A. Hierarchy, OTU, or Both
In Hierarchy mode, the data collapses into OTUs with triangular expansion buttons. Clicking on an expansion button expands the selected taxonomic level to the next lower level. This generates an easy-to-read display that can be expanded and compressed to investigate comparisons between libraries or metadata groups. The number of items in the lineage lines is determined by the input file, i.e. the depth of classification performed on raw sequence data. Clicking on the OTU category will highlight the line across the entire dataset. Hierarchy Level sets the default of how many levels of hierarchy are displayed.
In OTU mode, each taxonomic classification is listed individually, often resulting in very long OTU names that use large amounts of screen space. To make the data more tractable in this setting, use the OTU Start, OTU Width, and OTU Last functions. The number of categories in the lineage lines is determined by the input file, i.e., the depth of classification provided by the classification analysis tool run on the raw sequence data. Clicking on the OTU name will highlight the line across the entire dataset.
Selecting Both creates two panes on the workspace screen. The upper pane shows the Hierarchy, and the lower pane shows the OTUs; these are two views of the same dataset.
B. Raw and Normalized Data Options
- This option shows the raw sequence data counts (integers).
2. % of Library
- Data are normalized by column (library or metadata grouping). This is generally the most useful setting for comparing sequence distributions between samples or groups of samples. This is generally referred to as "relative abundance" in the literature.
3. % of Total
- Data are normalized to the entire OTU count for the total dataset.
C. OTU Displays
1. OTU Start
This allows the user to control the taxonomic level at which to start the lineage lines in the OTU pane display. For example, rather than every sequence starting at the domain level 1 for domain (Bacteria, Eukarya, or Archaea), the user can select OTU Start at level 2 for phylum (Acidobacteria/Proteobacteria/etc.), or at level 3 for subphylum (Alphaproteobacteria/Betaproteobacteria/etc.).
2. OTU Width
This option limits the number of taxonomic categories displayed in the OTU name. The number of taxonomic categories can be selected by entering the desired taxonomic levels to display. For example, setting OTU Start at level 1, and OTU Width at 3, would produce the following: “Bacteria/Proteobacteria/Alphaproteobacteria…”
3. OTU Show Last
This option appends the last item in the taxonomic line onto an OTU name, independent of the values of OTU Start and OTU Width. The depth of taxonomic classification is determined by the input file. In the example above, OTU Show Last could be the genus level: “Bacteria/Proteobacteria/Alphaproteobacteria…/Paracoccus”
All OTU names can be forced to display ONLY the terminal category by checking this option and setting OTU Width to zero. One item OTUs (such as OTU Group names or OTUs that can only be traced to domain level, like “Bacteria”) are considered the last OTU position; single name OTUs will disappear when OTU Show Last is deselected.
D. Hierarchy Level
- Sets the default number of taxonomic levels expanded in the hierarchy pane.
1. Show Libraries
- This option will display all libraries in a workspace by name, sorting them alphabetically by name (e.g., AL123, EM321, RA964) from left to right.
2. Show Sorted Libraries
- Sorted libraries have one or more metadata tag(s) pre-appended before the library name, separated by a colon. The libraries are then displayed alphabetically first by metadata tag(s), then by library name. This option allows the user to sort the library columns by metadata while still seeing all individual names as well (e.g., City1:AL124, City1:RA964, City2:EM321). Often libraries and sequences are named with obscure codes; therefore, this mechanism allows the introduction of human readable text which may ease the interpretation of the project data. This human readable metadata are carried forward into all of the analyses and graphical displays within Explicet.
a) Sort Libraries
View → Sort Libraries
- A pop-up window will open
- In left panel, select the metadata descriptor you wish to sort by
Click Add button between panels
- Name of metadata descriptor will appear in the right panel
To change the sort order of an individual metadata, click on the current Sort Order value (ascending/descending), and select the desired sort order from the dropdown menu.
- To sort by more than one metadata item, pick multiple metadata descriptors from the list. Metadata items are sorted in the order they appear in the list.
Click Sort at top right of window to accept library sorting
b) Remove Library Sorting
If you wish to remove a library sorting, follow the same procedure, except select the undesired metadata field in the right panel and click Remove button between panels.
3. Show Library Groups
This option displays the results of creating groups of libraries with the Group Libraries command. Groups are formed by selecting libraries based on a common metadata field. The relative abundances of each selected library are averaged to form the values for the group. The Counts values displayed for groups are computed by using the averaged relative abundances for each taxonomic categories. For more information on how the Counts in library groups are calculated, and to create library groups, see Select Data.
F. Sort OTUs
- Sorting OTUs will arrange the data columns in the OTU pane by ascending or descending OTU abundance.
1. Sort OTU
Click on the library name in the OTU pane to sort by descending order based on the contents of that column (or Total). A small arrow will appear above the column title which indicates sort order.
Click the library name again to flip the sort order. The small arrow above the column title will switch to pointing upward, indicating that the OTU pane has been sorted in ascending order based on the contents of that column (or Total).
2. Remove OTU Sorting
- There are 3 different ways to restore alphabetic sorting of the OTU pane:
1. Double click on the library name or Total
2. Hold Ctrl-click on the library name or Total
3. View → Unsort OTU