Using CINEMA

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Introduction

CINEMA is a Colour INteractive Editor for Multiple Alignments. The program allows visualisation and manipulation of both protein and DNA sequences. In Version 2.0, the functionality has been extended to include split-screen editing, and applet customisation through the use of "pluglets". This file describes the range of functions of the current CINEMA applet and how they can be employed to align your sequences. Inevitably, there will be teething problems - please consult the known problems before you start, and let us know what other difficulties you encounter.


Quick overview

CINEMA allows you to build alignments interactively, either using a free-format Cut and Paste facility to import your own protein or DNA sequences, or by adding sequences directly from the OWL composite database. Alternatively, alignments may be loaded from the PRINTS fingerprint database, and again may be customised by the addition of user-defined or OWL sequences. Alignments are edited by clicking on the sequences and dragging them to create gaps; whole sequences may be shifted to the left or right by clicking on the right mouse button and dragging.


Mouse buttons

Three mouse buttons are used by this applet - for those users who have only two, or fewer buttons, it is possible to simulate additional buttons through the use of modifier keys. Use the Meta key to simulate a right mouse button action, and the Alt key to simulate that of a middle mouse button.

The applets have not been tested on all platforms and browsers, so if you are experiencing difficulties using them on your platform please contact us.


Editing the alignment

The alignment can be edited by positioning the cursor on the coloured sequence of interest, clicking on the left or right mouse button and dragging the mouse: the left mouse button inserts or removes gaps; the right mouse button shifts the sequence to the left or right. Whilst using the right mouse button to shift sequences you will find that it is possible to keep dragging the sequence to the left, even though there are no leading gaps in the sequence. This is accomplished by placing leading gaps in all the other sequences and shifting the horizontal scroll bar. To stop the accumulation of leading gaps you may sometimes find that right-shifting a sequence causes the sequence being moved to remain stationary and all other sequences to move to the left.


File input/output

All files are stored on the server and not on the client.


Cutting, copying and pasting

Sequences may be moved within the alignment, or removed from it, using the Cut, Copy and Paste items in the Edit menu.

To remove a sequence from the alignment, click on the target sequence and use Edit:Cut. To restore the cut sequence, click on the position in the alignment where you wish to make the insertion and use Edit:Paste. To copy a sequence within the alignment, click on the target sequence and use Edit:Copy, then paste the sequence into the alignment by clicking on the desired insert position. (Cut and Copy are not enabled until you have clicked on a given sequence, and Paste will not be enabled until you have copied a sequence into the cut buffer and clicked on another sequence.)


Group editing

Group editing allows you to insert and delete gaps, and move a set of sequences, in a single operation. To invoke this facility, use the Edit:Groups option - this will pop up a Sequence groups window. Type a new group name into the Additional group box in the top left-hand corner and press the Add group button - the new group will appear at the bottom of the list of sequences. You can add sequences to a group in three ways:

  1. Drag and drop a sequence from the Ungrouped set onto the new group name
  2. Select a group by clicking on the group name and clicking on sequences that need to be moved to the group
  3. Drag and drop a group name onto another group to transfer all the sequences from the first group into the second.
Groups can be deleted by either typing the group name in the top left-hand corner of the window and then pressing the Delete group button, or by selecting the group with a single mouse click and pressing Delete group. When a group is deleted, all the sequences in the group are returned to a special group called Ungrouped, which cannot be deleted.

Each group has a toggle button to switch the Group Edit function for that group on or off. To enable group edits, toggle the Enable group edits button in the bottom left-hand corner of the window.


Selecting and editing motifs

To select short motifs, or larger alignment segments, use the Edit:Motif menu option. Define the motif by clicking and holding the middle mouse button and dragging a boundary or box over the region of interest. Upon release of the mouse button, the selected motif is displayed in the Region manager. The motif can then be saved directly using the Save button, or edited prior to saving using the Motif text option: this invokes the Motif Text Output editor, which allows the motif to be changed as desired (e.g., individual sequences may be removed, etc.).

Additional sequences can be added to a motif simply by dragging the edge of the bounding box, again using the middle mouse button. Multiple motifs can be defined in this way, and can be manipulated separately within the Region manager: e.g., motifs may be deleted by highlighting the relevant motif and clicking on the Delete button; all motifs may be purged using the Clear all option, and so on.

Motifs are saved with the default file extension .mot, requiring only the provision of a suitable file-name (this is the Save option) - the output format is suitable for input to database scanning software.


Editing and inserting sequences

Sequences may be edited using the Edit:Sequence option, which invokes a Sequence editor. Clicking on a sequence in the alignment makes a copy in the Editor, which may then be changed and added back into CINEMA - if a new ID code is not supplied, a ~ symbol is added to the existing code to indicate that the sequence has been altered.

New sequences may be added to the alignment, using the Sequence editor as a paste facility (this function is also available on the File menu). In addition, you can use Control-C and Control-V to transfer sections of a sequence between CINEMA and an editor on your client machine.

Remember, if you save an alignment containing a novel sequence into a public directory, it will be visible to all.


Finding where you are in a sequence

To help you understand where you are in a sequence, a scale bar is provided at the bottom of the main window. The scale bar shows the position of the amino acids in the gapped alignment. To the left of the scale bar, two numbers are shown: normally these show the visible start and end positions of the sequence last clicked; when the mouse button is held down, the numbers indicate the absolute and relative position of the cursor.


Retrieving information about a sequence

Each sequence in the alignment has a button labelled with the database ID code for the sequence; clicking on the button will pop up a new browser window containing text from its source database entry.


Colour schemes

The Colour scheme manager, invoked via the Colours menu, allows you to select between the available colour schemes, and/or to modify the background colours for each amino acid on a one-to-one basis. By default, the alignment is coloured crudely according to residue type (proline and glycine have special structural properties, particularly in membrane proteins, so are grouped separately; similarly for cysteine, which is often involved in disulphide bond formation):

Polar positiveH, K, RBlue
Polar negativeD, ERed
Polar neutralS, T, N, QGreen
Non-polar aliphaticA, V, L, I, MWhite
Non-polar aromaticF, Y, WPurple
P, GBrown
CYellow
Special charactersB, Z, X, -Grey

When the colour menu is invoked, four options now present themselves:

To swap between colour schemes, click on the Primary colour scheme option; this invokes the Primary colour scheme manager. In the top right- hand corner of the window is a Load button. Clicking on this presents a new window, with a range of colour schemes to choose from. Select an option and click the Load scheme button to import the new colour scheme into the Primary colour manager. If this is the scheme of choice, click on the Apply button to see the effects reflected in the alignment. In this release, we offer two additional schemes over and above the original Default scheme (with muted, gentle shades) and the Modified scheme (which is bolder and more intense). An example of the rather lurid Taylor colouring scheme is shown here.

To change the colour of an individual amino acid, click on the button labelled with the single-letter code and move the scroll bars at the bottom of the page. When you are happy with the choice of colour, again, use the Apply button to update the alignment. Note: user-defined colours cannot be saved in this release of CINEMA.

The secondary colouring option comes into play when using CINEMA in split-screen mode. In this case, invoking the Secondary colour scheme manager via the Colours menu allows a different colouring scheme to be applied to the second window. Load the colour scheme of choice into the manager and apply to the alignment as described above. This may have no effect, because the default mode is to use only a single colouring scheme. Therefore, return to the Colours menu and select the Use both colour schemes option. This should automatically apply the second scheme to the bottom window (if not, click on Apply in the Secondary colour scheme manager).

To return to a single colouring scheme for both alignments, choose the Use single colour scheme option of the Colors menu. Exit the Colour managers by clicking on the Close button in the bottom right- hand corner of the window.


Fonts

Five fonts are provided as standard. You can vary the size of the lettering depending on how you are using the applet: e.g.,large fonts for presentations, and small fonts for seeing as much as possible of the sequence in one window. The fonts are:


New interface

CINEMA 2.0 is an extensive rewrite of the alpha version, and although its method of use is essentially the same, the interface has been modified to reflect the new, more flexible, applet. By comparison with the first release, the principal change to the interface is an icon bar beneath the original menu bar. Clicking on the icons invokes commonly-used functions from the main menus. For example, icons are provided to:

cut sequences from the alignment

paste sequences into the alignment

copy sequences into the sequence editor

open the sequence editor

open the group editor

open the colour editor

import an alignment from a database

import a sequence from a database

open a file on the server

save an alignment file to the server

open a 3D skeleton viewer

open a six-frame translator

open a diagonal plot

When the cursor is place over one of the icons, its function is reflected in the status bar at the bottom of the window.


Split screen

The View:Split screen option allows the alignment screen to be split horizonally into two separate windows. In the current implementation, this extends the functionality in two ways:

Alternative alignment views
First, simultaneous views of separate parts of the alignment may now be seen in different windows. Thus, for example, the effects of manipulations made at the N-terminus may be seen simultaneously at the C-terminus. As before, navigation within the separate screens is controlled via the horizontal and vertical scroll-bars.

Hydropathy profiles
Second, stacked hydropathy profiles for each sequence may be viewed beneath the alignment by choosing the View:Hydrophobicity option. The profiles communicate with the alignment in such a way that effects of insertions or deletions made in a given sequence are seen as shifts in the corresponding profile. Conversely, shifts in a profile have the effect of opening or closing gaps in the corresponding sequence. The stacked profiles may therefore be used as a tool, in some cases, to guide alignment.

The screen may be restored to the split alignment by choosing the View: 2nd Alignment editor option. Split screen mode itself may be turned off by choosing the View:Unsplit screen option.


Pluglets

The Pluglet menu allows customisation of CINEMA, i.e. by allowing users to import their own pluglets. Clicking on the Load pluglets option causes the menu bar to be updated with a new menu relating to the imported applet, and a new icon to appear in the icon bar. Clicking on the icon invokes the new applet.

The Skeleton pluglet
For example, clicking on the Skeleton icon invokes a 3D backbone viewer. Within the alignment, clicking on a SWISS-PROT code (e.g. OPSD_SHEEP) sends the structure for that sequence to the viewer, provided a corresponding PDB file is available. If there is no PDB file, the applet will print a warning message. The skeleton is manipulated via click and drag mouse actions.

With mouse drag:

With mouse click:

Dragging a motif in the alignment (middle mouse button) colours the relevant part of the structure if there is a sequence match. The colours depicted in the 3D backbone reflect the residue colours in the alignment - these may be easier to see if you choose the bold colouring option.Several motifs may be picked from the alignment for 3D visualisation.

Using the SS colors menu option, secondary structure elements can be coloured within the structure if there is a secondary structure file available. The remaining menu options clear labels and colours from the display, and allow you to quit from the viewer.

The Six-frame pluglet
Clicking on the 6F icon six-frame translator. Nucleic acid sequences may be typed directly, or added by means of cut-and-paste, into the translator Sequence input box. Clicking on the Translate button provides translations in all 6 reading frames in the bottom window. Any of these may be selected by double clicking on the relevant sequence: the chosen translation may then be uploaded into CINEMA using the Add button.

It may be unclear from the results which is the correct translation. To verify which sequences might code for proteins, short fragments (up to 30 residues) can be used to perform rapid identity searches of OWL. Simply paste successive translations into the Sequence fragment input box and Send query. This is not a guarantee of success, but it can provide a worthwhile check.

Before adding a sequence to an alignment, it is useful to provide an identification or reference code, which may be typed into the Accession number input box. A Description input box is also provided for the addition of annotations.

Use the Clear button to reset the display for translation of further sequences. Exit the pluglet using the Close option on the File menu.

The DotPlot pluglet
Clicking on the icon invokes the diagonal plot pluglet; this allows comparison of the similarity of selected sequences within the alignment (identical sequences form an unbroken diagonal from the bottom left to the top right-hand corner of the plot). To read sequences into the pluglet, click on the ID codes (at the far left of the alignment) of the two sequences you wish to compare. The pluglet automatically computes the diagonal plot using the default values for word size and stringency.

To recalculate the plot with different stringency values, or with different word sizes, change the relevant defaults and click on the Compile button.

To read in new sequences, simply click on the ID codes of the desired sequences: the first click refreshes the y-axis sequence; the second click effectively shunts the new sequence to the x-axis and replaces that sequence at the y-axis.

To close the pluglet, select the File:Close option.

The ClustalW pluglet
The ClustalW pluglet is invoked by clicking on the Clustal interface option of the AutoAlign menu. This produces a separate window allowing submission of the alignment job to a background process. The particular interface implemented here assumes that all sequences in the applet window are to be aligned. Sequences that are not required in this process can be swiftly removed using the Cut icon.

The Interface window requires a job ID to be provided prior to submitting the process to a batch queue. Simply supply a relevant short job name in the input box provided and click on the Submit job button to set the job running - this should take a short while, depending on the load on the server.

As the job proceeds, the Interface will display information about the submission. Initially, the Interface screen is updated to reflect the name of the job, but none of the buttons are active. When the job is complete, Load Alignment and View Log buttons pop-up and become live. Load Alignment loads the result from ClustalW into the applet window: but remember, if you no longer wish to use them, clear the unaligned sequences from the alignment window first, using the File::Clear All menu option. View Log displays a log of the ClustalW job in a separate browser.

The Advanced button allows various alignment parameters to be controlled: for example, there are options to choose the scoring matrix, the input data type (i.e., protein or DNA), gap penalties, etc..

To close the Interface window, choose the File::Close menu option.

The Interactive Align pluglet
The Interactive Align pluglet is invoked by clicking on the Interactive Align option of the AutoAlign menu. This pops up an AutoAlignment Form with which to select the sequences for alignment. This provides greater flexibility than the Clustal interface, which automatically takes as input all sequences in the applet window.

By default, all sequences are highlighted for alignment. To weed out those sequences you do not wish to align, click on the relevant highlighted ID code/s (deselected sequences lose their highlights). Having selected/deselected the sequences of interest, click on the Align button to start the interactive alignment procedure. While calculating the alignment, the cursor changes to a "watch face"; when the calculation is complete, the alignment window is automatically updated with the newly-aligned sequences.

If you wish to change the alignment parameters, the default values for gap creation and extension may be changed by altering the numbers supplied in the appropriate boxes. Of course, given that this is an automatic alignment program, the alignments produced will rarely be perfect, no matter how much the various parameters are `tweaked'!

To close the AutoAlignment Form click on the Quit button.

The BLAST pluglet
The BLAST pluglet is invoked by clicking on the Blast Interface option of the Database menu. The first step is to select which sequence to search against the database. To do this, invoke the Sequence Editor via the main Edit:Sequence menu option. Clicking on different sequences within the applet window will read them directly into the editor. From here, the sequence (or, indeed, part of it) may be transferred into the Blast Interface input box using the mouse Cut and Paste facilities.

Having loaded the sequence, select the Database to search by clicking on the database of choice in the box provided (here, currently only OWL29.6). Then make sure that the correct BLAST program has been selected - remember, blastp for proteins. To run the job, click on the Start search button. The database search runs on-line, so the cursor changes to "watch face" to indicate that the job is running.

When the search is complete, the results are loaded into the bottom part of Blast Interface window. All the usual BLAST information is displayed, but in tabular form, beginning with the ID code of the retrieved match, its score, its probability value and a description (title) of the matched protein (high scores with very low P-values are considered by the program to be mathematically significant; as P tends to 1.0, the match is more likely to be random).

In addition to the information presented in the table, an alignment representation of each sequence pair is provided (the top is the query sequence and the bottom the target sequence)- this allows an instant visual diagnosis of the quality of the pairwise comparison.

Individual sequences may be uploaded into the alignment window by toggling the Add to alignment button on and clicking on the relevant hyperlinked ID code to the left of the BLAST output (unfortunately, this option has currently only been enabled for SWISS-PROT codes). Clicking on successive codes will add them to the alignment (although, they will not themselves be aligned). The other toggle options here are to Add to the 6-frame translator and/or to Add to the sequence editor.

The Advanced button allows the various BLAST parameters to be changed.

To close the window, select the File::Close menu option.


Future developments

CINEMA is in a process of evolution. The following improvements are planned:

  1. Alignment input from other Web resources (such as MIPS, Pfam, ProDom, etc.)
  2. Sequence input from other primary sources (such as SWISS-PROT, PIR, NRL-3D, etc.)
  3. ID code hot-links to other database types (such as scop, CATH, PROSITE, SBASE, etc.)
  4. A wider selection of alignment colouring attributes (such as hydropathy, positional variability, secondary structure prediction, etc.)
  5. Making new colour schemes persistent
  6. Block output in a format suitable for CINEMA input
  7. An undo method that will store changes in a stack and allow you to move through your edits
  8. Links to 2D graphics viewers (helical wheels, hydropathy plots, fingerprint profiles, etc.)
  9. A possible link to a genome browser
  10. A mechanism for integrating the application with CORBA environments
  11. Auto-positioning of new sequences added to the alignment
  12. And much, much more!

If you have thoughts on how you think the applet could develop, we would be delighted to hear from you. So please send us your comments.


Installation on your Intranet

For security reasons we recommend that you install this applet on your organisation's Intranet.

An installation kit is free to academic institutions.

Please use the contact form to request information on how to install this application on your Intranet.


Known problems

We are currently aware of certain `features' of this applet and are doing our best to address them:

  1. When the CINEMA window is partially obscured you will experience a reduction in performance of the scrolling
  2. Using Netscape2.0 on SGI boxes, error messages are generated concerning the scroll-bar that it attaches to a list
  3. Invoking new windows from the applet causes Netscape3.0 to crash
  4. On Netscape Navigator for Windows95, new windows/frames created by the applet always appear to be the same size and not the size specified by the applet
  5. On certain configurations of Netscape for Windows95, users will find that it is difficult to simulate the action of the middle mouse button
  6. BLAST is producing odd (duplicate) results with short query sequences. We are looking into this.
Please let us know of any other problems.


Authors

The CINEMA package was developed by:

CINEMA is currently managed by:


References

Parry-Smith, D.J., Payne, A.W.R, Michie, A.D. and Attwood, T.K. (1997)
"CINEMA - A novel Colour INteractive Editor for Multiple Alignments."
Gene, 211(2), GC45-56.

Attwood, T.K., Payne, A.W.R., Michie, A.D. and Parry-Smith, D.J. (1997)
"A Colour INteractive Editor for Multiple Alignments - CINEMA",
EMBnet.news, 3 (3).


Acknowledgements

We are grateful to Pfizer Ltd. and to the OMF for providing the initial funds to develop this software, and latterly to EMBnet for finding funds to continue its support.


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