Difference between revisions of "PAINT User Guide"

From GO Wiki
Jump to: navigation, search
m (Partial annotation of trees)
 
(489 intermediate revisions by 10 users not shown)
Line 1: Line 1:
 +
[[Category:PAINT]]
 
=Summary=
 
=Summary=
[[PAINT]] is a Java application for viewing and annotating phylogenetic trees. This document briefly describes how to set up and use the tool.
+
[[PAINT]] is a Java application for viewing and annotating phylogenetic trees. The present document describes how to set up and use the tool.
  
 
=Requirements=
 
=Requirements=
Java 1.6 must be installed.
+
Java 1.8 (aka Java 8 on a Macintosh) must be installed.
  
=Installing PAINT=
+
=Installing and configuring PAINT=
PAINT is a Java application, and can be run on a wide variety of platforms. To install PAINT on either Mac or Windows, follow these steps:
+
PAINT is a Java application, and can be run on a either Mac or Windows. To install PAINT, download the application from: http://paintcuration.usc.edu/
  
* Download the PAINT application at:
+
=Launching PAINT=
http://sourceforge.net/project/showfiles.php?group_id=184610
+
* On a Windows machine, run the program <code>lauchPAINT.bat</code>.
 +
* On a Mac, open a Unix terminal window, go to the directory containing the PAINT program, and execute the command:  
 +
<code>sh launchPAINT.sh</code>    OR      <code>./launchPAINT.sh</code>
  
* On a Mac, open a Unix terminal window, go to the directory containing the PAINT program, and execute the command: sh launchTAV.sh.  On a Windows machine, run the program lauchTAV.bat.
+
----
  
 
 
=Using PAINT=
 
=Using PAINT=
  
==Logging in==
+
==Login==
In the tree viewer, go to File->Login in the menu. Type in your user id and password.
+
You are required to login before you can open a tree. The purpose is to record proper acknowledgement for all the curated annotations (of tree nodes) created by you.
 +
 
 +
Go to <code>File -> Login</code>.
 +
 
 +
If you just want to view the tree and annotations, you can enter <code>gouser</code> as the username. The password is filled already. This is a read-only login.
 +
 
 +
If you want to curate trees, enter your username and password. If you don’t have a login and password, send an email to huaiyumi@usc.edu and request one.
 +
 
 +
==Curating a gene family==
 +
The analogy is to a library.  You will first find and check out (lock) the families you want to curate, and then select a family to curate from your list of locked families. All families now have a curation status (curated, partially curated, uncurated).
 +
 
 +
===Step 1: Find and "lock" families for curation===
 +
When you lock the family, other curators won’t be able to curate them. This is to prevent people from working on the same family.
 +
[[File:PAINT_search.png|thumb|Figure 1. PAINT family search box|450px]]
 +
* Go to <code>File</code> > <code>Manage and View Books...</code>
 +
**A window will pop up (Fig 1).
 +
***You can search for families by various identifiers: PTHR ID, PTN ID, Gene Symbol, Protein Identifier, Gene Identifier, or gene definition.
 +
***Retrieve a list of all families, or just the uncurated families.
 +
***Retrieve a list of families that require review (incompletely curated).
 +
**Press the "submit" button to launch search
 +
[[File:PAINT_family_search_results.png|thumb|Figure 2. PAINT family search results|450px]]
 +
* Select one or several families to lock. Fig 2 shows an example when all uncurated families are returned. There are 4 possible curation status states:
 +
**Manually curated – These are the families curated, and the curator believes that the curation is complete.
 +
**Locked – Those families are locked by a curator. The name of the curator who locks the family is shown in the Locked by column.
 +
**Partially curated – These are the families that have been curated. The curator can unlock the family and leave it as partially curated.
 +
**Require PAINT review – The previously curated paint annotations are changed due to updates in either PANTHER and GO.
 +
**Unknown – These are uncurated families.
 +
*Check the box in the <code>Lock/Unlock</code> column of the families you want to check out, and click <code>Lock or Unlock selected Books</code> button at the bottom of the panel.
 +
 
 +
===Step 2: Open a family to curate===
 +
* To open a family, click <code>View Locked Books</code>, and then click the <code>View</code> button (Fig 3).
 +
* A family can only be locked by a single user. If a family is already locked, you can open it in View Only mode.
 +
* You can only curate families you have locked.
 +
 
 +
[[File:PAINT_family_opening.png|thumb|Figure 3. Opening a previously locked family.|450px]]
 +
 
 +
===Step 3: Save your annotations===
 +
You can choose to save but keep the family locked so you can continue the curation later. You can also save and unlock the family.
 +
* Go to <code>File</code> > <code>Save to Database</code>. A window will pop up with the following options:
 +
**'''Cancel'''
 +
**'''Save and unlock:''' The family will be unlocked and marked as Partially Curated.
 +
**'''Save:''' The family will remain locked. The curator should do this as often as possible during the curation.
 +
**'''Save, unlock & set curated:''' The family will be marked as Manually Curated.
 +
 
 +
----
  
==Accessing a PANTHER family tree, and GO annotations for leaves==
+
==Appearance and Basic Operation==
Once you’re logged in, you can search for a tree by going to File->Open from database. You can enter a search term (wildcard characters won't work, though for some of the searches you can enter partial names with no wildcard characters). Alternatively, you can select a PANTHER family identifier from the drop-down menuA list of families will be returned. In the future, you will be able to "lock" the book to prevent others from having write access during curation, but for now this is disabled. Right now you can view a family by clicking on the family identifier in the search results box.
+
===Windows===
 +
PAINT is organized into three main panels (Fig 4): 
 +
[[File:PAINT-overview.png|thumb|Figure 4. Main PAINT window|500px]]
 +
* The '''upper left panel''' shows a '''[[PAINT_User_Guide#Phylogenetic_Tree |phylogenetic tree]]'''.
 +
 
 +
* The '''upper right panel''' allows you to switch back and forth between (i) the '''[[PAINT_User_Guide#Annotation_matrix |Annotation Matrix]]'''; (ii) the '''[[PAINT_User_Guide#Protein_Information_table |Protein Information Table]]''' and (iii) a multiple sequence alignment '''([[PAINT_User_Guide#Multiple_sequence_alignment_.28MSA.29 |MSA]])''' of all sequences.
 +
 
 +
* The '''bottom panel''' contains two tabs: '''[[PAINT_User_Guide#Annotations_window |Annotations]]''' and '''[[PAINT_User_Guide#Evidence_window |Evidence]]'''.
 +
 
 +
All the tabbed panes may be resized or split out into windows.
 +
   
 +
* Click on a tab (e.g., Protein Information, Evidence) to bring it to the front. 
 +
* Click the icons in the tabs or the upper right corner to Undock/Dock, Minimize, Maximize, or close individual tabs or groups of tabs.
 +
* Tabs and panes may also be rearranged within a window by dragging. 
 +
* Columns in the Protein Information Table can be resized.
 +
* Windows may be closed, arranged, or resized by dragging boundaries.
  
==Adjusting the view==
 
 
===Recommended configuration for curation===
 
===Recommended configuration for curation===
* The larger the better, so resize the entire window by dragging the lower right corner. Leave some space at the top of your screen, though, for the annotation matrix so you can read some of the vertical text in the matrix.
+
* Bigger is better.  Use as much of the monitor as you can afford.  If you are using a laptop, you may wish to attach an external monitor.
* Undock the "matrix" panel from the bottom panel, and drag and resize to correspond to the right ("grid") panel.  The first row of the matrix should line up with the first leaf node of the tree in the tree panel.
+
* Adjust the width of the window and the partition between the Tree and the Table until you are comfortable with them.
===Resizing the panel and column widths===
+
 
You can resize the tree panel by clicking and dragging the partition between the two panels. The right panel can be toggled between multiple sequence alignment ("MSA") and a table of information about each sequence in the tree ("Grid"). Table columns can also be resized.  
+
----
 +
 
 +
===Phylogenetic Tree===
 +
 
 +
A phylogenetic tree contains nodes and branches (Fig 5). There are three types of nodes, root, internal and leaf. Leaf nodes correspond to the proteins in the tree. Root and internal nodes represent the inferred most common ancestor of the descendants. Branch length may be interpreted as time estimates between the nodes.
 +
 
 +
[[File:Figure 2.png|thumb|Figure 5. PAINT phylogenetic tree|400px]]
 +
The root and internal nodes of the tree are shown as circles (speciation events) and squares (gene duplication events). If the tree has been previously curated, the nodes maybe colored in indicate the type of annotation (e.g., with inferred or experimental evidence). More details will be described in the "Making an inferrence" section of this guide. The nodes have unique identifiers that start with PTN (=PaNther Node), followed by 9 digits. Mouse over a node to see its identifier.  If you right-click on a node, a menu will appear with the options to:
 +
* '''Collapse node:''' - the entire clade is collapsed to a single node (rectangle). All the descendants are hidden, but the GO term assignments to them are still available for annotation. Right-click the node again and select "Expand node" to re-expand it.
 +
* '''Reroot to node:''' - make the selected node and the root, and hide the rest of the tree. This is useful when the tree is too large. To bring back the entire tree again, use menu "Tree -> Reset Root to Main".
 +
* '''Export seq ids from leaves:''' the ids of all leave sequences descended from the node are exported to a text file
 +
* '''Prune:''' All nodes descended from the node are removed from the tree.
 +
 
 +
The tree branches can be rescaled if they are too long for comfortable viewing or too short to distinguish individual nodes. The default branch scale is 50, which works for most trees. To rescale, select <code>Tree->Scale...</code> and enter a different number.
 +
 
 +
====Navigating within the tree====
 +
* Click on a protein name in the tree to highlight the protein in the tree and the table.
 +
* Left-click on a node in the tree to highlight the entire clade descended from it.
 +
 
 +
----
 +
 
 +
===Annotation matrix===
 +
'''Note: The colors refer to the default colors in PAINT'''
 +
 
 +
[[File:PAINT_main.png|thumb|Figure 6. Main PAINT window|500px]]
 +
 
 +
The matrix has a row for each gene/gene product in the tree, and a column for each GO term that is directly annotated to at least one gene/gene product in the tree. The annotation matrix gives an overview of the annotations associated with any proteins in table format. It displays one of the three Gene Ontologies at a time. You can switch to a different ontology by clicking the radio button on the upper left part of the window (red arrow, Fig 6). Mouse-over the downward triangle to see the GO term (yellow circle). The terms in the annotation matrix are grouped, with the most specific terms on the left. A few very broad terms such as “protein binding” are not shown, even though they are listed in the Annotations pane.
 +
 
 +
Proteins with experimental annotations (IDA, EXP, IMP, IGI, IPI, or IEP evidence codes) for a particular ontology are colored and shown in boldface (blue circles). You may select one ontology at a time to examine using the radio buttons (red arrow) at the top of the window.
 +
 
 +
* Click on a protein in the tree and the corresponding row will be highlighted in the matrix.   
 +
 
 +
[[File:annotation_matrix_color.png|thumb|500px|Figure 7. PAINT Annotation matrix]]
 +
 
 +
* The annotations of the corresponding proteins and GO terms in the matrix are shown in colored squares (Fig 7).
 +
** When you first open a tree, only the experimental annotations are shown.  These are the annotations than can be used for annotating ancestral genes.
 +
*** Experimental annotations are represented by green color.  If it is a direct annotation (i.e. the actual annotation is to that exact term in that column of the matrix), there is a black dot in the middle of the green square.  If it is an indirect annotation (i.e. the actual annotation is to a child of the term in that column of the matrix), there is a white dot in the middle of the square.
 +
*** NOT annotations are indicated with by a red circle with a white X.
 +
** When you have annotated an ancestral node, inferred annotations are also shown in the matrix.  This allows you to easily keep track of what you've already annotated.
 +
*** Inferred annotations are represented by blue color, with either a black (direct) or white (indirect) dot in the center, or X for NOT as above.
 +
 +
* Mouse-over an annotation square to see the tool tip of the protein name and the term.
 +
* Click on the annotation square to highlight the row. All the annotations to the protein, as well as the evidences and confidence codes will be displayed in the Annotation panel (see below for more details).
 +
* Right-click (or Command-click in Mac) on the experimental annotation (green square) in the matrix will automatically highlight the inferred most recent common ancestor (MRCA) node for the term.
 +
 
 +
----
 +
 
 +
===Protein Information table===
 +
[[File:PAINT_protein_info.png|thumb|450px|Figure 8. Protein information table]]
 +
The phylogenetic tree is aligned with a protein information table showing additional information and linkouts to various databases ([[Media:PAINT_protein_info.png|Fig 8]]).  You can adjust the relative sizes of each within the window by dragging the line in the partition separating them.  Note that the identifier table contains a lot of information that can be observed by scrolling to the right.
 +
 
 +
====Navigating withing the Protein Information table====
 +
 
 +
* Click anywhere within a row in the table to highlight the protein in the tree and the table.
 +
* Click on one of the blue linkouts will open a link in your web browser.
 +
 
 +
<br clear='all'/>
 +
----
 +
 
 +
===Multiple sequence alignment (MSA)===
 +
[[File:PAINT_msa.png|thumb|450px|Figure 9. Multiple Sequence Alignment view]]
 +
The trees were estimated from an MSA ([[Media:PAINT_msa.png|Figure 9]]). The evolutionarily conserved part of the alignment is indicated with uppercase letters. The other less conserved region is in lowercase letters. If a sequence misses a position in the matchstate, it is called a delete state and is designated by a ''dash''. If a sequence needs to insert a position in the less conserved region in order to keep the match state region aligned, it is called an insert state and is designated by a ''dot''.
 +
 
 +
The conserved columns are colored with dark blue, blue or light blue, which indicates the conservation of 80%, 60% or 40%, respectively, in the column.
 +
 
 +
Toggle back and forth between the table view (“Protein Information”) and the MSA view (“MSA”) using the buttons above the table/MSA panel.
 +
 
 +
Note: You can view the sequence of a hypothetical ancestral protein (node) by first collapsing the appropriate node.
 +
<br clear='all'/>
 +
----
 +
 
 +
===Annotations and Evidence windows===
 +
[[File:PAINT_annotation_panel.png|thumb|700px|Figure 10. The annotations window]]
 +
[[File:PAINT_evidence.jpg|thumb|400px|Figure 11. Evidence window]]
 +
To view the annotations associated with a specific protein, click on that protein in the tree or table. Annotations appear in the <code>Annotation pane</code> (Fig 10), containing the following information:
 +
* '''ECO (Evidence code):''' The type evidence code supporting the annotation.
 +
* '''Term name:''' The GO term name and accession. Clicking on the term links out to [http://amigo.geneontology.org/amigo AmiGO]. A term with a NOT annotation is displayed as strikethrough text.
 +
* '''Reference:''' The reference supporting the annotation. Clicking on the reference links out to PubMed. IBA annotations display an internal reference, PAINT_REF:00nnnnn, where nnnnn is the numerical part of the Panther family ID.
 +
* '''With:''' This column contains the evidence to support the inference.
 +
* '''Qualifiers:''' The qualifiers 'NOT', 'colocalizes_with' and 'contributes_to' each have a column in the annotation table, and a checkbox that is checked when the qualifier is present.
 +
* '''Delete:''' This is used to remove an inference made by PAINT (see [[PAINT_User_Guide#Removing_an_IBD_annotation|Removing an IBD annotation section]] below).
 +
 
 +
===Evidence window===
 +
 
 +
The evidence window displayed automatically generated logs of the tree curation, such as annotations, validation and changes made by the PAINT pipeline upon data release.
 +
 
 +
----
 +
 
 +
===Curator notes===
 +
 
 +
The <code>Curator notes</code> is a text editor used to record notes on the curation process. <code>Curator notes</code> can be modified  by going to the <code>File</code> > <code>Update comment</code> menu.
 +
'''NOTE: The purpose of the annotation notes is to convey important points about the annotations and the phylogenetic tree both to other annotators and to users''', so annotators should try to make the notes as clear as possible.
 +
 
 +
The annotator may use the Curator notes to describe important points in the annotation process, including:
 +
* References used to annotate the family (for example, a few major reviews)
 +
* Any important points about the family topology, including potential inconsistencies in the tree
 +
* Reasons for annotating to a different node than the MRCA (most common recent ancestor), ie the node that triangulation of annotation identifies.
 +
* Link to GitHub tickets leading to review of the tree annotation.
 +
 
 +
----
 +
==PAINT navigation functionality==
 +
 
 +
===“Find” function===
 +
[[File:PAINT_Find.jpg|thumb|400px|Figure 12. PAINT 'Find' functionality]]
 +
The Find function (<code>Edit</code> > <code>Find…</code>, Fig 12A) allows you to search for either a gene or a GO term.  Select a gene or term search using the radio buttons (Fig 12B).  Searches are case-insensitive.
  
===Viewing the multiple sequence alignment (MSA)===
+
A gene search matches against exact match of any text stored in the database, such as any sequence identifiers, gene symbol, or even gene name (red arrow, Fig 12C). The search does not return partial match (blue arrow, (Fig 12C). To do a partial match, wildcard character(s) (*) can be added before and/or after the search term. Scroll through the list of matches and click on a specific match to highlight it in the tree, table, and annotation matrix, and to display its annotations in the Annotations window.
The trees were estimated from an MSA. One can toggle back and forth between the table view (“Grid”) and the MSA view (“MSA”) using the buttons just below the menu, above the table/MSA panel.
 
You can view ancestral sequences, by first collapsing the appropriate node in the tree (right-click, or apple-click on Mac).
 
  
===Rescaling the tree branch lengths===
+
You may search GO terms using text, or you may use numbers to search for GO IDs.
You may also want to rescale the trees if the branches look too long for comfortable viewing. Go to Tree->Scale... and enter a different number. For most trees we find 50 works well, but it depends on the tree.
+
<br clear='all'/>
 +
----
  
==Importing an existing GAF file==
 
Gene Annotation Format (GAF) files can be imported, to add or modify existing annotations. Annotations that were stored as a GAF file can be read back in.
 
*Open the appropriate tree (see Accessing PANTHER trees above).
 
*Go to File->Import in the menu.
 
*Select the GAF file that contains the annotations, and click the Import button.
 
  
==Annotating the trees with GO terms==
+
 
Ancestral nodes in the tree can be annotated with any GO term that has been experimentally determined in one (or more) of its descendants, and then these “inferred” annotations can be propagated to its other descendants.
+
==Making an inference: Transferring annotations==
 +
Ancestral nodes in the tree can be annotated with any GO term that has been annotated to one (or more) of its descendants.  These “inferred” annotations can be propagated to its other descendants.
 
===Annotating an ancestral node, and propagating to descendants by inheritance===
 
===Annotating an ancestral node, and propagating to descendants by inheritance===
*Select the GO term from the matrix view, by clicking and holding.
+
[[File:Fig13 triangulation.jpg|thumb|450px|Figure 13A. PTHR43114 before annotation]]
*Drag to the ancestral node you wish to annotate. When the mouse is over the node, it will turn dark.  Release the mouse button to annotate.
+
 
*NOTE that you can only annotate a node if AT LEAST ONE descendant has that experimental annotation (or a more specific one), so if a node does not turn dark it cannot be annotated.
+
* In the example shown in Fig 13A, 5 proteins are annotated by EXP to the GO term <code>adenine deaminase activity</code> (green squares in the 1st column of the Annotation Matrix, indicated by the red downward arrow).
===Removing an annotation===
+
 
*Click on the desired node.  Nodes with annotations are colored orange.
+
'''Tip:''' To view the last common ancestor that can be annotated based on triangulation(*), right click on a GO term, or anywhere in the column that contains that GO term. An inferred node, as well as its descendants, will be highlighted in grey (blue arrow, Fig 13A).
*Go to the "annotations" panel (default position is at the bottom), find the annotation you wish to remove, and click on the trash can icon.
+
* (*) '''Triangulation''' is the calculation of the last common ancestor of two sequences; in this case PAINT calculates the last common ancestor supporting an annotation to the same GO term.
===Annotating a descendant as having lost an ancestral function===
+
* Note that the node calculated by triangulation may not be the best on to annotate: the curator can decide to annotate to an more ancient or a more recent ancestor, dependent on other evidence. For example, annotations in other GO aspects may support an earlier annotation than suggested by the triangulation.
*Select the node by clicking on it.
+
* The curator should not assume that the genes in the tree are fully curated with repsect to the primary literature. It may be useful to review other papers, including reviews, to ensure annotations found in the tree accurately represents the current state of knowledge. 
*Click on the "ECO/QUAL" column of the desired annotation.
+
 
*Select NOT by putting the mouse over, and then select the evidence for the NOT annotation.
+
====To annotate an ancestral node====
==Saving (exporting) your annotations==
+
[[File:Fig13B.jpg|thumb|450px|Figure 13B. PTHR43114 after annotation]]
Annotations can be saved as Gene Annotation Format (GAF) file.
+
# Click a GO term (green square) from the Annotation Matrix (Fig. 13B) (or anywhere in the column containing the GO term).
*Go to File->Export in the menu.
+
# Drag the term to the ancestral node you wish to annotate. This can be the inferred node or any other nodes. When you mouse over it, a <code>+ sign</code> will be visible next to the node.  Release the mouse button to annotate. Click here for a video demo of the procedure: http://youtu.be/8kHrdiuNfos.
*Enter the file name (must end in .gaf), and click on the Export button.
+
# The node is now annotated with that term using the evidence code “IBD” (“Inferred from Biological Descendant”) (Fig. 13B).
 +
# PAINT then automatically propagates the IBD annotation to all descendants of the PAINTEed node, such that all descendants of the node will now be annotated with that term using the evidence code “IBA” (“Inferred from Biological Ancestor”). (Proteins and nodes already annotated with the term or one of its descendant terms will remain unchanged.)
 +
 
 +
====Annotating an ancestral node with a qualifier====
 +
[[File:Propagating qualifiers.jpg|thumb|800px|Figure 14. Propagating qualifiers]]
 +
* If you propagate an experimental annotation that has a qualifier, ie. "NOT", "contributes_to" (for MF annotations), or "colocalizes_with" (for CC annotations), the qualifier will also get propagated, unless there are contradictory annotations, ie, annotations ''with'' and annotations ''without'' the qualifier (for the same or for different genes). In that case, a pop-up window will appear to specify whether the annotations with or without the qualifier(s) should be propagated. Click the appropriate radio button, and click on the <code>Continue</code> box to apply the selection (Fig 14).
 +
 
 +
=====Notes=====
 +
* You may only annotate a node with a given GO term if '''at least one descendant''' has an annotation to that term or a child termIf you try to propagate a term with no supporting annotation, the node will turn red, and the propagation cannot be made.
 +
* Effectively, the PAINT curator only makes IBD annotations; IBA annotations are generated automatically to all descendants of the node to which an IBD annotation is made.
 +
* The IBD annotation automatically includes evidence for each of the sequences having an EXP annotation to the term or one if its descendants; it is not necessary to propagate individual EXP to generate the evidence for the annotation.
 +
 
 +
===Negation of annotations: "NOT" statements===
 +
Background: Since PAINT is a model of the family's evolution, adding a NOT modifier to a descendant (either another node or a leaf) represents a loss of function during evolution, that is, we are stating that the specified function was present in an ancestral protein and has been ''lost'' in the indicated protein or clade. This is a special case of the GO guidelines for NOT, which state that a NOT annotation may be made in situations where a particular function may be expected but is absent.
 +
 
 +
PAINT defines two reasons for an evolutionary loss of function, described with two separate evidence codes (ECO):
 +
* IRD = '''I'''nferred from '''R'''apid '''D'''ivergence from ancestral sequence evidence used in manual assertion: Used when there is a long branch, often following a duplication, and significant sequence divergence. For very divergent sequences, predictions are less reliable, even in the presence of a common ancestor.
 +
* IKR = '''I'''nferred from phylogenetic determination of loss of '''K'''ey '''R'''esidues evidence: Used when a residue known to be required for the activity of the protein has mutated.
 +
 
 +
In both cases, the node (intermediate or leaf) on which the NOT annotation is placed gets the evidence code selected (IKR or IRD), and descendants, if any, are annotated with the IBA evidence.  
 +
 
 +
====To add the NOT qualifier to IBD annotations====
 +
[[File:Fig15A.jpg|thumb|450px|Figure 15A. Tree annotated with an IBD]]
 +
[[File:Fig15B.jpg|thumb|450px|Figure 15B. Pop up window to select NOT evidence]]
 +
# From a tree annotated with a IBD annotation (Fig 15A), select a node or protein to be negated.  This may be either a directly annotated node or one of its children.
 +
# Click the checkbox in the NOT column of the Annotations window.
 +
# A popup menu will appear (Fig 15B). In the menu <code>Select evidence code for NOT annotation</code>, select one of the radio buttons:
 +
#* NOT due to rapid divergence
 +
#* NOT due to change in key residue(s)
 +
# '''Optional:''' In the box labeled <code>Please enter PMID and select sequence(s) from descendants providing evidence</code>, you may enter data not captured by primary annotation that support the negation. For example if a paper shows that one of the descendants does not have an activity, you can enter the PMID and select which gene was
 +
# '''Optional:''' If appropriate, you may select from the list under <code>Annotate to an ancestor term?</code>, a more general GO term to propagate to the node or sequence instead of the term negated.
 +
* In addition to the annotation no longer propagating downward, a small hash mark will appear near the node in the tree to indicate that the block exists (visible in Fig 15C).  Note that a hash mark only indicates the existence of at least one NOT annotation, not that every annotation through that node is negated.
 +
 
 +
'''Annotations propagated:'''
 +
[[File:Fig15C.jpg|thumb|450px|Figure 15C. Intermediate node annotated with NOT by IRK. Descendants are annotated with a NOT qualifier and IBA evidence]]
 +
[[File:Fig15D.jpg|thumb|450px|Figure 15D. Leaf node annotated with NOT by IRK.]]
 +
* If the NOT is on a node with descendants, the node will get the evidence code select (IKR or IBD), and the descendants will get an IBA evidence (Fig 15C).
 +
* If the NOT is on a leaf node it will get the evidence code select (IKR or IBD) (Fig 15D).
 +
* Upon export of PAINT annotations:
 +
** Annotations with IKR and all NOT annotations to proteins descended from that node will have the NOT qualifier added (as these have good evidence for loss of function).
 +
** Annotations with IRD and all NOT annotations to proteins descended from that node, no annotation will be exported. Thus this acts like a STOP PROPAGATION.
 +
 
 +
==Removing IBD, IKR and IRD annotations==
 +
# Click on the desired node. Nodes with inferred annotations are colored orange.
 +
# Go to the Annotation tab and click the <code>Delete</code> in the Delete column (shown in a red square in Fig 13B).
 +
 
 +
'''Notes:'''
 +
* Annotations and qualifiers can only be removed from the specific node to which they were made.
 +
* Primary annotations may be be changed; they may be disputed in the [https://github.com/geneontology/go-annotation/issues GO GitHub go-annotation repo].
 +
 
 +
== Partial annotation of trees ==
 +
 
 +
{|align='right'
 +
|-
 +
|[[File:PTHR24073-RabFamily.jpg|thumb|500px|The RAB GTPase superfamily]]]
 +
|}
 +
 
 +
When you want to annotate a very large family, e.g. the RAB GTPase superfamily (PTHR24073), it may not be feasible to annotate all clades at the same time. In this kind of situation, you may choose to annotate only the clades you are knowledgeable and confident of, and leave other clades unexamined. When you do this, you should fully annotate the clades you choose to annotate. For example, if you choose to do the IFT27 clade, do it fully. Please don't do piecemeal annotations in various locations that may make it hard for a subsequent annotator to understand what has been done.
 +
 
 +
We also agreed at the July 2014 PAINT Jamboree that you can make propagations all the way to the root if you feel that there is an ancestral role, even if you think that some clades have lost this. For example, in the RAB GTPase superfamily, we think that it had an ancestral function as a GTPase, but it is possible that some clades, e.g. the IFT22 clade, have lost this ancestral activity. You can make these high level propagations as part of your initial annotation of the family. If there are clades where this is wrong, perhaps the IBA annotation from PAINT will generate feedback that will help us correct it.
 +
 
 +
=== Recording partial annotation in the notes file ===
 +
If you only partially annotate a tree, please record in the notes file which clades you have worked on using the node number, e.g. '''Eukaryota_PTN001180007''' as well as a common name, e.g. '''IFT27''', if it is helpful.
  
 +
==Recording trees examined, but not annotated==
 +
 +
When you examine a tree and feel that it should not be annotated for some reason, please record that in the Evidence Notes so that we can track the fact that the family has been examined. Please use one of these tags (in all caps) in the Notes section of the Evidence tab. You can additional information after the tag if you wish (syntax between tag and additional info not discussed or determined). Then, save your annotations as normal so that PAINT will save the notes file.
 +
 +
* '''MISSING ANNOTATION''' - Use this if the tree looks OK, but there are insufficient experimental annotations to propagate any annotations.
 +
* '''MISSING SEQUENCE''' - Use this if you feel that a specific sequence or sequences is missing. You can list the IDs of the sequence(s) after the tag.
 +
* '''BAD TREE''' - Use this if you feel that the tree has major problems beyond one or a few missing sequences.
 +
 +
----
 
=Interpreting the PANTHER trees=
 
=Interpreting the PANTHER trees=
==Speciation and duplication events==
+
==Speciation and duplication events, and horizontal transfer==
In the tree, a speciation node is shown with a circle, and a gene duplication node with an square.
+
In the tree, a speciation node is shown with a circle, and a gene duplication node with an square.  Horizontal transfer events also appear in the tree, though more rarely, and these are represented with a diamond.
  
 
==Branch lengths==
 
==Branch lengths==
Branch lengths show the relative rate of evolution.  Shorter branches indicate slower evolution and greater conservation of ancestral characters.  Note that sometimes there is not enough data to compare all branches that descend from a given node.  In this case, we have set all descendant branches to a length of 2.0 (very long branches). This just means we don’t have enough data to accurately estimate relative rates. It is often due to a sequence fragment, and at a duplication node it may also indicate a gene that has been incorrectly broken into two different genes by a gene prediction program. In the spirit of keeping the algorithm simple, we calculate the branch length by simply counting the fraction of different positions between an inferred ancestral sequence and its descendant (and applying the Jukes-Cantor correction).
+
* Branch lengths show the amount of sequence divergence that has occurred between a given node and its ancestral node, in terms of the average number of amino acid substitutions per site.  Shorter branches indicate less sequence divergence and therefore greater conservation of ancestral characters.  A branch might be shorter because of a slower evolutionary rate (greater negative selection), or because less "time" has gone by (actually a combination of number of generations and population dynamics), or both.
 +
* Very long branches indicate an unreliable divergence estimate, due to insufficient data.  Note that sometimes there is not enough data to compare all branches that descend from a given node.  In this case, we have set all descendant branches to a length of 2.0 (very long branches). Branch lengths of 2.0 are often due to a sequence fragment, and at a duplication node it may also indicate a gene that has been incorrectly broken into two different genes by a gene prediction program.
 +
* Following a gene duplication (after a square node), the relative branch lengths for descendant branches are particularly useful: the shortest branch (least diverged) is more likely to have greater functional conservation.
  
 
==Multiple sequence alignment (MSA)==
 
==Multiple sequence alignment (MSA)==
The MSA uses upper-case characters and dashes (‘-for insertions/deletions) to denote positions of the MSA that were used to estimate the phylogenetic tree. Lower-case characters and periods (‘.’ for insertions/deletions) denote positions that were ignored when estimating the phylogenetic tree.
+
* Some columns in the MSA have upper-case characters (and dashes '-' for insertions/deletions).  These columns were used to estimate the phylogenetic tree.
 +
* Lower-case characters and periods (‘.’ for insertions/deletions) denote positions that were ignored when estimating the phylogenetic tree. Sometimes, tree errors arise because not enough columns were used, and the phylogeny could not be reconstructed well based on the included columns.  Since they were not used in the phylogeny, lower-case characters can be particularly helpful in verifying the tree topology: any conserved insertions should be parsimoniously traceable to a common ancestor.
 +
 
 +
----
 +
=Reporting bugs or likely errors in the trees=
 +
 
 +
==Tree issues==
 +
Most often, the errors in phylogenetic trees are due to problems with the sequence alignment, or the specific MSA columns used to estimate the phylogeny. The phylogeny inference program performs fairly robust handling of sequence fragments, but sequence fragments still cause errors. Another source of error is when the sequences evolve very slowly, generating little variation from which to estimate phylogeny. In this case, the errors can usually be fixed by including additional alignment positions to consider in the phylogeny.
 +
 
 +
'''If a Panther tree needs to be reviewed, please create a ticket in the Panther GitHub tracker: https://github.com/pantherdb/Helpdesk/issues'''
 +
 
 +
==PAINT issues==
 +
'''Issues with the PAINT tools should be reported in this tracker: https://github.com/pantherdb/db-PAINT/issues'''
 +
 
 +
=Curation Guidelines=
  
==Known bugs==
+
'''Those guidelines have been published (Gaudet, Livestone, Lewis, Thomas, 2011) [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178059/?tool=pubmed]'''
The tree building program has some known bugs that are being fixed.  Most often, the errors are due to problems with the sequence alignment, or the specific MSA columns used to estimate the phylogeny. It performs fairly robust handling of sequence fragments, but sequence fragments still cause errors. Another source of error is when the sequences evolve very slowly, generating little variation from which to estimate phylogeny. In this case, the errors can usually be fixed by including additional alignment positions to consider in the phylogeny.
 
  
==Reporting bugs or likely errors in the trees==
+
Curation guidelines are described in detail on this page:
Please email Paul directly at pdthomas@usc.edu.
+
http://wiki.geneontology.org/index.php/PAINT_SOP

Latest revision as of 01:58, 14 April 2021

Summary

PAINT is a Java application for viewing and annotating phylogenetic trees. The present document describes how to set up and use the tool.

Requirements

Java 1.8 (aka Java 8 on a Macintosh) must be installed.

Installing and configuring PAINT

PAINT is a Java application, and can be run on a either Mac or Windows. To install PAINT, download the application from: http://paintcuration.usc.edu/

Launching PAINT

  • On a Windows machine, run the program lauchPAINT.bat.
  • On a Mac, open a Unix terminal window, go to the directory containing the PAINT program, and execute the command:

sh launchPAINT.sh OR ./launchPAINT.sh


Using PAINT

Login

You are required to login before you can open a tree. The purpose is to record proper acknowledgement for all the curated annotations (of tree nodes) created by you.

Go to File -> Login.

If you just want to view the tree and annotations, you can enter gouser as the username. The password is filled already. This is a read-only login.

If you want to curate trees, enter your username and password. If you don’t have a login and password, send an email to huaiyumi@usc.edu and request one.

Curating a gene family

The analogy is to a library. You will first find and check out (lock) the families you want to curate, and then select a family to curate from your list of locked families. All families now have a curation status (curated, partially curated, uncurated).

Step 1: Find and "lock" families for curation

When you lock the family, other curators won’t be able to curate them. This is to prevent people from working on the same family.

Figure 1. PAINT family search box
  • Go to File > Manage and View Books...
    • A window will pop up (Fig 1).
      • You can search for families by various identifiers: PTHR ID, PTN ID, Gene Symbol, Protein Identifier, Gene Identifier, or gene definition.
      • Retrieve a list of all families, or just the uncurated families.
      • Retrieve a list of families that require review (incompletely curated).
    • Press the "submit" button to launch search
Figure 2. PAINT family search results
  • Select one or several families to lock. Fig 2 shows an example when all uncurated families are returned. There are 4 possible curation status states:
    • Manually curated – These are the families curated, and the curator believes that the curation is complete.
    • Locked – Those families are locked by a curator. The name of the curator who locks the family is shown in the Locked by column.
    • Partially curated – These are the families that have been curated. The curator can unlock the family and leave it as partially curated.
    • Require PAINT review – The previously curated paint annotations are changed due to updates in either PANTHER and GO.
    • Unknown – These are uncurated families.
  • Check the box in the Lock/Unlock column of the families you want to check out, and click Lock or Unlock selected Books button at the bottom of the panel.

Step 2: Open a family to curate

  • To open a family, click View Locked Books, and then click the View button (Fig 3).
  • A family can only be locked by a single user. If a family is already locked, you can open it in View Only mode.
  • You can only curate families you have locked.
Figure 3. Opening a previously locked family.

Step 3: Save your annotations

You can choose to save but keep the family locked so you can continue the curation later. You can also save and unlock the family.

  • Go to File > Save to Database. A window will pop up with the following options:
    • Cancel
    • Save and unlock: The family will be unlocked and marked as Partially Curated.
    • Save: The family will remain locked. The curator should do this as often as possible during the curation.
    • Save, unlock & set curated: The family will be marked as Manually Curated.

Appearance and Basic Operation

Windows

PAINT is organized into three main panels (Fig 4):

Figure 4. Main PAINT window

All the tabbed panes may be resized or split out into windows.

  • Click on a tab (e.g., Protein Information, Evidence) to bring it to the front.
  • Click the icons in the tabs or the upper right corner to Undock/Dock, Minimize, Maximize, or close individual tabs or groups of tabs.
  • Tabs and panes may also be rearranged within a window by dragging.
  • Columns in the Protein Information Table can be resized.
  • Windows may be closed, arranged, or resized by dragging boundaries.

Recommended configuration for curation

  • Bigger is better. Use as much of the monitor as you can afford. If you are using a laptop, you may wish to attach an external monitor.
  • Adjust the width of the window and the partition between the Tree and the Table until you are comfortable with them.

Phylogenetic Tree

A phylogenetic tree contains nodes and branches (Fig 5). There are three types of nodes, root, internal and leaf. Leaf nodes correspond to the proteins in the tree. Root and internal nodes represent the inferred most common ancestor of the descendants. Branch length may be interpreted as time estimates between the nodes.

Figure 5. PAINT phylogenetic tree

The root and internal nodes of the tree are shown as circles (speciation events) and squares (gene duplication events). If the tree has been previously curated, the nodes maybe colored in indicate the type of annotation (e.g., with inferred or experimental evidence). More details will be described in the "Making an inferrence" section of this guide. The nodes have unique identifiers that start with PTN (=PaNther Node), followed by 9 digits. Mouse over a node to see its identifier. If you right-click on a node, a menu will appear with the options to:

  • Collapse node: - the entire clade is collapsed to a single node (rectangle). All the descendants are hidden, but the GO term assignments to them are still available for annotation. Right-click the node again and select "Expand node" to re-expand it.
  • Reroot to node: - make the selected node and the root, and hide the rest of the tree. This is useful when the tree is too large. To bring back the entire tree again, use menu "Tree -> Reset Root to Main".
  • Export seq ids from leaves: the ids of all leave sequences descended from the node are exported to a text file
  • Prune: All nodes descended from the node are removed from the tree.

The tree branches can be rescaled if they are too long for comfortable viewing or too short to distinguish individual nodes. The default branch scale is 50, which works for most trees. To rescale, select Tree->Scale... and enter a different number.

Navigating within the tree

  • Click on a protein name in the tree to highlight the protein in the tree and the table.
  • Left-click on a node in the tree to highlight the entire clade descended from it.

Annotation matrix

Note: The colors refer to the default colors in PAINT

Figure 6. Main PAINT window

The matrix has a row for each gene/gene product in the tree, and a column for each GO term that is directly annotated to at least one gene/gene product in the tree. The annotation matrix gives an overview of the annotations associated with any proteins in table format. It displays one of the three Gene Ontologies at a time. You can switch to a different ontology by clicking the radio button on the upper left part of the window (red arrow, Fig 6). Mouse-over the downward triangle to see the GO term (yellow circle). The terms in the annotation matrix are grouped, with the most specific terms on the left. A few very broad terms such as “protein binding” are not shown, even though they are listed in the Annotations pane.

Proteins with experimental annotations (IDA, EXP, IMP, IGI, IPI, or IEP evidence codes) for a particular ontology are colored and shown in boldface (blue circles). You may select one ontology at a time to examine using the radio buttons (red arrow) at the top of the window.

  • Click on a protein in the tree and the corresponding row will be highlighted in the matrix.
Figure 7. PAINT Annotation matrix
  • The annotations of the corresponding proteins and GO terms in the matrix are shown in colored squares (Fig 7).
    • When you first open a tree, only the experimental annotations are shown. These are the annotations than can be used for annotating ancestral genes.
      • Experimental annotations are represented by green color. If it is a direct annotation (i.e. the actual annotation is to that exact term in that column of the matrix), there is a black dot in the middle of the green square. If it is an indirect annotation (i.e. the actual annotation is to a child of the term in that column of the matrix), there is a white dot in the middle of the square.
      • NOT annotations are indicated with by a red circle with a white X.
    • When you have annotated an ancestral node, inferred annotations are also shown in the matrix. This allows you to easily keep track of what you've already annotated.
      • Inferred annotations are represented by blue color, with either a black (direct) or white (indirect) dot in the center, or X for NOT as above.
  • Mouse-over an annotation square to see the tool tip of the protein name and the term.
  • Click on the annotation square to highlight the row. All the annotations to the protein, as well as the evidences and confidence codes will be displayed in the Annotation panel (see below for more details).
  • Right-click (or Command-click in Mac) on the experimental annotation (green square) in the matrix will automatically highlight the inferred most recent common ancestor (MRCA) node for the term.

Protein Information table

Figure 8. Protein information table

The phylogenetic tree is aligned with a protein information table showing additional information and linkouts to various databases (Fig 8). You can adjust the relative sizes of each within the window by dragging the line in the partition separating them. Note that the identifier table contains a lot of information that can be observed by scrolling to the right.

Navigating withing the Protein Information table

  • Click anywhere within a row in the table to highlight the protein in the tree and the table.
  • Click on one of the blue linkouts will open a link in your web browser.



Multiple sequence alignment (MSA)

Figure 9. Multiple Sequence Alignment view

The trees were estimated from an MSA (Figure 9). The evolutionarily conserved part of the alignment is indicated with uppercase letters. The other less conserved region is in lowercase letters. If a sequence misses a position in the matchstate, it is called a delete state and is designated by a dash. If a sequence needs to insert a position in the less conserved region in order to keep the match state region aligned, it is called an insert state and is designated by a dot.

The conserved columns are colored with dark blue, blue or light blue, which indicates the conservation of 80%, 60% or 40%, respectively, in the column.

Toggle back and forth between the table view (“Protein Information”) and the MSA view (“MSA”) using the buttons above the table/MSA panel.

Note: You can view the sequence of a hypothetical ancestral protein (node) by first collapsing the appropriate node.


Annotations and Evidence windows

Figure 10. The annotations window
Figure 11. Evidence window

To view the annotations associated with a specific protein, click on that protein in the tree or table. Annotations appear in the Annotation pane (Fig 10), containing the following information:

  • ECO (Evidence code): The type evidence code supporting the annotation.
  • Term name: The GO term name and accession. Clicking on the term links out to AmiGO. A term with a NOT annotation is displayed as strikethrough text.
  • Reference: The reference supporting the annotation. Clicking on the reference links out to PubMed. IBA annotations display an internal reference, PAINT_REF:00nnnnn, where nnnnn is the numerical part of the Panther family ID.
  • With: This column contains the evidence to support the inference.
  • Qualifiers: The qualifiers 'NOT', 'colocalizes_with' and 'contributes_to' each have a column in the annotation table, and a checkbox that is checked when the qualifier is present.
  • Delete: This is used to remove an inference made by PAINT (see Removing an IBD annotation section below).

Evidence window

The evidence window displayed automatically generated logs of the tree curation, such as annotations, validation and changes made by the PAINT pipeline upon data release.


Curator notes

The Curator notes is a text editor used to record notes on the curation process. Curator notes can be modified by going to the File > Update comment menu. NOTE: The purpose of the annotation notes is to convey important points about the annotations and the phylogenetic tree both to other annotators and to users, so annotators should try to make the notes as clear as possible.

The annotator may use the Curator notes to describe important points in the annotation process, including:

  • References used to annotate the family (for example, a few major reviews)
  • Any important points about the family topology, including potential inconsistencies in the tree
  • Reasons for annotating to a different node than the MRCA (most common recent ancestor), ie the node that triangulation of annotation identifies.
  • Link to GitHub tickets leading to review of the tree annotation.

PAINT navigation functionality

“Find” function

Figure 12. PAINT 'Find' functionality

The Find function (Edit > Find…, Fig 12A) allows you to search for either a gene or a GO term. Select a gene or term search using the radio buttons (Fig 12B). Searches are case-insensitive.

A gene search matches against exact match of any text stored in the database, such as any sequence identifiers, gene symbol, or even gene name (red arrow, Fig 12C). The search does not return partial match (blue arrow, (Fig 12C). To do a partial match, wildcard character(s) (*) can be added before and/or after the search term. Scroll through the list of matches and click on a specific match to highlight it in the tree, table, and annotation matrix, and to display its annotations in the Annotations window.

You may search GO terms using text, or you may use numbers to search for GO IDs.



Making an inference: Transferring annotations

Ancestral nodes in the tree can be annotated with any GO term that has been annotated to one (or more) of its descendants. These “inferred” annotations can be propagated to its other descendants.

Annotating an ancestral node, and propagating to descendants by inheritance

Figure 13A. PTHR43114 before annotation
  • In the example shown in Fig 13A, 5 proteins are annotated by EXP to the GO term adenine deaminase activity (green squares in the 1st column of the Annotation Matrix, indicated by the red downward arrow).

Tip: To view the last common ancestor that can be annotated based on triangulation(*), right click on a GO term, or anywhere in the column that contains that GO term. An inferred node, as well as its descendants, will be highlighted in grey (blue arrow, Fig 13A).

  • (*) Triangulation is the calculation of the last common ancestor of two sequences; in this case PAINT calculates the last common ancestor supporting an annotation to the same GO term.
  • Note that the node calculated by triangulation may not be the best on to annotate: the curator can decide to annotate to an more ancient or a more recent ancestor, dependent on other evidence. For example, annotations in other GO aspects may support an earlier annotation than suggested by the triangulation.
  • The curator should not assume that the genes in the tree are fully curated with repsect to the primary literature. It may be useful to review other papers, including reviews, to ensure annotations found in the tree accurately represents the current state of knowledge.

To annotate an ancestral node

Figure 13B. PTHR43114 after annotation
  1. Click a GO term (green square) from the Annotation Matrix (Fig. 13B) (or anywhere in the column containing the GO term).
  2. Drag the term to the ancestral node you wish to annotate. This can be the inferred node or any other nodes. When you mouse over it, a + sign will be visible next to the node. Release the mouse button to annotate. Click here for a video demo of the procedure: http://youtu.be/8kHrdiuNfos.
  3. The node is now annotated with that term using the evidence code “IBD” (“Inferred from Biological Descendant”) (Fig. 13B).
  4. PAINT then automatically propagates the IBD annotation to all descendants of the PAINTEed node, such that all descendants of the node will now be annotated with that term using the evidence code “IBA” (“Inferred from Biological Ancestor”). (Proteins and nodes already annotated with the term or one of its descendant terms will remain unchanged.)

Annotating an ancestral node with a qualifier

Figure 14. Propagating qualifiers
  •  If you propagate an experimental annotation that has a qualifier, ie. "NOT", "contributes_to" (for MF annotations), or "colocalizes_with" (for CC annotations), the qualifier will also get propagated, unless there are contradictory annotations, ie, annotations with and annotations without the qualifier (for the same or for different genes). In that case, a pop-up window will appear to specify whether the annotations with or without the qualifier(s) should be propagated. Click the appropriate radio button, and click on the Continue box to apply the selection (Fig 14).
Notes
  • You may only annotate a node with a given GO term if at least one descendant has an annotation to that term or a child term. If you try to propagate a term with no supporting annotation, the node will turn red, and the propagation cannot be made.
  • Effectively, the PAINT curator only makes IBD annotations; IBA annotations are generated automatically to all descendants of the node to which an IBD annotation is made.
  • The IBD annotation automatically includes evidence for each of the sequences having an EXP annotation to the term or one if its descendants; it is not necessary to propagate individual EXP to generate the evidence for the annotation.

Negation of annotations: "NOT" statements

Background: Since PAINT is a model of the family's evolution, adding a NOT modifier to a descendant (either another node or a leaf) represents a loss of function during evolution, that is, we are stating that the specified function was present in an ancestral protein and has been lost in the indicated protein or clade. This is a special case of the GO guidelines for NOT, which state that a NOT annotation may be made in situations where a particular function may be expected but is absent.

PAINT defines two reasons for an evolutionary loss of function, described with two separate evidence codes (ECO):

  • IRD = Inferred from Rapid Divergence from ancestral sequence evidence used in manual assertion: Used when there is a long branch, often following a duplication, and significant sequence divergence. For very divergent sequences, predictions are less reliable, even in the presence of a common ancestor.
  • IKR = Inferred from phylogenetic determination of loss of Key Residues evidence: Used when a residue known to be required for the activity of the protein has mutated.

In both cases, the node (intermediate or leaf) on which the NOT annotation is placed gets the evidence code selected (IKR or IRD), and descendants, if any, are annotated with the IBA evidence.

To add the NOT qualifier to IBD annotations

Figure 15A. Tree annotated with an IBD
Figure 15B. Pop up window to select NOT evidence
  1.  From a tree annotated with a IBD annotation (Fig 15A), select a node or protein to be negated. This may be either a directly annotated node or one of its children.
  2. Click the checkbox in the NOT column of the Annotations window.
  3. A popup menu will appear (Fig 15B). In the menu Select evidence code for NOT annotation, select one of the radio buttons:
    • NOT due to rapid divergence
    • NOT due to change in key residue(s)
  4.  Optional: In the box labeled Please enter PMID and select sequence(s) from descendants providing evidence, you may enter data not captured by primary annotation that support the negation. For example if a paper shows that one of the descendants does not have an activity, you can enter the PMID and select which gene was
  5.  Optional: If appropriate, you may select from the list under Annotate to an ancestor term?, a more general GO term to propagate to the node or sequence instead of the term negated.
  • In addition to the annotation no longer propagating downward, a small hash mark will appear near the node in the tree to indicate that the block exists (visible in Fig 15C). Note that a hash mark only indicates the existence of at least one NOT annotation, not that every annotation through that node is negated.

Annotations propagated:

Figure 15C. Intermediate node annotated with NOT by IRK. Descendants are annotated with a NOT qualifier and IBA evidence
Figure 15D. Leaf node annotated with NOT by IRK.
  • If the NOT is on a node with descendants, the node will get the evidence code select (IKR or IBD), and the descendants will get an IBA evidence (Fig 15C).
  • If the NOT is on a leaf node it will get the evidence code select (IKR or IBD) (Fig 15D).
  • Upon export of PAINT annotations:
    • Annotations with IKR and all NOT annotations to proteins descended from that node will have the NOT qualifier added (as these have good evidence for loss of function).
    • Annotations with IRD and all NOT annotations to proteins descended from that node, no annotation will be exported. Thus this acts like a STOP PROPAGATION.

Removing IBD, IKR and IRD annotations

  1. Click on the desired node. Nodes with inferred annotations are colored orange.
  2. Go to the Annotation tab and click the Delete in the Delete column (shown in a red square in Fig 13B).

Notes:

  • Annotations and qualifiers can only be removed from the specific node to which they were made.
  • Primary annotations may be be changed; they may be disputed in the GO GitHub go-annotation repo.

Partial annotation of trees

The RAB GTPase superfamily
]

When you want to annotate a very large family, e.g. the RAB GTPase superfamily (PTHR24073), it may not be feasible to annotate all clades at the same time. In this kind of situation, you may choose to annotate only the clades you are knowledgeable and confident of, and leave other clades unexamined. When you do this, you should fully annotate the clades you choose to annotate. For example, if you choose to do the IFT27 clade, do it fully. Please don't do piecemeal annotations in various locations that may make it hard for a subsequent annotator to understand what has been done.

We also agreed at the July 2014 PAINT Jamboree that you can make propagations all the way to the root if you feel that there is an ancestral role, even if you think that some clades have lost this. For example, in the RAB GTPase superfamily, we think that it had an ancestral function as a GTPase, but it is possible that some clades, e.g. the IFT22 clade, have lost this ancestral activity. You can make these high level propagations as part of your initial annotation of the family. If there are clades where this is wrong, perhaps the IBA annotation from PAINT will generate feedback that will help us correct it.

Recording partial annotation in the notes file

If you only partially annotate a tree, please record in the notes file which clades you have worked on using the node number, e.g. Eukaryota_PTN001180007 as well as a common name, e.g. IFT27, if it is helpful.

Recording trees examined, but not annotated

When you examine a tree and feel that it should not be annotated for some reason, please record that in the Evidence Notes so that we can track the fact that the family has been examined. Please use one of these tags (in all caps) in the Notes section of the Evidence tab. You can additional information after the tag if you wish (syntax between tag and additional info not discussed or determined). Then, save your annotations as normal so that PAINT will save the notes file.

  • MISSING ANNOTATION - Use this if the tree looks OK, but there are insufficient experimental annotations to propagate any annotations.
  • MISSING SEQUENCE - Use this if you feel that a specific sequence or sequences is missing. You can list the IDs of the sequence(s) after the tag.
  • BAD TREE - Use this if you feel that the tree has major problems beyond one or a few missing sequences.

Interpreting the PANTHER trees

Speciation and duplication events, and horizontal transfer

In the tree, a speciation node is shown with a circle, and a gene duplication node with an square. Horizontal transfer events also appear in the tree, though more rarely, and these are represented with a diamond.

Branch lengths

  • Branch lengths show the amount of sequence divergence that has occurred between a given node and its ancestral node, in terms of the average number of amino acid substitutions per site. Shorter branches indicate less sequence divergence and therefore greater conservation of ancestral characters. A branch might be shorter because of a slower evolutionary rate (greater negative selection), or because less "time" has gone by (actually a combination of number of generations and population dynamics), or both.
  • Very long branches indicate an unreliable divergence estimate, due to insufficient data. Note that sometimes there is not enough data to compare all branches that descend from a given node. In this case, we have set all descendant branches to a length of 2.0 (very long branches). Branch lengths of 2.0 are often due to a sequence fragment, and at a duplication node it may also indicate a gene that has been incorrectly broken into two different genes by a gene prediction program.
  • Following a gene duplication (after a square node), the relative branch lengths for descendant branches are particularly useful: the shortest branch (least diverged) is more likely to have greater functional conservation.

Multiple sequence alignment (MSA)

  • Some columns in the MSA have upper-case characters (and dashes '-' for insertions/deletions). These columns were used to estimate the phylogenetic tree.
  • Lower-case characters and periods (‘.’ for insertions/deletions) denote positions that were ignored when estimating the phylogenetic tree. Sometimes, tree errors arise because not enough columns were used, and the phylogeny could not be reconstructed well based on the included columns. Since they were not used in the phylogeny, lower-case characters can be particularly helpful in verifying the tree topology: any conserved insertions should be parsimoniously traceable to a common ancestor.

Reporting bugs or likely errors in the trees

Tree issues

Most often, the errors in phylogenetic trees are due to problems with the sequence alignment, or the specific MSA columns used to estimate the phylogeny. The phylogeny inference program performs fairly robust handling of sequence fragments, but sequence fragments still cause errors. Another source of error is when the sequences evolve very slowly, generating little variation from which to estimate phylogeny. In this case, the errors can usually be fixed by including additional alignment positions to consider in the phylogeny.

If a Panther tree needs to be reviewed, please create a ticket in the Panther GitHub tracker: https://github.com/pantherdb/Helpdesk/issues

PAINT issues

Issues with the PAINT tools should be reported in this tracker: https://github.com/pantherdb/db-PAINT/issues

Curation Guidelines

Those guidelines have been published (Gaudet, Livestone, Lewis, Thomas, 2011) [1]

Curation guidelines are described in detail on this page: http://wiki.geneontology.org/index.php/PAINT_SOP