+\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 additional 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}{
+\label{msaex}
+\exstep{ Close all windows. Open the alignment at {\sf
+https://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) ({\sl with Defaults}), 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 (you may need the scroll down the 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 MAFFT alignment window in step (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{https://www.jalview.org/training/Training-Videos}.}
+}
+
+\section{Customising the Parameters used for Alignment}
+
+JABA web services allow you to vary the parameters used when performing a
+bioinformatics analysis. For JABA alignment services, this means you are
+usually able to modify the following types of parameters:
+\begin{list}{$\bullet$}{}
+\item{Amino acid or nucleotide substitution score matrix}
+\item{Gap opening and widening penalties}
+\item{Types of distance metric used to construct guide trees}
+\item{Number of rounds of re-alignment or alignment optimisation}
+\end{list}
+\begin{figure}[htbc]
+\center{
+\includegraphics[width=3in]{images/jvaliwsparamsbox.pdf}
+\caption{{\bf An alignment service's parameter editing dialog box}.}
+\label{jwsparamsdialog} }
+\end{figure}
+
+\subsection{Getting Help on the Parameters for a Service}
+
+Each parameter available for a method usually has a short description, which
+Jalview will display as a tooltip, or as a text pane that can be opened under
+the parameter's controls. In the parameter shown in Figure
+\ref{clustalwparamdetail}, the description was opened by selecting the button on the left hand side.
+
+\begin{figure}[htbp]
+\begin{center}
+\includegraphics[width=2.5in]{images/clustalwparamdetail.pdf}
+\caption{{\bf ClustalW parameter slider detail}. From the ClustalW {\sl Clustal $\Rightarrow$ Edit settings and run ...} dialog box. }
+\label{clustalwparamdetail}
+\end{center}
+\end{figure}
+
+\subsection{Alignment Presets}
+The different multiple alignment algorithms available from JABA vary greatly in
+the number of adjustable parameters, and it is often difficult to identify what
+are the best values for the sequences that you are trying to align. For these
+reasons, each JABA service may provide one or more presets -- which are
+pre-defined sets of parameters suited for particular types of alignment
+problem. For instance, the Muscle service provides the following presets:
+\begin{list}{$\bullet$}{}
+\item Large alignments (balanced)
+\item Protein alignments (fastest speed)
+\item Nucleotide alignments (fastest speed)
+\end{list}
+
+The presets are displayed in the JABA web services submenu, and can also be
+accessed from the parameter editing dialog box, which is opened by selecting
+the `{\sl Edit settings and run ...}' option from the web services menu. If you have used
+a preset, then it will be mentioned at the beginning of the job status file shown
+in the web service job progress window.
+
+\subsection{User Defined Presets}
+Jalview allows you to create your own presets for a particular service. To do
+this, select the `{\sl Edit settings and run ...}' option for your service,
+which will open a parameter editing dialog box like the one shown in Figure
+\ref{jwsparamsdialog}.
+
+The top row of this dialog allows you to browse the existing presets, and
+when editing a parameter set, allows you to change its nickname. As you
+adjust settings, buttons will appear at the top of the parameters dialog that
+allow you to Revert or Update the currently selected user preset with your changes, Delete the current preset, or Create a new preset, if none exists with the given name. In addition to the parameter set name, you can also provide a short
+description for the parameter set, which will be shown in the tooltip for the
+parameter set's entry in the web services menu.
+
+\subsubsection{Saving Parameter Sets}
+When creating a custom parameter set, you will be asked for a file name to save
+it. The location of the file is recorded in the Jalview user preferences in the
+same way as a custom alignment colourscheme, so when Jalview is launched again,
+it will show your custom preset amongst the options available for running the
+JABA service.
+
+%% TODO - reinstate this exercise about reinstating presets
+%
+% \exercise{Creating and using user defined presets}{\label{createandusepreseex}
+% \exstep{Import the file at
+% \textsf{http://www.jalview.org/tutorial/fdx\_unaligned.fa} into jalview.}
+% \exstep{Use the `{\slDiscover Database Ids}' function to recover the PDB cross
+% references for the sequences.}
+% \exstep{Align the sequences using the default ClustalW parameters.}
+% \exstep{Use the `{\sl Edit and run..}'
+% option to open the ClustalW parameters dialog box, and create a new preset using
+% the following settings:
+% \begin{list}{$\bullet$}{}
+% \item BLOSUM matrix (unchanged)
+% \item Gap Opening and End Gap penalties = 0.05
+% \item Gap Extension and Separation penalties = 0.05
+% \end{list}
+%
+% As you edit the parameters, buttons will appear on the dialog box
+% allowing you revert your changes or save your settings as a new parameter
+% set.
+%
+% Before you save your settings, remeber to give them a meaningful name by editing
+% the text box at the top of the dialog box.
+% }
+% \exstep{Repeat the alignment using your new parameter set by selecting it from
+% the {\sl ClustalW Presets menu}.}
+% \exstep{These sequences have PDB structures associated with them, so it is
+% possible to compare the quality of the alignments.
+%
+% Use the {\sl View all {\bf N}
+% structures} option to calculate the superposition of 1fdn on 1fxd for both
+% alignments (refer to section \ref{superposestructs} for instructions). Which
+% alignment gives the best RMSD ? }
+% \exstep{Apply the same alignment parameter settings to the example alignment
+% (available from \textsf{https://www.jalview.org/examples/uniref50.fa}).
+%
+% Are there differences ? If not, why not ?
+% }
+% }
+
+\section{Protein Alignment Conservation Analysis}
+\label{aacons}
+The {\sl Web Service $\Rightarrow$ Conservation} menu controls the computation
+of over 17 different amino acid conservation measures for the current alignment
+view. The JABAWS AACon Alignment Conservation Calculation Service, which is used
+to calculate these scores, provides a variety of standard measures described by
+Valdar in 2002\footnote{Scoring residue conservation. Valdar (2002) {\sl
+Proteins: Structure, Function, and Genetics} {\bf 43} 227-241.} as well as an efficient implementation of the SMERFs
+score developed by Manning et al. in 2008.\footnote{SMERFS Score Manning et al. {\sl BMC
+Bioinformatics} 2008, {\bf 9} 51 \href{http://dx.doi.org/10.1186/1471-2105-9-51}{doi:10.1186/1471-2105-9-51}}
+
+\subsection{Enabling and Disabling AACon Calculations}
+When the AACon Calculation entry in the {\sl Web Service $\Rightarrow$
+Conservation} menu is ticked, AACon calculations will be performed every time
+the alignment is modified. Selecting the menu item will enable or disable
+automatic recalculation.
+
+\subsection{Configuring which AACon Calculations are Performed}
+The {\sl Web Service $\Rightarrow$ Conservation $\Rightarrow$ Change AACon
+Settings ...} menu entry will open a web services parameter dialog for the
+currently configured AACon server. Standard presets are provided for quick and
+more expensive conservation calculations, and parameters are also provided to
+change the way that SMERFS calculations are performed.
+AACon settings for an alignment are saved in Jalview projects along with the
+latest calculation results.
+
+\subsection{Changing the Server used for AACon Calculations}
+If you are working with alignments too large to analyse with the public JABAWS
+server, then you will most likely have already configured additional JABAWS
+servers. By default, Jalview will chose the first AACon service available from
+the list of JABAWS servers available. You can change the AACon services by
+selecting it from the {\sl Web Service $\Rightarrow$
+Conservation $\Rightarrow$ Change AACon Settings} submenu.
+Alternatively to add new service, go to the desktop window menu and select {\sl Tools $\Rightarrow$
+Preferences $\Rightarrow$ Web Services tab} and add {\sl New Services URL}, then use the {\sl move up} or {\sl move down} buttons
+to reorder the services.
+
+
+\chapter{Analysis of Alignments}
+\label{alignanalysis}
+Jalview provides support for sequence analysis in two ways. A number of
+analytical methods are `built-in', these are accessed from the {\sl Calculate}
+alignment window menu. Computationally intensive analyses are run outside
+Jalview {\sl via} web services - and found under the
+{\sl Web Service} menu. In this section, we describe the built-in analysis
+capabilities common to both the Jalview Desktop and the JalviewJS.
+
+\section{PCA}
+Principal components analysis calculations create a spatial
+representation of the similarities within the current selection or the whole alignment if no selection has been made. After
+the calculation finishes, a 3D viewer displays each sequence as a point in
+3D `similarity space'. Sets of similar sequences tend to lie near each other in
+this space.
+Note: The calculation is computationally expensive, and may fail for very large
+sets of sequences - because the JVM has run out of memory. Memory issues, and
+how to overcome them, were discussed in Section \ref{memorylimits}.
+
+\subsubsection{What is PCA?}
+Principal components analysis is a technique for examining the structure of
+complex data sets. The components are a set of dimensions formed from the
+measured values in the data set, and the principal component is the one with the
+greatest magnitude, or length. The sets of measurements that differ the most
+should lie at either end of this principal axis, and the other axes correspond
+to less extreme patterns of variation in the data set.
+In this case, the components are generated by an eigenvector decomposition of
+the matrix formed from the sum of pairwise substitution scores at each aligned
+position between each pair of sequences. The basic method is described in the
+1995 paper by {\sl G. Casari, C. Sander} and {\sl A. Valencia} \footnote{{\sl
+Nature Structural Biology} (1995) {\bf 2}, 171-8.
+PMID: 7749921} and implemented at the SeqSpace server at the EBI.\footnote{See \url{https://www.jalview.org/help/html/calculations/pca.html}.}
+%
+% Jalview provides two different options for the PCA calculation: SeqSpace and
+% Jalview mode. In SeqSpace mode, PCAs are computed using the identity matrix, and
+% gaps are treated as 'the unknown residue' (this actually differs from the
+% original SeqSpace paper, and will be adjusted in a future version of Jalview).
+% In Jalview mode, PCAs are computed using the chosen score matrix - which for
+% protein sequences, defaults to BLOSUM 62, and for nucleotides, is the
+% DNA identity matrix that also treats Us and Ts as identical, to support analysis
+% of both RNA and DNA alignments.
+
+\subsubsection{The PCA Viewer}
+
+PCA analysis can be launched from the {\sl Calculate $\Rightarrow$ Tree or PCA} menu option.
+{\bf PCA requires a selection containing at
+least 4 sequences}. In the Choose Calculation window, select the {\sl Principal Components Analysis} button and then select {\sl Calculate}
+(Figure \ref{PCA}).
+Each sequence is represented by a small square, coloured by the background
+colour of the sequence ID label. The axes can be rotated by clicking and
+dragging the left mouse button and zoomed using the $\uparrow$ and $\downarrow$
+keys or the scroll wheel of the mouse (if available). A tool tip appears if the
+cursor is placed over a sequence. Sequences can be selected by clicking on them.
+[CTRL]-Click can be used to select multiple sequences.
+
+Labels will be shown for each sequence by toggling the {\sl View $\Rightarrow$
+Show labels} menu option, and the plot background colour changed {\sl via} the
+{\sl View $\Rightarrow$ Background Colour..} dialog box. A graphical
+representation of the PCA plot can be exported as an EPS or PNG image {\sl via}
+the {\sl File $\Rightarrow$ Save as $\Rightarrow$ \ldots } submenu.
+
+\exercise{Principal Component Analysis}
+{\label{pcaex}
+\exstep{Load the alignment at
+\textsf{https://www.jalview.org/tutorial/alignment.fa}.}
+\exstep{Select the menu option {\sl Calculate $\Rightarrow$ Tree or PCA..}. in the alignment
+window and a dialogue box will open. Select the Principal Component Analysis option
+and then click the Calculate button.}
+\exstep{Move
+this window within the desktop so that the alignment and PCA viewer windows are visible.
+Try rotating the plot by clicking and dragging the mouse on the plot in the PCA window.
+Note that clicking on points in the plot will highlight the sequences on the
+alignment.}
+\exstep{Use the [ESC] key to deselect sequence selection.
+Select {\sl Calculate $\Rightarrow$ Tree or PCA..}. in the alignment window. In dialogue box select Neighbour
+Joining and in the drop-down list select BLOSUM62. Click the Calculate button
+and a tree window will open.}
+\exstep{Place the mouse cursor on the tree so that the
+tree partition divides the tree into a number of groups, each with a
+different (arbitrarily selected) colour.
+Note how the colour of the sequence ID label matches both the colour of
+the partitioned tree and the points in the PCA plot.}
+{\bf See the video at:
+\url{https://www.jalview.org/training/Training-Videos}.}
+}
+
+\begin{figure}[hbtp]
+\begin{center}
+\includegraphics[width=2in]{images/PCA1.pdf}
+\includegraphics[width=3in]{images/PCA3.pdf}
+\caption{{\bf PCA Analysis.} }
+\label{PCA}
+\end{center}
+\end{figure}
+
+
+
+\subsubsection{PCA Data Export}
+Although the PCA viewer supports export of the current view, the plots produced
+are rarely suitable for direct publication. The PCA viewer's {\sl File} menu
+includes a number of options for exporting the PCA matrix and transformed points
+as comma separated value (CSV) files. These files can be imported by tools such
+as {\bf R} or {\bf gnuplot} in order to graph the data.
+
+\section{Trees}
+\label{trees}
+
+Jalview can calculate and display trees, providing interactive tree-based
+grouping of sequences though a tree viewer. All trees are calculated {\sl via}
+the {\sl Calculate $\Rightarrow$ Tree or PCA \ldots} menu option.
+Trees can be calculated from distance matrices determined from \% identity or
+aggregate BLOSUM 62 score using either {\sl Average Distance} (UPGMA) or {\sl
+Neighbour Joining} algorithms. The input data for a tree is either the selected
+region or the whole alignment, excluding any hidden regions.
+
+On calculating a tree, a new window opens (Figure \ref{trees1}) which contains
+the tree. Various display settings can be found in the tree window {\sl View}
+menu, including font, scaling and label display options. The {\sl File
+$\Rightarrow$ Save As} submenu contains options for image and Newick file
+export. Newick format is a standard file format for trees which allows them to
+be exported to other programs. Jalview can also read in external trees in
+Newick format {\sl via} the {\sl File $\Rightarrow$ Load Associated Tree} menu
+option. Leaf names on imported trees will be matched to the associated alignment
+- unmatched leaves will still be displayed, and can be highlighted using the
+{\sl View $\Rightarrow$ Mark Unlinked Leaves} menu option.
+
+
+
+\begin{figure}
+\begin{center}
+\includegraphics[width=2.5in]{images/trees1.pdf}
+\includegraphics[width=2.5in]{images/trees2.pdf}
+\includegraphics[width=1.25in]{images/trees4.pdf}
+\caption{{\bf Calculating Trees} Jalview provides a range of options
+for calculating trees.
+Jalview can also load precalculated trees in Newick format (right).}
+\label{trees1}
+\end{center}
+\end{figure}
+
+\begin{figure}
+\begin{center}
+\includegraphics[width=5in]{images/trees3.pdf}
+\caption{{\bf Interactive Trees} The tree level cutoff can be used to designate
+groups in Jalview.}
+\label{trees2}
+\end{center}
+\end{figure}
+
+Clicking on the tree brings up a cursor across the height of the tree. The
+sequences are automatically partitioned and coloured (Figure \ref{trees2}). To
+group them together, select the {\sl Calculate $\Rightarrow$ Sort $\Rightarrow$
+By Tree Order $\Rightarrow$ \ldots} alignment window menu option and choose the
+correct tree. The sequences will then be sorted according to the leaf order
+currently shown in the tree view. The coloured background to the sequence IDs
+can be removed with {\sl Select $\Rightarrow$ Undefine Groups} from the
+alignment window menu. Note that tree partitioning will also remove any groups
+and colourschemes on a view, so create a new view ([CTRL-T]) if you wish to
+preserve these.
+
+
+\exercise{Trees}
+{\label{treeex}
+
+\exstep{Open the alignment at
+\textsf{https://www.jalview.org/tutorial/alignment.fa}.}
+
+\exstep{Select {\sl Calculate $\Rightarrow$ Tree or PCA..}. in the alignment
+window menu and a dialogue box opens. In the tree section select Neighbour
+Joining, in the drop-down list select BLOSUM62 and click the Calculate
+button. A tree window will open.}
+
+\exstep{Click on the
+tree window, a cursor will appear as a vertical line. Note that clicking will
+place this cursor, and divides the tree into a number of groups, each highlighted
+with a different colour. Place the cursor to give about 4 groups.}
+
+\exstep{Place the mouse cursor on a node of the tree to open a tool tip. Double click the node to invert the leaves.
+}
+
+\exstep{In the tree window, select {\sl View $\Rightarrow$ Sort Alignment
+by Tree}. The sequences are reordered to match the order in the tree and groups
+ are formed implicitly. Alternatively in the alignment window, select
+{\sl Calculate $\Rightarrow$ Sort $\Rightarrow$ By Tree Order $\Rightarrow$
+ Neighbour Joining Tree using BLOSUM62 from...}.}
+
+\exstep{Select {\sl Calculate $\Rightarrow$ Tree or PCA..}. in the alignment
+window. In the dialogue box, select Average Distance and in the drop down
+list select BLOSUM62. Click the Calculate button and a new
+tree window will appear. The group colouring makes it easy to see the differences between the two
+trees calculated by the different methods.}
+
+\exstep{With no groups selected in the alignment window, select sequence 2 from
+column 60 to sequence 12 and column 123. Select {\sl Calculate $\Rightarrow$
+Tree or PCA..}. , in the dialogue box select Neighbour Joining and
+BLOSUM62, then click the Calculate button.
+ A tree will appear containing 11 sequences. It has been coloured
+ according to the already selected groups from the first tree and is calculated purely from the residues
+ in the selection.}
+
+Comparing the location of individual sequences between the three trees illustrates the importance of selecting appropriate regions of the
+alignment for the calculation of trees.