organism category using the Categories Filter. For example, you can explore viruses detected across samples separate from cellular organisms (i.e., bacteria and eukaryotes) or you may want to focus on bacteria alone. 

The heatmap below was filtered to show viruses detected in cat samples from the Feline Gingivostomatitis project. To do this, we adjusted the Categories filter to Viruses-Non Phage. We kept the suggested Threshold Filters (see Setting Initial Filters) but, under View Options, we set Taxon Level to Species. Note that we also sorted the samples by "Diseases and Conditions" to explore if there were viruses found only in cats with stomatitis (see Metadata in the Heatmap to learn how to add metadata).