Cinema 5 (Colour INteractive Editor for Multiple Alignments) is an interactive visual tool for the interpretation and manipulation of protein and DNA sequences. Cinema 5 allows the user to interactively construct and edit and visualise multiple sequence alignments. Cinema 5 provides a highly intuitive, flexible and user-friendly interface with many features:

• Several different alignments may be opened for editing simultaneously.
• Flexible zoom controls allow an alignment to be visualised at varying levels of detail.
• Several views can be created simultaneously onto a single alignment.
• Intuitive mouse-controlled interface for navigating and editing an alignment.
• Sequences may be grouped to simplify editing tasks.
• Breakpoints may be inserted into alignments to protect aligned sections whist other regions are edited.
• A simple, yet powerful, mechanism for defining, compiling and managing motifs and fingerprints
• A highly flexible sub-sequence search tool that allows simple regular expressions and fuzzy searching using substitution matrices.
• Advanced editing tools, such a hierarchical sequence groups & search-guided alignment.
• Integrated access to the UniProt Universal Protein Resource.

** IMPORTANT NOTE Cinema 5 BETA release **

This is a BETA release, and as such is not guaranteed to be bug-free. If you discover a bug, have any suggestions for future releases of Cinema, or just have a comment then please email us at utopia-project@yahoogroups.co.uk. Thank you!

Cinema 5 can open and import sequences and multiple sequence alignments either stored locally on your system, or from the UniProt universal protein resource (the Swiss-Prot and trEMBL databases)[1]. Sequences and alignments can be also be saved to local files, in a number of command file formats.


Supported file formats

The following table illustrates the formats currently supported:

The My Sequences folder

Cinema 5 uses a standard folder for locally stored sequence alignments, My Sequences. This folder will be created for you if not found on your system, and is located in the My Documents folder on Windows systems, and in the top-level directory of your user account on Linux and Apple OS X systems.

UniProt access

Cinema 5 also provides integrated access to the UniProt protein resource, enabling you to fetch a sequence from either the Swiss-Prot or trEMBL databases and then:

(a) create a new alignment containing the sequence, or
(b) import the sequence into an existing alignment.

By default, access to UniProt is provided via the internet, from a copy of the database hosted on the UTOPIA server. This copy of UniProt is updated frequently and *should* represent the most recently-published version.

No guarantee, however, can be made that the UTOPIA server will always be available for access, and so it is also possible to configure Cinema 5 to access a local copy of UniProt. You can, if you prefer, download your own copy of the Swiss-Prot/trEMBL databases and configure Cinema 5 to work with them instead. See Configuring Cinema 5 for details of setting up Cinema 5 to access a local copy of UniProt on your system.

To open a locally stored sequence or sequence alignment:

1. Click on the icon in the toolbar, or choose Open from the File menu.
2. The open file dialog appears, displaying the contents of the default sequences/alignments folder, My Sequences.
3. If necessary, use the dialog to locate the folder containing the desired sequence alignment file.
4. Either double-click the alignment file, or select it and click on OK to open the file.

There are two options for saving a locally stored sequence or sequence alignment. To save an alignment file under its existing name:

1. Click on the icon in the toolbar, or choose Save from the File menu.

To save an alignment file under a new name:

1. choose Save as... from the File menu.
2. The save as dialog appears. If necessary, use the dialog to locate the folder where the alignment file is to be saved.
3. In the file name box, enter a new name for the alignment file.
4. Click Save to save the alignment file under its new name.

Cinema 5 allows you to search for a particular sequence in UniProt (i.e. the Swiss-Prot and trEMBL databases) and, if found, fetch the sequence and open it as a new alignment. You can search for a sequence either in terms of its Accession number or its ID.

To open a sequence from UniProt, choose Open a sequence from UniProt from the File menu. The Open from UniProt dialog will appear:

1. Decide whether you wish to retrieve a sequence from Swiss-Prot or trEMBL, and select the appropriate radio button.
2. Enter the Accession number or ID of the sequence in the Fetch Sequence box.
3. Press the Get sequence button.

Cinema 5 allows you to import single or multiple sequences into any open alignment. Sequences can be imported from either locally-stored files, or from the UniProt protein database. The imported sequence(s) can then either be appended to the end of the alignment, or inserted into it at a specific point.

To import sequences into an alignment, choose Import sequence(s)... from either the File menu or the Context menu (accessible by right-clicking the mouse over the alignment area). The Import sequence(s) dialog will appear:

Cinema 5 provides a native file format for storing sequence alignments. Files stored in this format are identified by a .c5a suffix.

The advantage of using Cinema 5's native file format is that extra, Cinema-specific information is stored along with the standard sequence alignment data. In particular, motifs/fingerprints that have been defined and breakpoints that have been set will all be stored in a Cinema 5 format file. It is therefore recommended that the Cinema 5 format be used for saving alignments between sessions.

There are a number of situations in which Cinema 5 may be unable to access the UniProt databases. Whether, and when, these situations arise depends on how Cinema 5 is configured to access UniProt, either via the UTOPIA server, or from a local copy of the database (see Configuring Cinema 5 for full details of the different access methods). In each case, Cinema 5 will try to determine the cause of the access problem and provide a sensible response to the user.

If Cinema 5 is configured to access UniProt via the UTOPIA server, the following access problems might arise:

The following diagram illustrates the main Cinema 5 toolbar and identifies the function of each icon. Further details of the operation of each of these tools can be found elsewhere in this help file.

The central tool in Cinema 5 is the alignment view, a powerful and flexible interface for visualising and editing a multiple sequence alignment. With an alignment view, it is possible to:

• Navigate an alignment in a fast, simple and intuitive manner
• Visualise an alignment at different levels-of-detail
• Open multiple views onto a single alignment, each capable of showing different regions of the alignment, at different levels of detail and with different residue colour-schemes
• Edit an alignment directly using the mouse
• Open different alignments, each in their own view, for side-by-side comparison.

1. The sequence alignment itself, which fills the main central region of the view.
2. The sequence labels, to the left of the sequences. The sequence labels give an identifying name for each sequence (usually the accession number or other database ID), and illustrate any sequence groupings that have been assigned.
3. A numerical key, above the alignment, illustrating the start and end indices of the section of the alignment currently on display.
4. A consensus line, below the alignment, which by default shows the modal residue for each 'column' of the alignment.

In general, a multiple sequence alignment will be considerably larger in area than the view onto it; only small sub-sections of such an alignment will thus be visible at a time. The alignment view offers a number of simple methods for navigating a large alignment, so that different sections of it can be displayed.

Scroll bars

Scroll bars are provided at the bottom and right edges of the alignment view window when necessary, and allow the current view to be scrolled left/right or up/down in the standard manner using the mouse.


Keyboard navigation

The alignment may also be navigated using a number of keyboard short-cuts:

• The cursor keys can be used to move the current view left or right a residue position at a time, and up or down a sequence at a time.
• The Home and End keys move the view to the beginning and end of the alignment respectively, without changing the vertical position of the view in any way. Holding down the control key while pressing Home or End moves the view to the top-left or bottom-right edges of the alignment.
• The Page Up and Page Down keys move the view up or down, by the number of sequences that fill the height of the current view window (a 'page' of sequences). Holding down the control key while pressing Page Up or Page Down moves the view directly to the top or bottom of the alignment.

• Mouse-wheel alone scrolls the alignment up/down on a per-sequence basis, or, when all sequences are currently displayed, scrolls the alignment left/right on a per-residue basis.
• Mouse-wheel & Ctrl key scrolls the alignment right/left a screen-width at a time.
• Mouse-wheel & Shift key zooms the view in/out, in the same manner as the free zoom control.

While working with a sequence alignment, it may be desirable to visualise it at different levels of detail, providing, for example:

• A general overview of the entire alignment.
• A close-up view onto a specific section of the alignment for detailed editing.

Cinema 5 allows an alignment to be visualised at a wide range of different levels of detail, and provides a simple, intuitive method for switching between these levels using interactive 'zoom' controls. There are three basic zoom controls on the main tool bar:

• Zoom in will move the view 'closer' to the alignment, increasing the level-of-detail but decreasing the effective size of the area being visualised.
• Zoom out will move the view 'further away' from the alignment, decreasing the level-of-detail and increasing the effective size of the area being visualised.
• Free zoom provides an intuitive technique for selecting the desired level of detail using a mouse-operated slider.

It is possible to have several alignment views open onto the same alignment, allowing:

• Different regions of the same alignment to be visualised simultaneously, at different levels-of-detail if required.
• Different regions of the same alignment to be edited without the need to repeatedly shift the view between each region.

In order to create a new view onto the current alignment, select the icon from the main toolbar, or the New view window option from the View menu.

When there are two or more view windows open for the same alignment, Cinema 5 provides a visual cue that illustrates if, and where, the region of the alignment shown in the current view coincides with the regions shown in the other views. All views, except that which is currently active, are dimmed slightly, with only the regions of the alignment coinciding with that displayed in the active view shown at full intensity. The following image illustrates this behaviour:

This option may be turned off, see the section on Configuring Cinema 5.

If the mouse pointer is positioned over the alignment area or the sequence labels, and the 't' key pressed, a 'tool-tip' will be displayed providing information about that particular region of the alignment:

If the mouse pointer is over the alignment, the tool-tip displays:

• The sequence the pointer is currently over.
• The particular residue of that sequence the pointer is over.
• The actual position of this residue along the sequence.
• The relative position of the residue along the sequence, i.e. ignoring any gaps.

Automatic alignment tips

If desired, Cinema 5 can display alignment tips automatically, whenever the mouse pointer is left idle for a few seconds over the alignment or label areas. To turn automatic alignment tips on, see the section on Configuring Cinema 5.

Most operations that can be performed on a sequence alignment are accessed via the context menu, which appears when the right hand mouse button is clicked in an alignment view window. The context menu adapts to the current task: the options it displays will depend upon which region of the alignment view the mouse was clicked in and what operation the user was performing when the mouse was clicked.

It is important that the user becomes familiar with the context menu, as many of cinema 5's functions are only accessible through it.

Cinema 5 provides a number of tools for the basic editing of a multiple sequence alignment. These tools allow for the insertion into, and removal of, gaps from sequences, and the sliding of sequences left or right, relative to the rest of the alignment. Such operations may be applied to individual sequences or, through the creation of temporary and/or fixed groups, to multiple sequences simultaneously. A simple undo facility is also provided to correct mistakes.

Note that Cinema 5 maintains an overall consistency of sequence length within an alignment, i.e. all sequences are kept the same length as each other at all times. To ensure this consistency, extra gaps may be automatically added to, or removed from, the leading/trailing edges of one or more sequences following each interaction.

1. Move the mouse pointer to the position in the alignment at which the gaps are to be inserted. The insertion point will be to the left of the residue the pointer is over.
2. Press and hold down the left mouse button.
3. Drag the mouse to the right to insert one or more gaps.
4. If, during the insertion procedure, too many gaps are inserted, these gaps may be removed by dragging the mouse back to the left.
5. Release the left mouse button once the desired number of gaps has been inserted.

1. Move the mouse pointer over to the position in the alignment from which the gaps are to be removed. This can either be over one of the gaps, or over the residue directly to the right of a block of one or more gaps.
2. Press and hold down the left mouse button.
3. Drag the mouse to the left to remove the desired number of gaps.
4. If, during the removal procedure, too many gaps are removed, gaps may be re-inserted by dragging the mouse to back the right.
5. Release the left mouse button once the desired number of gaps has been removed.

Sequence sliding is the second basic interaction mode provided by Cinema 5. During interaction, an entire sequence may be shifted to the left or the right, in relation to the rest of the alignment. This enables sequences to be aligned without the insertion/removal of internal gaps. To slide a sequence, first ensure that the 'slide sequences' icon, found on the main Cinema 5 toolbar, is selected; then:

1. Position the mouse pointer over the desired sequence.
2. Press and hold down the left mouse button.
3. Drag the mouse to the left or right to slide the sequence in either direction
4. Release the mouse button once the sequence is in the correct position.

Both the insert/remove gaps and sequence sliding operations may also be applied to groups of multiple sequences simultaneously. A temporary group of sequences is be created by holding down the shift key and/or the control key, and then selecting the desired group of sequences with the left mouse button:

• Holding down the shift key allows a contiguous block of sequences to be grouped, by selecting the first and last sequence in the block.
• Holding down the control key allows a non-contiguous set of sequences to be grouped, by selecting each one in turn.
• The shift and control keys may be combined to allow the selection of more complicated groups.

Once a temporary group of sequences has been selected, an insert/remove gaps or sequence sliding operation may be performed on the entire group simply by interacting with one of the grouped sequences. If a sequence that is not a member of the temporary group is selected at any point then it will be disbanded.

Sequences can be grouped together on a more long-term basis to simplify group-based interaction. If it is likely that a number of operations are to be performed on the same group of sequences, it would be sensible to create a persistent sequence group for them. To create a sequence group:

1. Use the left mouse button and the shift and/or control keys to select the sequences to be grouped.
2. Right-click one of the selected sequences; the sequence context-menu will pop up.
3. Choose Group selected sequences from the menu.

The selected sequences will now be combined as a sequence group. The effect of this grouping can be seen as a little coloured block in the label area of each sequence contained in the group. Multiple sequence groups may be created in this way, and each group is assigned a particular colour, to enable easy identification its members. If one sequence belonging to a group is selected with the mouse, all other members are automatically selected as well. Any editing operation applied to one member of the group will be automatically applied to all others.

Although grouping sequences is a useful facility for applying the same operation to several sequences at once, there may reach a point where it is necessary to remove one or more of the sequences that have been added to a sequence group, or even to disband a sequence group entirely.

Removing a sequence from a sequence group

A sequence may be removed from a sequence group by right-clicking over the sequence and choosing Ungroup sequence from the context menu. The coloured block will be removed from the sequence's label area, indicating it is no longer a member of the sequence group.

Disbanding a sequence group

It may be necessary to disband a sequence group entirely, so that all of its members may be operated upon individually, rather than the group as a whole. A sequence group may be disbanded by right-clicking over any of the member sequences and choosing Disband sequence group from the context menu. The coloured block will be removed from all of the member sequences' label areas, indicating they are no longer a member of a sequence group.

Temporarily overriding a sequence group

It is possible to temporally override a sequence group so that one of its member sequences may be operated upon individually. By selecting a sequence with the middle mouse button, rather than the left, any interaction will only be applied to that sequence, rather than all of the members of its parent sequence group. Once the interaction is complete, the original sequence grouping is restored.

Existing sequences groups can also be grouped together with other groups and/or individual sequences, creating more complicated hierarchical sequence groups. In such cases, all sequences will be re-coloured to illustrate their current grouping at the top level of the hierarchy. The Sequence Group Manager topic has full details of how hierarchical groups may be constructed and managed.

Cinema 5 provides a very basic one-action undo feature. It is possible to undo the most recent insert/remove gaps and sequence-sliding interaction by selecting the icon on the main toolbar.

Cinema 5 provides a number of advanced tools for constructing and editing multiple sequence alignments:

• Breakpoints
• Dealing with redundant gaps
• Inserting multiple gaps
• Repositioning sequences
• Deleting whole sequences from an alignment
• Copying and pasting sequences between alignments
• Deleting columns from an alignment
• Textual sequence editing
• Saving the entire alignment as an image
• Printing the entire alignment

A Breakpoint is a vertical divider that may be inserted into an alignment, along a 'column' of residues. A breakpoint inserted into an alignment has the effect of splitting it into two sub-alignments at that point. For subsequent editing operations, Cinema 5 treats the sub-alignments either side of the breakpoint as if they were separate entities. If, e.g., a sequence sliding operation is performed, only the section of the sequence contained between the nearest breakpoints to the left and right of the mouse pointer will be effected.

Breakpoints can thus be used to preserve sections of an alignment while other sections are being edited. For example, if one region of an alignment has been successfully aligned, it is possible to add a breakpoint at one, or both sides of it. Subsequent editing to other areas of the alignment will not effect the region contained within the breakpoints, which has effectively been 'fenced off'.

To add a breakpoint:

1. Move the mouse pointer to the position in the alignment at which the breakpoint is to be added. Note that the breakpoint will be set to the left of the residue the pointer is over.
2. Right-click to pop-up the context menu and select Insert breakpoint.

To remove an existing breakpoint:

1. Position the mouse over the breakpoint to be removed.
2. Right-click to pop-up the context menu and select Remove breakpoint.

A redundant gap is a contiguous column of gaps in an alignment. Cinema 5 automatically removes redundant gaps when they occur at either the left or right edges of an alignment, or at the breakpoints defining a sub-alignment. However, any 'internal' redundant gaps that may occur as a result of editing are left alone. Redundant gaps can be easily identified: a non-redundant gap is illustrated by the '-' symbol on a grey background, whereas a gap belonging to a redundant gap column is just a grey box, with no symbol at all.

If necessary, there are two methods for removing redundant gaps: individually, or collectively.

To remove a specific redundant gap:

1. Position the mouse over the redundant gap.
2. Right-click to pop-up the context menu and select Remove redundant gap.

1. Position the mouse anywhere over the alignment.
2. Right-click to pop-up the context menu and select Purge redundant gaps.

In certain situations, it may be desirable to insert a large number of gaps in one operation, over a region of the alignment far larger than that displayed in the current view. Potentially, this could involve a tedious procedure of repeatedly using the standard insert/remove gaps tool and shifting the view left or right, in order to accommodate the number of gaps to be inserted.

Cinema 5 provides a specific interaction mode for the insertion of large contiguous blocks of gaps in a single operation. First, ensure that the icon, found on the main Cinema 5 toolbar, is selected; then:

1. Move the mouse pointer to the position in the alignment at which the operation is to start and click the left mouse button once.
2. Move the mouse to the right; a line will be drawn connecting the initial insertion point to the mouse pointer position, illustrating how many gaps are to be inserted.
3. The standard mouse/keyboard navigation tools can now be used to locate the region of the alignment to which gaps are to be inserted or removed.
4. Once the desired region of the alignment has been located, move the pointer to the position up to which gaps are to be inserted and click the left mouse button again. The gaps will be inserted requested.

It may be desirable to change the order of the sequences that make up an alignment. Cinema 5 provides a simple mechanism for repositioning a sequence within an alignment:

1. Position the mouse over the label of the sequence that is to be moved.
2. Press and hold down the middle mouse button.
3. Drag the mouse up or down to reposition the sequence, releasing the mouse button when finished.

During the construction of a multiple sequence alignment, it may be necessary to remove existing sequences from the alignment.

To remove one or more sequences from the alignment:

1. Select the sequence(s) to be removed with the left mouse button, using the shift and/or control keys to select multiple sequences.
2. Right-click one of the selected sequences; the sequence context-menu will pop up.
3. Choose Remove selected sequences from the menu.

Note that any breakpoints are ignored when sequences are removed in the manner described above.

Sequences can be easily copied and pasted between the alignments that are open in Cinema 5. A copied sequence can be pasted either at the end of an alignment, or into it at a specific point.

• To copy a sequence, right click the mouse over either the sequence or its label and select Copy sequence "XXXX" from the context menu. The sequence will now be copied into Cinema 5's clipboard, ready for pasting.
  
  
• To paste a copied sequence at the end of an alignment, right click the mouse button anywhere over the sequence labels, or the sequences themselves, and select Paste sequence "XXXX" at end of alignment from the context menu.
  
  
• To paste a copied sequence into an alignment at a specific point, right click the mouse button over the position in the alignment/labels you wish to paste the sequence and select Paste sequence "XXXX" here from the context menu.

It may be desirable to remove one or more entire "columns" of residues from an alignment, i.e. to remove a residue from the same position in every sequence of an alignment. For example, this might be useful for cutting out areas of an alignment you are not interested in during a particular task.

To remove one or more columns from the alignment:

1. Move the mouse pointer up to the numerical key at the top of the alignment, and position the pointer above the column (or the first column) you wish to delete.
2. Select the column(s) to be deleted by clicking on the numerical key with the left mouse button, using the shift and/or control keys to select multiple columns. You can also click and drag the mouse to select a contiguous block of columns. The portion of the numerical key above the selected columns will be highlighted to reflect the selection.
3. Right-click the mouse in one of these selected regions of the numerical key; a menu will pop up.
4. Choose delete selected columns from the menu.

Note that any breakpoints that were set at columns that have been deleted will also be removed.

Cinema 5 provides a simple text editor for direct manual editing of sequences. To edit a sequence in this manner, position the mouse pointer over the sequence, right-click to bring up the context menu and select Edit sequence text for the menu. The sequence text editor dialog will be displayed. From this dialog, the Accession name, sequence description and the string of residue symbols that make up the sequence may be altered. Once the required changes have been made, there are three options available for returning the new sequence to the alignment:

1. Update sequence, to replace the existing sequence with the modified version.
2. Insert as new sequence, the new sequence will be inserted below the existing sequence.
3. Append as new sequence, the new sequence will be appended to the bottom of the alignment.

Note that if the sequence is inserted or appended as new, but the original Accession name has not been changed, it will automatically be modified to distinguish it from the original sequence.

An alignment may be saved in its entirety as a bitmap image file, for use in document processing software, graphics software or for printing. A number of standard image file formats are supported: bmp, xpm, xbm, png. You can specify the size of the image to be saved in terms of pixels or millimetres.

To save an alignment as an image, right-click over the alignment to pop-up the context menu and choose Save alignment as an image, or choose Save as an image from the File menu. The Alignment output options dialog will appear:

From this dialog, you can determine the appearance and output size of the alignment. The first group of options controls whether the labels or the position key will be included in the image, and whether any motifs or breakpoints that are set will be drawn. To omit any of these features from the final image, un-check the appropriate box.

The second group of options controls the size of the image to be saved. You can specify an appropriate image size in either pixels or mm by:

1. Choosing the desired units from the drop-down box.
2. Entering a value in either the width or height boxes.

An alignment may be printed out to any standard printer attached to your system. Alignments may be printed in colour, and on any paper format your printer supports. You can specify the output size of the alignment in terms of pixels or millimetres. If the alignment is too large to fit the chosen paper size, Cinema 5 will print it in sections spread over an appropriate number of pages. These pages may subsequently be joined to recreate the entire alignment.

To print an alignment, right-click over the alignment to pop-up the context menu and choose Print entire alignment, or choose Print from the File menu. The Alignment output options dialog will appear:

From this dialog, you can determine the appearance and output size of the alignment. The first group of options controls whether the labels or the position key will be included, and whether any motifs or breakpoints that are set will be printed. To omit any of these features from the alignment, un-check the appropriate box.

The second group of options controls the size at which the alignment will be printed. You can specify an appropriate print size in either pixels or mm by:

1. Choosing the desired units from the drop-down box.
2. Entering a value in either the width or height boxes.

1. Wrap the alignment over the page: the alignment is split into page-wide sections, which are printed vertically down the page. This style is the most appropriate for long, thin alignments, and makes the best use of the available page space.
2. Tile the alignment: the alignment is printed "as is", being split up into a set of page-sized tiles, each printed on a separate page. This style is most appropriate for alignments with very large numbers of sequences, and for producing posters - the tiles may subsequently be arranged, jigsaw-fashion, to recreate the alignment.

Cinema 5 provides a highly flexible tool for searching for occurrences of a particular sub-sequence, either over the entire alignment, or a specific set of sequences. A search is initiated in one of two ways: (1) selecting a particular sub-sequence in the alignment and then searching for all other occurrences, or (2) manually specifying a sub-sequence to search for. Simple regular expressions may be used to construct a search string. Searches can be for exact matches to the sub-sequence, or a "fuzzy" search can be made, using a substitution matrix to score the potential matches. An intuitive search-result navigation system provides a simple and fast method for finding the required occurrence of the sub-sequence.

Cinema 5 also provides some extra facilities for working with search results. Following a sub-sequence search, a simple form of automatic alignment is possible by choosing pairs of results and having Cinema 5 align them.

To begin searching for a sub-sequence, launch the Find sub-sequence dialog by clicking on the icon on the main Cinema 5 toolbar, by choosing Find sub-sequence from the Edit menu, or right-click the mouse over the alignment and select Find sub-sequence from the context menu. To search for a sub-sequence:

1. Enter the sub-sequence text in the search field and press the Enter key, or click on the Find button
2. All search results found, i.e. regions of the alignment that exactly match the search string, will be circled in black. The view will be automatically shifted to display the first search result found, which is the 'active' search result and circled in red.

Note that if no occurrences of the sub-sequence are found, a message box will de displayed to confirm the failure of the search.

To search for all other occurrences of a particular subsequence in the alignment, first you must select the region of the alignment containing the sub-sequence.

Selecting a sub-sequence in an alignment

1. Position the mouse pointer over the first residue in the sub-sequence and then press and hold down the left mouse button to begin selecting a region.
2. Drag the mouse pointer to the right; a dashed outline will be drawn from the initially selected residue to the residue under the mouse pointer, illustrating the region of the alignment that is selected.
3. Once the required sub-sequence is fully enclosed by the dashed outline, release the left mouse button to select this region of the alignment.

Searching for all occurrences of the selected sub-sequence

All search results found, i.e. regions of the alignment that exactly match the search string, will be circled in black. The original sub-sequence is now the 'active' search result and is circled in red.

Note that if no occurrences of the sub-sequence are found, a message will de displayed in the status bar to confirm the failure of the search.

By default sub-sequence searching is performed over the entire alignment. However, it is possible to restrict the search to one or more specific sequences:

1. In the Alignment view, use the left mouse button to selected the sequence(s) to search over, using the shift and/or control keys to select multiple sequences.
2. In the Find sub-sequence dialog, un-check the Search over entire alignment box:
3. Enter a search string and press Find perform the search.

Cinema 5 allows the use of regular expressions to search for sub-sequences. A regular expression is a formula for matching strings that follow some pattern. In order to use regular expressions, you must check the Allow regular expressions box:

By default sub-sequence searching finds only exact matches to the search string. It is, however, possible to perform a 'fuzzy' search for a sub-sequence, using a substitution matrix to score potential matches and a user-controlled threshold to determine which of these matches are accepted. A number of common substitution matrices are supplied by default: BLOSUM62, BLOSUM80, PAM120 & PAM250.

To perform a fuzzy search:

1. Check the Use fuzzy search box in the Find sub-sequence dialog.
2. Use the combo-box to choose an appropriate substitution matrix with which to score the search results.
3. Enter a search string and press Find to perform the fuzzy search.
4. The search results may be refined by using the Score threshold slider to alter the threshold used to accept/reject potential matches. After every change to the threshold, press Find to re-run the search.

Note that Fuzzy searching will not be available if the Allow regular expressions box is checked. This box must be unchecked before the fuzzy searching controls can be used.

The results of a sub-sequence search may be navigated using the four arrow buttons in the bottom-half of the Find sub-sequence dialog:

Following a sub-sequence search, it is possible to use the search results to guide the sequence alignment process. Cinema 5 can be instructed to align pairs of sequences, according to the relative positions of specific search results along those sequences.

The basic approach is to anchor one search result so that its sequence remains in a fixed position, and then align other sequences to it, according to the positions of the search results on those sequences.

The first step in search result-guided sequence alignment is to nominate one of the sequence results to be anchored:

1. Navigate to the search result that is to be anchored, either by using the arrow buttons, or by clicking directly onto the result.
2. Click on the Anchor result button, the outline of the current result changes to indicate it is now anchored.
3. Now use the result navigation buttons to locate a search result on another sequence that you wish to align with the anchored result. Note that only the search results between nearest breakpoint either side of the anchored search result are considered during this process.
4. To align the current search result with the anchored result, click on the Align result to anchor button. The sequence containing the current result will be slid in the appropriate direction to that so that the result lines up with the anchored result.

During this procedure, it is possible to re-nominate the anchored search result at any point, by clicking on the Anchor result button. The previous anchored result will become a standard search result again.

Cinema 5 provides a simple, yet powerful, mechanism for defining, compiling and managing motifs within a multiple sequence alignment.

In Cinema 5, the motifs defined in an alignment are grouped together into fingerprints . By default, each alignment has a main family fingerprint automatically created for it. It is also possible, however, to define further sub-family fingerprints and add motifs to these.

Once defined, a number of operations may be performed on motifs and fingerprints, and Cinema 5 also provides a tool for managing them, called the Motif Manager.

Motifs are defined by first selecting a rectangular region of the alignment and then instructing Cinema 5 to convert this region into a motif.

Selecting regions of an alignment

1. Position the mouse pointer over the residue that is to be the top-left corner of the selected region, then press and hold down the left mouse button to begin selecting a region.
2. Drag the mouse pointer down and/or to the right; a dashed outline will be drawn from the initially selected residue to the residue under the mouse pointer, illustrating the region of the alignment that is selected.
3. Once the required region of the alignment is fully enclosed by the dashed outline, release the left mouse button to select this region of the alignment.

Selecting 'alignment-high' regions

Converting the selected region into a motif

Adding a motif to the main family fingerprint

1. Position the mouse pointer within the selected region and right-click to pop-up the context menu. Choose the Convert to motif and add to main family fingerprint menu option.
2. The outline of the selected region will change to a thick solid line, indicating that it has now been converted into a motif.
3. An entry for this motif will automatically be added to the Motif Manager, in the main family fingerprint folder.

Adding a motif to a sub-family fingerprint

1. Position the mouse pointer within the selected region and right-click to pop-up the context menu. Choose the Convert to motif and add to sub-family "XXXX" fingerprint menu option, where "XXXX" is the name of the sub-family fingerprint. If this fingerprint has not been previously identified or renamed, then its name will be automatically generated from the first and last sequences that the sub-family covers.
2. The outline of the selected region will change to a thick solid line, indicating that it is now a motif.
3. An entry will be added to the Motif Manager for the new motif, in a folder for the sub-family fingerprint. If the fingerprint has not been previously identified, then a folder will be automatically created for it.

Cinema provides a number of tools for working with motifs, either individually or as an entire fingerprint. In particular:

A motif may: Be resized. Have breakpoints added to it. Be saved as a .mot file. Be saved as a sequence alignment. Be saved as an image. Be printed. Be removed.

A fingerprint may: Be renamed. Have all motifs within it saved. Have all motifs removed from it. Be deleted from the alignment.

Once a motif has been defined, it may, if required, be resized. Only the span of residues the motif covers may be changed, however. To alter the span of sequences a motif covers (and thus the fingerprint it belongs to), the motif must be removed and then redefined.
To resize a motif, first make sure that the icon is selected from the tool bar, then:

1. Position the mouse pointer inside the motif to be edited and click the middle mouse button. A number of small square handles will appear in the corners and along the left and right edges of the motif. These handles can be used to resize the motif.
2. Position the mouse pointer over the most appropriate of these handles; press and hold the middle button.
3. Drag the mouse left or right to resize the motif.
4. Release the middle mouse button when finished.

In order to preserve the region of an alignment defined by a motif, it is possible to automatically add a breakpoint to its left and right edges. To add breakpoints to a motif:

1. Position the mouse within the motif area and right-click to pop-up the context menu; select Add breakpoints to motif.
2. A break point will be added to the alignment, coinciding with both the left and right edges of the motif. Any subsequent changes made to the alignment outside of the motif will not affect the region inside it.

Removing breakpoints from a motif

If a pair of breakpoints have been added to a motif in this way, a corresponding Remove breakpoints from motif option will appear in the context menu, which may be used to automatically remove these breakpoints.

The region of an alignment defined by a motif may be saved in a number of different formats, for a variety of uses:

• As a .mot motif file, for use in fingerprint scanning and similar applications.
• In a standard sequence alignment format.
• As a bitmap image.

    % [comment]
    [number of sequences in motif]
    [name of motif]
    GTEGPETUCVPTEGPETUCVP
    GTEGPETUCVPTEGPETUCVP
    GTEGPETUCVPTEGPETUCVP
    GTEG...
    *

1. Position the mouse within the motif area and right-click to pop-up the context menu.
2. Open the Motif "motif-name" sub-menu and then select Save motif "motif-name".
3. The file save dialog will appear.
4. The name of the motif will automatically appear in the dialog: either accept this name or enter a suitable replacement, and then click on the Save button to save the motif.

The region of an alignment defined by a motif may be saved as a separate multiple sequence alignment file. This file can subsequently be opened as a stand-alone sequence alignment in Cinema 5. To save a motif as an alignment:

1. Position the mouse within the motif area and right-click to pop-up the context menu.
2. Open the Motif "motif-name" sub-menu and then select Save motif "motif-name" as sequence alignment.
3. The file save dialog will appear; enter a suitable name to save the sequence alignment file as and click on the Save button.

The region of an alignment defined by a motif may be saved as a bitmap image file, for use in document processing software, graphics software or for printing out. A number of standard image file formats are supported: bmp, xpm, xbm, png. You can specify the size of the image to be saved in terms of pixels or millimetres.

To save a motif as an image, first position the mouse within the appropriate alignment area and right-click to pop-up the context menu. Open the Motif "motif-name" sub-menu and then select Save motif "motif-name" as an image. The output options dialog will appear:

From this dialog, you can specify the output size of the motif in either pixels or mm by:

1. Choosing the desired units from the drop-down box.
2. Entering a value in either the width or height boxes.

The region of an alignment defined by a motif may be printed out to any standard printer attached to your system. Motifs may be printed in colour, and on any paper format your printer supports. You can specify the print size in terms of pixels or millimetres.

To print a motif, first position the mouse within the appropriate alignment area and right-click to pop-up the context menu. Open the Motif "motif-name" sub-menu and then select Print motif "motif-name". The output options dialog will appear:

From this dialog, you can specify the print size of the motif in either pixels or mm by:

1. Choosing the desired units from the drop-down box.
2. Entering a value in either the width or height boxes.

If motif has been defined incorrectly, or is no longer needed it may be removed from the alignment (and its fingerprint):

1. Position the mouse within the motif area and right-click to pop-up the context menu.
2. Open the Motif "motif-name" sub-menu and then select Remove motif "motif-name".
3. The motif will be removed from the alignment, and its corresponding entry will be removed from the Motif Manager.

When a fingerprint is first created it is automatically given a name by Cinema 5, and the name of a fingerprint is subsequently used to generate names for all the motifs it contains. In the case of the main family fingerprint, its name is taken directly from the name of the alignment it belongs to. Sub-family fingerprints, on the other hand, are given names taken from the first and last sequences in the subset of sequences they cover.

If the generated name is not suitable, it is possible to choose a new name for a fingerprint. After changing the name of a fingerprint, all of the motifs it contains will subsequently be renamed to reflect this change. To rename a fingerprint:

1. Position the mouse within one of the motifs contained in the fingerprint and right-click to pop-up the context menu.
2. Open the Fingerprint "fingerprint-name" sub-menu and then select Rename fingerprint "fingerprint-name".
3. The Propose fingerprint name dialog will appear; enter a suitable name for the fingerprint in the edit box.
4. Once a new name has been chosen, click the OK button to accept it. Clicking the Cancel button will restore the original name.

After compiling a fingerprint, it may be desirable to save each of the motifs within it. Rather than saving each motif individually, an entire fingerprint of motifs can be saved at the same time. To save all of the motifs in a fingerprint:

1. Position the mouse within one of the motifs contained in the fingerprint and right-click to pop-up the context menu.
2. Open the Fingerprint "fingerprint-name" sub-menu and then select Save all motifs in fingerprint "fingerprint-name".
3. You will now be prompted to save each motif in turn. For each motif, the file save dialog will appear; either accept the suggested name, or enter a new one and click on the Save button.

To remove all motifs defined in a fingerprint:

1. Position the mouse within one of the motifs contained in the fingerprint and right-click to pop-up the context menu.
2. Open the Fingerprint "fingerprint-name" sub-menu and then select Remove all motifs from fingerprint "fingerprint-name".
3. All motifs will be removed from the selected fingerprint. Their corresponding entries will be removed from the Motif Manager.

To delete a fingerprint, and delete all motifs defined in it:

1. Position the mouse within one of the motifs contained in the fingerprint and right-click to pop-up the context menu.
2. Open the Fingerprint "fingerprint-name" sub-menu and then select Delete fingerprint "fingerprint-name".
3. The fingerprint and all motifs defined within it will be deleted from the alignment. All corresponding entries will be removed from the Motif Manager.

In general, we do not recommend that you import sequences into, or delete sequences from, an alignment after motifs have been defined, as it is highly likely that the motifs will be affected in some way by the change. If, however, it becomes necessary to add/remove sequences to an alignment in which motifs have been defined, Cinema 5 will either attempt to adjust the motifs to accommodate the change, or display a warning that some motifs may need to be re-defined.

Importing sequences

• Main-family fingerprint motifs are expanded to accommodate the new sequence(s).
• Sub-family fingerprint motifs that straddle the newly inserted sequence(s) are expanded to accommodate them.
• Sub-family fingerprint motifs below the newly inserted sequence(s) are shifted down to accommodate them.

Deleting sequences

The Sequence Group Manager is a tool that enables the user to create, organise and visualise hierarchical sequences groups.

Sequence groups provide a convenient mechanism for simplifying the task of editing a multiple sequence alignment. By combining two or more sequences together in a group, editing operations may be applied to all the sequences simultaneously. Cinema 5 allows complex hierarchical sequence groups to be created, through the grouping together of existing sequences groups and/or individual sequences.

Simple hierarchical sequence groups may be created directly within an alignment view, but the underlying hierarchical structure cannot be seen, as all sequence labels are coloured to reflect the top level of any group hierarchy they belong to. In order to have full control over hierarchical sequence groups, and to visualise them, the Sequence Group Manager must be used.

The Sequence Group Manager lists the sequences belonging to each alignment that is currently open, and groups are created within these lists, which correspond to sequence groups in the alignments. A simple and intuitive drag-and-drop mechanism can be used to organise the resulting group structures. Sequence groups can be customized with user-specified names and colours.

To create a group in the sequence group manager:

1. Select the sequences to be grouped, using the left mouse button, and the shift and/or control keys.
2. Right-click one of the selected sequences to pop-up the context menu; choose Group selected item(s).
3. A new sequence group will be created, and the selected sequences will be arranged in the group. The group will be automatically named 'new group x', and a new colour generated for it.

Hierarchical sequence groups are created in the same manner, by selecting a combination of sequence groups and/or individual sequences then choosing Group selected item(s) from the context menu. Note that when selecting a sequence group, click on the group name, rather than the component sequences.

When a sequence group is created directly from within the alignment view, a corresponding group is automatically setup in the Sequence Group Manager.

A simple drag-and-drop mechanism may be used to edit sequence groups from within the Sequence Group Manager:

• A sequence can be dragged and dropped over a group to include it in that group.
• A sequence can be dragged out of a group and dropped into the set of ungrouped sequences to remove it from the group.
• Groups can be dragged and dropped in or out of other groups to alter the hierarchical group structure.

When a sequence group is created, it is automatically given a name and a colour chosen for it. Using the Sequence Group Manager, it is possible to both rename and to choose a new colour for a sequence group. To rename a sequence group:

1. Right click the sequence group to popup the context menu; Choose Rename from the menu.
2. Alter the group name as required.

To change the colour of a sequence group:

1. Right click the sequence group to popup the context menu; Choose Change colour from the menu.
2. The Select Colour dialog appears. Use this dialog to select or generate the required colour.
3. Click on OK to accept the new colour. The sequence group will be re-coloured in both the Sequence Group Manager, and all appropriate alignment views.

To remove an existing sequence group:

1. Right click the sequence group to popup the context menu; Choose Delete group from the menu.
2. The group will be disbanded. All members of the group, sequences or sub-groups, will become children of the immediate parent of the deleted group. If the deleted group had no parent, the members will be returned to the set of ungrouped sequences.

The Motif Manager is a tool for managing and operating on motifs and fingerprints.

Each alignment that is open has, by default, an entry in the Motif Manager with a folder representing the main family fingerprint. All motifs subsequently defined in an alignment and added to its main family fingerprint will a have corresponding entry in this folder. Any sub-family fingerprints that are defined will have their own corresponding folders in the Motif Manager, and motifs belonging to such fingerprints are added to the appropriate folder.

Using the Motif Manager, it is possible to quickly locate specific motifs in an alignment, save, print or delete individual motifs and rename, save or delete entire fingerprints of motifs.

One of the most useful features of the motif manager is the ability to locate a specific motif defined in an alignment. If there are many motifs defined in different regions of a large alignment, Cinema 5 can be easily instructed to centre the view on a particular motif:

1. Position the mouse over the motif entry in the Motif Manager that corresponds to the desired motif. Right-click to call up the context menu
2. Select Show motif in view from the menu.
3. The currently active view onto this alignment will be updated so that the selected motif is visible.

Individual motifs may be saved as *.mot files, as sequence alignment files or as bitmap image files.

1. Position the mouse over the motif entry in the Motif Manager that corresponds to the desired motif. Right-click to call up the context menu
2. Select the appropriate Save motif... option from the menu.
3. A file save dialog will appear.
4. Cinema 5 will automatically suggested a filename for the motif, if this is unsuitable then enter an alternative name
5. If save the motif as a sequence alignment, choose an appropriate output format for the file
6. Finally, click on the Save button to save the motif.

From this dialog, you can specify the output size of the motif in either pixels or mm by:

1. Choosing the desired units from the drop-down box.
2. Entering a value in either the width or height boxes.

Individual motifs may be printed out to any standard printer attached to your system. Motifs may be printed in colour, and on any paper format your printer supports. You can specify the print size in terms of pixels or millimetres.

To print a motif, first position the mouse over the motif entry in the Motif Manager that corresponds to the desired motif. Right-click to call up the context menu and selected the Print motif option. The output options dialog will appear:

From this dialog, you can specify the print size of the motif in either pixels or mm by:

1. Choosing the desired units from the drop-down box.
2. Entering a value in either the width or height boxes.

If motif has been defined incorrectly, or is no longer needed it may be deleted from the alignment (and its fingerprint):

1. Position the mouse over the motif entry in the Motif Manager that corresponds to the desired motif. Right-click to call up the context menu
2. Select Delete motif from the menu.
3. The Selected motif will be deleted from the alignment. Its Motif Manager entry will also be removed.

When a fingerprint is first created it is automatically given a name by Cinema 5, and the name of a fingerprint is subsequently used to generate names for all the motifs it contains. In the case of the main family fingerprint, its name is taken directly from the name of the alignment it belongs to. Sub-family fingerprints, on the other hand, are given names taken from the first and last sequences in the subset of sequences they cover.

If the generated name is not suitable, it is possible to choose a new name for a fingerprint. After changing the name of a fingerprint, all of the motifs it contains will subsequently be renamed to reflect this change. To rename a fingerprint:

1. Position the mouse over the fingerprint folder in the Motif Manager that corresponds to the desired fingerprint. Right-click to call up the context menu
2. Select the Rename fingerprint option from the menu.
3. Edit the name as appropriate and press the return key when done.

1. Position the mouse over the fingerprint folder in the Motif Manager that corresponds to the desired fingerprint. Right-click to call up the context menu
2. Select the Save all motifs in fingerprint option from the menu.
3. You will now be prompted to save each motif in turn. For each motif, the file save dialog will appear; either accept the suggested name, or enter a new one and click on the Save button.

Using the Motif Manager, it is possible to remove all of the motifs that may have been defined in a particular fingerprint in one go:

1. Position the mouse over the fingerprint folder in the Motif Manager that corresponds to the desired fingerprint. Right-click to call up the context menu
2. Select the Remove all motifs from fingerprint option from the menu.
3. All motifs will be removed from the selected fingerprint, along with all corresponding motif entries in the Motif Manager. The fingerprint's folder will not be removed from the Motif Manager.

To delete a fingerprint, and delete all motifs defined in it:

1. Position the mouse over the fingerprint folder in the Motif Manager that corresponds to the desired fingerprint. Right-click to call up the context menu
2. Select the Delete fingerprint option from the menu.
3. The fingerprint, along with any motifs defined within it, will be delete from the alignment. All corresponding entries will be removed from the Motif Manager.

A colour scheme is the set of colours used for the visualisation of either a protein or a DNA sequence alignment.

In Cinema 5, each alignment view has a unique colour scheme associated with it, by default either the standard protein or DNA sequence colour schemes that are provided. Using the Colour Scheme Manager, however, it is possible to customize the colour scheme for a particular alignment view, load an existing alternative colour scheme and save any customizations for future use. Thus, with Cinema 5 it is possible to visualise a sequence alignment using a number of different colour schemes simultaneously.

To start the Colour Scheme Manager, make sure the correct alignment view window is active and choose Edit colour scheme from the Edit menu. The Colour Scheme Manager dialog will appear:

This dialog shows a list of the residue symbols used in the current alignment, and the colour associated with each symbol. It is possible to change the colour associated with a residue:

1. Right-click the block of colour next to the residue symbol and choosing Change colour from the pop-up menu.
2. The Select Colour dialog appears. Use this dialog to select or generate the required colour.
3. Click on OK to accept the new colour. The current alignment view will be updated to reflect this change in colour scheme.

Customized colour schemes may be saved for subsequent use by clicking on the Save button and choosing a suitable name. Similarly, an existing colour scheme may be opened for the current alignment view by clicking on the Load button and choosing the appropriate colour scheme file.

[1]	Apweiler R, Bairoch A, Wu CH, Barker WC, Boeckmann B, Ferro S, Gasteiger E, Huang H, Lopez R, Magrane M, Martin MJ, Natale DA, O'Donovan C, Redaschi N, Yeh LS (2004) UniProt: the Universal Protein Knowledgebase Nucleic Acids Res. 32: D115-D119.