+ \exercise{Graphical Output}{
+\exstep{Load the example Jalview Jar file in Exercise \ref{exscreen}.
+Customise it how you wish but leave it unwrapped.
+Select {\sl File $\Rightarrow$ Export Image $\Rightarrow$ HTML} from the alignment menu.
+Save the file and open it in your favoured web browser. }
+\exstep{Wrap the alignment and export the image to HTML again. Compare the two
+images. (Note that the exported image matches the format displayed in the
+alignment window but {\bf annotations are not exported}).}
+\exstep{Export the alignment using the {\sl File $\Rightarrow$ Export Image $\Rightarrow$ PNG} menu option.
+Open the file in an image viewer that allows zooming such as Paint or Photoshop (Windows), or Preview (Mac OS X) and zoom in.
+Notice that the image is a bitmap and it becomes pixelated when zoomed.
+(Note that the {\bf annotation lines are included} in the image.)}
+\exstep{Export the alignment using the {\sl File $\Rightarrow$ Export Image
+$\Rightarrow$ EPS} menu option. Open the file in a suitable program such as
+Photoshop, Illustrator, Inkscape, Ghostview, Powerpoint (Windows), or
+Preview (Mac OS X). Zoom in and note that the image has near-infinite
+resolution.}
+}
+
+% left out for Glasgow 2016
+% \newpage
+%
+% \section{Summary - the rest of the manual}
+%
+% The first few chapters have covered the basics of Jalview operation: from
+% starting the program, importing, exporting, selecting, editing and colouring
+% aligments, to the generation of figures for publication, presentation and web
+% pages.
+%
+% The remaining chapters in the manual cover:
+%
+% \begin{list}{$\circ$}{}
+% \item{Chapter \ref{featannot} covers the creation, manipulation and visualisation
+% of sequence and alignment annotation, and retrieval of sequence and feature data
+% from databases.}
+% \item {Chapter \ref{msaservices} explores the range of multiple alignment
+% programs offered via Jalview's web services, and introduces the use of
+% AACon for protein multiple alignment conservation analysis.}
+% \item {Chapter \ref{alignanalysis} introduces Jalview's built in tools for
+% multiple sequence alignment analysis, including trees, PCA, and alignment
+% conservation analysis. }
+% \item {Chapter \ref{3Dstructure} demonstrates the structure visualization
+% capabilities of Jalview.}
+% \item {Chapter \ref{proteinprediction} introduces protein sequence based
+% secondary structure and disorder prediction tools, including JPred.}
+% \item {Chapter \ref{dnarna} covers the special functions and
+% visualization techniques for working with RNA alignments and protein coding
+% sequences.}
+% \item {Chapter \ref{jvwebservices} provides instructions on the
+% installation of your own Jalview web services.}
+% \end{list}
+
+\chapter{Annotation and Features}
+\label{featannot}
+Annotations and features are additional information that is
+overlaid on the sequences and the alignment.
+Generally speaking, annotations reflect properties of the alignment as a
+whole, often associated
+with columns in the alignment. Features are often associated with specific
+residues in the sequence.
+
+Annotations are shown below the alignment in the annotation panel, the
+properties are often based on the alignment.
+Conversely, sequence features are properties of the individual sequences, so they do not change with the alignment,
+but are shown mapped on to specific residues within the alignment.
+
+Features and annotation can be interactively created, or retrieved from external
+data sources. Webservices like JPred (see \ref{jpred} above) can be used to
+analyse a given sequence or alignment and generate annotation for it.
+
+
+\section{Conservation, Quality and Consensus Annotation}
+\label{annotationintro}
+Jalview automatically calculates several quantitative alignment annotations
+which are displayed as histograms below the multiple sequence alignment columns.
+Conservation, quality and consensus scores are examples of dynamic
+annotation, so as the alignment changes, they change along with it.
+The scores can be used in the hybrid colouring options to shade the alignments.
+Mousing over a conservation histogram reveals a tooltip with more information.
+
+These annotations can be hidden and deleted via the context menu linked to the
+annotation row; but they are only created on loading an alignment. If they are
+deleted then the alignment should be saved and then reloaded to restore them.
+Jalview provides a toggle to autocalculate a consensus sequence upon editing. This is normally selected by default, but can be turned off for
+large alignments {\sl via} the {\sl Calculate $\Rightarrow$ Autocalculate
+Consensus} menu option if the interface is too slow.
+
+\subsubsection{Conservation Annotation}
+
+Alignment conservation annotation is quantitative numerical index reflecting the
+conservation of the physico-chemical properties for each column of the alignment.
+The calculation is based on AMAS method of multiple sequence alignment analysis (Livingstone C.D. and Barton G.J. (1993) CABIOS Vol. 9 No. 6 p745-756),
+with identities scoring highest, and amino acids with substitutions in the same physico-chemical class have next highest score.
+The score for each column is shown below the histogram.
+The conserved columns with a score of 11 are indicated by '*'.
+Columns with a score of 10 have mutations but all properties are conserved are marked with a '+'.
+
+\subsubsection{Consensus Annotation}
+
+Alignment consensus annotation reflects the percentage of the different residue
+per column. By default this calculation includes gaps in columns, gaps can be ignored via the Consensus label context
+menu to the left of the consensus bar chart.
+The consensus histogram can be overlaid
+with a sequence logo that reflects the symbol distribution at each column of
+the alignment. Right click on the Consensus annotation row and select the {\sl Show
+Logo} option to display the Consensus profile for the group or alignment.
+Sequence logos can be enabled by default for all new alignments {\sl via} the
+Visual tab in the Jalview desktop's preferences dialog box.
+
+\subsubsection{Quality Annotation}
+
+Alignment quality annotation is an ad-hoc measure of the likelihood of observing
+the mutations (if any) in a particular column of the alignment. The quality score is calculated for each column in an alignment by summing,
+for all mutations, the ratio of the two BLOSUM 62 scores for a mutation pair and each residue's conserved BLOSUM62 score (which is higher).
+This value is normalised for each column, and then plotted on a scale from 0 to 1.
+
+\subsubsection{Group Associated Annotation}
+\label{groupassocannotation}
+Group associated consensus and conservation annotation rows reflect the
+sequence variation within a particular group. Their calculation is enabled
+by selecting the {\sl Group Conservation} or {\sl Group Consensus} options in
+the {\sl Annotation $\Rightarrow$ Autocalculated Annotation } submenu of the
+alignment window.
+
+\subsection{Creating User Defined Annotation}
+
+To create a new annotation row, right click on the annotation label panel and select the {\sl Add New Row} menu option (Figure \ref{newannotrow}).
+A dialog box appears. Enter the label to use for this row and a new row will appear.
+
+To create a new annotation, first select all the positions to be annotated on the appropriate row.
+Right-clicking on this selection brings up the context menu which allows the insertion of graphics for secondary structure ({\sl Helix} or {\sl Sheet}),
+text {\sl Label} and the colour in which to present the annotation (Figure \ref{newannot}). On selecting {\sl Label} a dialog box will appear,
+requesting the text to place at that position. After the text is entered, the selection can be removed and the annotation becomes clearly
+visible\footnote{When annotating a block of positions, the text can be partly obscured by the selection highlight. Pressing the [ESC] key clears
+the selection and the label is then visible.}. Annotations can be coloured or deleted as desired.
+
+\begin{figure}[htbp]
+\begin{center}
+\includegraphics[width=1.3in]{images/annots1.pdf}
+\includegraphics[width=2in]{images/annots2.pdf}
+\caption{{\bf Creating a new annotation row.} Annotation rows can be reordered by dragging them to the desired place.}
+\label{newannotrow}
+\end{center}
+\end{figure}
+
+\begin{figure}[htbp]
+\begin{center}
+\includegraphics[width=2in]{images/annots3.pdf}
+\includegraphics[width=2in]{images/annots4.pdf}
+\includegraphics[width=2in]{images/annots5.pdf}
+\caption{{\bf Creating a new annotation.} Annotations are created from a selection on the annotation row and can be coloured as desired.}
+\label{newannot}
+\end{center}
+\end{figure}
+
+\subsection{Automated Annotation of Alignments and Groups}
+
+On loading a sequence alignment, Jalview will normally\footnote{Automatic
+annotation can be turned off in the {\sl Visual } tab in the {\sl Tools
+$\Rightarrow$ Preferences } dialog box.} calculate a set of automatic annotation
+rows which are shown below the alignment. For nucleotide sequence alignments,
+only an alignment consensus row will be shown, but for amino acid sequences,
+alignment quality (based on BLOSUM 62) and physicochemical conservation will
+also be shown. Conservation is calculated according to Livingstone and
+Barton\footnote{{\sl ``Protein Sequence Alignments: A Strategy for the
+Hierarchical Analysis of Residue Conservation." } Livingstone C.D. and Barton
+G.J. (1993) {\sl CABIOS } {\bf 9}, 745-756}.
+Consensus is the modal residue (or {\tt +} where there is an equal top residue).
+The inclusion of gaps in the consensus calculation can be toggled by
+right-clicking on the Consensus label and selecting {\sl Ignore Gaps in
+Consensus} from the pop-up context menu located with consensus annotation row.
+Quality is a measure of the inverse likelihood of unfavourable mutations in the alignment. Further details on these
+calculations can be found in the on-line documentation.
+
+
+\exercise{Annotating Alignments}{
+ \label{annotatingalignex}
+\exstep{Load the alignment at \textsf{http://www.jalview.org/tutorial/alignment.fa}.
+Right-click on the {\sl Conservation} annotation row to
+bring up the context menu and select {\sl Add New Row}. A dialog box will
+appear asking for {\sl Annotation Name} and {\sl Annotation Description}.
+Enter ``Iron binding site" and click {\sl OK}. A new, empty, row appears.
+}
+\exstep{
+Navigate to column 97. Move down and on the new annotation row called
+``Iron binding site, select column 97.
+Right click at this selection and select {\sl Label} from the context menu.
+Enter ``Fe" in the box and click {\sl OK}. Right-click on the selection again
+and select {\sl Colour}.
+Choose a colour from the colour chooser dialog
+and click {\sl OK}. Press [ESC] to remove the selection.
+
+{\sl Note: depending on your Annotation sort settings, your newly
+created annotation row might 'jump' to the top or bottom of the annotation
+panel. Just scroll up or down to find it again - the column you marked will
+still be selected. }
+
+}
+\exstep{ Select columns 70-77 on the annotation row. Right-click and choose {\sl Sheet} from the
+ context menu. You will be prompted for a label. Enter ``B" and press {\sl OK}. A new line showing the
+ sheet as an arrow appears. The colour of the label can be changed but not the colour of the sheet
+ arrow.
+}
+\exstep{Right click on the title text of annotation row that you just created.
+Select {\sl Export Annotation} in context menu and, in the Export Annotation
+dialog box that will open, select the Jalview format and click the {\sl [To Textbox]} button.
+
+The format for this file is given in the Jalview help. Press [F1] to open it, and find
+the ``Annotations File Format'' entry in the ``Alignment Annotations'' section of the contents
+pane. }
+
+\exstep{Export the file to a text editor and edit the file to change the name of the annotation
+row. Save the file and drag it onto the alignment view.}
+\exstep{Add an additional helix somewhere along the row by editing the file and
+re-importing it.
+
+{\sl Hint: Use the Export Annotation function to view what helix annotation looks like in
+a Jalview annotation file.}}
+\exstep{Use the {\sl Alignment Window $\Rightarrow$ File $\Rightarrow$ Export Annotations...}
+function to export all the alignment's annotation to a file.}
+\exstep{Open the exported annotation in a text editor, and use the Annotation File Format
+documentation to modify the style of the Conservation, Consensus and Quality annotation rows so
+they appear as several lines on a single line graph.
+
+{\sl Hint: You need to change the style of annotation row in the first field of the annotation
+row entry in the file, and create an annotation row grouping to overlay the three quantitative
+annotation rows.}
+}
+\exstep{{\bf Homework for after you have completed exercise \ref{secstrpredex}:}
+\label{viewannotfileex}
+
+Recover or recreate the secondary structure predictions that you made from
+JPred. Use the {\sl File $\Rightarrow$ Export Annotation} function to view the Jnet secondary structure prediction annotation row.
+
+Note the
+SEQUENCE\_REF statements surrounding the row specifying the sequence association for the
+annotation.
+}}
+
+
+\section{Importing Features from Databases}
+\label{featuresfromdb}
+Jalview supports feature retrieval from public databases.
+It includes built in parsers for Uniprot and ENA (or EMBL) records retrieved
+from the EBI. Sequences retrieved from these sources using the sequence fetcher (see
+Section \ref{fetchseq}) will already possess features.
+
+\subsection{Sequence Database Reference Retrieval}
+\label{fetchdbrefs}
+Jalview maintains a list of external database references for each sequence in
+an alignment. These are listed in a tooltip when the mouse is moved over the
+sequence ID when the {\sl View $\Rightarrow$ Sequence ID Tooltip $\Rightarrow$
+Show Database Refs } option is enabled. Sequences retrieved using the sequence
+fetcher will always have at least one database reference, but alignments
+imported from an alignment file generally have no database references.
+
+\subsubsection{Database References and Sequence Coordinate Systems}
+
+Jalview displays features in the local sequence's coordinate system which is
+given by its `start' and `end'. Any sequence features on the sequence will be
+rendered relative to the sequence's start position. If the start/end positions
+do not match the coordinate system from which the features were defined, then
+the features will be displayed incorrectly.
+
+\subsubsection{Viewing and Exporting a Sequence's Database Annotation}
+
+You can export all the database cross references and annotation terms shown in
+the sequence ID tooltip for a sequence by right-clicking and selecting the {\sl
+[Sequence ID] $\Rightarrow$ Sequence details \ldots} option from the popup
+menu.
+A similar option is provided in the {\sl Selection} sub-menu allowing you to
+obtain annotation for the sequences currently selected.
+
+\parbox[l]{3.4in}{
+The {\sl Sequence Details
+\ldots} option will open a window containing the same text as would be shown in
+the tooltip window, including any web links associated with the sequence. The
+text is HTML, and options on the window allow the raw code to be copied and
+pasted into a web page.}
+\parbox[c]{3in}{
+\centerline{\includegraphics[width=2.2in]{images/seqdetailsreport.pdf}}}
+
+\subsubsection{Automatically Discovering a Sequence's Database References}
+Jalview includes a function to automatically verify and update each sequence's
+start and end numbering against any of the sequence databases that the {\sl
+Sequence Fetcher} has access to. This function is accessed from the {\sl
+Webservice $\Rightarrow$ Fetch DB References} sub-menu in the Alignment
+window. This menu allows you to query either the set of {\sl Standard
+Databases}, which includes EMBL, Uniprot, the PDB, or just a specific datasource from one of the submenus.
+When one of the entries from this menu is selected, Jalview will use the ID
+string from each sequence in the alignment or in the currently selected set to
+retrieve records from the external source. Any sequences that are retrieved are
+matched against the local sequence, and if the local sequence is found to be a
+sub-sequence of the retrieved sequence then the local sequence's start/end
+numbering is updated. A new database reference mapping is created, mapping the
+local sequence to the external database, and the local sequence inherits any
+additional annotation retrieved from the database sequence.
+
+The database retrieval process terminates when a valid mapping is found for a
+sequence, or if all database queries failed to retrieve a matching sequence.
+Termination is indicated by the disappearance of the moving progress indicator
+on the alignment window. A dialog box may be shown once it completes which
+lists sequences for which records were found, but the sequence retrieved from
+the database did not exactly contain the sequence given in the alignment (the
+{\sl ``Sequence not 100\% match'' dialog box}).
+
+
+\subsubsection{The Fetch Uniprot IDs Dialog Box}
+\label{discoveruniprotids}
+If any sources are selected which refer to Uniprot coordinates as their reference system,
+then you may be asked if you wish to retrieve Uniprot IDs for your sequence. Pressing OK instructs Jalview to verify the sequences against Uniprot records retrieved using the sequence's ID string. This operates in much the same way as the {\sl Web Service $\Rightarrow$ Fetch Database References } function described in Section \ref{fetchdbrefs}.
+If a sequence is verified, then the start/end numbering will be adjusted to match the Uniprot record.
+
+\subsubsection{Rate of Feature Retrieval}
+Feature retrieval can take some time if a large number of sources are selected
+and if the alignment contains a large number of sequences.
+As features are retrieved, they are immediately added to the current alignment view.
+The retrieved features are shown on the sequence and can be customised as described previously.
+
+
+\subsection{Colouring Features by Score or Description
+Text}
+\label{featureschemes}
+Sometimes, you may need to visualize the differences in information carried by
+sequence features of the same type. This is most often the case when features
+of a particular type are the result of a specific type of database query or calculation. Here, they may also carry information within their textual description, or most commonly for calculations, a score related to the property being investigated. Jalview can shade sequence
+features using a graduated colourscheme in order to highlight these variations.
+In order to apply a graduated scheme to a feature type, select the `Graduated
+colour' entry in the Sequence {\sl Feature Type}'s popup menu, which is opened by
+right-clicking the {\sl Feature Type} or {\sl Color} in the {\sl Sequence Feature Settings} dialog box. Two types
+of colouring styles are currently supported: the default is quantitative
+colouring, which shades each feature based on its score, with the highest
+scores receiving the `Max' colour, and the lowest scoring features coloured
+with the `Min' colour. Alternately, you can select the `Colour by label'
+option to create feature colours according to the description text associated
+with each feature. This is useful for general feature types - such as
+Uniprot's `DOMAIN' feature - where the actual type of domain is given in the
+feature's description.
+
+Graduated feature colourschemes can also be used to exclude low or
+high-scoring features from the alignment display. This is done by choosing your
+desired threshold type (either above or below), using the drop-down menu in the
+dialog box. Then, adjust the slider or enter a value in the text box to set the
+threshold for displaying this type of feature.
+
+The feature settings dialog box allows you to toggle between a graduated and
+simple feature colourscheme using the pop-up menu for the feature type. When a
+graduated scheme is applied, it will be indicated in the colour column for
+that feature type - with coloured blocks or text to indicate the colouring
+style and a greater than ($>$) or less than ($<$) symbol to indicate when a
+threshold has been defined.
+
+\subsection{Using Features to Re-order the Alignment}
+\label{featureordering}
+The presence of sequence features on certain sequences or in a particular
+region of an alignment can quantitatively identify important trends in
+the aligned sequences. In this case, it is more useful to
+re-order the alignment based on the number of features or their associated scores, rather than simply re-colour the aligned sequences. The sequence feature settings
+dialog box provides two buttons: `Seq sort by Density' and `Seq sort by
+Score', that allow you to reorder the alignment according to the number of
+sequence features present on each sequence, and also according to any scores
+associated with a feature. Each of these buttons uses the currently displayed
+features to determine the ordering, but
+if you wish to re-order the alignment using a single type of feature, then you can do this from the {\sl Feature Type}'s
+popup menu. Simply right-click the type's style in the Sequence Feature Settings dialog
+box, and select one of the {\sl Sort by Score} and {\sl Sort by Density}
+options to re-order the alignment. Finally, if a specific region is selected,
+then only features found in that region of the alignment will be used to
+create the new alignment ordering.
+% \exercise{Shading and Sorting Alignments using Sequence Features}{
+% \label{shadingorderingfeatsex}
+%
+% This exercise is currently not included in the tutorial because no DAS servers
+% currently exist that yield per-residue features for any Uniprot sequence.
+%
+% \exstep{Re-load the alignment from \ref{dasfeatretrexcercise}.
+% }
+% \exstep{Open the
+% feature settings panel, and, after first clearing the current
+% selection, press the {\em Seq Sort by Density} button a few times.}
+% \exstep{Use the DAS fetcher to retrieve the Kyte and Doolittle Hydrophobicity
+% scores for the protein sequences in the alignment.
+% {\sl Hint: the nickname for the das source is `KD$\_$hydrophobicity'.}}
+% \exstep{Change the feature settings so only the hydrophobicity features are
+% displayed. Mouse over the annotation and also export and examine the GFF and
+% Jalview features file to better understand how the hydrophobicity measurements
+% are recorded.}
+% \exstep{Apply a {\sl Graduated Colour} to the hydrophobicity annotation to
+% reveal the variation in average hydrophobicity across the alignment.}
+% \exstep{Select a range of alignment columns, and use one of the sort by feature buttons to order the alignment according to that region's average
+% hydrophobicity.}
+% \exstep{Save the alignment as a project, for use in exercise
+% \ref{threshgradfeaturesex}.} }
+%
+% \exercise{Shading alignments with combinations of graduated feature
+% colourschemes}{
+% \label{threshgradfeaturesex}
+% \exstep{Reusing the annotated alignment from exercise
+% \ref{shadingorderingfeatsex}, experiment with the colourscheme threshold to
+% highlight the most, or least hydrophobic regions. Note how the {\sl Colour} icon for the {\sl Feature Type} changes when you change the threshold type and press OK.}
+% \exstep{Change the colourscheme so
+% that features at the threshold are always coloured grey, and the most
+% hydrophobic residues are coloured red, regardless of the threshold value
+% ({\em hint - there is a switch on the dialog to do this for you}).}
+% \exstep{Enable the Uniprot {\em chain} annotation in the feature settings
+% display and re-order the features so it is visible under the hydrophobicity
+% annotation.}
+% \exstep{Apply a {\sl Graduated Colour} to the {\em chain}
+% annotation so that it distinguishes the different canonical names associated
+% with the mature polypeptide chains.}
+% \exstep{Export the alignment's sequence features using the Jalview and GFF file formats, to see how the different types of graduated feature
+% colour styles are encoded. }
+% }
+
+\subsection{Creating Sequence Features}
+Sequence features can be created simply by selecting the area in a sequence (or sequences) to form the feature and selecting {\sl Selection $\Rightarrow$ Create Sequence Feature } from the right-click context menu (Figure \ref{features}). A dialog box allows the user to customise the feature with respect to name, group, and colour. The feature is then associated with the sequence. Moving the mouse over a residue associated with a feature brings up a tool tip listing all features associated with the residue.
+
+\begin{figure}[htbp]
+\begin{center}
+\includegraphics[width=2in]{images/feature1.pdf}
+\includegraphics[width=2.5in]{images/feature2.pdf}
+\includegraphics[width=1.5in]{images/feature3.pdf}
+\caption{{\bf Creating sequence features.} Features can readily be created from selections via the context menu and are then displayed on the sequence. }
+\label{features}
+\end{center}
+\end{figure}
+
+Creation of features from a selection spanning multiple sequences results in the creation of one feature per sequence.
+Each feature remains associated with its own sequence.
+
+\subsection{Customising Feature Display}
+
+Feature display can be toggled on or off by selecting the {\sl View
+$\Rightarrow$ Show Sequence Features} menu option. When multiple features are
+present it is usually necessary to customise the display. Jalview allows the
+display, colour, rendering order and transparency of features to be modified
+{\sl via} the {\sl View $\Rightarrow$ Feature Settings\ldots} menu option. This
+brings up a dialog window (Figure \ref{custfeat}) which allows the
+visibility of individual feature types to be selected, colours changed (by
+clicking on the colour of each sequence feature type) and the rendering order
+modified by dragging feature types to a new position in the list. Dragging the
+slider alters the transparency of the feature rendering. The Feature
+Settings dialog also includes functions for more advanced feature shading
+schemes and buttons for sorting the alignment according to the distribution of
+features. These capabilities are described further in sections
+\ref{featureschemes} and \ref{featureordering}.
+
+\begin{figure}[htbp]
+\begin{center}
+\includegraphics[width=4in]{images/features4.pdf}
+\caption{{\bf Multiple sequence features.} An alignment with JPred secondary structure prediction annotation below it, and many sequence features overlaid onto the aligned sequences. The tooltip lists the features annotating the residue below the mouse-pointer.}
+\end{center}
+\end{figure}
+
+\begin{figure}[htbp]
+\begin{center}
+\includegraphics[width=4in]{images/features5.pdf}
+\caption{{\bf Customising sequence features.} Features can be recoloured, switched on or off and have the rendering order changed. }
+\label{custfeat}
+\end{center}
+\end{figure}
+
+\subsection{Sequence Feature File Formats}
+
+Jalview supports the widely used GFF tab delimited format\footnote{see
+http://www.sanger.ac.uk/resources/software/gff/spec.html} and its own Jalview
+Features file format for the import of sequence annotation. Features and
+alignment annotation are also extracted from other formats such as Stockholm,
+and AMSA. URL links may also be attached to features. See the online
+documentation for more details of the additional capabilities of the Jalview
+features file.
+
+\exercise{Creating Features}{
+\exstep{Open the alignment at \textsf{http://www.jalview.org/tutorial/alignment.fa}.
+We know that the Cysteine residues at columns 97, 102, 105 and 135 are involved in
+iron binding so we will create them as features. Navigate to column 97, sequence 1.
+Select the entire column by clicking in the ruler bar. Then right-click on the selection
+to bring up the context menu and select {\sl Selection $\Rightarrow$ Create Sequence Feature}.
+A dialog box will appear.
+}
+\exstep{
+Enter a suitable Sequence Feature Name (e.g. ``Iron binding site") in the
+appropriate box. Click on the Feature Colour bar to change the colour if
+desired, add a short description (``One of four Iron binding Cysteines") and
+press {\sl OK}. The features will then appear on the sequences. } \exstep{Roll
+the mouse cursor over the new features.
+Note that the position given in the tool tip is the residue number, not the column number.
+To demonstrate that there is one feature per sequence, clear all selections by pressing [ESC] then insert a gap in sequence 3 at column 95.
+Roll the mouse over the features and you will see that the feature has moved with the sequence. Delete the gap you created.
+}
+\exstep{
+Add a similar feature to column 102. When the feature dialog box appears, clicking the Sequence Feature
+Name box brings up a list of previously described features. Using the same Sequence Feature Name allows the features to be grouped.}
+\exstep{Select {\sl View $\Rightarrow$ Feature Settings\ldots} from the
+alignment window menu. The Sequence Feature Settings window will appear. Move
+this so that you can see the features you have just created. Click the check
+box for ``Iron binding site" under {\sl Display} and note that display of this
+feature type is now turned off. Click it again and note that the features are
+now displayed. Close the sequence feature settings box by clicking {\sl OK} or
+{\sl Cancel}.} }
+
+\chapter{Multiple Sequence Alignment}
+\label{msaservices}
+Sequences can be aligned using a range of algorithms provided by JABA web
+services, including ClustalW\footnote{{\sl ``CLUSTAL W:
+improving the sensitivity of progressive multiple sequence alignment through sequence
+weighting, position specific gap penalties and weight matrix choice."} Thompson
+JD, Higgins DG, Gibson TJ (1994) {\sl Nucleic Acids Research} {\bf 22},
+4673-80}, Muscle\footnote{{\sl ``MUSCLE: a multiple sequence alignment method
+with reduced time and space complexity"} Edgar, R.C.
+(2004) {\sl BMC Bioinformatics} {\bf 5}, 113}, MAFFT\footnote{{\sl ``MAFFT: a
+novel method for rapid multiple sequence alignment based on fast Fourier
+transform"} Katoh, K., Misawa, K., Kuma, K. and Miyata, T. (2002) {\sl Nucleic
+Acids Research} {\bf 30}, 3059-3066. and {\sl ``MAFFT version 5:
+improvement in accuracy of multiple sequence alignment"} Katoh, K., Kuma, K.,
+Toh, H. and Miyata, T. (2005) {\sl Nucleic Acids Research} {\bf 33}, 511-518.},
+ProbCons,\footnote{PROBCONS: Probabilistic Consistency-based Multiple Sequence
+Alignment.
+Do, C.B., Mahabhashyam, M.S.P., Brudno, M., and Batzoglou, S.
+(2005) {\sl Genome Research} {\bf 15} 330-340.} T-COFFEE\footnote{T-Coffee:
+A novel method for multiple sequence alignments. (2000) Notredame, Higgins and
+Heringa {\sl JMB} {\bf 302} 205-217} and Clustal Omega.\footnote{Fast, scalable
+generation of high-quality protein multiple sequence alignments using Clustal
+Omega. Sievers F, Wilm A, Dineen DG, Gibson TJ, Karplus K, Li W, Lopez R,
+McWilliam H, Remmert M, Soding J, Thompson JD, Higgins DG (2011) {\sl Molecular
+Systems Biology} {\bf 7} 539
+\href{http://dx.doi.org/10.1038/msb.2011.75}{doi:10.1038/msb.2011.75}} Of these,
+T-COFFEE is slow but accurate. ClustalW is historically
+the most widely used. Muscle is fast and probably best for
+smaller alignments. MAFFT is probably the best for large alignments,
+however Clustal Omega, released in 2011, is arguably the fastest and most
+accurate tool for protein multiple alignment.
+
+\section{Performing a multiple sequence alignment}
+To run an alignment web service, select the appropriate method from the {\sl
+Web Service $\Rightarrow$ Alignment $\Rightarrow$ \ldots} submenu (Figure
+\ref{webservices}). For each service you may either perform an alignment with
+default settings, use one of the available presets, or customise the parameters
+with the `{\sl Edit and Run ..}' dialog box. Once the job is submitted, a
+progress window will appear giving information about the job and any errors that
+occur. After successful completion of the job, a new alignment window is opened
+with the results, in this case an alignment. By default, the new alignment will be
+ordered in the same way as the input sequences. Note: many alignment
+programs re-order the input during their analysis and place homologous
+sequences close together, the MSA algorithm ordering can be recovered
+using the `Algorithm ordering' entry within the {\sl Calculate $\Rightarrow$
+Sort } sub menu.
+
+\subsection{Realignment to add sequences to an existing alignment}
+The re-alignment option is currently only supported by Clustal
+Omega and ClustalW. When performing a re-alignment, Jalview submits the
+current selection to the alignment service complete with any existing gaps.
+Realignment with ClustalW is useful when one wishes to align
+additional sequences to an existing alignment without any further optimisation
+to the existing alignment. ClustalO's realignment works by generating a
+probabilistic model (a.k.a HMM) from the original alignment, and then realigns
+{\bf all} sequences to this profile. For a well aligned MSA, this process
+will simply reconstruct the original alignment (with additonal sequences), but
+in the case of low quality MSAs, some differences may be introduced.
+
+\begin{figure}[htbp]
+\begin{center}
+\parbox[c]{1.5in}{\includegraphics[width=1.5in]{images/ws1.pdf}}
+\parbox[c]{2.5in}{\includegraphics[width=2.5in]{images/ws2.pdf}}
+\parbox[c]{2in}{\includegraphics[width=2in]{images/ws3.pdf}}
+\caption{{\bf Multiple alignment via web services} The appropriate method is
+selected from the menu (left), a status box appears (centre), and the results
+appear in a new window (right).}
+\label{webservices}
+\end{center}
+\end{figure}
+
+\subsection{Alignments of Sequences that include Hidden Regions}
+If the view or selected region submitted for alignment contains hidden
+regions, then {\bf only the visible sequences will be submitted to the service}.
+Furthermore, each contiguous segment of sequences will be aligned independently
+(resulting in a number of alignment `subjobs' appearing in the status window).
+Finally, the results of each subjob will be concatenated with the hidden regions
+in the input data prior to their display in a new window. This approach ensures
+that 1) hidden column boundaries in the input data are preserved in the
+resulting alignment - in a similar fashion to the constraint that hidden columns
+place on alignment editing (see Section \ref{lockededits} and 2) hidden
+columns can be used to preserve existing parts of an alignment whilst the
+visible parts are locally refined.
+
+\subsection{Alignment Service Limits}
+Multiple alignment is a computationally intensive calculation. Some JABA server
+services and service presets only allow a certain number of sequences to be
+aligned. The precise number will depend on the server that you are using to
+perform the alignment. Should you try to submit more sequences than a service
+can handle, then an error message will be shown informing you of the maximum
+number allowed by the server.
+
+\exercise{Multiple Sequence Alignment}{
+\exstep{ Close all windows and open the alignment at {\sf
+http://www.jalview.org/tutorial/unaligned.fa}. Select {\sl
+Web Service $\Rightarrow$ Alignment $\Rightarrow$ Muscle with Defaults}.
+A window will open giving the job status. After a short time, a second window will open
+ with the results of the alignment.}
+ \exstep{Return to the first sequence alignment window by clicking on
+ the window, and repeat using {\sl ClustalO} (Omega) and {\sl MAFFT}, from the
+ {\sl Web Service $\Rightarrow$ Alignment} menu, using the same initial
+ alignment.
+ Compare them and you should notice small differences. }
+\exstep{Select the last three sequences in the MAFFT alignment, and de-align them
+with {\sl Edit $\Rightarrow$ Remove All Gaps}. Press [ESC] to deselect these
+sequences. Then submit this view for re-alignment with {\sl ClustalO}.}
+\exstep{Return to the alignment window in section (c), use [CTRL]-Z (undo) to
+recover the alignment of the last three sequences in this MAFFT alignment.
+Once the ClustalO re-alignment has completed, compare the results of
+re-alignment of the three sequences with their alignment in the original MAFFT result.}
+\exstep{Select columns 60 to 125 in the original MAFFT alignment and hide them,
+by right clicking the mouse to bring up context menu.
+Select {\sl Web Service $\Rightarrow$ Alignment $\Rightarrow$ Mafft with Defaults} to
+submit the visible portion of the alignment to MAFFT. When the web service job pane appears,
+note that there are now two alignment job status panes shown in the window.}
+\exstep{When the MAFFT job has finished, compare the alignment of the N-terminal visible
+region in the result with the corresponding region of the original alignment.}
+\exstep {If you wish,
+select and hide a few more columns in the N-terminal region, and submit the alignment to the
+service again and explore the effect of local alignment on the non-homologous parts of the
+N-terminal region.}
+{\bf See the video at:
+\url{http://www.jalview.org/training/Training-Videos}.}