1 %\documentclass[a4paper,11pt]{report}
2 \documentclass[a4paper,11pt]{book}
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25 \newtheorem{theorem}{Theorem}
26 \newtheorem{corollary}[theorem]{Corollary}
27 \newtheorem{definition}{Definition}
30 \title{Jalview 2.5: A manual and introductory tutorial }
31 \author{David Martin, James Procter, Andrew Waterhouse and Geoff Barton}
32 \date{Manual version 1.2.3 6th May 2011}
34 \newcommand{\clearemptydoublepage}{\newpage{\pagestyle{empty}}\cleardoublepage}
36 % how the hell do we add another panel with text like : This tutorial introduces
37 % the user to the features of Jalview, a multiple sequence alignment editor and
38 % viewer available from http://www.jalview.org to the title page.
40 % \renewcommand{menustyle}{\tt} %do something more advanced here.
43 \newcounter{exstep}[ecount]
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47 \newcommand{\exercise}[2] {
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49 \begin{center} \fbox{\parbox[b][\height]{6in}{
51 % this doesn't work - page refs are off
52 % \mbox{\addcontentsline{toc}{subsection}{ Exercise \theecount : #1 } }
53 Exercise \theecount : #1 }
54 \par #2 }} \end{center}
57 \newcommand{\exstep}[1]{ \stepcounter{exstep} {\sl \theexstep.} \begin{minipage}[t]{5.5in} #1 \end{minipage} \par \vspace *{1mm} }
58 %%% Remove the % on the next 2 lines to hide tutorials.
59 %\renewcommand{\exercise}[2]{}
60 %\renewcommand{\exstep}[1]{}
68 % we make our own title because JBP is not clever enough to work out how
82 A manual and introductory tutorial }
88 David Martin, James Procter, Andrew Waterhouse, Saif Shehata and Geoff Barton
90 With additional material by Nancy Giang.
95 College of Life Sciences, University of Dundee
97 Dundee, Scotland DD1 5EH, UK
103 % post CLS lifesci course on 15th January
104 % draft. Remaining items are AACon, RNA visualization/editing and Protein disorder analysis exercises.
112 \clearemptydoublepage
114 % ($Revision$) 11th October 2010.}
115 % TODO revise for 2.6
117 \pagenumbering{roman}
120 \clearemptydoublepage
125 \pagenumbering{arabic}
129 \label{jalviewbasics}
130 \section{Introduction}
132 Jalview is a multiple sequence alignment viewer, editor and analysis tool.
133 Jalview is designed to be platform independent (running on Mac, MS Windows, Linux
134 and any other platform that supports Java), capable of editing and analysing
135 large alignments (thousands of sequences) with minimal degradation in
136 performance, and able to show multiple integrated views of the alignment and
137 other data. Jalview can read and write many common sequence formats including
138 FASTA, Clustal, MSF(GCG) and PIR.
141 There are two types of Jalview program. The {\bf Jalview Desktop} is a stand
142 alone application that provides powerful editing, visualization, annotation and
143 analysis capabilities. The {\bf JalviewLite} applet has the same core
144 visualization, editing and analysis capabilities as the desktop, without the
145 desktop's webservice and figure generation capabilities. It is designed to be
146 embedded in a web page,\footnote{A demonstration version of Jalview (Jalview Micro
147 Edition) also runs on a mobile phone but the functionality is limited to sequence
148 colouring.} and includes a javascript API to allow customisable display of alignments for web sites such as
149 {\bf pfam}.\footnote{\url{http://pfam.sanger.ac.uk}}
152 Jalview 2.8 was released in November 2012. The Jalview Desktop in this version
153 provides access to protein and nucleic acid sequence, alignment and structure
154 databases, and includes the Jmol\footnote{ Provided under the LGPL licence at
155 \url{http://www.jmol.org}} viewer for molecular structures, and the VARNA\footnote{Provided under GPL licence at \url{http://varna.lri.fr}} program for the visualization of RNA secondary structure. A
156 Distributed Annotation System (DAS) client\footnote{jDAS - released under Apache license (v2.0) at \url{http://code.google.com/p/jdas}} which facilitates the retrieval and display of third party sequence
157 annotation in association with sequences and any associated structure. It also
158 provides a graphical user interface for the multiple sequence alignment, conservation analysis and protein disorder prediction methods provided as {\bf Ja}va {\bf B}ioinformatics
159 {\bf A}nalysis {\bf W}eb {\bf S}ervices (JABAWS). JABAWS\footnote{released under GPL at \url{http://www.compbio.dundee.ac.uk/jabaws}} is a system for running bioinformatics programs that you can download and run on your own machine or cluster, or install on compute clouds.
161 \subsection{Jalview's Capabilities}
162 % TODO add references to appropriate sections for each capability described here.
163 Figure \ref{jvcapabilities} gives an overview of the main features of the Jalview desktop application. Its primary function is the editing and visualization of sequence alignments, and their interactive analysis. Tree building, principal components analysis, physico-chemical property conservation and sequence consensus analyses are built in to the program. Web services enable Jalview to access remote alignment and secondary structure prediction programs, as well as to retrieve protein and nucleic acid sequences, alignments, protein structures and sequence annotation. Sequences, alignments, trees, structures, features and alignment annotation may also be exchanged with the local filesystem. Multiple visualizations of an alignment may be worked on simultaneously, and the user interface provides a comprehensive set of controls for colouring and layout. Alignment views are dynamically linked with Jmol structure displays, a tree viewer and spatial cluster display, facilitating interactive exploration of the alignment's structure. The application provides its own Jalview project file format in order to store the current state of an alignment and analysis windows. Jalview also provides WYSIWIG\footnote{WYSIWIG: What You See Is What You Get.} style figure generation capabilities for the preparation of alignments for publication.
166 \includegraphics[width=5.8in]{images/jvcapabilities.pdf}
167 \caption{{\bf Capabilities of the Jalview Desktop.} The Jalview Desktop Application provides a stable environment for the creation, editing and analysis of alignments and the generation of figures.}
168 \label{jvcapabilities}
172 \subsubsection{Jalview History}
173 Jalview was initially developed in 1996 by Michele Clamp, James Cuff, Steve
174 Searle and Geoff Barton at the University of Oxford and then the European
175 Bioinformatics Institute. Development of Jalview 2 was made possible with
176 eScience funding from the BBSRC\footnote{Biotechnology and Biological Sciences
177 Research Council grant {\sl ``VAMSAS: Visualization and Analysis of Molecules,
178 Sequence Alignments and Structures"}, a joint project to enable interoperability
179 between Jalview, TOPALi and AstexViewer.} in 2004, enabling Andrew Waterhouse and
180 Jim Procter to re-engineer the original program to introduce contemporary developments
181 in bioinformatics and take advantage of the latest web and Java technology.
182 Jalview's development is now supported for a further 5 years from October 2009
183 by an award from the BBSRC's Tools and Resources fund. In 2010, 2011, and 2012, Jalview
184 benefitted from the \href{http://code.google.com/soc/}{Google Summer of Code},
185 when Lauren Lui and Jan Engelhardt introduced new features for handling RNA
186 alignments and secondary structure annotation, in collaboration with Yann Ponty.\footnote{\url{http://www.lix.polytechnique.fr/~ponty/}}
189 %TODO describe future plans in history ? not a good idea.
190 % Jalview continues to be one of the worlds most popular\footnote{and in the authors opinion, the best.} sequence alignment and analysis tools.
192 \subsubsection{Citing Jalview}
193 If you use Jalview in your work you should cite:\newline
194 {\sl ``Jalview Version 2 - a multiple sequence alignment editor and analysis workbench"}\newline
195 Waterhouse, A.M., Procter, J.B., Martin, D.M.A, Clamp, M. and Barton, G. J. (2009) \newline {\sl Bioinformatics} doi: 10.1093/bioinformatics/btp033
197 This paper supersedes the original Jalview publication:\newline {\sl ``The Jalview Java alignment
198 editor"} \newline Michele Clamp, James Cuff, Stephen M. Searle and Geoffrey J. Barton (2004) \newline {\sl Bioinformatics} {\bf 20} 426-427.
201 \subsection{About this tutorial }
203 This tutorial is written in a manual format with short exercises where
204 appropriate, typically at the end of each section. This chapter concerns the
205 basic operation of Jalview and should be sufficient for those who just want to
206 load Jalview (Section \ref{startingjv}), open an alignment (Section \ref{loadingseqs}), perform basic editing and colouring (Section \ref{selectingandediting} and Section \ref{colours}), and produce publication
207 and presentation quality graphical output (Section \ref{layoutandoutput}).
209 Chapter \ref{analysisannotation} covers the additional visualization and
210 analysis techniques that Jalview provides. This includes working with the
211 embedded Jmol molecular structure viewer, building and viewing trees and PCA
212 plots, and using trees for sequence conservation analysis. An overview of
213 the Jalview Desktop's webservices is given in Section \ref{jvwebservices}, and
214 the alignment and secondary structure prediction services are described
215 in detail in Sections \ref{msaservices} and \ref{protsspredservices}. Following
216 this, Section \ref{featannot} details the creation and visualization of sequence
217 and alignment annotation, and the retrieval of sequences and annotation from
218 databases and DAS Servers. Finally, Section \ref{workingwithnuc} discusses
219 specific features of use when working with nucleic acid sequences, such as translation and linking to protein
220 coding regions, and the display and analysis of RNA secondary structure.
222 %^Chapter \ref{jalviewadvanced} The third chapter covers the detail^ of Jalview and is aimed at the user who is
223 %already familiar with Jalview operation but wants to get more out of their
226 \subsubsection{Typographic Conventions}
228 Keystrokes using the special non-symbol keys are represented in the tutorial by
229 enclosing the pressed keys with square brackets ({\em e.g.} [RETURN] or [CTRL]).
230 Keystroke combinations are combined with a `-' symbol ({\em e.g.} [CTRL]-C means
231 press [CTRL] and the `C' key). Menu options are given as a path from the menu
232 that contains them - for example {\sl File $\Rightarrow$ Input Alignment
233 $\Rightarrow$ From URL} means to select the `From URL' option from the `Input
234 Alignment' submenu of a window's `File' dropdown menu.
236 \section{Obtaining and starting The Jalview Desktop Application}
240 \includegraphics[width=4.5in]{images/download.pdf}
241 \caption{{\bf Download page on the Jalview web site}}
246 This tutorial is based on the application version of Jalview, the Jalview
247 Desktop. Much of the information will also be useful for users of the JalviewLite
248 applet, which has the same core editing, analysis and visualization capabilities (see the
249 \href{http://www.jalview.org/examples/applets.html}{JalviewLite Applet Examples}
250 page for examples). The Jalview Desktop, however, is much more powerful, and
251 includes additional support for interaction with external web services, and
252 production of publication quality graphics.
254 The Jalview Desktop can be run in two ways; as an application launched from the
255 web {\sl via} Java webstart, or as an application loaded onto your hard drive.
256 The webstart version is launched from the {\bf Launch Jalview Desktop} link at
257 the top-right of pages at \url{http://www.jalview.org}. To download the locally
258 installable version, follow the links from the
259 \href{http://www.jalview.org/download}{Download page} (Figure \ref{download}).
260 These links will always launch the latest stable release of Jalview.\par
262 When the application is launched with webstart, two dialogs may appear before
263 the application starts. If your browser is not set up to handle webstart, then
264 clicking the launch link may download a file that needs to be opened
265 manually, or prompt you to select the correct program to handle the webstart
266 file. If that is the case, then you will need to locate the {\bf javaws} program
267 on your system\footnote{The file that is downloaded will have a type of {\bf
268 application/x-java-jnlp-file} or {\bf .jnlp}. The {\bf javaws} program that can run
269 this file is usually found in the {\bf bin} directory of your Java
270 installation}. Once java webstart has been launched, you may also be prompted to
271 accept a security certificate signed by the Barton Group.\footnote{On some
272 systems, the certificate may be signed by 'UNKNOWN'. In this case, clicking
273 through the dialogs to look at the detailed information about the certificate
274 should reveal it to be a Barton group certificate.} You can always trust us, so
275 click trust or accept as appropriate. The splash screen (Figure \ref{splash})
276 gives information about the version and build date that you are running,
277 information about later versions (if available), and the paper to cite in your
278 publications. This information is also available on the Jalview web site and
279 from the Desktop's {\sl Help $\Rightarrow$ About} menu option.
285 \includegraphics[width=4.5in]{images/splash.pdf}
286 \caption{{\bf Jalview splash screen}}
291 When Jalview starts it will automatically load an example alignment from the
292 Jalview site. This behaviour can be changed in the Jalview Desktop preferences
293 dialog opened from the Desktop's {\sl Tools $\Rightarrow$ Preferences..} menu.
294 This alignment will look like the one in Figure \ref{startpage} (this is taken
295 from Jalview version 2.7).
300 \includegraphics[width=4in]{images/start.pdf}
301 \caption{{\bf Default startup for Jalview}}
307 \subsubsection{Jalview News RSS Feed}
309 From time to time, important announcements are made available to users of the
310 Jalview Desktop {\sl via} the Jalview News reader. This window will open
311 automatically when new news is available, and can also be accessed {\sl via} the
312 Desktop's {\sl Tools $\Rightarrow$ Show Jalview News} menu entry.
316 \includegraphics[height=3in]{images/jvrssnews.pdf}
317 \caption{{\bf The Jalview News Reader.} The news reader opens automatically
318 when new articles are available from the Jalview Desktop's news channel.}
319 \label{jalviewrssnews}
324 \exercise{Starting Jalview}{
326 \exstep{Point your web browser at the \href{http://www.jalview.org}{Jalview web site} and start Jalview by clicking on the `Launch Jalview Desktop' button.}
327 \exstep{Open the Jalview Desktop's user preferences dialog (from the Tools
328 menu), and untick the checkbox adjacent to the `Open file' entry in the
329 `Visual' preferences tab.}
330 \exstep{Click OK to save the preferences, then \em{launch {\bf another} Jalview
331 instance from the web site}. The example alignment should not be
332 loaded when the new Jalview instance starts up.}
333 {\sl Note: Should you want to reload the example alignment, then select the
334 {\em File$\Rightarrow$ From URL} entry from the Desktop menu, and click on the
335 URL history button on the right hand side of the dialog box that opens.
336 You can then select the example file's URL, followed by OK to open the file. } }
338 \subsection{Getting Help}
340 \subsubsection{Built in documentation}
341 Jalview has comprehensive on-line help documentation. Select {\sl Help
342 $\Rightarrow$ Documentation} from the main window menu and a new window will
343 open (Figure \ref{help}). The appropriate topic can then be selected from the
344 navigation panel on the left hand side. To search for a specific topic, click
345 the `search' tab and enter keywords in the box which appears.
350 \parbox[c]{2in}{\centerline{\includegraphics[width=2.75in]{images/help1.pdf}}}
351 \parbox[c]{4in}{\includegraphics[width=4in]{images/help2.pdf}}
352 \caption{{\bf Accessing the built in Jalview documentation}}
357 \subsubsection{Email lists}
359 The Jalview Discussion list {\tt jalview-discuss@jalview.org} provides a forum
360 for Jalview users and developers to raise problems and exchange ideas - any
361 problems, bugs, and requests for help should be raised here. The {\tt
362 jalview-announce@jalview.org} list can also be subscribed to if you wish to be
363 kept informed of new releases and developments.
365 Archives and mailing list
366 subscription details can be found in the Jalview web site's \href{http://www.jalview.org/community}{community section}.
370 The major features of the Jalview Desktop are illustrated in Figure \ref{anatomy}. The alignment window is the primary window for editing and visualization, and can contain several independent views of the alignment being worked with. The other windows (Trees, Structures, PCA plots, etc) are linked to a specific alignment view. Each area of the alignment window has a separate context menu accessed by clicking the right mouse button.
372 Jalview has two navigation and editing modes: normal mode, where editing and navigation is performed using the mouse, and cursor mode where editing and navigation are performed using the keyboard. The F2 key is used to switch between these two modes.
376 \includegraphics[width=6.5in]{images/jalview_anatomy.pdf}
377 \caption{{\bf The anatomy of Jalview.} The major features of the Jalview Desktop Application are labelled.}
378 % TODO: modify text labels to be clearer - black on grey and black border for clarity
383 \subsection{Navigation in Normal mode}
385 Jalview always starts up in Normal mode, where the mouse is used to interact
386 with the displayed alignment view. You can move about the alignment by clicking
387 and dragging the ruler scroll bar to move horizontally, or by clicking and
388 dragging the alignment scroll bar to the right of the alignment to move
389 vertically. If all the rows or columns in the alignment are displayed, the
390 scroll bars will not be visible.
392 Each alignment view shown in the alignment window presents a window onto the
393 visible regions of the alignment. This means that with anything more than a few
394 residues or sequences, alignments can become difficult to visualize on the
395 screen because only a small area can be shown at a time. It can help,
396 especially when examining a large alignment, to have an overview of the whole
397 alignment. Select {\sl View $\Rightarrow$ Overview Window} from the Alignment
398 window menu bar (Figure \ref{overview}\footnote{the menu shown in this figure
399 is from Jalview 2.2, later versions have more options.}).
403 \includegraphics[width=4.5in]{images/overview.pdf}
404 \caption{{\bf Alignment Overview Window.} The overview window for a view is opened from the {\em View} menu.}
409 The red box in the overview window shows the current view in the alignment
410 window. A percent identity histogram is plotted below the alignment overview.
411 Shaded parts indicate rows and columns of the alignment that are hidden (in this
412 case, a single row at the bottom of the alignment - see Section
413 \ref{hidingregions}). You can navigate around the alignment by dragging the red
416 %Try this now and see how the view in the alignment window changes.
418 \parbox[c]{3in}{Alignment and analysis windows are closed by clicking on the
419 usual `close' icon (indicated by arrows on Mac OS X). If you want to close all
420 the alignments and analysis windows at once, then use the {\sl Window
421 $\Rightarrow$ Close All} option from the Jalview desktop.
423 {\bf \em{Warning: make sure you have saved your work because this cannot be
425 \parbox[c]{3in}{\centerline{\includegraphics[width=2.5in]{images/start_closeall.pdf}
428 \subsection{Navigation in Cursor mode}
430 \parbox[c]{5in}{Cursor mode navigation enables the experienced user to quickly
431 and precisely navigate, select and edit parts of an alignment. On pressing F2 to
432 enter cursor mode the position of the cursor is indicated by a black background
433 and white text. The cursor can be placed using the mouse or moved by pressing
434 the arrow keys ($\uparrow$, $\downarrow$, $\leftarrow$, $\rightarrow$).
435 }\parbox[c]{1.25in}{\centerline{\includegraphics[width=0.8in]{images/cursor1.pdf}}}
437 Rapid movement to specific positions is accomplished as listed below:
438 \begin{list}{$\circ$}{}
439 \item {\bf Jump to Sequence {\sl n}:} Type a number {\sl n} then press [S] to move to sequence (row) {\sl n}
440 \item {\bf Jump to Column {\sl n}:} Type a number {\sl n} then press [C] to move to column {\sl n} in the alignment.
441 \item {\bf Jump to Residue {\sl n}:} Type a number {\sl n} then press [P] to move to residue number {\sl n} in the current sequence.
442 \item {\bf Jump to column {\sl m} row {\sl n}:} Type the column number {\sl m}, a comma, the row number {\sl n} and press [RETURN].
445 \exercise{Navigation}{
447 \exstep{Reload the example file by accessing the Desktop's
448 {\sl File $\Rightarrow$ Input Alignment $\Rightarrow$ From URL} dialog and
449 clicking on the {\sl down arrow} to retrieve the example file URL stored in its history
450 (\url{http://www.jalview.org/examples/exampleFile_2_7.jar})}
451 \exstep{Scroll around the alignment using the alignment (vertical) and ruler (horizontal) scroll bars.}
452 \exstep{Find and open the Overview Window. Move around the alignment by clicking and dragging the red box in the overview window.}
453 \exstep{Look at the status bar as you move the mouse over the alignment. It
454 should indicate information about the sequence and residue under the cursor.
455 } \exstep{Press [F2] to enter Cursor mode. Use the arrow keys to move the cursor
456 around the alignment.
457 Move to sequence 7 by pressing {\sl 7 S}. Move to column 18 by pressing {\sl 1 8
458 C}. Move to residue 18 by pressing {\sl 1 8 P}. Note that these can be two
459 different positions if gaps are inserted into the sequence. Move to sequence 5,
460 column 13 by typing {\sl 1 3 , 5 [RETURN]}.
462 \subsection{The Find Dialog Box}
463 \label{searchfunction}
464 A further option for navigation is to use the {\sl Select $\Rightarrow$
465 Find\ldots} function. This opens a dialog box into which can be entered regular
466 expressions for searching sequences and sequence IDs, or sequence numbers.
467 Hitting the [Find next] button will highlight the first (or next) occurrence of
468 that pattern in the sequence ID panel or the alignment, and will adjust the view
469 in order to display the highlighted region. The Jalview help provides
470 comprehensive documentation for this function, and a quick guide to the regular
471 expressions that can be used with it.
473 %TODO insert a figure for the Find dialog box
476 \section{Loading your own sequences}
478 Jalview provides many ways to load your own sequences. %For this section of the tutorial you will need to download the file http://www.jalview.org/tutorial/alignment.fa to a suitable location on your hard drive.
480 \subsection{Drag and Drop}
481 In most operating systems you can just drag a file icon from a file browser
482 window and drop it on an open Jalview application window. The file will then be opened as a new alignment window. %You can try this with the tutorial file you have downloaded. When you have opened the file, close it again by selecting the close control on the window. If you drop an alignment file onto an open alignment window it will be appended
483 to that alignment. Drag and drop also works when loading data from a URL -
484 simply drag the link or url from the address panel of your browser on to an
485 alignment or the Jalview desktop background and Jalview will load data from the
487 % (Figure \ref{drag})
489 % \begin{figure}[htbp]
491 % \includegraphics[width=2in]{images/drag2.pdf}
492 % \includegraphics[width=2in]{images/drag3.pdf}
493 % \caption{{\bf An alignment can be opened by dragging the file onto the Jalview window. }}
498 % %\includegraphics[width=2in]{images/drag1.pdf}
501 \subsection{From a File}
502 Jalview can read sequence alignments from a sequence alignment file. This is a
503 text file, not a word processor document. For entering sequences from a
504 wordprocessor document see Cut and Paste (Section \ref{cutpaste}) below. Select
505 {\sl File $\Rightarrow$ Input Alignment $\Rightarrow$ From File} from the main
506 menu (Figure \ref{loadfile}). You will then get a file selection window where
507 you can choose the file to open. Remember to select the appropriate file type.
508 Jalview can automatically identify some sequence file formats.
513 \includegraphics[width=2in]{images/loadfilebox.pdf}
514 %\includegraphics[width=2in]{images/loadfilebox2.pdf}
515 \includegraphics[width=3in]{images/loadfileboxdrop.pdf}
516 \caption{{\bf Opening an alignment from a file saved on disk. }}
521 \subsection{From a URL}
522 Jalview can read sequence alignments directly from a URL. Please note that the
523 files must be in a sequence alignment format - an HTML alignment or graphics
524 file cannot be read by Jalview.
525 Select {\sl File $\Rightarrow$ Input Alignment $\Rightarrow$ From URL} from the
526 main menu and a window will appear asking you to enter the URL (Figure
527 \ref{loadurl}). Jalview will attempt to automatically discover the file format.
532 \includegraphics[width=2in]{images/menuloadurl.pdf}
533 \includegraphics[width=3in]{images/loadurlbox.pdf}
534 \caption{{\bf Opening an alignment from a URL }}
539 \subsection{Cut and Paste}
541 Documents such as those produced by Microsoft Word cannot be readily understood
542 by Jalview. The way to read sequences from these documents is to select the
543 data from the document and copy it to the clipboard. There are two ways to
544 do this. One is to right-click on the desktop background, and select the
545 `Paste to new alignment' option in the menu that appears. The other is to select
546 {\sl File $\Rightarrow$ Input Alignment $\Rightarrow$ From Textbox} from the
547 main menu, and paste the sequences into the textbox window that will appear
548 (Figure \ref{loadtext}). In both cases, presuming that they are in the right
549 format, Jalview will happily read them into a new alignment window.
554 \includegraphics[width=2in]{images/menuloadtext.pdf}
555 \includegraphics[width=3in]{images/loadtextbox.pdf}
556 \caption{{\bf Opening an alignment from pasted text }}
562 \subsection{From a public database}
564 Jalview can retrieve sequences and sequence alignments from the public databases
565 housed at the European Bioinformatics Institute, including Uniprot, Pfam, Rfam
566 and the PDB, as well as any DAS sequence server registered at the configured DAS
567 registry. Jalview's sequence fetching capabilities allow you to avoid having to
568 manually locate and save sequences from a web page before loading them into
569 Jalview. It also allows Jalview to gather additional metadata provided by the
570 source, such as annotation and database cross references.
571 Select {\sl File $\Rightarrow$ Fetch Sequence(s) \ldots} from the main menu and
572 a window will appear (Figure \ref{loadseq}). Pressing the database selection
573 button in the dialog box opens a new window showing all the database sources
574 Jalview can access (grouped by the type of database). Once you've selected the
575 appropriate database, hit OK close the database selection window, and then enter
576 one or several database IDs or accession numbers separated by a semicolon and
577 press OK. Jalview will then attempt to retrieve them from the chosen database.
578 Example queries are provided for some databases to test that a source is
579 operational, and can also be used as a guide for the type of accession numbers
580 understood by the source.\footnote{Most DAS sources support {\em range queries}
581 that can be used to download just a particular range from a sequence database
586 \includegraphics[width=5in]{images/fetchseq.pdf}
587 \caption{{\bf Retrieving sequences from a public database}}
593 \exercise{Loading sequences}{
595 \exstep{Start Jalview then close all windows (if necessary) by selecting {\sl Window $\Rightarrow$ Close All} from the Desktop window.}
596 \exstep{Select {\sl File $\Rightarrow$ Input Alignment $\Rightarrow$ From URL} from the Desktop and enter \textsf{http://www.jalview.org/tutorial/alignment.fa} in the box. Click OK
597 and the alignment should load.
599 \exstep{Close all windows using the {\sl Window $\Rightarrow$ Close All} menu option from the Desktop.
601 Point your web browser at the same URL (\url{http://www.jalview.org/tutorial/alignment.fa}) and save the file to your desktop.
603 Open this file in Jalview by selecting {\sl File $\Rightarrow$ Input Alignment $\Rightarrow$ From File} from the main menu and selecting the file from your desktop. Click OK and load the alignment.}
604 \exstep{Select {\sl Desktop $\Rightarrow$ Window $\Rightarrow$ Close All} and drag the alignment.fa file from the desktop onto the Jalview window. The alignment should open. Try dragging onto an empty Jalview and onto an existing
605 alignment and observe the results. You can also try dragging the
606 URL directly onto Jalview.
608 \exstep{ Select {\sl File $\Rightarrow$ Fetch Sequence(s)..} from the Desktop. Select the PFAM seed database and enter the accession number PF03460. Click OK. An alignment of about 107 sequences should load. }
609 \exstep{Open \url{http://www.jalview.org/tutorial/alignment.fa} in a
612 {\bf Note:} If the URL is downloaded instead of opened in the browser, then locate the file in your download directory and open it in a text editor.
614 \exstep{Select and copy the entire text to the clipboard (usually {\sl via} the
615 browser's {\sl Edit $\Rightarrow$ Copy} menu option). Ensure Jalview is running
616 and select {\sl File $\Rightarrow$ Input Alignment $\Rightarrow$ From Textbox}.
617 Paste the clipboard into the large window using the {\sl Edit $\Rightarrow$
618 Paste} text box menu option. Click {\sl New Window} and the alignment will be
621 \subsection{Memory Limits}
623 Jalview is a Java program. One unfortunate implication of this is that Jalview
624 cannot dynamically request additional memory from the operating system. It is
625 important, therefore, that you ensure that you have allocated enough memory to
626 work with your data. On most occasions, Jalview will warn you when you have
627 tried to load an alignment that is too big to fit in to memory (for instance,
628 some of the PFAM alignments are {\bf very} large). You can find out how much
629 memory is available to Jalview with the desktop window's {\sl $\Rightarrow$
630 Tools $\Rightarrow$ Show Memory Usage} function, which enables the display of
631 the currently available memory at the bottom left hand side of the Desktop
632 window's background. Should you need to increase the amount of memory available
633 to Jalview, full instructions are given in the built in documentation (opened by
634 selecting {\sl Help $\Rightarrow$ Documentation}) and on the JVM memory
635 parameters page (\url{http://www.jalview.org/jvmmemoryparams.html}).
638 \section{Writing sequence alignments}
639 \label{savingalignments} \subsection{Saving the alignment} Jalview allows the
640 current sequence alignments to be saved to file so they can be restored at a
641 later date, passed to colleagues or analysed in other programs. From the
642 alignment window menu select {\sl File $\Rightarrow$ Save As} and a dialog box
643 will appear (Figure \ref{savealign}). You can navigate to an appropriate
644 directory in which to save the alignment. Jalview will remember the last filename
645 and format used to save (or load) the alignment, enabling you to quickly save
646 the file during or after editing by using the {\sl File $\Rightarrow$ Save} entry.
648 Jalview offers several different formats in which an alignment can be saved. The
649 jalview format (.jar) is the only one which will preserve the colours, groupings
650 and similar information in the alignment. The other formats produce text files
651 containing just the sequences with no visualization information, although some
652 allow limited annotation and sequence features to be stored (e.g. AMSA).
653 Unfortunately only Jalview can read Jalview files. The {\sl File $\Rightarrow$
654 Output To Textbox} menu option allows the alignment to be copied and pasted into
655 other documents or web servers.
661 \includegraphics[width=1.0in]{images/saveas.pdf}
664 \includegraphics[width=4in]{images/saveas2.pdf}
666 \caption{{\bf Saving alignments in Jalview to disk}}
671 \subsection{Jalview Projects}
672 \parbox[c]{4in}{If you wish to save the complete Jalview session rather than
673 just one alignment (e.g. because you have calculated trees or multiple different
674 alignments) then your work should be saved as a Jalview Project
675 file.\footnote{Tip: Ensure that you have allocated plenty of memory to Jalview
676 when working with large alignments in Jalview projects. See Section \protect
677 \ref{memorylimits} above for how to do this.}
678 From the main menu select {\sl File $\Rightarrow$ Save Project} and a file save
679 dialog box will appear. Loading a project will restore Jalview to exactly the
680 view at which the file was saved, complete with all alignments, trees,
681 annotation and displayed structures rendered appropriately.
682 } \parbox[c]{2in}{ \centerline {
683 \includegraphics[width=1.5in]{images/saveproj.pdf} }}
685 \exercise{Saving Alignments}{
687 \exstep{Start Jalview, close all windows and load the ferredoxin alignment from
688 PFAM (PFAM seed accession number PF03460 (see Exercise \ref{load}). }
690 Select {\sl File $\Rightarrow$ Save As} from the alignment window menu. Choose a location into which to save the alignment and select a format. All formats except {\sl Jalview } can be viewed in a normal text editor (e.g. Notepad) or in a web browser. Enter a file name and click {\sl Save}. Check this file by closing all windows and opening it with Jalview or by
691 browsing to it with your web browser.
693 \exstep{ Repeat the previous step trying different file formats.}
694 \exstep{Select {\sl File $\Rightarrow$ Output to Textbox $\Rightarrow$ FASTA}. You can select and copy this alignment to the clipboard using the textbox menu options {\sl Edit $\Rightarrow$ Select All} followed by {\sl Edit $\Rightarrow$ Copy}. The alignment can then be pasted into any application of choice, e.g. a word processor or web form.
696 \exstep{Ensure at least one alignment window is shown in Jalview. Open the
697 overview window and scroll to any part of the alignment. Select {\sl File
698 $\Rightarrow$ Save Project} from the main menu and save in a suitable place.
699 Close all windows and then load the project {\sl via} the {\sl File $\Rightarrow$ Load Project} menu option. Note how all the windows and positions are exactly as they were when they were saved. } }
702 \section{Selecting and editing sequences}
703 \label{selectingandediting}
704 Jalview makes extensive use of selections - most of the commands available from
705 its menus operate on the {\sl currently selected region} of the alignment, either to
706 change their appearance or perform some kind of analysis. This section
707 illustrates how to make and use selections and groups.
709 \subsection{Selecting parts of an alignment}
710 Selections can be of arbitrary regions in an alignment, one or more complete columns, or one or more complete sequences.
712 A selected region can be copied and pasted as a new alignment using the {\sl Edit $\Rightarrow$ Copy} and {\sl Edit $\Rightarrow$ Paste $\Rightarrow$ To New Alignment} in the alignment window menu options.
714 To clear (unselect) the selection press the [ESC] (escape) key.
716 \subsubsection{Selecting arbitrary regions}
717 To select part of an alignment, place the mouse at the top left corner of the region you wish to select. Press and hold the mouse button and drag the mouse to the bottom right corner of the chosen region then release the mouse button. A dashed red box appears around the selected region (Figure \ref{select}).
722 \includegraphics[width=4in]{images/select1.pdf}
723 \caption{{\bf Selecting a region in an alignment}}
728 \subsubsection{Selecting columns}
729 To select the same residues in all sequences, click and drag along the alignment ruler. This selects the entire height of the alignment. Ranges of positions can also be selected by clicking on the first position then holding down the [SHIFT] key whilst clicking the other end of the selection. Discontinuous regions can be selected by holding down [CTRL] and clicking on positions to add to the column selection. Note that each [CTRL]-Click changes the current selected sequence region to that column, but adds to the column selection. Selected columns are indicated by red highlighting in the ruler bar (Figure \ref{selectcols}).
734 \includegraphics[width=4in]{images/select2.pdf}
735 \caption{{\bf Selecting multiple columns in an alignment.} The red highlighting
736 on the alignment ruler marks the selected columns. Note that only the most
737 recently selected column has a dashed-box around it to indicate a region
743 \subsubsection{Selecting sequences}
747 \includegraphics[width=4in]{images/select3.pdf}
748 \caption{{\bf Selecting multiple sequences in an alignment.} Use [CTRL] or [SHIFT] to select many sequences at once.}
753 To select multiple complete sequences, click and drag the mouse down the
754 sequence ID panel. The same technique as used for columns above can be used with [SHIFT]-Click for continuous and [CTRL]-Click to select discontinuous ranges of sequences (Figure \ref{selectrows}).
757 \subsubsection{Making selections in Cursor mode}
759 To define a selection in cursor mode (which is enabled by pressing [F2] when the alignment window is selected),
760 navigate to the top left corner of the proposed selection (using the mouse, the arrow keys, or the keystroke
761 commands described in Section \ref{cursormode}). Pressing the [Q] key marks this as the
762 corner. A red outline appears around the cursor (Figure \ref{cselect})
764 Navigate to the bottom right corner of the proposed selection and press the [M] key. This marks the bottom right corner of the selection. The selection can then be treated in the same way as if it had been created in normal mode.
768 \includegraphics[width=1.5in]{images/csel1.pdf}
769 \includegraphics[width=1.5in]{images/csel2.pdf}
770 \includegraphics[width=1.5in]{images/csel3.pdf}
771 \includegraphics[width=1.5in]{images/csel4.pdf}
772 \caption{{\bf Making a selection in cursor mode.} Navigate to the top left corner (left), press [Q] (left center), navigate to the bottom right corner (right center) and press [M] (right)}
777 \subsubsection{Inverting the current selection}
779 The current sequence or column selection can be inverted, using {\sl Select
780 $\Rightarrow$ Invert Sequence/Column Selection} in the alignment window.
781 Inverting the selection is useful when selecting large regions in an alignment,
782 simply select the region that is to be kept unselected, and then invert the selection.
783 This may also be useful when hiding large regions in an alignment (see Section \ref{hidingregions} below).
784 Instead of selecting the columns and rows that are to be hidden, simply select the region that is to be kept
785 visible, invert the selection, then select {\sl View $\Rightarrow$ Hide
786 $\Rightarrow$ Selected Region }.
788 \subsection{Creating groups}
789 Selections are lost as soon as a different region is selected. Groups can be
790 created which are labeled regions of the alignment. To create a group, first
791 select the region which is to comprise the group. Then click the right mouse
792 button on the selection to bring up a context menu. Select {\sl Selection
793 $\Rightarrow$ Group $\Rightarrow$ Edit name and description of
794 current group}\footnote{In earlier versions of Jalview, this entry was variously
795 `Group', `Edit Group Name', or `JGroupXXXXX' (Where XXXXX was some serial
796 number).} then enter a name for the group in the dialogue box which appears.
800 \includegraphics[width=3in]{images/group1.pdf}
801 \includegraphics[width=1.5in]{images/newgroup.pdf}
802 \caption{{\bf Creating a new group from a selection}}
807 By default the new group will have a box drawn around it. The appearance of the group can be changed (see Section \ref{colours} below). This group will stay defined even when the selection is removed.
809 \subsection{Exporting the current selection}
811 The current selection can be copied to the clipboard (in PFAM format). It can
812 also be output to a textbox using the output functions in the pop-up menu
813 obtained by right clicking the current selection. The textbox enables quick
814 manual editing of the alignment prior to importing it into a new window (using
815 the [New Window] button) or saving to a file with the {\sl File $\Rightarrow$
816 Save As } pulldown menu option from the text box.
819 \exercise{Making selections and groups}{
821 \exstep{Close all windows in Jalview and load the ferredoxin alignment (PFAM
822 ID PF03460). Choose a residue and place the mouse cursor on it. Click and drag the mouse cursor to create a selection. As you drag, a red box will `rubber band' out to show the extent of the selection. Release the mouse button and a red box should border the selected region. Now press [ESC] to clear the selection.} \exstep{
823 Select one sequence by clicking on the ID panel. Note that the sequence ID takes on a highlighted background and a red box appears around the selected sequence. Now hold down [SHIFT] and click another sequence ID a few positions above or below. Note how the selection expands to include all the sequences between the two positions on which you clicked.
824 Now hold down [CTRL] and click on several sequences ID's both selected and unselected. Note how unselected IDs are individually added to the selection and previously selected IDs are individually deselected.
826 \exstep{ Repeat the step above but selecting columns by clicking on the ruler bar instead of selecting rows by clicking on the sequence ID.
828 \exstep{Press [F2] to enter Cursor mode. Navigate to column 59, row 1 by pressing {\sl 5 9 , 1 [RETURN]}. Press {\sl Q} to mark this position. Now navigate to column 65, row 8 by pressing {\sl 6 5 , 8 [RETURN]}. Press {\sl M} to complete the selection.}
829 \exstep{\label{exselectgrpcolour}Open the popup menu by right-clicking the
830 selected region with the mouse. Open the {\sl Selection $\Rightarrow$ Group $\Rightarrow$ Group Colour } menu and select `Percentage Identity'. This will turn the selected region into a group and colour it accordingly.}
831 \exstep{Hold down [CTRL] and use the mouse to select and deselect sequences by clicking on their Sequence ID label. Note how the group expands to include newly selected sequences, and the `Percentage Identity' colouring changes. }
833 Use the mouse to click and drag the right-hand edge of the selected group. Note again how the group resizes.}
836 Right click on the text area to open the selection popup-menu. Follow the menus and pick an output format from the {\sl Selection $\Rightarrow$ Output to Textbox \ldots} submenu.
838 \exstep{Try manually editing the alignment and then press the [New Window] button to import the file into a new alignment window.}
840 % more? change colouring style. set border colour.
843 \subsection{Reordering the alignment}
844 Sequence reordering is simple. Highlight the sequences to be moved then press the up or down arrow keys as appropriate (Figure \ref{reorder}). If you wish to move a sequence up past several other sequences it is often quicker to select the group past which you want to move it and then move the group rather than the individual sequence.
848 \includegraphics[width=3in]{images/move1.pdf}
849 \includegraphics[width=3in]{images/move2.pdf}
850 \caption{{\bf Reordering the alignment.} The selected sequence moves up one position on pressing the $\uparrow$ key}
855 \exercise{Reordering the alignment}{
856 \exstep{Open an alignment (e.g.the PFAM domain PF03460). Select one sequence. Using the up and down arrow keys, alter its position in the alignment. Note that this will not work in cursor mode.
858 \exstep{Hold [CTRL] and select two sequences separated by one or more un-selected sequences. Note how multiple sequences are grouped together when they are re-ordered using the up and down arrow keys.
863 \subsection{Hiding regions}
864 \label{hidingregions}
865 It is sometimes convenient to exclude some sequences or residues in the alignment without actually deleting them. Jalview allows sequences or alignment columns within a view to be hidden, and this facility has been used to create the several different views in the example alignment file that is loaded when Jalview is first started (See Figure \ref{startpage}).
867 To hide a set of sequences, select them and right-click the mouse on the selected sequence IDs to bring up the context menu. Select {\sl Hide Sequences} and the sequences will be concealed, with a small blue triangle indicating their position (Figure \ref{hideseq}). To unhide (reveal) the sequences, right click on the triangle and select {\sl Reveal Sequences} from the context menu.
872 \includegraphics[width=1.5in]{images/hide1.pdf}
873 \includegraphics[width=2.5in]{images/hide2.pdf}
874 \includegraphics[width=1.75in]{images/hide3.pdf}
875 \caption{{\bf Hiding Sequences} Hidden sequences are represented by a small blue triangle in the sequence ID panel}
880 A similar mechanism applies to columns (Figure \ref{hidecol}). Selected columns (indicated by a red marker) can be hidden and revealed in the same way {\sl via} the context menu by right clicking on the ruler bar. The hidden column selection is indicated by a small blue triangle in the ruler bar.
884 \includegraphics[width=2in]{images/hide4.pdf}
885 \includegraphics[width=3in]{images/hide5.pdf}
886 \includegraphics[width=1in]{images/hide6.pdf}
887 \caption{{\bf Hiding Columns} Hidden columns are represented by a small blue triangle in the ruler bar}
892 It is often easier to select the region that you intend to work with, rather
893 than the regions that you want to hide. In this case, select the required region and use the {\sl View $\Rightarrow$ Hide
894 $\Rightarrow$ All but Selected Region } menu entry, or press [Shift]+[Ctrl]+H
895 to hide the unselected region.
897 \subsubsection{Representing a group with a single sequence}
899 Instead of hiding a group completely, it is sometimes useful to work with just one representative sequence. The {\sl $<$Sequence ID$>$ $\Rightarrow$ Represent group with $<$Sequence ID$>$ } option from the sequence ID pop-up menu enables this variant of the hidden groups function. The remaining representative sequence can be visualized and manipulated like any other. However, any alignment edits that affect the sequence will also affect the whole sequence group.
901 \exercise{Hiding and revealing regions}{
902 \exstep{Close all windows then open the PFAM accession PF03460. Select a contiguous set of sequences by clicking and dragging on the sequence ID panel. Right click on the selected sequence IDs and select {\sl Hide Sequences}.
905 Right click on the blue triangle indicating hidden sequences and select {\sl Reveal Sequences}. (If you have hidden all sequences then you will need to use the alignment window menu option {\sl View $\Rightarrow$ Show $\Rightarrow$ All Sequences.})
908 Repeat but using a non-contiguous set of sequences. Note that when multiple regions are hidden there are two options, {\sl Reveal Sequences} and {\sl Reveal All}.
910 \exstep{Repeat the above but hiding and revealing columns instead of sequences.
912 \exstep{Select a region of the alignment, add in some additional columns to the
913 selection, and experiment with the `Hide all but selected region' function. }
914 \exstep{Select some sequences and pick one to represent the rest. Bring up the sequence ID pop-up menu for that sequence and select the {\sl Represent group with $<$Sequence ID$>$ } option. Use the pop-up menu again to reveal the hidden sequences that you just picked a representative for.}
920 \subsection{Introducing and removing gaps}
922 The alignment view provides an interactive editing interface, allowing gaps to be inserted or deleted to the left of any position in a sequence or sequence group. Alignment editing can only be performed whilst in keyboard editing mode (entered by pressing [F2]) or by clicking and dragging residues with the mouse when [SHIFT] or [CTRL] is held down (which differs from earlier versions of Jalview).
924 \subsubsection{Locked Editing}
926 The Jalview alignment editing model is different to that used in other alignment
927 editors. Because edits are restricted to the insertion and deletion of gaps to the left of a particular sequence position, editing has the effect of shifting the rest of the sequence(s) being edited down or up-stream with respect to the rest of alignment. The {\sl Edit $\Rightarrow$ Pad Gaps} option can be enabled to eliminate `ragged edges' at the end of the alignment, but does not avoid the `knock-on' effect which is sometimes undesirable. However, its effect can be limited by performing the edit within a selected region. In this case, gaps will only be removed or inserted within the selected region. Edits are similarly constrained when they occur adjacent to a hidden column.
929 \subsubsection{Introducing gaps in a single sequence}
931 To introduce a gap, place the cursor on the residue to the immediate right of where the gap should appear. Hold down the SHIFT key and the left mouse button, then drag the sequence to the right till the required number of gaps has been inserted.
933 One common error is to forget to hold down [SHIFT]. This results in a selection which is one sequence high and one residue long. Gaps cannot be inserted in such a selection. The selection can be cleared and editing enabled by pressing the [ESC] key.
935 \subsubsection{Introducing gaps in all sequences of a group}
937 To insert gaps in all sequences in a selection or group, place the mouse cursor on any residue in the selection or group to the immediate right of the position in which a gap should appear. Hold down the CTRL key and the left mouse button, then drag the sequences to the right until the required number of gaps has appeared.
939 Gaps can be removed by dragging the residue to the immediate right of the gap
940 leftwards whilst holding down [SHIFT] (for single sequences) or [CTRL] (for a group of sequences).
941 \subsubsection{Sliding Sequences}
943 Pressing the [$\leftarrow$] or [$\rightarrow$] arrow keys when one or more
944 sequences are selected will ``slide'' the selected sequences to the left or
945 right (respectively). Slides occur regardless of the region selection -
946 which, for example, allows you to easily reposition misaligned subfamilies
947 within a larger alignment.
949 \subsubsection{Undoing edits}
950 Jalview supports the undoing of edits {\sl via} the {\sl Edit $\Rightarrow$ Undo Edit}
951 alignment window menu option, or CTRL-Z. An edit, if undone, may be re-applied
952 with {\sl Edit $\Rightarrow$ Redo Edit}, or CTRL-Y. Note, however, that the
953 {\sl Undo} function only works for edits to the alignment or sequence ordering.
954 Colouring of the alignment, showing and hiding of sequences or modification of
955 annotation cannot be undone.
958 % % better idea to introduce hiding sequences, and use the invert selection, hide
959 % others, to simplify manual alignment construction
960 \exercise{Editing alignments}{
962 % TODO: VERIFY FOR 2.6.1 and 2.7 - NUMBERING/INSTRUCTIONS APPEAR OFF
963 {You are going to manually reconstruct part of the example Jalview alignment
965 \href{http://www.jalview.org/examples/exampleFile.jar}{http://www.jalview.org/examples/exampleFile.jar}.
967 \item{Remember to use [CTRL]+Z to undo an edit, or the {\sl File $\Rightarrow$
968 Reload } function to revert the alignment back to the original version if you
969 want to start again.}
970 \item{ If you are using OSX, and a key combination - such as [CTRL]+A - does
971 not work, then try pressing the [CMD] key instead of [CTRL].
974 \exstep{ Load the URL
975 \textsf{http://www.jalview.org/tutorial/unaligned.fa} which contains part of the
976 ferredoxin alignment from PF03460.
979 \exstep{ Select the first 7 sequences, and press H to hide them (or right click
980 on the sequence IDs to open the sequence ID popup menu, and select {\sl Hide
983 \exstep{ Select FER3\_RAPSA and FER\_BRANA. Slide the sequences to
984 the left so the initial {\bf A} lies at column 57 using the $\Rightarrow$ key.}
986 \exstep{ Select FER1\_SPIOL, FER1\_ARATH, FER2\_ARATH, Q93Z60\_ARATH and
987 O80429\_MAIZE (Hint: you can do this by pressing [CTRL]-I to invert the sequence selection and then
988 deselect FER1\_MAIZE), and use the $\Rightarrow$ key to slide them to so they
989 begin at column 5 of the alignment view.}
991 \exstep{ Select all the visible
992 sequences in the block by pressing [CTRL]-A. Insert a single gap in all selected
993 sequences at column 38 by holding [CTRL] and clicking on the R in FER1\_SPIOL and
994 dragging one column to right. Insert another gap at column 47 in all sequences in
997 \exstep{ Correct the ferredoxin domain alignment for FER1\_SPIOL by
998 inserting two additional gaps after the gap at column 47: First press [ESC] to
999 clear the selection, then hold [SHIFT] and click and drag on the G and move it
1000 two columns to the right.}
1002 \exstep{ Now complete the
1003 alignment of FER1\_SPIOL with a {\bf locked edit} by pressing [ESC] and select
1004 columns 47 to 57 of the FER1\_SPIOL row. Move the mouse onto the G at column 50,
1005 hold [SHIFT] and drag the G to the left by one column to insert a gap at column
1008 \exstep{ In the next two steps you will complete the alignment of the last two sequences.
1010 Select the last two sequences (FER1\_MAIZE and O80429\_MAIZE), then press [SHIFT]
1011 and click and drag the initial methionine of O80429\_MAIZE 5 columns to the right
1012 so it lies at column 10. Keep holding [SHIFT] and click and drag to insert
1013 another gap at the proline at column 25 (25C in cursor mode). Remove the gap at
1014 column 44, and insert 4 gaps at column 47 (after AAPM).}
1016 \exstep{ Hold [SHIFT] and drag the I at column 39 of FER1\_MAIZE 2 columns to the
1017 right. Remove the gap at FER1\_MAIZE column 49 by [SHIFT]+click and drag left by
1018 one column. Press [ESC] to clear the selection, and then insert three gaps in
1019 FER1\_MAIZE at column 47 by holding [SHIFT] and click and drag the S in FER1\_MAIZE to the right by three columns. Finally,
1020 remove the gap in O80429\_MAIZE at column 56 using [SHIFT]-drag to the left on
1024 {\sl Edit $\Rightarrow$ Undo Edit} and {\sl Edit $\Rightarrow$ Redo Edit} menu
1025 option, or their keyboard shortcuts ([CTRL]+Z and [CTRL]+Y) to step
1026 backwards and replay the edits you have made.}
1032 \includegraphics[width=3in]{images/edit1.pdf}
1033 \includegraphics[width=3in]{images/edit2.pdf}
1034 \caption{{\bf Introducing gaps in a single sequence.} Gaps are introduced as the
1035 selected sequence is dragged to the right while pressing and holding [SHIFT].}
1042 \includegraphics[width=3in]{images/edit3.pdf}
1043 \includegraphics[width=3in]{images/edit4.pdf}
1044 \caption{{\bf Introducing gaps in a group.} Gaps are introduced as the selected
1045 group is dragged to the right with [CTRL] pressed.}
1051 \subsubsection{Editing in Cursor mode}
1053 Gaps can be be easily inserted when in cursor mode (toggled with [F2]) by
1054 pressing [SPACE]. Gaps will be inserted at the cursor, shifting the residue
1055 under the cursor to the right. To insert {\sl n} gaps type {\sl n} and then
1056 press [SPACE]. To insert gaps into all sequences of a group, use [CTRL]-[SPACE]
1057 or [SHIFT]-[SPACE] (both keys held down together).
1059 Gaps can be removed in cursor mode by pressing [BACKSPACE]. First make sure you
1060 have everything unselected by pressing ESC. The gap under the cursor will be
1061 removed. To remove {\sl n} gaps, type {\sl n} and then press [BACKSPACE]. Gaps
1062 will be deleted up to the number specified. To delete gaps from all sequences of
1063 a group, press [CTRL]-[BACKSPACE] or [SHIFT]-[BACKSPACE] (both keys held down
1064 together). Note that the deletion will only occur if the gaps are in the same
1065 columns in all sequences in the selected group, and those columns are to the
1066 right of the selected residue.
1068 \exercise{Keyboard edits}{ \item{This continues on from exercise
1069 \ref{mousealedit}, and recreates the final part of the example ferredoxin
1070 alignment from the unaligned sequences using Jalview's keyboard editing mode.
1072 {\bf {\sl Note for Windows Users:}} The [SHIFT]-[SPACE] command has the same effect as
1073 the [CTRL]-[SPACE] command mentioned in this exercise, and you should use
1074 [SHIFT]-[SPACE] in order to avoid opening the window menu.}
1076 \exstep{Load the sequence alignment at
1077 \textsf{http://www.jalview.org/tutorial/unaligned.fa}, or continue using the
1078 edited alignment from exercise \ref{mousealedit}. If you continue from the
1079 previous exercise, then first right click on the sequence ID panel and select
1082 Now, enter cursor mode by pressing [F2]}
1083 % TODO: BACKSPACE or DELETE WHEN SEQS ARE SELECTED WILL DELETE ALL SEQS JAL-783
1084 \exstep{Insert 58 gaps at the start of the first sequence (FER\_CAPAA). Press {\sl 58} then {\sl [SPACE]}. }
1085 \exstep{Go down one sequence and select rows 2-5 as a block. Click on the second sequence ID (FER\_CAPAN). Hold down shift and click on the fifth (FER1\_PEA). }
1086 \exstep{Insert 6 gaps at the start of this group. Go to column 1 row 2 by typing {\sl 1,2} then pressing {\sl [RETURN]}. Now insert 6 gaps. Type {\sl 6} then hold down {\sl [CTRL]} and press {\sl [SPACE]}.}
1087 \exstep{Now insert one gap at column 34 and another at 38. Insert 3 gaps at 47.
1088 Press {\sl 34C} then {\sl [CTRL]-[SPACE]}. Press {\sl 38C} then [CTRL]-[SPACE].
1089 Press {\sl 47C} then {\sl 3 [CTRL-SPACE]} the first through fourth sequences are
1091 \exstep{The fifth sequence (FER1\_PEA) is poorly aligned. We will delete some gaps and add some new ones. Press {\sl [ESC]} to clear the selection. Navigate to the start of sequence 5 and delete 3 gaps. Press {\sl 1,5 [RETURN]} then {\sl 3 [BACKSPACE]} to delete three gaps. Go to column 31 and delete the gap. Press {\sl 31C [BACKSPACE]} .}
1092 \exstep{ Similarly delete the gap now at column 34, then insert two gaps at column 38. Press {\sl 34C [BACKSPACE] 38C 2 [SPACE]}. Delete three gaps at column 44 and insert one at column 47 by pressing {\sl 44C 3 [BACKSPACE] 47C [SPACE]}. The top five sequences are now aligned.}
1095 \section{Colouring sequences}
1098 Colouring sequences is a key aspect of alignment presentation. Jalview allows
1099 you to colour the whole alignment, or just specific groups. Alignment and
1100 group colours are rendered
1101 {\sl below} any other colours, such as those arising from sequence features
1102 (these are described in Section \ref{featannot}). This means that if you
1103 try to apply one of the colourschemes described in this section, and nothing
1104 appears to happen, it may be that you have sequence feature annotation
1105 displayed, and you may have to disable it using the {\sl View $\Rightarrow$
1106 Show Features} option before you can see your colourscheme.
1108 There are two main types of colouring styles: simple static residue colourschemes and dynamic schemes which use conservation and consensus analysis to control colouring. A hybrid colouring is also possible, where static residue schemes are modified using a dynamic scheme. The individual schemes are described in Section \ref{colscheme} below.
1110 \subsection{Colouring the whole alignment}
1112 \parbox[c]{3.75in}{The alignment can be coloured {\sl via} the {\sl Colour} menu option in the alignment window. Selecting the colour scheme causes all residues to be coloured. The menu is divided into three sections. The first section gives options for the behaviour of the menu options, the second lists static and dynamic colourschemes available for selection. The last gives options for making hybrid colourschemes using conservation shading or colourscheme thresholding.
1116 \includegraphics[width=2.5in]{images/colour2.pdf}
1120 \subsection{Colouring a group or selection}
1122 Selections or groups can be coloured in two ways. The first is {\sl via} the Alignment Window's {\sl Colour} menu as stated above, after first ensuring that the {\sl Apply Colour To All Groups} flag is not selected. This must be turned {\sl off} specifically as it is {\sl on} by default. When unticked, selections from the Colours menu will only change the colour for residues in the current selection, or the alignment view's ''background colourscheme'' when no selection exists.
1124 The second method is to use the {\sl Selection $\Rightarrow$ Group $\Rightarrow$ Group Colour} context menu option obtained by right clicking on the group (Figure \ref{colgrp}). This only changes the colour of the current selection or group.
1126 \begin{figure}[htbp]
1128 %TODO update group_col.pdf to show latest jalview group edit submenu
1129 \includegraphics[width=4in]{images/group_col.pdf}
1130 \caption{{\bf Colouring a group via the context menu.}}
1135 \subsection{Shading by conservation}
1136 For many colour schemes, the intensity of the colour in a column can be scaled
1137 by the degree of amino acid property conservation. Selecting {\sl Colour
1138 $\Rightarrow$ By Conservation} enables this mode, and {\sl Modify
1139 Conservation Threshold...} brings up a selection box (the {\sl Conservation
1140 Colour Increment} dialog box) allowing the alignment colouring to be modified.
1141 Selecting a higher value limits colouring to more highly conserved columns (Figure \ref{colcons}).
1143 \begin{figure}[htbp]
1145 \includegraphics[width=2in]{images/colourcons1.pdf}
1146 \includegraphics[width=2in]{images/colourcons3.pdf}
1147 \includegraphics[width=2in]{images/colourcons5.pdf}
1148 \caption{{\bf Conservation Shading} The density of the ClustalX style residue colouring is controlled by the conservation threshold. The effect of 0\% (left), 50\% (center) and 100\% (right) thresholds are shown.
1154 \subsection{Thresholding by percentage identity}
1156 `Thresholding' is another hybrid colour model where a residue is only coloured
1157 if it is not excluded by an applied threshold. Selecting {\sl Colour $\Rightarrow$ Above Identity Threshold} brings up a selection box with a slider controlling the minimum percentage identity threshold to be applied. Selecting a higher threshold (by sliding to the right) limits the colouring to columns with a higher percentage identity (as shown by the Consensus histogram in the annotation panel).
1159 \subsection{Colouring by Annotation}
1160 \label{colourbyannotation}
1162 Any of the {\bf quantitative} annotations shown on an alignment can be used to
1163 threshold or shade the whole alignment.\footnote{Please remember to turn off
1164 Sequence Feature display to see the shading}
1166 The {\sl Colour $\Rightarrow$ By
1167 Annotation} option opens a dialog which allows you to select which annotation
1168 to use, the minimum and maximum shading colours or whether the original
1169 colouring should be thresholded (the `Use original colours' option).
1171 Default settings for minimum and maximum colours can be set in the Jalview
1172 Desktop's preferences.
1174 \centerline{\includegraphics[width=2.8in]{images/col_byannot.pdf}}}
1176 The {\bf per Sequency only} option in the {\bf Colour By Annotation} dialog
1177 allows each sequence to be shaded according to sequence associated annotation
1178 rows, such as protein disorder scores. This functionality is described futher in
1179 Section \ref{protdisorderpred}.
1181 \subsection{Colour schemes}
1184 Full details on each colour scheme can be found in the Jalview on-line help. A brief description of each one is provided below:
1186 \subsubsection{ClustalX}
1189 \parbox[c]{3.5in}{This is an emulation of the default colourscheme used for alignments in ClustalX, a graphical interface for the ClustalW multiple sequence alignment program. Each residue in the alignment is assigned a colour if the amino acid profile of the alignment at that position meets some minimum criteria specific for the residue type. }
1190 \parbox[c]{3in}{\includegraphics[width=2.75in]{images/col_clustalx.pdf}}
1192 \subsubsection{Blosum62}
1194 \parbox[c]{3.5in}{Gaps are coloured white. If a residue matches the consensus sequence residue at that position it is coloured dark blue. If it does not match the consensus residue but the Blosum62 matrix gives a positive score, it is coloured light blue.}
1196 \includegraphics[width=2.75in]{images/col_blosum62.pdf}
1199 \subsubsection{Percentage Identity}
1201 The Percent Identity option colours the residues (boxes and/or text) according to the percentage of the residues in each column that agree with the consensus sequence. Only the residues that agree with the consensus residue for each column are coloured.
1204 \includegraphics[width=2.75in]{images/col_percent.pdf}
1207 \subsubsection{Zappo}
1209 The residues are coloured according to their physicochemical properties. The physicochemical groupings are Aliphatic/hydrophobic, Aromatic, Positive, Negative, Hydrophillic, conformationally special, and Cyst(e)ine.
1212 \includegraphics[width=2.75in]{images/col_zappo.pdf}
1215 \subsubsection{Taylor}
1218 This colour scheme was devised by Willie Taylor and an entertaining description of it's origin can be found in Protein Engineering, Vol 10 , 743-746 (1997)
1221 \includegraphics[width=2.75in]{images/col_taylor.pdf}
1224 \subsubsection{Hydrophobicity}
1226 Residues are coloured according to the hydrophobicity table of Kyte, J., and Doolittle, R.F., J. Mol. Biol. 1157, 105-132, 1982. The most hydrophobic residues are coloured red and the most hydrophilic ones are coloured blue.
1229 \includegraphics[width=2.75in]{images/col_hydro.pdf}
1232 \subsubsection{Helix Propensity}
1235 The residues are coloured according to their Chou-Fasman\footnote{\label{chou-fasman}Chou, PY and Fasman, GD. Annu Rev Biochem. 1978;47:251-76.} helix propensity. The highest propensity is magenta, the lowest is green.
1238 \includegraphics[width=2.75in]{images/col_helix.pdf}
1241 \subsubsection{Strand Propensity}
1244 The residues are coloured according to their Chou-Fasman\textsuperscript{\ref{chou-fasman}} Strand propensity. The highest propensity is Yellow, the lowest is blue.
1247 \includegraphics[width=2.75in]{images/col_strand.pdf}
1252 \subsubsection{Turn Propensity}
1254 The residues are coloured according to their Chou-Fasman\textsuperscript{\ref{chou-fasman}} turn propensity. The highest propensity is red, the lowest is cyan.
1257 \includegraphics[width=2.75in]{images/col_turn.pdf}
1260 \subsubsection{Buried Index}
1262 The residues are coloured according to their Chou-Fasman\textsuperscript{\ref{chou-fasman}} burial propensity. The highest propensity is blue, the lowest is green.
1265 \includegraphics[width=2.75in]{images/col_buried.pdf}
1269 \subsubsection{Nucleotide}
1270 \parbox[c]{3.5in}{ Residues are coloured with four colours corresponding to the
1271 four nucleotide bases. All non ACTG residues are uncoloured. See Section
1272 \ref{workingwithnuc} for further information about working with nucleic acid
1273 sequences and alignments.
1274 } \parbox[c]{3in}{ \includegraphics[width=2.75in]{images/col_nuc.pdf} }
1276 \subsubsection{Purine Pyrimidine}
1277 \parbox[c]{3.5in}{ Residues are coloured according to whether the corresponding
1278 nucleotide bases are purine (magenta) or pyrimidine (cyan) based. All non ACTG
1279 residues are uncoloured. For further information about working with nucleic acid
1280 sequences and alignments, see Section \ref{workingwithnuc}
1281 %and Section \ref{workingwithrna}
1284 } \parbox[c]{3in}{ \includegraphics[width=2.75in]{images/col_purpyr.pdf} }
1286 \subsubsection{RNA Helix colouring}
1287 \parbox[c]{3.5in}{ Columns are coloured according to their assigned RNA helix as
1288 defined by a secondary structure annotation line on the alignment. Colours for
1289 each helix are randomly assigned, and option only available when an RNA
1290 secondary structure row is present on the alignment.
1291 % For more details see Section \ref{workingwithrna}
1293 \includegraphics[width=2.75in]{images/col_rnahelix.pdf} }
1295 \exercise{Colouring Alignments}{
1297 Open a sequence alignment, for example the PFAM domain PF03460. Select the alignment menu option {\sl Colour $\Rightarrow$ ClustalX}. Note the colour change. Now try all the other colour schemes in the {\sl Colour} menu. Note that some colour schemes do not colour all residues.
1300 Colour the alignment using {\sl Colour $\Rightarrow$ Blosum62}. Select a group of around 4 similar sequences. Use the context menu (right click on the group) option {\sl Selection $\Rightarrow$ Group $\Rightarrow$ Group Colour $\Rightarrow$ Blosum62} to colour the selection. Notice how some residues which were not coloured are now coloured. The calculations performed for dynamic colouring schemes like Blosum62 are based on the group being coloured, not the whole alignment (this also explains the colouring changes observed in exercise \ref{exselectgrpcolour} during the group selection step).
1303 Keeping the same selection as before, colour the complete alignment using {\sl Colour $\Rightarrow$ Taylor}. Select the menu option {\sl Colour $\Rightarrow$ By Conservation}. Slide the selector from side to side and observe the changes in the alignment colouring in the selection and in the complete alignment.
1307 \subsubsection{User Defined}
1308 This dialogue allows the user to create any number of named colour schemes at will. Any residue may be assigned any colour. The colour scheme can then be named. If you save the colour scheme, this name will appear on the Colour menu (Figure \ref{usercol}).
1311 \begin{figure}[htbp]
1313 \includegraphics[width=2.5in]{images/col_user1.pdf}
1314 \includegraphics[width=2in]{images/col_user2.pdf}
1315 \includegraphics[width=1.75in]{images/col_user3.pdf}
1316 \caption{{\bf Creation of a user defined colour scheme.} Residue types are assigned colours (left). The profile is saved (center) and can then be accessed {\sl via} the {\sl Colour} menu (right).}
1322 \exercise{User defined colour schemes}{
1323 \exstep{Load a sequence alignment. Select the alignment menu option {\sl Colour $\Rightarrow$ User Defined}. A dialogue window will open.
1325 \exstep{Click on an amino acid button, then select a colour for that amino acid. Repeat till all amino acids are coloured to your liking.
1327 \exstep{ Insert a name for the colourscheme in the appropriate field and click {\sl Save Scheme}. You will be prompted for a file name in which to save the colour scheme. The dialogue window can now be closed.
1330 The new colour scheme appears in the list of colour schemes in the {\sl Colour} menu and can be selected in future Jalview sessions.
1334 \section{Alignment formatting and graphics output}
1335 \label{layoutandoutput}
1336 Jalview is a WYSIWIG alignment editor. This means that for most kinds of graphics output, the layout that is seen on screen will be the same as what is output in an exported graphics file. It is therefore important to pick the right kind of display layout prior to generating figures.
1338 \subsection{Multiple Alignment Views}
1340 Jalview is able to create multiple independent visualizations of the same underlying alignment - these are called {\sl Views}. Because each view displays the same underlying data, any edits performed in one view will update the alignment or annotation visible in all views.
1342 \parbox[c]{4in}{Alignment views are created using the {\sl View $\Rightarrow$ New View} option of the alignment window or by Pressing [CTRL]-T. This will create a new view with the same groups, alignment layout and display options as the current one. Pressing G will gather together Views as named tabs on the alignment window, and pressing X will expand gathered Views so they can be viewed simultaneously in their own separate windows. To delete a group, press [CTRL]-W.}\parbox[c]{2.75in}{
1343 \begin{center}\centerline{
1344 \includegraphics[width=2.5in]{images/mulv_tabs.pdf}}
1348 % JBPNote make an excercise on views ?
1350 \subsection{Alignment layout}
1351 Jalview provides two screen layout modes, unwrapped (the default) where the alignment is in one long line across the window, and wrapped, where the alignment is on multiple lines, each the width of the window. Most layout options are controlled by the Format menu option in the alignment window, and control the overall look of the alignment in the view (rather than just a selected region).
1353 \subsubsection{Wrapped alignments}
1354 Wrapped alignments can be toggled on and off using the {\sl Format $\Rightarrow$ Wrap} menu option (Figure \ref{wrap}). Note that the annotation lines are also wrapped. Wrapped alignments are great for publications and presentations but are of limited use when working with large numbers of sequences.
1356 If annotations are not all visible in wrapped mode, expand the alignment window to view them. Note that alignment annotation (see Section \ref{featannot}) cannot be interactively created or edited in wrapped mode, and selection of large regions is difficult.
1357 \begin{figure}[htbp]
1359 \parbox[c]{2in}{\includegraphics[width=2in]{images/wrap1.pdf}}
1360 \parbox[c]{4in}{\includegraphics[width=4in]{images/wrap2.pdf}}
1361 \caption{{\bf Wrapping the alignment}}
1367 \subsubsection{Fonts}
1369 \parbox[c]{4in}{The text appearance in a view can be modified {\sl via} the {\sl Format $\Rightarrow$ Font\ldots} alignment window menu. This setting applies for all alignment and annotation text except for that displayed in tool-tips. Additionally, font size and spacing can be adjusted rapidly by clicking the middle mouse button and dragging across the alignment window.}
1370 \parbox[c]{2in}{\centerline{\includegraphics[width=1.75in]{images/font.pdf}}}
1372 \subsubsection{Numbering and label justification}
1373 Options in the {\sl Format} menu are provided to control the alignment view, and provide a range of options to control the display of sequence and alignment numbering, the justification of sequence IDs and annotation row column labels on the annotation rows shown below the alignment.
1375 \subsubsection{Alignment and Group colouring and appearance}
1376 The display of hidden row/column markers and gap characters can be turned off with {\sl Format $\Rightarrow$ Hidden Markers} and {\sl Format $\Rightarrow$ Show Gaps}, respectively. The {\sl Text} and {\sl Colour Text} option controls the display of sequence text and the application of alignment and group colouring to it. {\sl Boxes } controls the display of the background area behind each residue that is coloured by the applied coloursheme.
1378 \subsubsection{Highlighting nonconserved symbols}
1379 The alignment layout and group sub-menu both contain an option to hide
1380 conserved symbols from the alignment display ({\sl Format $\Rightarrow$ Show
1381 nonconserved } in the alignment window or {\sl Selection $\Rightarrow$ Group
1382 $\Rightarrow$ Show Nonconserved} by right clicking on a group). This mode is useful when working with
1383 alignments that exhibit a high degree of homology, because Jalview will only
1384 display gaps or sequence symbols that differ from the consensus for each
1385 column, and render all others with a `.'.
1386 %TODO add a graphic to illustrate this.
1387 \subsection{Annotation ordering and display}
1388 % TODO: describe consensus, conservation, quality user preferences, and group
1389 % annotation preferences.
1390 The annotation lines which appear below the sequence alignment are described in
1391 detail in Section \ref{featannot}. They can be hidden by toggling the {\sl View
1392 $\Rightarrow$ Show Annotations} menu option. Additionally, each annotation line
1393 can be hidden and revealed in the same way as sequences {\sl via} the context menu on
1394 the annotation name panel (Figure \ref{annot}). Annotations can be reordered by
1395 dragging the annotation line label on the annotation label panel. Placing the
1396 mouse over the top annotation label brings up a resize icon on the left. When this is
1397 displayed, Click-dragging up and down provides more space in the alignment window for viewing the annotations, and less space for the sequence alignment.
1401 \includegraphics[width=2.5in]{images/annot1.pdf}
1402 \includegraphics[width=2.5in]{images/annot2.pdf}
1403 \caption{{\bf Hiding Annotations} Annotations can either be hidden from the {\sl View} menu (left) or individually from the context menu (right)}
1408 \exercise{Alignment Layout}{
1410 \exstep{Start Jalview and open the URL \textsf{http://www.jalview.org/examples/exampleFile.jar}. Select {\sl Format $\Rightarrow$ Wrap} from the alignment window menu. Experiment with the various options from the {\sl Format} menu. to adjust the ruler placement, sequence ID format and so on. }
1411 \exstep{Hide all the annotation rows by selecting {\sl View $\Rightarrow$ Show Annotations} from the alignment window menu. Reveal the annotations by selecting the same menu option.}
1412 \exstep{{\bf Deselect} {\sl Format $\Rightarrow$ Wrap}, and right click on the annotation row labels to bring up the context menu. Select {\sl Hide This Row}. Bring up the context menu again and select {\sl Show All Hidden Rows} to reveal them}
1413 \exstep{Annotations can be reordered by clicking and dragging the row to the desired position. Click on the {\sl Consensus} row and drag it upwards to just above {\sl Quality}. The rows should now be reordered. Features and annotations are covered in more detail in Section \ref{featannot} below.}
1414 \exstep{Move the mouse to the top left hand corner of the Secondary Structure annotation row label - a grey up/down arrow symbol should appear - when this is shown, the height of the {\sl Annotation Area} can be changed by Clicking and dragging the mouse up or down.}
1417 \subsection{Graphical output}
1419 \parbox[c]{4in}{Jalview allows alignments figures to be exported in three different formats, each of which is suited to a particular purpose. Image export is {\sl via} the {\sl File $\Rightarrow$ Export Image $\Rightarrow$ \ldots } alignment window menu option. }
1420 \parbox[c]{2in}{\centerline{\includegraphics[width=1.25in]{images/image.pdf}}}
1422 \subsubsection{HTML}
1424 \parbox[c]{3in}{HTML is the format used by web pages. Jalview outputs the alignment as an HTML table with all the colours and fonts as seen. Any additional annotation will also be embedded as sensitive areas on the page, such as URL links for each sequence's ID label. This file can then be viewed directly with any web browser. Each residue is placed in an individual table cell. Unwrapped alignments will produce a very wide page.}
1425 \parbox[c]{3.5in}{\centerline{\includegraphics[width=3in]{images/image_html.pdf}}}
1428 \parbox[c]{3in}{EPS is Encapsulated Postscript. {\bf It is the format of choice
1429 for publications and posters} as it gives the highest quality output of any of
1430 the image types. It can be scaled to any size, so will still look good on an A0
1432 This format can be read by most good presentation and graphics packages such as Adobe Illustrator or Inkscape.
1434 \parbox[c]{3.5in}{\centerline{\includegraphics[width=3in]{images/image_eps.pdf}} \par \centerline{Zoom Detail of EPS image.}}
1438 PNG is Portable Network Graphics. This output option produces an image that can be easily included in web pages and incorporated in presentations using e.g. Powerpoint or Open Office. It is a bitmap image so does not scale and is unsuitable for use on posters, or in publications.
1440 For submission of alignment figures to journals, please use EPS\footnote{If the journal complains, {\em insist}.}.
1442 \parbox[c]{3.5in}{\centerline{\includegraphics[width=3in]{images/image_png.pdf}} \par \centerline{Zoom Detail of PNG image.}}
1443 \exercise{Graphical Output}{
1444 \exstep{Load the example Jalview Jar file in Exercise \ref{exscreen}. Customise it how you wish but leave it unwrapped. Select {\sl File $\Rightarrow$ Export Image $\Rightarrow$ HTML} from the alignment menu. Save the file and open it in your favourite web browser. }
1445 \exstep{Now wrap the alignment and export the image to HTML again. Compare the two images. Note that the exported image matches the format displayed in the alignment window but {\bf annotations are not exported}.}
1446 \exstep{Export the alignment using the {\sl File $\Rightarrow$ Export Image $\Rightarrow$ PNG} menu option. Open the file in an image viewer that allows zooming such as Paint or Photoshop (Windows), or Preview (Mac OS X) and zoom in. Notice that the image is a bitmap and it becomes pixelated very quickly. Note also that the {\bf annotation lines are included} in the image.}
1447 \exstep{Export the alignment using the {\sl File $\Rightarrow$ Export Image
1448 $\Rightarrow$ EPS} menu option. Open the file in a suitable program such as
1449 Photoshop, Illustrator, Inkscape, Ghostview, Powerpoint (Windows), or
1450 Preview (Mac OS X). Zoom in and note that the image has near-infinite
1453 \chapter{Analysis and Annotation}
1454 \label{analysisannotation}
1456 This chapter describes the annotation, analysis, and visualization tasks that
1457 the Jalview Desktop can perform.
1459 Section \ref{wkwithstructure} introduces the structure visualization
1460 capabilities of Jalview. In Section \ref{alignanalysis}, you will find
1461 descriptions and exercises on building and displaying trees, PCA analysis,
1462 alignment redundancy removal, pairwise alignments and alignment conservation
1463 analysis. Section \ref{jvwebservices} introduces the various web based services
1464 available to Jalview users, and Section \ref{jabaservices} explains how to
1465 configure the Jalview Desktop for access to new JABAWS servers.
1466 Section \ref{msaservices} describes how to use the range of multiple alignment
1467 programs provided by JABAWS, and Section \ref{aacons} introduces JABAWS' AACon
1468 service for protein multiple alignment conservation analysis.
1469 Section \ref{protsspredservices} explains how to perform protein secondary
1470 structure predictions with JPred, and JABAWS' protein disorder prediction
1471 services are introduced in Section \ref{protdisorderpred}.
1473 Section \ref{featannot} describes the mechanisms provided by Jalview for
1474 interactive creation of sequence and alignment annotation, and how they can be
1475 displayed, imported and exported and used to reorder the alignment. Section
1476 \ref{featuresfromdb} discusses the retrieval of database references and
1477 establishment of sequence coordinate systems for the retrieval and display of
1478 features from databases and DAS annotation services. Section
1479 \ref{workingwithnuc} describes functions and visualization techniques relevant
1480 to working with nucleotide sequences, coding region annotation and nucleotide
1481 sequence alignments.
1482 % and Section \ref{workingwithrna} covers the visualization,
1483 % editing and analysis of RNA secondary structure.
1485 \section{Working with structures}
1486 \label{wkwithstructure}
1487 Jalview facilitates the use of protein structures for the analysis of alignments
1488 by providing a linked view of structures associated with sequences in
1489 the alignment. The Java based molecular viewing program Jmol\footnote{See the
1490 Jmol homepage \url{http://www.jmol.org} for more information.} has been
1491 incorporated\footnote{Earlier versions of Jalview included MCView - a simple
1492 main chain structure viewer. Structures are visualized as an alpha carbon trace
1493 and can be viewed, rotated and coloured in a structure viewer and the results
1494 interpreted on a sequence alignment.} which enables sophisticated molecular
1495 visualizations to be prepared and investigated alongside an analysis of
1496 associated sequences.
1497 PDB format files can be imported directly or structures can be retrieved from
1498 the European Protein Databank (PDBe) using the Sequence Fetcher (see
1501 \subsection{Automatic association of PDB structures with sequences}
1502 Jalview can automatically determine which structures are associated with a
1503 sequence in a number of ways.
1504 \subsubsection{Discovery of PDB IDs from sequence database cross-references}
1505 If a sequence has an ID from a public database that contains cross-references to
1506 the PDB, such as Uniprot. Right-click on any sequence ID and select {\sl Structure $\Rightarrow$
1507 Associate Structure with Sequence $\Rightarrow$ Discover PDB IDs } from the context menu (Figure \ref{auto}). Jalview will attempt to associate the
1508 sequence with a Uniprot sequence and from there discover any associated PDB
1509 structures. This takes a few seconds and applies to all sequences in the
1510 alignment which have valid Uniprot IDs. On moving the cursor over the sequence
1511 ID the tool tip\footnote{Tip: The sequence ID tooltip can often become large for
1512 heavily cross referenced sequence IDs. Use the {\sl View $\Rightarrow$ Sequence
1513 ID Tooltip $\Rightarrow$ } submenu to disable the display of database cross
1514 references or non-positional features. } now shows the Uniprot ID and any
1515 associated PDB structures.
1517 \begin{figure}[htbp]
1519 %TODO fix formatting
1523 \includegraphics[width=1.5in]{images/auto1.pdf}
1528 \includegraphics[scale=0.5]{images/auto2.pdf}
1534 \includegraphics[width=1.5in]{images/auto3.pdf}
1539 \caption{{\bf Automatic PDB ID discovery.} The tooltip (left) indicates that no PDB structure has been associated with the sequence. After PDB ID discovery (center) the tool tip now indicates the Uniprot ID and any associated PDB structures (right)}
1544 \subsubsection{Drag-and-drop association of PDB files with sequences by filename
1546 \label{multipdbfileassoc}
1547 If one or more PDB files stored on your computer are dragged from their location
1548 on the file browser onto an alignment window, Jalview will search the alignment
1549 for sequences with IDs that match any of the files dropped onto the alignment.
1550 If it discovers matches, a dialog like the one in Figure
1551 \ref{multipdbfileassocfig} is shown, giving the option of creating associations
1554 If no associations are made, then sequences extracted
1555 from the structure will be simply added to the alignment. However, if only
1556 some of the PDB files are associated, jalview will raise another dialog box giving
1557 you the option to add any remaining sequences from the PDB structure files not present in
1558 the alignment. This allows you to easily decorate sequences in a newly imported
1559 alignment with any corresponding structures you've already collected in a directory
1560 accessible from your computer.\footnote{We plan to extend this facility in
1561 future so Jalview will automatically search for PDB files matching your
1562 sequence within a local directory. Check out
1563 \href{http://issues.jalview.org/browse/JAL-801}{Jalview issue 801}}
1565 % there is no mention of the other footnote (#3) that appears saying: Tip: The sequence ID tooltip can often become large for heavily cross-referenced sequence IDs. Use the ...
1566 % JBP: yes there is - under 'Discovery of ' subsection.
1567 \begin{figure}[htbp]
1569 \includegraphics[]{images/pdbdragdropassoc.pdf}
1571 \caption{{\bf Associating PDB files with sequences by drag-and-drop.} Dragging
1572 PDB files onto an alignment of sequences with names matching the dragged files
1573 names (A), results in a dialog box (B) that gives the option to associate each
1574 file with any sequences with matching IDs. }
1575 \label{multipdbfileassocfig}
1580 \subsection{Viewing Structures}
1581 The structure viewer can be launched in two ways from the sequence ID context
1582 menu. To view a particular structure associated with a sequence in the
1583 alignment, simply select it from popup menu's associated structures submenu in
1584 {\sl Structure $\Rightarrow$ View Structure $\Rightarrow$ $<$PDB ID$>$}. The
1585 second way is most useful if you want to view all structural data available for
1586 a set of sequences in an alignment. If any of the {\bf currently selected}
1587 sequences have structures associated, the {\sl Structure } submenu of the
1588 sequence ID popup menu will include an option to {\sl View {\bf N}
1589 structures}. Selecting this option will open a new structure view containing
1590 the associated structures superposed according to the alignment.
1592 In both cases, each structure to be displayed will be downloaded or loaded from
1593 the local file system, and shown as a ribbon diagram coloured according to the
1594 associated sequence in the current alignment view (Figure \ref{structure}
1595 (right)). The structure can be rotated by clicking and dragging in the structure
1596 window. The structure can be zoomed using the mouse scroll wheel or by
1597 [SHIFT]-dragging the structure.
1598 Moving the mouse cursor over a sequence to which the structure is linked in the
1599 alignment view highlights the respective residue's sidechain atoms. The
1600 sidechain highlight may be obscured by other parts of the molecule. Similarly,
1601 moving the cursor over the structure shows a tooltip and highlights the
1602 corresponding residue in the alignment. Clicking the alpha carbon or phosphorous
1603 backbone atom will toggle the highlight and residue label on and off. Often, the
1604 position highlighted in the sequence may not be in the visible portion of the
1605 current alignment view and the sliders will scroll automatically to show the
1606 position. If the alignment window's {\sl View $\Rightarrow$ Automatic Scrolling
1607 } option is not selected, however, then the automatic adjustment will be
1608 disabled for the current view.
1610 \begin{figure}[htbp]
1615 \includegraphics[scale=0.5]{images/structure1.pdf}
1622 \includegraphics[width=3in]{images/structure2.pdf}
1626 \caption{{\bf Structure visualization} The structure viewer is launched from the sequence ID context menu (left) and allows the structure to be visualized using the embedded Jmol molecular viewer (right). }
1631 \subsection{Customising structure display}
1633 Structure display can be modified using the {\sl Colour} and {\sl View} menus
1634 in the structure viewer. The background colour can be modified by selecting the
1635 {\sl Colours $\Rightarrow$ Background Colour\ldots} option.
1637 By default, the structure will be coloured in the same way as the associated
1638 sequence(s) in the alignment view from which it was launched. The structure can
1639 be coloured independently of the sequence by selecting an appropriate colour
1640 scheme from the {\sl Colours} menu. It can be coloured according to the
1641 alignment using the {\sl Colours $\Rightarrow$ By Sequence } option. The image
1642 in the structure viewer can be saved as an EPS or PNG with the {\sl File
1643 $\Rightarrow$ Save As $\Rightarrow$ \ldots} submenu, which also allows the raw
1644 data to be saved as PDB format. The mapping between the structure and the
1645 sequence (How well and which parts of the structure relate to the sequence) can
1646 be viewed with the {\sl File $\Rightarrow$ View Mapping} menu option.
1648 \subsubsection{Using the Jmol visualization interface }
1650 Jmol has a comprehensive set of selection and visualization functions that are
1651 accessed from the Jmol popup menu (by right-clicking in the Jmol window or by
1652 clicking the Jmol logo). Molecule colour and rendering style can be manipulated,
1653 and distance measurements and molecular surfaces can be added to the view. It
1654 also has its own ``Rasmol\footnote{See \url{http://www.rasmol.org}}-like'' scripting
1655 language, which is described elsewhere\footnote{Jmol Wiki:
1656 \url{http://wiki.jmol.org/index.php/Scripting}
1658 Jmol Scripting reference:
1659 \url{http://www.stolaf.edu/academics/chemapps/jmol/docs/}}. Jalview utilises the
1660 scripting language to interact with Jmol and to store the state of a Jmol
1661 visualization within Jalview archives, in addition to the PDB data file
1662 originally loaded or retrieved by Jalview. To access the Jmol scripting
1663 environment directly, use the {\sl Jmol $\Rightarrow$ Console} menu option.
1665 If you would prefer to use Jmol to manage structure colours, then select the
1666 {\sl Colours $\Rightarrow$ Colour with Jmol} option. This will disable any
1667 automatic application of colour schemes when new structure data is added, or
1668 when associated alignment views are modified.
1671 \exercise{Viewing Structures}{\label{viewingstructex}
1672 \exstep{Load the alignment at
1673 \textsf{http://www.jalview.org/examples/exampleFile.jar}. Right-click on the
1674 sequence ID label for any of the sequences (e.g. {\sl FER1\_SPIOL}) to bring up
1675 the context menu. Select {\sl FER1\_SPIOL $\Rightarrow$ Structure $\Rightarrow$
1676 Associate Structure with Sequence $\Rightarrow$ Discover
1677 PDB IDs}. Jalview will now attempt to find PDB structures for the sequences in
1679 % JBP Note: Bug JAL-1238 needs to be fixed ASAP
1681 {\bf Note:} If you are using Jalview v2.8 - use the {\sl Uniprot } source from the {\sl Web services $\Rightarrow$ Fetch DB References $\Rightarrow$ ..} submenu of the Alignment Window to retrieve the PDB IDs. }
1682 \exstep{ Right-click on the sequence ID for {\sl FER1\_SPIOL}.
1683 Select { \sl FER1\_SPIOL $\Rightarrow$ Structure $\Rightarrow$ View Structure
1684 $\Rightarrow$ 1A70}. A structure viewing window appears. Rotate the molecule by clicking and dragging in the structure viewing box. Zoom with the mouse scroll wheel. } \exstep{Roll the mouse cursor along the {\sl FER1\_SPIOL} sequence in the alignment. Note that if a residue in the sequence maps to one in the structure, a label will appear next to that residue in the structure viewer. Move the mouse over the structure. Placing the mouse over a part of the structure will bring up a tool tip indicating the name and number of that residue. The corresponding residue in the sequence is highlighted in black. Clicking the alpha carbon toggles the highlight and residue label on and off. Try this by clicking on a set of three or four adjacent residues so that the labels are persistent, then finding where they are in the sequence. }
1685 \exstep{Select {\sl Colours $\Rightarrow$ Background Colour\ldots} from the structure viewer menu and choose a suitable colour. Press OK to apply this. Select {\sl File $\Rightarrow$ Save As $\Rightarrow$ PNG} and save the image. View this with a suitable program. }
1686 \exstep{Select {\sl File $\Rightarrow$ View Mapping} from the structure viewer menu. A new window opens showing the residue by residue alignment between the sequence and the structure.}
1687 \exstep{Select {\sl File $\Rightarrow$ Save $\Rightarrow$ PDB file} and choose a new filename to save the PDB file. Once the file is saved, open the location in your file browser (or explorer window) and drag the PDB file that you just saved on to the Jalview desktop (or load it from the {\sl Jalview Desktop $\Rightarrow$ Input Alignment $\Rightarrow$ From File } menu). Verify that you can open and view the associated structure from the sequence ID pop-up menu's {\sl Structure } submenu in the new alignment window.}
1689 \exstep{Right click on the structure to bring up the Jmol window. Explore the menu options. Try to change the style of molecular display - by first using the {\sl Jmol $\Rightarrow$ Select (n) $\Rightarrow$ All} command (where {\sl n} is the number of residues selected), and then the {\sl Jmol $\Rightarrow$ Style $\Rightarrow$ Scheme $\Rightarrow$ Ball and Stick} command.}
1690 \exstep{Use the {\sl File $\Rightarrow$ Save As .. } function to save the alignment as a Jalview Project. Now close the alignment and the structure view, and load the project file you just saved.
1692 Verify that the Jmol display is as it was when you just saved the file.}
1695 \subsection{Superimposing structures}
1696 \label{superposestructs}
1697 Many comparative biomolecular analysis investigations aim to determine if the
1698 biochemical properties of a given molecule are significantly different to its
1699 homologues. When structure data is available, comparing the shapes of molecules
1700 by superimposing them enables substructure that may impart different behaviour
1701 to be quickly identified. The identification of optimal 3D superpositions
1702 involves aligning 3D data rather than sequence symbols, but the result can
1703 still be represented as a sequence alignment, where columns indicate positions
1704 in each molecule that should be superposed to recreate the optimal 3D alignment.
1706 Jalview can employ Jmol's 3D fitting routines\footnote{See
1707 \href{http://chemapps.stolaf.edu/jmol/docs/?ver=12.2$\#$compare}{http://chemapps.stolaf.edu/jmol/docs/?ver=12.2$\#$compare}
1708 for more information.} to recreate 3D structure superpositions based on the
1709 correspondences defined by one or more sequence alignments involving structures shown in the Jmol display. Superposition based on the currently displayed alignment view happens automatically if a
1710 structure is added to an existing Jmol display using the {\sl Structure
1711 $\Rightarrow$ View PDB Structure $\Rightarrow$ ..}. A new Jmol view containing
1712 superposed structures can also be created using the {\sl Structure
1713 $\Rightarrow$ View all {\bf N} PDB Structures} option (when {\bf {\sl N}}
1714 $>$ 1) if the current selection contains two or more sequences with associated
1717 \subsubsection{Obtaining the RMSD for a superposition}
1718 The RMSD (Root Mean Square Deviation) is a measure of how similar the structures
1719 are when they are superimposed. Figure \ref{mstrucsuperposition} shows a
1720 superposition created during the course of Exercise \ref{superpositionex}. The
1721 parts of each molecule used to construct the superposition are rendered using
1722 the cartoon style, with other parts of the molecule drawn in wireframe. The Jmol
1723 console, which has been opened after the superposition was performed, shows the
1724 RMSD report for the superposition.
1725 Full information about the superposition is also output to the Jalview
1726 console.\footnote{The Jalview Java Console is opened from {\sl Tools
1727 $\Rightarrow$ Java Console} option in the Desktop's menu bar} This output also
1728 includes the precise atom pairs used to superpose structures.
1730 \subsubsection{Choosing which part of the alignment is used for structural
1731 superposition} Jalview uses the visible part of each alignment view to define
1732 which parts of each molecule are to be superimposed. Hiding a column in a view
1733 used for superposition will remove that correspondence from the set, and will
1734 exclude it from the superposition and RMSD calculation.
1735 This allows the selection of specific parts of the alignment to be used for
1736 superposition. Only columns that define a complete set of correspondences for
1737 all structures will be used for structural superposition, and as a consequence,
1738 the RMSD values generated for each pair of structures superimposed can be
1741 In order to recompute a superposition after changing a view or editing the
1742 alignment, select the {\sl Jmol $\Rightarrow$ Align sequences } menu option. The {\sl
1743 Jmol $\Rightarrow$ Superpose with ..} submenu allows you to choose which of the
1744 associated alignments and views are to be used to create the set of
1745 correspondences. This menu is useful when composing complex superpositions
1746 involving multi-domain and multi-chain complexes, when correspondences may be
1747 defined by more than one alignment.
1749 Note that these menu options appear when you have two or more structures in one Jmol viewer.
1753 \begin{figure}[htbp]
1755 \includegraphics[width=5.5in]{images/fdxsuperposition.pdf}
1756 \caption{{\bf Superposition of two ferredoxin structures.} The alignment on the
1757 left was used by jalview to superpose structures associated with the
1758 FER1\_SPIOL and FER1\_MAIZE sequences in the alignment. Parts of each structure
1759 used for superposition are rendered as a cartoon, the remainder rendered in
1760 wireframe. The RMSD between corresponding positions in the structures before and
1761 after the superposition is shown in the Jmol console.}
1762 \label{mstrucsuperposition}
1766 \exercise{Aligning structures using the ferredoxin
1767 sequence alignment.}{\label{superpositionex}
1769 \exstep{Continue with the Jalview project created in exercise
1770 \ref{viewingstructex}. Use the {\sl Discover PDB IDs} function to retrieve PDB
1771 IDs associated with the FER1\_MAIZE sequence.}
1772 \exstep{Once discovery has completed, use the {\sl
1773 View PDB Structure} submenu to view the PDB file associated with FER1\_MAIZE.
1774 Jalview will give you the option of aligning the structure to the one already
1775 open. To superimpose the structure associated with FER1\_MAIZE with the one
1776 associated with FER1\_SPIOL, press the {\bf Yes} button.
1778 {\sl The Jmol view will update to show both structures, and one will be
1779 moved on to the other. If this doesn't happen, use the Align function in the Jmol submenu}}
1780 \exstep{Create a new view on the alignment, and hide all but columns 121
1782 \exstep{Use the {\sl Jmol} submenu to
1783 recompute the superposition using just columns 121-132 of the alignment.
1785 {\sl Note how the molecules shift position when superposed using a short part of
1786 the two structures.}}
1787 \exstep{Compare the initial and final RMSDs for superimposing molecules with
1788 the small section and with the whole alignment. Which view do you think give the
1789 best 3D superposition, and why ?} }
1791 \subsection{Colouring structure data associated with multiple alignments and views}
1792 Normally, the original view from which a particular structure view was
1793 opened will be the one used to colour structure data. If alignments involving
1794 sequences associated with structure data shown in a Jmol have multiple views, Jalview gives you full control
1795 over which alignment, or alignment view, is used to colour the structure
1796 display. Sequence-structure colouring associations are
1797 changed {\sl via} the {\sl View $\Rightarrow$ Colour by ..} menu, which lists all
1798 views associated with data shown in the embedded Jmol view. A tick is shown beside
1799 views currently used as colouring source, and moving the
1800 mouse over each view will bring it to the front of the alignment display,
1801 allowing you to browse available colour sources prior to selecting one. If the
1802 {\sl Select many views} option is selected, then multiple views can be selected as sources for colouring the
1803 structure data. {\sl Invert selection} and {\sl Select all views} options are also provided to quickly change between multi-view selections.
1805 Note that the {\sl Select many views} option is useful if you have different
1806 views that colour different areas or domains of the alignment. This option is
1807 further explored in Section \ref{complexstructurecolours}.
1809 \begin{figure}[htbp]
1811 \includegraphics[width=5.5in]{images/mviewstructurecol.pdf}
1812 \caption{{\bf Choosing a different view for colouring a structure display}
1813 Browsing the {\sl View $\Rightarrow$ Colour by ..} menu provides full control
1814 of which alignment view is used to colour structures when the {\sl Colours
1815 $\Rightarrow$ By Sequence} option is selected.}
1816 \label{mviewstructurecol}
1820 \subsubsection{Colouring complexes}
1821 \label{complexstructurecolours}
1822 The ability to control which multiple alignment view is used to colour
1823 structural data is essential when working with data relating to
1824 multidomain biomolecules and complexes.
1826 In these situations, each chain identified in the structure may have a different
1827 evolutionary history, and a complete picture of functional variation can
1828 only be gained by integrating data from different alignments on the same
1829 structure view. An example of this is shown in Figure
1830 \ref{mviewalcomplex}, based on data from Song et. al\footnote{Structure of
1831 DNMT1-DNA Complex Reveals a Role for Autoinhibition in Maintenance DNA Methylation. Jikui Song, Olga Rechkoblit, Timothy H. Bestor, and Dinshaw J. Patel.
1832 {\sl Science} 2011 {\bf 331} 1036-1040
1833 \href{http://www.sciencemag.org/content/331/6020/1036}{DOI:10.1126/science.1195380}}
1835 \begin{figure}[htbp]
1837 \includegraphics[]{images/mchainstructureview.pdf}
1838 \caption{{\bf The biological assembly of Mouse DNA Methyltransferase-1 coloured
1839 by Pfam alignments for its major domains} Alignments for each domain within the
1840 Uniprot sequence DNMT1\_MOUSE have been used to visualise sequence conservation
1841 in each component of this protein-DNA complex. Instructions for recreating this figure are given in exercise \ref{dnmtcomplexex}. }
1842 \label{mviewalcomplex}
1846 \exercise{Colouring a protein complex to explore domain-domain interfaces}{\label{dnmtcomplexex}
1848 \exstep{Download the PDB file at
1849 \textsf{\url{http://www.jalview.org/tutorial/DNMT1\_MOUSE.pdb}} to your desktop. This
1850 is the biological unit for PDB ID 3pt6, as identified by the PDBe's PISA server.}
1852 \exstep{Launch the Jalview desktop and ensure you have at least 256MB of
1853 free memory available.
1855 {\sl Use the following webstart link:
1856 \href{http://www.jalview.org/webstart/jalview_1G.jnlp}{http://www.jalview.org/webstart/jalview\_1G.jnlp}}.}
1857 \exstep{Retrieve the following
1858 {\bf full} PFAM alignments: PF02008, PF00145, PF01426 (make sure you select the {\sl PFAM {\bf (Full)}} source). These will each be retrieved into their own alignment window.}
1859 \exstep{Drag the structure you downloaded in
1860 step 1 onto one of the alignments to associate it with the mouse sequence in
1861 that Pfam domain family.}
1862 \exstep{For every DNMT1\_MOUSE sequence in the alignment, use the sequence
1863 ID popup menu's {\sl Structure} submenu to view the DNMT1\_MOUSE structure for the associated mouse sequence. When given the option, {\bf view all of the structures in the same Jmol viewer}. Check the contents of the {\sl View $\Rightarrow$ Colour by ..} submenu to see what alignments can be used to
1864 colour the sequence.}
1865 \exstep{Repeat the previous two steps for each of
1866 the other alignments. In each case, when performing the `View DNMT1\_MOUSE.pdb'
1867 step, Jalview will ask if you wish to create a new Jmol view. You should
1868 respond `No', {\bf ensuring that each sequence fragment is associated with the same Jmol view}.}
1869 \exstep{Pick a different colourscheme for each alignment, and use the {\sl
1870 Colour by ..} submenu to ensure they are all used to colour the complex shown
1871 in the Jmol window.}
1872 \exstep{The final step needed to reproduce the shading in Figure
1873 \ref{mviewalcomplex} is to use the {\sl Colour $\Rightarrow$ By
1874 Annotation } option in each alignment window to shade the alignment by the
1875 {\bf Conservation} annotation row. This function was described in section
1876 \ref{colourbyannotation}.
1878 Ensure that you first disable the {\sl View $\Rightarrow$ Show Features} menu option, or you may not see any colour changes in the associated structure.
1880 {\sl Note: Choose a different shading scheme for each
1881 alignment so that the regions of strong physicochemical conservation are highlighted. This
1882 kind of shading will reveal conserved regions of interaction between domains
1884 \exstep{Save your work as a Jalview project and verify that it can be opened again by starting another Jalview Desktop instance, and dragging the saved project into the desktop window.}
1886 {\sl Note: This exercise relies on new features introduced in Jalview 2.7. If
1887 you notice any strange behaviour when trying out this exercise, it may be a
1889 \href{http://issues.jalview.org/browse/JAL-1008}{http://issues.jalview.org/browse/JAL-1008}
1890 for one relating to highlighting of positions in the alignment window).} }
1892 \section{Analysis of alignments}
1893 \label{alignanalysis}
1894 Jalview provides support for sequence analysis in two ways. A number of
1895 analytical methods are `built-in', these are accessed from the {\sl Calculate}
1896 alignment window menu. Computationally intensive analyses are run outside
1897 Jalview {\sl via} web services - these are typically accessed {\sl via} the {\sl
1898 Web Service} menu, and described in \ref{jvwebservices} and subsequent sections.
1899 In this section, we describe the built-in analysis capabilities common to both
1900 the Jalview Desktop and the JalviewLite applet.
1903 This calculation creates a spatial representation of the similarities within the
1904 current selection or the whole alignment if no selection has been made. After
1905 the calculation finishes, a 3D viewer displays the each sequence as a point in
1906 3D `similarity space'. Sets of similar sequences tend to lie near each other in
1908 Note: The calculation is computationally expensive, and may fail for very large
1909 sets of sequences - because the JVM has run out of memory. Memory issues, and
1910 how to overcome them, were discussed in Section \ref{memorylimits}.
1912 \subsubsection{What is PCA?}
1913 Principal components analysis is a technique for examining the structure of
1914 complex data sets. The components are a set of dimensions formed from the
1915 measured values in the data set, and the principle component is the one with the
1916 greatest magnitude, or length. The sets of measurements that differ the most
1917 should lie at either end of this principle axis, and the other axes correspond
1918 to less extreme patterns of variation in the data set.
1919 In this case, the components are generated by an eigenvector decomposition of
1920 the matrix formed from the sum of pairwise substitution scores at each aligned
1921 position between each pair of sequences. The basic method is described in the
1922 1995 paper by {\sl G. Casari, C. Sander} and {\sl A. Valencia} \footnote{{\sl
1923 Nature Structural Biology} (1995) {\bf 2}, 171-8.
1924 PMID: 7749921} and implemented at the SeqSpace server at the EBI.
1926 Jalview provides two different options for the PCA calculation. Protein PCAs are
1927 by default computed using BLOSUM 62 pairwise substitution scores, and nucleic
1928 acid alignment PCAs are computed using a score model based on the identity
1929 matrix that also treats Us and Ts as identical, to support analysis of both RNA
1930 and DNA alignments. The {\sl Change Parameters} menu also allows the calculation
1931 method to be toggled between SeqSpace and a variant calculation that is detailed
1932 in Jalview's built in documentation.\footnote{See
1933 \url{http://www.jalview.org/help/html/calculations/pca.html}.}
1935 \subsubsection{The PCA Viewer}
1937 PCA analysis can be launched from the {\sl Calculate $\Rightarrow$ Principle
1938 Component Analysis} menu option. {\bf PCA requires a selection containing at
1939 least 4 sequences}. A window opens containing the PCA tool (Figure \ref{PCA}).
1940 Each sequence is represented by a small square, coloured by the background
1941 colour of the sequence ID label. The axes can be rotated by clicking and
1942 dragging the left mouse button and zoomed using the $\uparrow$ and $\downarrow$
1943 keys or the scroll wheel of the mouse (if available). A tool tip appears if the
1944 cursor is placed over a sequence. Sequences can be selected by clicking on them.
1945 [CTRL]-Click can be used to select multiple sequences.
1946 \begin{figure}[hbtp]
1948 \includegraphics[width=2in]{images/PCA1.pdf}
1949 \includegraphics[width=3in]{images/PCA3.pdf}
1950 \caption{{\bf PCA Analysis} }
1955 Labels will be shown for each sequence by toggling the {\sl View $\Rightarrow$
1956 Show Labels} menu option, and the plot background colour changed {\sl via} the
1957 {\sl View $\Rightarrow$ Background Colour..} dialog box. A graphical
1958 representation of the PCA plot can be exported as an EPS or PNG image {\sl via}
1959 the {\sl File $\Rightarrow$ Save As $\Rightarrow$ \ldots } submenu.
1961 \exercise{Principle Component Analysis}{ \exstep{Load the alignment at
1962 \textsf{http://www.jalview.org/examples/exampleFile.jar} and press [ESC] to clear any selections. Alternatively, select {\sl Select $\Rightarrow$ Undefine Groups} to remove all groups and colourschemes. } \exstep{Select the menu option {\sl Calculate $\Rightarrow$ Principle Component Analysis}. A new window will open. Move this window so that the tree, alignment and PCA viewer window are all 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 them on the alignment and tree. }
1963 \exstep{ Click on the tree window. Careful selection of the tree partition
1964 location will divide the alignment into a number of groups, each of a different
1965 colour. Note how the colour of the sequence ID label matches both the colour of
1966 the partitioned tree and the points in the PCA plot.
1969 \subsubsection{PCA data export}
1970 Although the PCA viewer supports export of the current view, the plots produced
1971 are rarely suitable for direct publication. The PCA viewer's {\sl File} menu
1972 includes a number of options for exporting the PCA matrix and transformed points
1973 as comma separated value (CSV) files. These files can be imported by tools such
1974 as {\bf R} or {\bf gnuplot} in order to graph the data.
1978 Jalview can calculate and display trees, providing interactive tree-based
1979 grouping of sequences though a tree viewer. All trees are calculated {\sl via}
1980 the {\sl Calculate $\Rightarrow$ Calculate Tree $\Rightarrow$ \ldots} submenu.
1981 Trees can be calculated from distance matrices determined from \% identity or
1982 aggregate BLOSUM 62 score using either {\sl Average Distance} (UPGMA) or {\sl
1983 Neighbour Joining} algorithms. The input data for a tree is either the selected
1984 region or the whole alignment, excluding any hidden regions.
1986 On calculating a tree, a new window opens (Figure \ref{trees1}) which contains
1987 the tree. Various display settings can be found in the tree window {\sl View}
1988 menu, including font, scaling and label display options, and the {\sl File
1989 $\Rightarrow$ Save As} submenu contains options for image and Newick file
1990 export. Newick format is a standard file format for trees which allows them to
1991 be exported to other programs. Jalview can also read in external trees in
1992 Newick format {\sl via} the {\sl File $\Rightarrow$ Load Associated Tree} menu
1993 option. Leaf names on imported trees will be matched to the associated alignment
1994 - unmatched leaves will still be displayed, and can be highlighted using the
1995 {\sl View $\Rightarrow$ Mark Unlinked Leaves} menu option.
2000 \includegraphics[width=2.5in]{images/trees1.pdf}
2001 \includegraphics[width=2.5in]{images/trees2.pdf}
2002 \includegraphics[width=1.25in]{images/trees4.pdf}
2003 \caption{{\bf Calculating Trees} Jalview provides four built in models for calculating trees. Jalview can also load precalculated trees in Newick format (right).}
2009 Clicking on the tree brings up a cursor across the height of the tree. The
2010 sequences are automatically partitioned and coloured (Figure \ref{trees2}). To
2011 group them together, select the {\sl Calculate $\Rightarrow$ Sort $\Rightarrow$
2012 By Tree Order $\Rightarrow$ \ldots} alignment window menu option and choose the
2013 correct tree. The sequences will then be sorted according to the leaf order
2014 currently shown in the tree view. The coloured background to the sequence IDs
2015 can be removed with {\sl Select $\Rightarrow$ Undefine Groups} from the
2016 alignment window menu. Note that tree partitioning will also remove any groups
2017 and colourschemes on a view, so create a new view ([CTRL-T]) if you wish to
2022 \includegraphics[width=5in]{images/trees3.pdf}
2023 \caption{{\bf Interactive Trees} The tree level cutoff can be used to designate groups in Jalview}
2028 %\subsubsection{Multiple Views and Input Data recovery from PCA and Tree Viewers}
2030 %Both PCA and Tree viewers are linked analysis windows. This means that their selection and display are linked to a particular alignment, and control and reflect the selection state for a particular view.
2032 \subsubsection{Recovering input data for a tree or PCA plot calculation}
2034 The {\sl File $\Rightarrow$ Input Data } option will open a new alignment window containing the original data used to calculate the tree or PCA plot (if available). This function is useful when a tree has been created and then the alignment subsequently changed.
2036 \parbox[c]{1.25in}{\centerline{\includegraphics[width=1.25in]{images/pca_fmenu.pdf}
2039 \subsubsection{Changing the associated view for a tree or PCA viewer}
2041 The {\sl View $\Rightarrow$ Associated Nodes With $\Rightarrow$ .. } submenu is shown when the viewer is associated with an alignment that is involved in multiple views. Selecting a different view does not affect the tree or PCA data, but will change the colouring and display of selected sequences in the display according to the colouring and selection state of the newly associated view.
2042 } \parbox[c]{3in}{\centerline{
2043 \includegraphics[width=2.5in]{images/pca_vmenu.pdf} }}
2047 \exstep{Ensure that you have at least 1G memory available in Jalview (start with this link: \href{http://www.jalview.org/webstart/jalview_1G.jnlp}{http://www.jalview.org/webstart/jalview\_1G.jnlp}).}
2048 \exstep{Open the alignment at \textsf{http://www.jalview.org/tutorial/alignment.fa}. Select {\sl Calculate $\Rightarrow$ Calculate Tree $\Rightarrow$ Neighbour Joining Using BLOSUM62}. A new tree window will appear.}
2049 \exstep{Click on the tree window. A cursor will appear. Note that placing this cursor divides the tree into a number of groups by colour. Place the cursor to give about 4 groups, then select {\sl Calculate $\Rightarrow$ Sort $\Rightarrow$ By Tree Order $\Rightarrow$ Neighbour Joining Tree using BLOSUM62 from ... }. The sequences are reordered to match the order in the tree and groups are formed implicitly.}
2050 \exstep{Select {\sl Calculate $\Rightarrow$ Calculate Tree $\Rightarrow$ Neighbour Joining Using \% Identity}. A new tree window will appear. The group colouring makes it easy to see the diferences between the two trees, calculated using different methods.}
2051 \exstep{Select from sequence 2 column 60 to sequence 12 column 123. Select {\sl Calculate $\Rightarrow$ Calculate Tree $\Rightarrow$ Neighbour Joining Using BLOSUM62}. A new tree window will appear. It can be seen that the tree contains 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.
2052 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.
2054 \exstep{Recover the {\sl Input Data} for the tree you just calculated from the {\sl File} menu. Check the {\sl Edit $\Rightarrow$ Pad Gaps } option is {\sl not} ticked, and insert one gap anywhere in the alignment. Now select {\sl Calculate $\Rightarrow$ Calculate Tree $\Rightarrow$ Neighbour Joining Using BLOSUM62}.
2056 A warning dialog box {\bf ``Sequences not aligned'' } appears because the sequences input to the tree calculation are of different lengths. }
2058 \exstep{Now select {\sl Edit $\Rightarrow$ Pad Gaps } and try to perform the tree calculation again - this time a new tree should appear.
2060 This demonstrates the use of the {\sl Pad Gaps } editing preference, which ensures that all sequences are the same length after editing. }
2064 \subsection{Tree Based Conservation Analysis}
2065 \label{treeconsanaly}
2067 Trees reflect the pattern of global sequence similarity exhibited by the
2068 alignment, or region within the alignment, that was used for their calculation.
2069 The Jalview tree viewer enables sequences to be partitioned into groups based
2070 on the tree. This is done by clicking within the tree viewer window. Once subdivided, the
2071 conservation between and within groups can be visually compared in order to
2072 better understand the pattern of similarity revealed by the tree and the
2073 variation within the clades partitioned by the grouping. The conservation based
2074 colourschemes and the group associated conservation and consensus annotation
2075 (enabled using the alignment window's {\sl View $\Rightarrow$ Autocalculated
2076 Annotation $\Rightarrow$ Group Conservation} and {\sl Group Consensus} options)
2077 can help when working with larger alignments.
2079 \exercise{Tree Based Conservation Analysis}{
2080 \label{consanalyexerc}
2081 \exstep{Load the PF03460 PFAM seed alignment using the sequence fetcher. Colour it with the {\sl Taylor colourscheme}, and apply {\sl Conservation } shading. }
2082 \exstep{Build a Neighbourjoining tree using BLOSUM62 and use the {\sl Sort Alignment By Tree} option in the tree viewer submenu to order alignment using the calculated tree.}
2083 \exstep{Select a point on the tree to partition the alignment, and examine the variation in colouring between different groups.
2085 You may find it easier to browse the alignment if you first uncheck the {\sl View $\Rightarrow$ Show Annotations} option, and open the Overview Window to aid navigation.}
2086 \exstep{Try changing the colourscheme to BLOSUM62 (whilst ensuring that {\sl Apply Colour to All Groups} is selected)}
2087 {\sl Note: You may want to save the alignment and tree as a project file, since
2088 it is used in the next few exercises. } }
2090 \subsection{Redundancy Removal}
2092 The redundancy removal dialog box is opened using the {\sl Edit $\Rightarrow$ Remove Redundancy\ldots} option in the alignment menu. As its menu option placement suggests, this is actually an alignment editing function, but it is convenient to describe it here. The redundancy removal dialog box presents a percentage identity slider which sets the redundancy threshold. Aligned sequences which exhibit a percentage identity greater than the current threshold are highlighted in black. The [Remove] button can then be used to delete these sequences from the alignment as an edit operation\footnote{Which can usually be undone. A future version of Jalview may allow redundant sequences to be hidden, or represented by a chosen sequence, rather than deleted.}.
2095 \includegraphics[width=5.5in]{images/redundancy.pdf}
2097 \label{removeredundancydialog}
2098 \caption{The Redundancy Removal dialog box opened from the edit menu. Sequences that exceed the current percentage identity threshold and are to be removed are highlighted in black.}
2101 \exercise{Remove redundant sequences}{
2103 {\sl Note: Jalview 2.8 users - bugs in this version mean that the 'Unlinked leaves' markings will not be shown when sequences are removed during this exercise.}
2105 \exstep{Re-use or recreate the alignment and tree which you worked with in the
2106 tree based conservation analysis exercise (exercise \ref{consanalyexerc})}
2107 \exstep{Open the Remove Redundancy dialog and adjust the threshold to 90\%. Remove the sequences that are more than 90\% similar under this alignment.}
2108 \exstep{Select the Tree viewer's {\sl View $\Rightarrow$ Mark Unlinked Leaves} option, and note that the removed sequences are now prefixed with a * in the tree view.}
2109 \exstep{Use the [Undo] button on the dialog to recover the sequences. Note that the * symbols disappear from the tree display.}
2110 \exstep{Experiment with the redundancy removal and observe the relationship between the percentage identity threshold and the pattern of unlinked nodes in the tree display.}
2113 \subsection{Subdividing the alignment according to specific mutations}
2115 It is often necessary to explore variations in an alignment that may correlate
2116 with mutations observed in a particular region; for example, sites exhibiting
2117 single nucleotide polymorphism, or residues involved in substrate recognition in
2118 an enzyme. One way to do this would be to calculate a tree using the specific
2119 region, and subdivide it in order to partition the alignment.
2120 However, calculating a tree can be slow for large alignments, and the tree may
2121 be difficult to partition when complex mutation patterns are being analysed. The
2122 {\sl Select $\Rightarrow$ Make groups for selection } function was introduced to
2123 make this kind of analysis easier. When selected, it will use the characters in
2124 the currently selected region to subdivide the alignment. For example, if a
2125 single column is selected, then the alignment (or each group defined on the
2126 alignment) will be divided into groups based on the residue or nucleotide found
2127 at that position. These new groups are annotated with the characters in the
2128 selected region, and Jalview's group based conservation analysis annotation and
2129 colourschemes can then be used to reveal any associated pattern of sequence
2130 variation across the whole alignment.
2132 \subsection{Automated annotation of Alignments and Groups}
2134 On loading a sequence alignment, Jalview will normally\footnote{Automatic
2135 annotation can be turned off in the {\sl Visual } tab in the {\sl Tools
2136 $\Rightarrow$ Preferences } dialog box.} calculate a set of automatic annotation
2137 rows which are shown below the alignment. For nucleotide sequence alignments,
2138 only an alignment consensus row will be shown, but for amino acid sequences,
2139 alignment quality (based on BLOSUM 62) and physicochemical conservation will
2140 also be shown. Conservation is calculated according to Livingstone and
2141 Barton\footnote{{\sl ``Protein Sequence Alignments: A Strategy for the
2142 Hierarchical Analysis of Residue Conservation." } Livingstone C.D. and Barton
2143 G.J. (1993) {\sl CABIOS } {\bf 9}, 745-756}.
2144 Consensus is the modal residue (or {\tt +} where there is an equal top residue).
2145 The inclusion of gaps in the consensus calculation can be toggled by
2146 right-clicking on the the Consensus label and selecting {\sl Ignore Gaps in
2147 Consensus} from the context menu. Quality is a measure of the inverse likelihood
2148 of unfavourable mutations in the alignment. Further details on these
2149 calculations can be found in the on-line documentation.
2151 These annotations can be hidden and deleted but are only created on loading an
2152 alignment. If they are deleted then the alignment should be saved and reloaded
2153 to restore them. Jalview provides a toggle to autocalculate a consensus sequence
2154 upon editing. This is normally selected by default, but can be turned off for
2155 large alignments {\sl via} the {\sl Calculate $\Rightarrow$ Autocalculate
2156 Consensus} menu option if the interface is too slow.
2158 \subsubsection{Group Associated Annotation}
2159 \label{groupassocannotation}
2160 Group associated consensus and conservation annotation rows reflect the
2161 sequence variation within a particular group. Their calculation is enabled
2162 by selecting the {\sl Group Conservation} or {\sl Group Consensus} options in
2163 the {\sl View $\Rightarrow$ Autocalculated Annotation } submenu of the alignment
2166 \subsubsection{Alignment and Group Sequence Logos}
2169 The consensus annotation row that is shown below the alignment can be overlaid
2170 with a sequence logo that reflects the symbol distribution at each column of
2171 the alignment. Right click on the Consensus annotation row and select the {\sl Show
2172 Logo} option to display the Consensus profile for the group or alignment.
2173 Sequence logos can be enabled by default for all new alignments {\sl via} the
2174 Visual tab in the Jalview desktop's preferences dialog box.
2176 \exercise{Group conservation analysis}{
2177 \exstep{Re-use or recreate the alignment and tree which you worked with in the
2178 tree based conservation analysis exercise (exercise \ref{consanalyexerc})}
2179 \exstep{Create a new view, and ensure the annotation panel is displayed, and
2180 enable the display of {\sl Group Consensus} and the display of sequence
2181 logos to make it easier to see the different residue populations within each group.}
2182 \exstep{Select a column exhibiting about 50\% conservation that lies within the
2183 central conserved region of the alignment. Subdivide the alignment according to
2184 this selection using {\sl Select $\Rightarrow$ Make groups for selection}.}
2185 \exstep{Re-order the alignment according to the new groups that have been
2186 defined. Click on the group annotation row IDs to select groups exhibiting a
2188 \exstep{Select another column exhibiting about 50\% conservation
2189 overall, and subdivide the alignment further. Note that the new groups
2190 inherit the names of the original groups, allowing you to identify the
2191 combination of mutations that resulted in the subdivision.
2193 \exstep{Clear the groups, and try to subdivide the alignment using two
2194 non-adjacent columns.
2196 {\sl Hint: You may need to hide the intervening columns before you can select
2197 both of the columns that you wish to use to subdivide the alignment.}}
2198 \exstep{Switch back to the original view, and experiment with subdividing
2199 the tree groups made in the previous exercise.}
2202 \subsection{Other Calculations}
2205 \subsubsection{Pairwise Alignments}
2207 Jalview can calculate optimal pairwise alignments between arbitrary sequences {\sl via} the {\sl Calculate $\Rightarrow$ Pairwise Alignments\ldots} menu option. Global alignments of all pairwise combinations of the selected sequences are performed and the results returned in a text box.
2211 \includegraphics[width=4in]{images/pairwise.pdf}
2212 \caption{{\bf Pairwise alignment of sequences.} Pairwise alignments of three selected sequences are shown in a textbox.}
2219 \section{Webservices}
2220 \label{jvwebservices}
2221 The term ``Webservices'' refers to a variety of data exchange
2222 mechanisms based on HTTP.\footnote{HTTP: Hyper-Text Transfer Protocol.}
2224 \parbox[c]{4.5in}{Jalview can exploit public webservices to access databases
2225 remotely, and also submit data to public services by opening pages with your web browser. These types of
2226 services are `one-way', {\sl i.e.} data is either sent to the webservice or
2227 retrieved from it by Jalview. The desktop application can also interact
2228 with `two-way' remote analysis services in order to offload computationally
2229 intensive tasks to High Performance Computing facilities. Most of these two-way
2230 services are provided by {\bf Ja}va {\bf B}ioinformatics {\bf A}nalysis {\bf
2231 W}eb {\bf S}ervice (JABAWS) servers\footnote{See
2232 http://www.compbio.dundee.ac.uk/jabaws for more information and to download
2233 your own server.}, which provides an easily installable system for performing
2234 a range of bioinformatics analysis tasks. }
2235 \parbox[c]{1.75in}{\includegraphics[width=1.65in]{images/wsmenu.pdf}}
2237 \subsection{One-way web services}
2239 There are three types of one way service in jalview. Database services,
2240 which were introduced in in Section \ref{fetchseq}, provide sequence and
2241 alignment data. They can also be used to add sequence IDs to an alignment
2242 imported from a local file, prior to further annotation retrieval, as described
2243 in Section \ref{featuresfromdb}. A second type of one way service is provided
2244 by Jalview's DAS sequence feature retrieval system, which is described
2245 in Section \ref{dasfretrieval}. The final type of one way service are sequence
2246 and ID submission services, exemplified by the `Envision2 Services' provided
2247 by the ENFIN Consortium\footnote{ENFIN is the European Network for Functional
2248 INtegration. Please see http://www.enfin.org for more information. }.
2250 \subsubsection{One-way submission services}
2251 Jalview can use the system's web browser to submit sets of sequences and
2252 sequence IDs to web based applications. Single sequence IDs can be passed to
2253 a web site using the user definable URL links listed under the {\sl
2254 Links} submenu of the sequence ID popup menu. These are configured
2255 in the {\sl Connections} tab of the {\sl Preferences} dialog box.
2257 The Envision 2 services presented in the webservice menu provides are the first
2258 example of one-way services where multiple sequences or sequence IDs can be
2259 sent. The {\sl Web service $\Rightarrow$ Envision 2 Services} menu entry
2260 provides two sub-menus that enable you to submit the sequences or IDs
2261 associated with the alignment or just the currently selected sequences to one
2262 of the Envision2 workflows. Selecting any one will open a new browser window on
2263 the Envision2 web application. The menu entries and their tooltips provide
2264 details of the Envision2 workflow and the dataset set that will be submitted
2265 ({\sl i.e.} the database reference type, or associated sequence subset). Please
2266 note, due to technical limitations, Jalview can currently only submit small
2267 numbers of sequences to the workflows - if no sequence or ID submissions are
2268 presented in the submenus, then try to select a smaller number of sequences to
2271 \subsection{Remote Analysis Web Services}
2272 Remote analysis services enable Jalview to use external computational
2273 facilities. There are curently three types of service - multiple sequence
2274 alignment, protein secondary structure prediction, and alignment analysis.
2275 Many of these are provided by JABA servers, which are described at the end of
2276 this section. In all cases, Jalview will construct a job based on the alignment
2277 or currently selected sequences, ask the remote server to run the job, monitor
2278 status of the job and, finally, retrieve the results of the job and display
2279 them. The Jalview user is kept informed of the progress of the job through a
2282 Currently, web service jobs and their status windows are not stored in Jalview
2283 Project Files\footnote{This may be rectified in future versions.}, so it is
2284 important that you do not close Jalview whilst a job is running. It is also
2285 essential that you have a continuous network connection in order to
2286 successfully use web services from Jalview, since it periodically checks the
2287 progress of running jobs.
2290 \subsection{JABA Web Services for sequence alignment and analysis}
2291 \label{jabaservices}
2292 JABA stands for ``JAva Bioinformatics Analysis'', which is a system developed
2293 by Peter Troshin and Geoff Barton at the University of Dundee for running
2294 computationally intensive bioinformatics analysis programs. A JABA installation
2295 typically provides a range of JABA web services (JABAWS) for use by other
2296 programs, such as Jalview.
2298 Exercises in the remainder of this section will demonstrate the simplest way of
2299 installing JABA on your computer, and configuring Jalview so it can access the JABA services. If you
2300 need any further help or more information about the services, please go to the
2301 \href{http://www.compbio.dundee.ac.uk/jabaws}{JABAWS home page}.
2302 %% \subsubsection{Aims}
2303 %% \begin{list}{$\bullet$}{}
2304 %% \item Gain experience using the different alignment services provided by
2306 %%\item Learn about the way that Jalview stores user presets for JABA services
2307 %%\item Learn how to install JABA services and configure Jalview to access them
2310 \subsection{Changing the Web Services menu layout}
2311 \label{changewsmenulayout}
2312 If you are working with a lot of different JABA services, you may wish to change
2313 the way Jalview lays out the web services menu. You can do this from the Web
2314 Services tab of the {\sl Preferences} dialog box.
2316 \exercise{Changing the Layout of the Web Services Menu}{
2317 \label{changewsmenulayoutex}
2318 \exstep{Make sure you have loaded an alignment into Jalview, and examine the
2319 current layout of the alignment windowÔøΩs {\sl Web Service} menu.}
2320 \exstep{Open the preferences dialog box and select the web services tab.}
2321 \exstep{Ensure the {\sl Enable JABAWS services} checkbox is selected, and unselect
2322 the {\sl Enable Enfin Services} checkboxes.}
2323 \exstep{Hit {\sl Refresh Services} to update the web services menu -- once the
2324 progress bar has completed, open the {\sl Web Service} menu to view the changes.}
2325 \exstep{Select the {\sl Index by host} checkbox and refresh the services once again.
2327 {\sl Observe the way the layout of the JABAWS Alignment submenu changes.}
2329 \exstep{Do the same with the {\sl Index by type} checkbox.}
2332 Jalview provides these options for configuring the layout of the {\sl Web Service}
2333 menu because different Jalview users may have access to a different number of
2334 JABA services, and each will have their own preference regarding the layout of
2337 \begin{figure}[htbc]
2339 \includegraphics[width=3in]{images/jvjabawsconfig.pdf}
2340 \caption{{\bf The Jalview Web Services preferences panel.} Options are provided
2341 for configuring the list of JABA servers that Jalview will use, enabling and
2342 disabling Enfin services, and configuring the layout of the web services
2344 \label{jvjabawsconfig}
2349 \subsubsection{Testing JABA services}
2350 The JABAWS configuration dialog shown in Figure \ref{jvjabawsconfig} has colour
2351 codes to indicate whether the Desktop can access the server, and whether all
2352 services advertised by the server are functional. The colour codes are:
2354 \begin{list}{$\bullet$}{}
2355 \item Red - Server cannot be contacted or reports a connection error.
2356 \item Amber - Jalview can connect, but one or more services are non-functional.
2357 \item Green - Server is functioning normally.
2359 %TODO green and a tick, red and a cros, amber and a value indicating whether its all going
2361 Test results from JABAWS are reported on Jalview's console output (opened from
2362 the Tools menu). Tests are re-run every time Jalview starts, and when the
2363 [Refresh] button is pressed on the Jalview JABAWS configuration panel.
2365 \subsubsection{Resetting the JABA services setting to their defaults}
2366 Once you have configured a JABAWS server and selected the OK button of the
2367 preferences menu, the settings will be stored in your Jalview preferences file,
2368 along with any preferences regarding the layout of the web services menu. If
2369 you should ever need to reset the JABAWS server list to its defaults, use the
2370 `Reset Services' button on the Web Services preferences panel.
2372 \subsection{Running your own JABA server}
2373 You can download and run JABA on your own machine using the `VMWare' or
2374 VirtualBox virtual machine environments. If you would like to learn how to do
2375 this, there are full instructions at the
2376 \href{http://www.compbio.dundee.ac.uk/jabaws/}{JABA web site}.
2378 \exercise{Installing a JABA Virtual Machine on your computer}{
2379 \label{jabawsvmex}{\sl This tutorial will demonstrate the simplest way of
2380 installing JABA on your computer, and configuring Jalview so it can access the JABA services.
2384 You will need a copy of VMWare Player/Workstation/Fusion on your machine.
2387 \exstep{If you do not have VMWare player installed, download it from
2388 www.vmware.com (this takes a few minutes -- you will need to register and wait
2389 for an email with a download link).}
2390 \exstep{Download the JABA virtual appliance archive called `jaba-vm.zip' from
2391 \textsf{http://www.compbio.dundee.ac.uk/jabaws/archive/jabaws-vm.zip}
2393 WARNING: This is large (about 300MB) and will take some time to download.
2395 \exstep{Unpack the archive's contents to a place on your machine with at least
2398 (On Windows, right click on the archive, and use the 'Extract archive..' option).
2400 \exstep{Open the newly extracted directory and double click the VMWare virtual
2401 machine configuration file (jabaws.vcf). This will launch the VMWare player.
2403 \exstep{Once VMWare player has started up, it may ask the question ``Did you move or copy
2404 this virtual appliance?'' -- select `Copy'.}
2405 \exstep{You may be prompted to download the VMWare linux tools. These are not
2406 necessary, so close the window or click on `Later'.}
2407 \exstep{You may also be prompted to install support for one or more devices (USB
2408 or otherwise). Say `No' to these options.}
2409 \exstep{Once the machine has loaded, it will display a series of IP addresses
2410 for the different services provided by the VM. Make a note of the JABAWS URL --
2411 this will begin with `http:' and end with `/jabaws''.}
2414 \exercise{Configuring Jalview to access your new JABAWS virtual appliance}{
2415 \label{confnewjabawsappl}
2416 \exstep{Start Jalview (If you have not done so already).}
2417 \exstep{Enable the Jalview Java Console by selecting its option from the Tools
2420 {\sl Alternately, use the System Java console if you have
2421 configured it to open when Jalview is launched, {\sl via} your system's Java
2422 preferences (under the `Advanced' tab on Windows).}}
2423 \exstep{Open the {\sl Preferences} dialog and locate the Web Services tab.}
2424 \exstep{Add the URL for the new JABAWS server you started in Exercise
2425 \ref{jabawsvmex} to the list of JABAWS urls using the `New Service
2427 \exstep{You will be asked if you want to test the service. Hit `Yes' to do this
2428 -- you should then see some output in the console window.
2430 {\sl Take a close look at the output in the console. What do you think is
2433 \exstep{Hit OK to save your preferences -- you have now added a new JABA
2434 service to Jalview!}
2435 \exstep{Try out your new JABA services by loading the ferredoxin sequences from
2436 http://www.jalview.org/tutorial/alignment.fa}
2437 \exstep{Launch an alignment using one
2438 of the JABA methods provided by your server. It will be listed under the JABAWS Alignment submenu of the {\sl Web Service} menu on the alignment window.
2440 {\sl Note: You can watch the JABA VM appliance's process working by opening the
2441 process monitor on your system. (On Windows XP, this involves right-clicking the
2442 system clock and opening the task manager -- then selecting the 'Processes' tab
2447 \section{Multiple Sequence Alignment}
2449 Sequences can be aligned using a range of algorithms provided by JABA web
2450 services. These include ClustalW\footnote{{\sl ``CLUSTAL W: improving the
2451 sensitivity of progressive multiple sequence alignment through sequence
2452 weighting, position specific gap penalties and weight matrix choice."} Thompson
2453 JD, Higgins DG, Gibson TJ (1994) {\sl Nucleic Acids Research} {\bf 22},
2454 4673-80}, Muscle\footnote{{\sl ``MUSCLE: a multiple sequence alignment method
2455 with reduced time and space complexity"} Edgar, R.C.
2456 (2004) {\sl BMC Bioinformatics} {\bf 5}, 113}, MAFFT\footnote{{\sl ``MAFFT: a
2457 novel method for rapid multiple sequence alignment based on fast Fourier
2458 transform"} Katoh, K., Misawa, K., Kuma, K. and Miyata, T. (2002) {\sl Nucleic
2459 Acids Research} {\bf 30}, 3059-3066. and {\sl ``MAFFT version 5:
2460 improvement in accuracy of multiple sequence alignment"} Katoh, K., Kuma, K.,
2461 Toh, H. and Miyata, T. (2005) {\sl Nucleic Acids Research} {\bf 33}, 511-518.},
2462 ProbCons,\footnote{PROBCONS: Probabilistic Consistency-based Multiple Sequence
2464 Do, C.B., Mahabhashyam, M.S.P., Brudno, M., and Batzoglou, S.
2465 (2005) {\sl Genome Research} {\bf 15} 330-340.} T-COFFEE\footnote{T-Coffee:
2466 A novel method for multiple sequence alignments. (2000) Notredame, Higgins and
2467 Heringa {\sl JMB} {\bf 302} 205-217} and Clustal Omega.\footnote{Fast, scalable
2468 generation of high-quality protein multiple sequence alignments using Clustal
2469 Omega. Sievers F, Wilm A, Dineen DG, Gibson TJ, Karplus K, Li W, Lopez R,
2470 McWilliam H, Remmert M, Soding J, Thompson JD, Higgins DG (2011) {\sl Molecular
2471 Systems Biology} {\bf 7} 539
2472 \href{http://dx.doi.org/10.1038/msb.2011.75}{doi:10.1038/msb.2011.75}} Of these,
2473 T-COFFEE is the slowest, but also the most accurate. ClustalW is historically
2474 the most widely used. Muscle is faster than ClustalW and probably the most
2475 accurate for smaller alignments and MAFFT is probably the best for large
2476 alignments, however Clustal Omega, which was released in 2011, is arguably the
2477 fastest and most accurate tool for protein multiple alignment.
2480 To run an alignment web service, select the appropriate method from the {\sl
2481 Web Service $\Rightarrow$ Alignment $\Rightarrow$ \ldots} submenu (Figure
2482 \ref{webservices}). For each service you may either perform an alignment with
2483 default settings, use one of the available presets, or customise the parameters
2484 with the `{\sl Edit and Run ..}' dialog box. Once the job is submitted, a
2485 progress window will appear giving information about the job and any errors that
2486 occur. After successful completion of the job, a new window is opened with the
2487 results, in this case an alignment. By default, the new alignment will be
2488 ordered in the same way as the input sequences; however, many alignment programs
2489 re-order the input to place homologous sequences close together. This ordering
2490 can be recovered using the `Original ordering' entry within the {\sl Calculate
2491 $\Rightarrow$ Sort } sub menu.
2493 \begin{figure}[htbp]
2495 \parbox[c]{1.5in}{\includegraphics[width=1.5in]{images/ws1.pdf}}
2496 \parbox[c]{2.5in}{\includegraphics[width=2.5in]{images/ws2.pdf}}
2497 \parbox[c]{2in}{\includegraphics[width=2in]{images/ws3.pdf}}
2498 \caption{{\bf Multiple alignment via web services} The appropriate method is
2499 selected from the menu (left), a status box appears (centre), and the results
2500 appear in a new window (right)}
2505 \subsubsection{Realignment}
2507 The re-alignment option is currently only supported by ClustalW and Clustal
2508 Omega. When performing a re-alignment, Jalview submits the current selection to
2509 the alignment service complete with any existing gaps. This approach is useful
2510 when one wishes to align additional sequences to an existing alignment without
2511 any further optimisation to the existing alignment. The Re-alignment service
2512 provided by ClustalW in this case is effectively a simple form of profile
2515 \subsubsection{Alignments of sequences that include hidden regions}
2517 If the view or selected region that is submitted for alignment contains hidden
2518 regions, then {\bf only the visible sequences will be submitted to the service}.
2519 Furthermore, each contiguous segment of sequences will be aligned independently
2520 (resulting in a number of alignment `subjobs' appearing in the status window).
2521 Finally, the results of each subjob will be concatenated with the hidden regions
2522 in the input data prior to their display in a new window. This approach ensures
2523 that 1) hidden column boundaries in the input data are preserved in the
2524 resulting alignment - in a similar fashion to the constraint that hidden columns
2525 place on alignment editing (see Section \ref{lockededits}), and 2) hidden
2526 columns can be used to preserve existing parts of an alignment whilst the
2527 visible parts are locally refined.
2529 \exercise{Multiple Sequence Alignment}{
2530 \exstep{ Close all windows and open the alignment at {\sf
2531 http://www.jalview.org/tutorial/unaligned.fa}. Select {\sl
2532 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{Select the first sequence set by clicking on the window and try running ClustalW and MAFFT (from the {\sl Web Service $\Rightarrow$ Alignment} menu) on the same initial alignment. Compare them and you should notice small differences. }
2533 \exstep{Select the last three sequences in the MAFFT alignment, and de-align them with {\sl Edit $\Rightarrow$ Remove All Gaps}. Press [ESC] to deselect them and then submit the view for re-alignment with ClustalW.}
2534 \exstep{Use [CTRL]-Z to recover the alignment of the last three sequences in the MAFFT alignment. Once the ClustalW re-alignment has completed, compare the results of re-alignment of the three sequences with their alignment in the original MAFFT result.}
2535 \exstep{Select columns 60 to 125 in the original MAFFT alignment and hide them. Select {\sl Web Services $\Rightarrow$ 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.}
2536 \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. 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.}
2540 \subsection{Customising the parameters used for alignment}
2542 JABA web services allow you to vary the parameters used when performing a
2543 bioinformatics analysis. For JABA alignment services, this means you are
2544 usually able to modify the following types of parameters:
2545 \begin{list}{$\bullet$}{}
2546 \item Amino acid or nucleotide substitution score matrix
2547 \item Gap opening and widening penalties
2548 \item Types of distance metric used to construct guide trees
2549 \item Number of rounds of re-alignment or alignment optimisation
2553 \subsubsection{Getting help on the parameters for a service}
2554 Each parameter available for a method usually has a short description, which
2555 jalview will display as a tooltip, or as a text pane that can be opened under
2556 the parameter's controls. In the parameter shown in Figure
2557 \ref{clustalwparamdetail}, the description was opened by selecting the button on the left hand side. Online help for the
2558 service can also be accessed, by right clicking the button and selecting a URL
2559 from the pop-up menu that will open.
2561 \begin{figure}[htbp]
2563 \includegraphics[width=2.5in]{images/clustalwparamdetail.pdf}
2564 \caption{{\bf ClustalW parameter slider detail}. From the ClustalW {\sl Clustal $\Rightarrow$ Edit settings and run ...} dialog box. }
2565 \label{clustalwparamdetail}
2569 \subsection{Alignment Presets}
2570 The different multiple alignment algorithms available from JABA vary greatly in
2571 the number of adjustable parameters, and it is often difficult to identify what
2572 are the best values for the sequences that you are trying to align. For these
2573 reasons, each JABA service may provide one or more presets -- which are
2574 pre-defined sets of parameters suited for particular types of alignment
2575 problem. For instance, the Muscle service provides the following presets:
2576 \begin{list}{$\bullet$}{}
2578 \item Protein alignments (fastest speed)
2579 \item Nucleotide alignments (fastest speed)
2582 The presets are displayed in the JABA web services submenu, and can also be
2583 accessed from the parameter editing dialog box, which is opened by selecting
2584 the `{\sl Edit settings and run ...}' option from the web serviceÔøΩs menu. If you have used
2585 a preset, then it will be mentioned at the beginning of the job status file shown
2586 in the web service job progress window.
2588 \subsubsection{Alignment Service Limits}
2589 Multiple alignment is a computationally intensive calculation. Some JABA server
2590 services and service presets only allow a certain number of sequences to be
2591 aligned. The precise number will depend on the server that you are using to
2592 perform the alignment. Should you try to submit more sequences than a service
2593 can handle, then an error message will be shown informing you of the maximum
2594 number allowed by the server.
2596 \subsection{User defined Presets}
2597 Jalview allows you to create your own presets for a particular service. To do
2598 this, select the `{\sl Edit settings and run ...}' option for your service,
2599 which will open a parameter editing dialog box like the one shown in Figure
2600 \ref{jwsparamsdialog}.
2602 The top row of this dialog allows you to browse the existing presets, and
2603 when editing a parameter set, allows you to change its nickname. As you
2604 adjust settings, buttons will appear at the top of the parameters dialog that
2605 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
2606 description for the parameter set, which will be shown in the tooltip for the
2607 parameter set's entry in the web services menu.
2609 \begin{figure}[htbc]
2611 \includegraphics[width=3in]{images/jvaliwsparamsbox.pdf}
2612 \caption{{\bf Jalview's JABA alignment service parameter editing dialog box}.}
2613 \label{jwsparamsdialog} }
2616 \subsubsection{Saving parameter sets}
2617 When creating a custom parameter set, you will be asked for a file name to save
2618 it. The location of the file is recorded in the Jalview user preferences in the
2619 same way as a custom alignment colourscheme, so when Jalview is launched again,
2620 it will show your custom preset amongst the options available for running the
2624 % \exercise{Creating and using user defined presets}{\label{createandusepreseex}
2625 % \exstep{Import the file at
2626 % \textsf{http://www.jalview.org/tutorial/fdx\_unaligned.fa} into jalview.}
2627 % \exstep{Use the `{\slDiscover Database Ids}' function to recover the PDB cross
2628 % references for the sequences.}
2629 % \exstep{Align the sequences using the default ClustalW parameters.}
2630 % \exstep{Use the `{\sl Edit and run..}'
2631 % option to open the ClustalW parameters dialog box, and create a new preset using
2632 % the following settings:
2633 % \begin{list}{$\bullet$}{}
2634 % \item BLOSUM matrix (unchanged)
2635 % \item Gap Opening and End Gap penalties = 0.05
2636 % \item Gap Extension and Separation penalties = 0.05
2639 % As you edit the parameters, buttons will appear on the dialog box
2640 % allowing you revert your changes or save your settings as a new parameter
2643 % Before you save your settings, remeber to give them a meaningful name by editing
2644 % the text box at the top of the dialog box.
2646 % \exstep{Repeat the alignment using your new parameter set by selecting it from
2647 % the {\sl ClustalW Presets menu}.}
2648 % \exstep{These sequences have PDB structures associated with them, so it is
2649 % possible to compare the quality of the alignments.
2651 % Use the {\sl View all {\bf N}
2652 % structures} option to calculate the superposition of 1fdn on 1fxd for both
2653 % alignments (refer to section \ref{superposestructs} for instructions). Which
2654 % alignment gives the best RMSD ? }
2655 % \exstep{Apply the same alignment parameter settings to the example alignment
2656 % (available from \textsf{http://www.jalview.org/examples/uniref50.fa}).
2658 % Are there differences ? If not, why not ?
2662 \section{Protein alignment conservation analysis}
2664 The {\sl Web Service $\Rightarrow$ Conservation} menu controls the computation
2665 of up to 17 different amino acid conservation measures for the current alignment
2666 view. The JABAWS AACon Alignment Conservation Calculation Service, which is used
2667 to calculate these scores, provides a variety of standard measures described by
2668 Valdar in 2002\footnote{Scoring residue conservation. Valdar (2002) {\sl
2669 Proteins: Structure, Function, and Genetics} {\bf 43} 227-241.} as well as an efficient implementation of the SMERFs
2670 score developed by Manning et al. in 2008.\footnote{SMERFS Score Manning et al. {\sl BMC
2671 Bioinformatics} 2008, {\bf 9} 51 \href{http://dx.doi.org/10.1186/1471-2105-9-51}{doi:10.1186/1471-2105-9-51}}
2673 \subsubsection{Enabling and disabling AACon calculations}
2674 When the AACon Calculation entry in the {\sl Web Services $\Rightarrow$
2675 Conservation} menu is ticked, AACon calculations will be performed every time
2676 the alignment is modified. Selecting the menu item will enable or disable
2677 automatic recalculation.
2679 \subsubsection{Configuring which AACon calculations are performed}
2680 The {\sl Web Services $\Rightarrow$ Conservation $\Rightarrow$ Change AACon
2681 Settings ...} menu entry will open a web services parameter dialog for the
2682 currently configured AACon server. Standard presets are provided for quick and
2683 more expensive conservation calculations, and parameters are also provided to
2684 change the way that SMERFS calculations are performed.
2685 AACon settings for an alignment are saved in Jalview projects along with the
2686 latest calculation results.
2688 \subsubsection{Changing the server used for AACon calculations}
2689 If you are working with alignments too large to analyse with the public JABAWS
2690 server, then you will most likely have already configured additional JABAWS
2691 servers. By default, Jalview will chose the first AACon service available from
2692 the list of JABAWS servers available. If available, you can switch to use
2693 another AACon service by selecting it from the {\sl Web Services $\Rightarrow$
2694 Conservation $\Rightarrow$ Switch Server} submenu.
2697 \section{Protein Secondary Structure Prediction}
2698 \label{protsspredservices}
2699 Protein secondary structure prediction is performed using the
2700 Jpred\footnote{{\sl ``The Jpred 3 Secondary Structure Prediction Server''} Cole, C., Barber, J. D. and Barton, G. J. (2008) {\sl Nucleic Acids Research} {\bf 36}, (Web Server Issue) W197-W201
2702 {\sl ``Jpred: A Consensus Secondary Structure Prediction Server''} Cuff, J. A.,
2703 Clamp, M. E., Siddiqui, A. S., Finlay, M. and Barton, G. J. (1998) {\sl
2704 Bioinformatics} {\bf 14}, 892-893} server at the University of
2705 Dundee\footnote{http://www.compbio.dundee.ac.uk/www-jpred/}. The behaviour of
2706 this calculation depends on the current selection:
2707 \begin{list}{$\circ$}{}
2708 \item If nothing is selected, Jalview will check the length of each alignment row to determine if the visible sequences in the view are aligned.
2710 \item If all rows are the same length (often due to the
2711 application of the {\sl Edit $\Rightarrow$ Pad Gaps} option), then
2712 a JPred prediction will be run for the first sequence in the
2713 alignment, using the current alignment as the profile to use for prediction.
2714 \item Otherwise, just the first sequence will be submitted for a
2715 full JPred prediction.
2717 \item If just one sequence (or a region in one sequence) has been selected, it
2718 will be submitted to the automatic JPred prediction server for homolog detection
2720 \item If a set of sequences are selected, and they appear to be aligned using
2721 the same criteria as above, then the alignment will be used for a JPred
2722 prediction on the first sequence in the set (that is, the one that appears first in the alignment window).
2724 Jpred is launched in the same way as the other web services. Select {\sl Web
2725 Services $\Rightarrow$ Secondary Structure Prediction $\Rightarrow$ JNet
2726 Secondary Structure Prediction}\footnote{JNet is the Neural Network based
2727 secondary structure prediction method that the JPred server uses.} from the
2728 alignment window menu (Figure \ref{jpred}).
2729 A status window opens to inform you of the progress of the job. Upon completion, a new alignment window opens and the Jpred
2730 predictions are included as annotations. Consult the Jpred documentation for
2731 information on interpreting these results.
2733 \begin{figure}[htbp]
2735 \includegraphics[width=2.25in]{images/jpred1.pdf}
2736 \includegraphics[width=3in]{images/jpred2.pdf}
2737 \caption{{\bf Secondary Structure Prediction} Status (left) and results (right) windows for JNet predictions. }
2742 \subsubsection{Hidden Columns and JNet Predictions}
2744 Hidden columns can be used to exclude parts of a sequence or profile from the
2745 input sent to the JNet service. For instance, if a sequence is known to include
2746 a large loop insertion, hiding that section prior to submitting the JNet
2747 prediction can produce different results. In some cases, these secondary structure predictions can be more reliable for sequence on either side of the insertion\footnote{This, of course, cannot be guaranteed.}. Prediction results returned from the service will
2748 be mapped back onto the visible parts of the sequence, to ensure a single frame
2749 of reference is maintained in your analysis.
2751 \exercise{Secondary Structure Prediction}{
2752 \label{secstrpredex}
2753 \exstep{ Open the alignment at \url{http://www.jalview.org/tutorial/alignment.fa}. Select the sequence {\sl FER\_MESCR} by clicking on the sequence ID. Then select {\sl Web Services $\Rightarrow$ Secondary Structure Prediction $\Rightarrow$ JNet Secondary Structure Prediction} from the alignment window menu. A status window will appear and after some time (about 2-4 min) a new window with the JPred prediction will appear. Note that the number of sequences in the results window is many more than in the original alignment as JNet performs a PSI-BLAST search to expand the prediction dataset.
2755 % TODO: check how long this takes - about 2 mins once it gets on the cluster.
2757 Select a different sequence and perform a JNet prediction in the same way. There will probably be minor differences in the predictions.
2760 Select the second sequence prediction, and copy and paste it into the first
2761 prediction window. You can now compare the two predictions. Jnet secondary structure prediction annotations are examples of {\bf sequence-associated alignment annotation}.
2762 % which is described in Section \ref{seqassocannot} below.
2765 Select and hide some columns in one of the profiles that were returned from the JNet service, and then submit the profile for prediction again.
2768 When you get the result, verify that the prediction has not been made for the
2769 hidden parts of the profile, and that the JPred reliability scores differ from the prediction made on the full profile.
2771 {\sl Note: you may want to keep this data for use in exercise \ref{viewannotfileex}.}
2775 \section{Protein Disorder Prediction}
2776 \label{protdisorderpred}
2778 Disordered regions in proteins were classically thought to correspond to
2779 'linkers' between distinct protein domains, but disorder can also play a role in
2780 function. The {\sl Web Services $\Rightarrow$ Disorder} menu in the alignment window
2781 allows access to protein disorder prediction services provided by the configured
2784 \subsection{Disorder prediction results}
2785 Each service operates on sequences in the alignment to identify regions likely
2786 to be unstructured or flexible, or alternately, fold to form globular domains.
2787 As a consequence, disorder predictor results include both sequence features and
2788 sequence associated alignment annotation rows. Section \ref{featannot} describes
2789 the manipulation and display of these data in detail, and {\bf Figure
2790 \ref{alignmentdisorder}} demonstrates how sequence feature shading and
2791 thresholding (described in Section \ref{featureschemes}) can be used to
2792 highlight differences in disorder prediction across aligned sequences.
2794 \begin{figure}[htbp]
2796 \includegraphics[width=5in]{images/disorderpred.pdf}
2797 \caption{{\bf Shading alignment by sequence disorder}. Alignment of Interleukin IV homologs coloured with Blosum62 with protein disorder prediction sequence features overlaid, shaded according to their score. Borderline disordered regions appear white, reliable predictions are either Green or Brown depending on the type of disorder prediction. }
2798 \label{alignmentdisorder}
2802 \subsubsection{Navigating large sets of disorder predictions}
2804 {\bf Figure \ref{alignmentdisorderannot}} shows a single sequence annotated with
2805 a range of disorder predictions. Disorder prediction annotation rows are
2806 associated with a sequence in the same way as secondary structure prediction
2807 results. When browsing an alignment containing large numbers of disorder
2808 prediction annotation rows, clicking on the annotation row label will highlight
2809 the associated sequence in the alignment display, and double clicking will
2810 select that sequence.
2812 \begin{figure}[htbp]
2814 \includegraphics[width=5in]{images/disorderpredannot.pdf}
2815 \caption{{\bf Annotation rows for several disorder predictions on a sequence}. A zoomed out view of a prediction for a single sequence. The sequence is shaded to highlight disorderd regions (brown and grey), and the line plots below the Sequence show the raw scores for various disorder predictors. Horizontal lines on each graph mark the level at which disorder predictions become significant. }
2816 \label{alignmentdisorderannot}
2821 \subsection{Disorder predictors provided by JABAWS 2.0}
2822 For full details of each predictor and the results that Jalview can display,
2824 \href{http://www.jalview.org/help/html/webServices/proteinDisorder.html}{Jalview's
2825 protein disorder service documentation}. Short descriptions of the methods provided in JABAWS 2.0 are given below:
2827 \subsubsection{DisEMBL}
2828 \href{http://dis.embl.de/}{DisEMBL (Linding et al., 2003)} is a set of machine-learning based predictors trained to
2829 recognise disorder-related annotation found on PDB structures.
2831 \textbf{COILS} Predicts
2832 loops/coils according to DSSP
2833 definitions\footnote{DSSP Classifications of secondary structure are: $\alpha$-helix (H), 310-helix (G), $\beta$-strand (E)
2834 are ordered, and all other states ($\beta$-bridge (B), $\beta$-turn (T), bend (S),
2835 $\pi$-helix (I), and coil (C)) considered loops or coils.}. Features mark range(s) of
2836 residues predicted as loops/coils, and annotation row gives raw value
2837 for each residue. Value over 0.516 indicates loop/coil.
2839 \textbf{HOTLOOPS} constitute a refined subset of \textbf{COILS}, namely those loops with
2840 a high degree of mobility as determined from C$\alpha$ temperature factors (B
2841 factors). It follows that highly dynamic loops should be considered
2842 protein disorder. Features mark range(s) of residues predicted to
2843 be hot loops and annotation row gives raw value for each
2844 residue. Values over 0.6 indicates hot loop.
2846 \textbf{REMARK465} ``Missing
2847 coordinates in X-ray structure as defined by remark465 entries in PDB.
2848 Nonassigned electron densities most often reflect intrinsic disorder,
2849 and have been used early on in disorder prediction.'' Features give
2850 range(s) of residues predicted as disordered, and annotation rows gives
2851 raw value for each residue. Values over 0.1204 indicates disorder.
2853 \subsubsection{RONN {\sl a.k.a.} Regional Order Neural Network}
2854 \href{http://www.strubi.ox.ac.uk/RONN}{RONN} employs an approach
2855 known as the `bio-basis' method to predict regions of disorder in
2856 sequences based on their local similarity with a gold-standard set of
2857 disordered protein sequences. It yields a set of disorder prediction
2858 scores, which are shown as sequence annotation below the alignment.
2860 \textbf{JRonn}\footnote{JRonn denotes the score for this server because JABAWS
2861 runs a Java port of RONN developed by Peter Troshin and distributed as
2862 part of \href{http://www.biojava.org/}{Biojava 3}} Annotation Row gives RONN score for each residue in
2863 the sequence. Scores above 0.5 identify regions of the protein likely
2866 \subsubsection{IUPred}
2867 \href{http://iupred.enzim.hu/Help.php}{IUPred} employs
2868 an empirical model to estimate likely regions of disorder. There are
2869 three different prediction types offered, each using different
2870 parameters optimized for slightly different applications. It provides
2871 raw scores based on two models for predicting regions of `long
2872 disorder' and `short disorder'. A third predictor identifies regions
2873 likely to form structured domains.
2875 \textbf{Long disorder} Annotation rows predict
2876 context-independent global disorder that encompasses at least 30
2877 consecutive residues of predicted disorder. A 100 residue
2878 window is used for calculation. Values above 0.5 indicates the residue is
2879 intrinsically disordered.
2881 \textbf{Short disorder} Annotation rows predict for short, (and
2882 probably) context-dependent, disordered regions, such as missing
2883 residues in the X-ray structure of an otherwise globular protein.
2884 Employs a 25 residue window for calculation, and includes adjustment
2885 parameter for chain termini which favors disorder prediction at the
2886 ends. Values above 0.5 indicate short-range disorder.
2888 \textbf{Structured domains} are marked with sequence Features. These highlight
2889 likely globular domains useful for structure genomics investigation. Post-analysis of disordered region profile to find continuous regions
2890 confidently predicted to be ordered. Neighbouring regions close to
2891 each other are merged, while regions shorter than the minimal domain
2892 size of at least 30 residues are ignored.
2894 \subsubsection{GLOBPLOT}
2895 \href{http://globplot.embl.de/}{GLOBPLOT} defines regions of
2896 globularity or natively unstructured regions based on a running sum of
2897 the propensity of residues to be structured or unstructured. The
2898 propensity is calculated based on the probability of each amino acid
2899 being observed within well defined regions of secondary structure or
2900 within regions of random coil. The initial signal is smoothed with a
2901 Savitzky-Golay filter, and its first order derivative
2902 computed. Residues for which the first order derivative is positive
2903 are designated as natively unstructured, whereas those with negative
2904 values are structured.
2906 {\bf Disordered region} sequence features are created marking mark range(s) of residues with positive first order derivatives, and
2907 \textbf{Globular Domain} features mark long stretches of order. \textbf{Dydx} annotation rows gives the first order derivative of smoothed score. Values above 0 indicates
2908 residue is disordered.
2910 \textbf{Smoothed Score and Raw Score} annotation rows give the smoothed and raw scores used to create the differential signal that
2911 indicates the presence of unstructured regions. These are hidden
2912 by default, but can be shown by right-clicking on the alignment
2913 annotation panel and selecting \textbf{Show hidden annotation}.
2915 \section{Features and Annotation}
2917 Features and annotations are additional information that is overlaid on the sequences and the alignment. Generally speaking, annotations are associated with columns in the alignment. Features are associated with specific residues in the sequence.
2919 Annotations are shown below the alignment in the annotation panel, and often reflect properties of the alignment as a whole. The Conservation, Consensus and Quality scores are examples of dynamic annotation, so as the alignment changes, they change along with it. Conversely, sequence features are properties of the individual sequences, so they do not change with the alignment, but are shown mapped on to specific residues within the alignment.
2921 Features and annotation can be interactively created, or retrieved from external
2922 data sources. DAS (the Distributed Annotation System) is the primary source of
2923 sequence features, whilst webservices like JNet (see \ref{jpred} above) can be used to analyse a given sequence or alignment and generate annotation for it.
2926 \subsection{Creating sequence features}
2927 Sequence features can be created simply by selecting the area in a sequence (or sequences) to form the feature and selecting {\sl Selection $\Rightarrow$ Create Sequence Feature } from the right-click context menu (Figure \ref{features}). A dialogue box allows the user to customise the feature with respect to name, group, and colour. The feature is then associated with the sequence. Moving the mouse over a residue associated with a feature brings up a tool tip listing all features associated with the residue.
2929 \begin{figure}[htbp]
2931 \includegraphics[width=2in]{images/feature1.pdf}
2932 \includegraphics[width=2.5in]{images/feature2.pdf}
2933 \includegraphics[width=1.5in]{images/feature3.pdf}
2934 \caption{{\bf Creating sequence features.} Features can readily be created from selections via the context menu and are then displayed on the sequence. }
2939 Creation of features from a selection spanning multiple sequences results in the creation of one feature per sequence. Each feature remains associated with it's own sequence.
2941 \subsection{Customising feature display}
2943 Feature display can be toggled on or off by selecting the {\sl View
2944 $\Rightarrow$ Show Sequence Features} menu option. When multiple features are
2945 present it is usually necessary to customise the display. Jalview allows the
2946 display, colour, rendering order and transparency of features to be modified
2947 {\sl via} the {\sl View $\Rightarrow$ Feature Settings\ldots} menu option. This
2948 brings up a dialogue window (Figure \ref{custfeat}) which allows the
2949 visibility of individual feature types to be selected, colours changed (by
2950 clicking on the colour of each sequence feature type) and the rendering order
2951 modified by dragging feature types to a new position in the list. Dragging the
2952 slider alters the transparency of the feature rendering. The Feature
2953 Settings dialog also includes functions for more advanced feature shading
2954 schemes and buttons for sorting the alignment according to the distribution of
2955 features. These capabilities are described further in sections
2956 \ref{featureschemes} and \ref{featureordering}.
2958 \begin{figure}[htbp]
2960 \includegraphics[width=4in]{images/features4.pdf}
2961 \caption{{\bf Multiple sequence features.} An alignment with JPred secondary structure prediction annotation below it, and many sequence features overlaid onto the aligned sequences. The tooltip lists the features annotating the residue below the mouse-pointer.}
2965 \begin{figure}[htbp]
2967 \includegraphics[width=4in]{images/features5.pdf}
2968 \caption{{\bf Customising sequence features.} Features can be recoloured, switched on or off and have the rendering order changed. }
2973 \subsection{Sequence Feature File Formats}
2975 Jalview supports the widely used GFF tab delimited format\footnote{see
2976 http://www.sanger.ac.uk/resources/software/gff/spec.html} and its own Jalview
2977 Features file format for the import of sequence annotation. Features and
2978 alignment annotation are also extracted from other formats such as Stockholm,
2979 and AMSA. URL links may also be attached to features. See the online
2980 documentation for more details of the additional capabilities of the jalview
2983 \exercise{Creating features}{
2984 \exstep{Open the alignment at \textsf{http://www.jalview.org/tutorial/alignment.fa}. We know that the Cysteine residues at columns 97, 102, 105 and 135 are involved in iron binding so we will create them as features. Navigate to column 97, sequence 1. Select the entire column by clicking in the ruler bar. Then right-click on the selection to bring up the context menu and select {\sl Selection $\Rightarrow$ Create Sequence Feature}. A dialogue box will appear.
2987 Enter a suitable Sequence Feature Name (e.g. ``Iron binding site") in the
2988 appropriate box. Click on the Feature Colour bar to change the colour if
2989 desired, add a short description (``One of four Iron binding Cysteines") and press OK. The features will then appear on the sequences. } \exstep{Roll the mouse cursor over the new features. Note that the position given in the tool tip is the residue number, not the column number. To demonstrate that there is one feature per sequence, clear all selections by pressing [ESC] then insert a gap in sequence 3 at column 95. Roll the mouse over the features and you will see that the feature has moved with the sequence. Delete the gap you created.
2992 Add a similar feature to column 102. When the feature dialogue box appears, clicking the Sequence Feature Name box brings up a list of previously described features. Using the same Sequence Feature Name allows the features to be grouped.}
2993 \exstep{Select {\sl View $\Rightarrow$ Feature Settings\ldots} from the
2994 alignment window menu. The Sequence Feature Settings window will appear. Move
2995 this so that you can see the features you have just created. Click the check
2996 box for ``Iron binding site" under {\sl Display} and note that display of this
2997 feature type is now turned off. Click it again and note that the features are
2998 now displayed. Close the sequence feature settings box by clicking OK or
3001 \subsection{Creating user defined annotation}
3003 Annotations are properties that apply to the alignment as a whole and are visualized on rows in the annotation panel.
3004 To create a new annotation row, right click on the annotation label panel and select the {\sl Add New Row} menu option (Figure \ref{newannotrow}). A dialogue box appears. Enter the label to use for this row and a new row will appear.
3006 \begin{figure}[htbp]
3008 \includegraphics[width=1.3in]{images/annots1.pdf}
3009 \includegraphics[width=2in]{images/annots2.pdf}
3010 \caption{{\bf Creating a new annotation row.} Annotation rows can be reordered by dragging them to the desired place.}
3015 To create a new annotation, first select all the positions to be annotated on the appropriate row. Right-clicking on this selection brings up the context menu which allows the insertion of graphics for secondary structure ({\sl Helix} or {\sl Sheet}), text {\sl Label} and the colour in which to present the annotation (Figure \ref{newannot}). On selecting {\sl Label} a dialogue box will appear, requesting the text to place at that position. After the text is entered, the selection can be removed and the annotation becomes clearly visible\footnote{When annotating a block of positions, the text can be partly obscured by the selection highlight. Pressing the [ESC] key clears the selection and the label is then visible.}. Annotations can be coloured or deleted as desired.
3017 \begin{figure}[htbp]
3019 \includegraphics[width=2in]{images/annots3.pdf}
3020 \includegraphics[width=2in]{images/annots4.pdf}
3021 \includegraphics[width=2in]{images/annots5.pdf}
3022 \caption{{\bf Creating a new annotation.} Annotations are created from a selection on the annotation row and can be coloured as desired.}
3027 \exercise{Annotating alignments}{
3028 \exstep{Load the alignment at \textsf{http://www.jalview.org/tutorial/alignment.fa}. Right-click on the annotation label for {\sl Conservation} to bring up the context menu and select {\sl Add New Row}. A dialogue box will appear asking for {\sl Label for annotation}. Enter ``Iron binding site" and click OK. A new, empty, row appears.
3031 Navigate to column 97. Select column 97 on the new annotation row. Right click on the selection and select {\sl Label} from the context menu. Enter ``Fe" in the box and click OK. Right-click on the selection again and select {\sl Colour}. Choose a colour from the colour chooser dialogue and click OK. Press [ESC] to remove the selection.
3033 \exstep{ Select columns 70-77 on the annotation row. Right-click and choose {\sl Sheet} from the context menu. You will be prompted for a label. Enter ``B" and press OK. A new line showing the sheet as an arrow appears. The colour of the label can be changed but not the colour of the sheet arrow.
3035 \exstep{Right click on the annotation row that you just created. Select {\sl Export Annotation} and, in the {\bf Export Annotation} dialog box that will open, select the Jalview format and click the [To Textbox] button.
3037 The format for this file is given in the Jalview help. Press [F1] to open it, and find the ``Annotations File Format'' entry in the ``Alignment Annotations'' section of the contents pane. }
3039 \exstep{Export the file to a text editor and edit the file to change the name of the annotation row. Save the file and drag it onto the alignment view.}
3040 \exstep{Try to add an additional helix somewhere along the row by editing the file and re-importing it.
3041 {\sl Hint: Use the {\bf Export Annotation} function to view what helix annotation looks like in a jalview annotation file.}}
3042 \exstep{Use the {\sl Alignment Window $\Rightarrow$ File $\Rightarrow$ Export Annotations...} function to export all the alignment's annotation to a file.}
3043 \exstep{Open the exported annotation in a text editor, and use the {\bf Annotation File Format} documentation to modify the style of the Conservation, Consensus and Quality annotation rows so they appear as several lines on a single line graph.
3044 {\sl Hint: You need to change the style of annotation row in the first field of the annotation row entry in the file, and create an annotation row grouping to overlay the three quantitative annotation rows.}
3046 \label{viewannotfileex}\exstep{Recover or recreate the secondary structure
3047 prediction that you made in exercise \ref{secstrpredex}. Use the {\sl File $\Rightarrow$ Export Annotation} function to view the Jnet secondary structure prediction annotation row. Note the {\bf SEQUENCE\_REF} statements surrounding the row specifying the sequence association for the annotation. } }
3049 \section{Importing features from databases}
3050 \label{featuresfromdb}
3051 Jalview supports feature retrieval from public databases either directly or {\sl
3052 via} the Distributed Annotation System (DAS\footnote{http://www.biodas.org/}).
3053 It includes built in parsers for Uniprot and EMBL records retrieved from the
3054 EBI. Sequences retrieved from these sources using the sequence fetcher (see
3055 Section \ref{fetchseq}) will already possess features.
3057 \subsection{Sequence Database Reference Retrieval}
3059 Jalview maintains a list of external database references for each sequence in
3060 an alignment. These are listed in a tooltip when the mouse is moved over the
3061 sequence ID when the {\sl View $\Rightarrow$ Sequence ID Tooltip $\Rightarrow$
3062 Show Database Refs } option is enabled. Sequences retrieved using the sequence
3063 fetcher will always have at least one database reference, but alignments
3064 imported from an alignment file generally have no database references.
3066 \subsubsection{Database References and Sequence Coordinate Systems}
3068 Jalview displays features in the local sequence's coordinate system which is
3069 given by its `start' and `end'. Any sequence features on the sequence will be
3070 rendered relative to the sequence's start position. If the start/end positions
3071 do not match the coordinate system from which the features were defined, then
3072 the features will be displayed incorrectly.
3074 \subsubsection{Viewing and exporting a sequence's database annotation}
3076 You can export all the database cross references and annotation terms shown in
3077 the sequence ID tooltip for a sequence by right-clicking and selecting the {\sl
3078 [Sequence ID] $\Rightarrow$ Sequence details \ldots} option from the popup menu.
3079 A similar option is provided in the {\sl Selection} sub-menu allowing you to
3080 obtain annotation for the sequences currently selected.
3083 The {\sl Sequence Details
3084 \ldots} option will open a window containing the same text as would be shown in
3085 the tooltip window, including any web links associated with the sequence. The
3086 text is HTML, and options on the window allow the raw code to be copied and
3087 pasted into a web page.}
3089 \centerline{\includegraphics[width=2.2in]{images/seqdetailsreport.pdf}}}
3091 \subsubsection{Automatically discovering a sequence's database references}
3092 Jalview includes a function to automatically verify and update each sequence's
3093 start and end numbering against any of the sequence databases that the {\sl
3094 Sequence Fetcher} has access to. This function is accessed from the {\sl
3095 Webservice $\Rightarrow$ Fetch DB References} sub-menu in the Alignment
3096 window. This menu allows you to query either the set of {\sl Standard
3097 Databases}, which includes EMBL, Uniprot, the PDB, and the currently selected
3098 DAS sequence sources, or just a specific datasource from one of the submenus.
3099 When one of the entries from this menu is selected, Jalview will use the ID
3100 string from each sequence in the alignment or in the currently selected set to
3101 retrieve records from the external source. Any sequences that are retrieved are
3102 matched against the local sequence, and if the local sequence is found to be a
3103 sub-sequence of the retrieved sequence then the local sequence's start/end
3104 numbering is updated. A new database reference mapping is created, mapping the
3105 local sequence to the external database, and the local sequence inherits any
3106 additional annotation retrieved from the database sequence.
3108 The database retrieval process terminates when a valid mapping is found for a
3109 sequence, or if all database queries failed to retrieve a matching sequence.
3110 Termination is indicated by the disappearance of the moving progress indicator
3111 on the alignment window. A dialog box may be shown once it completes which
3112 lists sequences for which records were found, but the sequence retrieved from
3113 the database did not exactly contain the sequence given in the alignment (the
3114 {\sl ``Sequence not 100\% match'' dialog box}).
3116 \exercise{Retrieving Database References}{
3117 \exstep{Load the example alignment at http://www.jalview.org/tutorial/alignment.fa}
3118 \exstep{Verify that there are no database references for the sequences by first
3119 checking that the {\sl View $\Rightarrow$ Sequence ID Tooltip $\Rightarrow$ Show
3120 Database IDs} option is selected, and then mousing over each sequence's ID.}
3121 \exstep{Use the {\sl Webservice $\Rightarrow$ Fetch DB References} menu option to retrieve database IDs for the sequences.}
3122 \exstep{Examine the tooltips for each sequence in the alignment as the retrieval progresses - note the appearance of new database references.}
3123 \exstep{Once the process has finished, save the alignment as a Jalview Project.}
3124 \exstep{Now close all the windows and open the project again, and verify that the database references and sequence features are still present on the alignment}
3126 \exstep{View the {\sl Sequence details \ldots} report for the FER1\_SPIOL sequence and for the whole alignment. Which sequences have web links associated with them ?}
3130 \subsection{Retrieving Features {\sl via} DAS}
3131 \label{dasfretrieval}
3132 Jalview includes a client to retrieve features from DAS annotation servers. To
3133 retrieve features, select {\sl View $\Rightarrow$ Feature Settings\ldots} from the alignment window menu. Select the {\sl DAS Settings} tab in the Sequence Feature Settings Window (Figure \ref{das}). A list of DAS sources compiled from the currently configured DAS registry\footnote{By default, this will be the major public DAS server registry maintained by the Sanger Institute: http://www.dasregistry.org} is shown in the left hand pane. Highlighting an entry on the left brings up information about that source in the right hand panel.
3135 \begin{figure}[htbp]
3137 \includegraphics[width=2.5in]{images/das1.pdf}
3138 \includegraphics[width=2.5in]{images/das2.pdf}
3139 \caption{{\bf Retrieving DAS annotations.} DAS features are retrieved using the {\sl DAS Settings} tab (left) and their display customised using the {\sl Feature Settings} tab (right).}
3144 Select appropriate DAS sources as required then click on {\sl Fetch DAS
3145 Features}. If you know of additional sources not listed in the configured
3146 registry, then you may add them with the {\sl Add Local Source} button. Use
3147 the {\sl Authority},{\sl Type}, and {\sl Label} filters to restrict the list
3148 of sources to just those that will return features for the sequences in the
3151 Following DAS feature retrieval, the {\sl Feature Settings} panel takes on a
3152 slightly different appearance (Figure \ref{das} (right)). Each data source is
3153 listed and groups of features from one data source can be selected/deselected
3154 by checking the labeled box at the top of the panel.
3157 \subsubsection{The Fetch Uniprot IDs dialog box}
3158 \label{discoveruniprotids}
3159 If any sources are selected which refer to Uniprot coordinates as their reference system, then you may be asked if you wish to retrieve Uniprot IDs for your sequence. Pressing OK instructs Jalview to verify the sequences against Uniprot records retrieved using the sequence's ID string. This operates in much the same way as the {\sl Web Service $\Rightarrow$ Fetch Database References } function described in Section \ref{fetchdbrefs}. If a sequence is verified, then the start/end numbering will be adjusted to match the Uniprot record to ensure that features retrieved from the DAS source are rendered at the correct position.
3161 \subsubsection{Rate of feature retrieval}
3162 Feature retrieval can take some time if a large number of sources is selected and if the alignment contains a large number of sequences. This is because Jalview only queries a particular DAS source with one sequence at a time, to avoid overloading it. As features are retrieved, they are immediately added to the current alignment view. The retrieved features are shown on the sequence and can be customised as described previously.
3165 \exercise{Retrieving features with DAS}{
3166 \label{dasfeatretrexcercise}
3167 \exstep{Load the alignment at
3168 \textsf{http://www.jalview.org/tutorial/alignment.fa}. Select {\sl View
3169 $\Rightarrow$ Feature Settings \ldots} from the alignment window menu. Select
3170 the {\sl DAS Settings} tab. A long list of available DAS sources is listed. Select a small number, eg Uniprot, DSSP, signalP and netnglyc. Click OK. A window may prompt whether you wish Jalview to map the sequence IDs onto Uniprot IDs. Click {\sl Yes}. Jalview will start retrieving features. As features become available they will be mapped onto the alignment. } \exstep{If Jalview is taking too long to retrieve features, the process can be cancelled with the {\sl Cancel Fetch} button. Rolling the mouse cursor over the sequences reveals a large number of features annotated in the tool tip. Close the Sequence Feature Settings window. }
3171 \exstep{Move the mouse over the sequence ID panel. Non-positional features such as literature references and protein localisation predictions are given in the tooltip, below any database cross references associated with the sequence.}
3172 \exstep{Search through the alignment to find a feature with a link symbol next to it. Right click to bring up the alignment view popup menu, and find a corresponding entry in the {\sl Link } sub menu. }
3173 % TODO this doesn't work ! \includegraphics[width=.3in]{images/link.pdf}
3176 Select {\sl View $\Rightarrow$ Feature Settings\ldots} to reopen the Feature Settings window. All the loaded feature types should now be displayed. Those at the top of the list are drawn on top of those below, obscuring them in the alignment view where they overlap. Move the feature settings window so that the alignment is visible and uncheck some of the feature types by clicking the tick box in the display column. Observe how the alignment display changes. Note that unselected feature types do not appear in the tool tip.
3178 \exstep{Reorder the features by dragging feature types up and down the order in the Feature Settings panel. e.g. Click on {\sl CHAIN} then move the mouse downwards to drag it below {\sl DOMAIN}. Note that {\sl DOMAIN} is now shown on top of {\sl CHAIN} in the alignment window. Drag {\sl METAL} to the top of the list. Observe how the cysteine residues are now highlighted as they have a {\sl METAL} feature associated with them.
3181 \exstep{Press the {\sl Optimise Order} button. The features will be ordered according to increasing length, placing features that annotate shorter regions of sequence higher on the display stack.}
3183 \exstep{Select {\sl File $\Rightarrow$ Export Features\ldots} from the Alignment window. You can choose to export the retrieved features as a GFF file, or Jalview's own Features format.
3184 % TODO: describe working with features files and GFF
3188 \subsection{Colouring features by score or description
3190 \label{featureschemes}
3191 Sometimes, you may need to visualize the differences in information carried by
3192 sequence features of the same type. This is most often the case when features
3193 of a particular type are the result of a specific type of database query or calculation. Here, they may also carry information within their textual description, or most commonly for calculations, a score related to the property being investigated. Jalview can shade sequence
3194 features using a graduated colourscheme in order to highlight these variations.
3195 In order to apply a graduated scheme to a feature type, select the `Graduated
3196 colour' entry in the Sequence {\sl Feature Type}'s popup menu, which is opened by
3197 right-clicking the {\sl Feature Type} or {\sl Color} in the {\sl Sequence Feature Settings} dialog box. Two types
3198 of colouring styles are currently supported: the default is quantitative
3199 colouring, which shades each feature based on its score, with the highest
3200 scores receiving the `Max' colour, and the lowest scoring features coloured
3201 with the `Min' colour. Alternately, you can select the `Colour by label'
3202 option to create feature colours according to the description text associated
3203 with each feature. This is useful for general feature types - such as
3204 Uniprot's `DOMAIN' feature - where the actual type of domain is given in the
3205 feature's description.
3207 Graduated feature colourschemes can also be used to exclude low or
3208 high-scoring features from the alignment display. This is done by choosing your
3209 desired threshold type (either above or below), using the drop-down menu in the
3210 dialog box. Then, adjust the slider or enter a value in the text box to set the
3211 threshold for displaying this type of feature.
3213 The feature settings dialog box allows you to toggle between a graduated and
3214 simple feature colourscheme using the pop-up menu for the feature type. When a
3215 graduated scheme is applied, it will be indicated in the colour column for
3216 that feature type - with coloured blocks or text to indicate the colouring
3217 style and a greater than ($>$) or less than ($<$) symbol to indicate when a
3218 threshold has been defined.
3220 \subsection{Using features to re-order the alignment}
3221 \label{featureordering}
3222 The presence of sequence features on certain sequences or in a particular
3223 region of an alignment can quantitatively identify important trends in
3224 the aligned sequences. In this case, it is more useful to
3225 re-order the alignment based on the number of features or their associated scores, rather than simply re-colour the aligned sequences. The sequence feature settings
3226 dialog box provides two buttons: `Seq sort by Density' and `Seq sort by
3227 Score', that allow you to reorder the alignment according to the number of
3228 sequence features present on each sequence, and also according to any scores
3229 associated with a feature. Each of these buttons uses the currently displayed
3230 features to determine the ordering, but
3231 if you wish to re-order the alignment using a single type of feature, then you can do this from the {\sl Feature Type}'s
3232 popup menu. Simply right-click the type's style in the Sequence Feature Settings dialog
3233 box, and select one of the {\sl Sort by Score} and {\sl Sort by Density}
3234 options to re-order the alignment. Finally, if a specific region is selected,
3235 then only features found in that region of the alignment will be used to
3236 create the new alignment ordering.
3238 \exercise{Shading and sorting alignments using sequence features}{
3239 \label{shadingorderingfeatsex}
3240 \exstep{Re-load the alignment from \ref{dasfeatretrexcercise}.
3243 feature settings panel, and, after first clearing the current
3244 selection, press the {\em Seq Sort by Density} button a few times.}
3245 \exstep{Use the DAS fetcher to retrieve the Kyte and Doolittle Hydrophobicity
3246 scores for the protein sequences in the alignment.
3247 {\sl Hint: the nickname for the das source is `KD$\_$hydrophobicity'.}}
3248 \exstep{Change the feature settings so only the hydrophobicity features are
3249 displayed. Mouse over the annotation and also export and examine the GFF and
3250 Jalview features file to better understand how the hydrophobicity measurements
3252 \exstep{Apply a {\sl Graduated Colour} to the hydrophobicity annotation to
3253 reveal the variation in average hydrophobicity across the alignment.}
3254 \exstep{Select a range of alignment columns, and use one of the sort by feature buttons to order the alignment according to that region's average
3256 \exstep{Save the alignment as a project, for use in exercise
3257 \ref{threshgradfeaturesex}.} }
3259 \exercise{Shading alignments with combinations of graduated feature
3261 \label{threshgradfeaturesex}
3262 \exstep{Reusing the annotated alignment from exercise
3263 \ref{shadingorderingfeatsex}, experiment with the colourscheme threshold to
3264 highlight the most, or least hydrophobic regions. Note how the {\sl Colour} icon for the {\sl Feature Type} changes when you change the threshold type and press OK.}
3265 \exstep{Change the colourscheme so
3266 that features at the threshold are always coloured grey, and the most
3267 hydrophobic residues are coloured red, regardless of the threshold value
3268 ({\em hint - there is a switch on the dialog to do this for you}).}
3269 \exstep{Enable the Uniprot {\em chain} annotation in the feature settings
3270 display and re-order the features so it is visible under the hydrophobicity
3272 \exstep{Apply a {\sl Graduated Colour} to the {\em chain}
3273 annotation so that it distinguishes the different canonical names associated
3274 with the mature polypeptide chains.}
3275 \exstep{Export the alignment's sequence features using the Jalview and GFF file formats, to see how the different types of graduated feature
3276 colour styles are encoded. }
3278 \section{Working with DNA}
3279 \label{workingwithnuc}
3280 Jalview was originally developed for the analysis of protein sequences, but
3281 now includes some specific features for working with nucleic acid sequences
3282 and alignments. Jalview recognises nucleotide sequences and alignments based on
3283 the presence of nucleotide symbols [ACGT] in greater than 85\% of the
3284 sequences. Built in codon-translation tables can be used to translate ORFs
3285 into peptides for further analysis. EMBL nucleotide records retrieved {\sl via} the
3286 sequence fetcher (see Section \ref{fetchseq}) are also parsed in order to
3287 identify codon regions and extract peptide products. Furthermore, Jalview
3288 records mappings between protein sequences that are derived from regions of a
3289 nucleotide sequence. Mappings are used to to transfer annotation between
3290 nucleic acid and protein sequences, and to dynamically highlight regions in
3291 one sequence that correspond to the position of the mouse pointer in another.
3292 %TODO Working with Nucleic acid sequences and structures.
3293 \subsection{Alignment and Colouring}
3295 Jalview provides a simple colourscheme for DNA bases, but does not apply any
3296 specific conservation or substitution score model for the shading of
3297 nucleotide alignments. However, pairwise alignments performed using the {\sl Calculate $\Rightarrow$ Pairwise Alignment
3298 \ldots} option will utilise an identity score matrix to calculate alignment
3299 score when aligning two nucleotide sequences.
3301 \subsubsection{Aligning Nucleic Acid Sequences}
3303 Jalview has limited knowledge of the capabilities of the programs that
3304 are made available to it {\sl via} web services, so it is up to you, the user,
3305 to decide which service to use when working with nucleic acid sequences. The
3306 table below shows which alignment programs are most appropriate
3307 for nucleotide alignment. Generally, all will work, but some may be more suited
3308 to your purposes than others. We also note that none of these include
3309 support for taking RNA secondary structure prediction into account when aligning
3310 sequences. We expect that in the future, Jalview will fully support secondary
3311 structure aware RNA alignment.
3315 \begin{tabular}{|l|c|l|}
3317 Program& NA support& Notes\\
3319 ClustalW& Yes&\begin{minipage}[f]{3in}
3320 Default is to autodetect nucleotide
3321 sequences. Editable parameters include nucleotide substitution matrices and
3328 Muscle& Yes (treat U as T)&\begin{minipage}[f]{3in}
3329 Default is to autodetect nucleotide
3330 sequences. Editable parameters include nucleotide substitution matrices and
3337 MAFFT& Yes&\begin{minipage}[f]{3in}
3338 Will autodetect nucleotide sequences and use a hardwired substitution model
3339 (all amino-acid sequence related parameters are ignored). Unknown whether
3340 substitution model treats Uracil specially.
3346 ProbCons& No&\begin{minipage}[f]{3in}
3347 ProbCons has no special support for aligning nucleotide sequences. Whilst an
3348 alignment will be returned, it is unlikely to be reliable.
3354 T-COFFEE& Yes&\begin{minipage}[f]{3in}
3355 Sequence type is automatically detected and an appropriate
3356 parameter set used as required. A range of nucleotide specific
3357 score models are available.\end{minipage}
3361 \caption{{\bf JABAWS Alignment programs suitable for aligning nucleic acid
3362 sequences.} All JABAWS alignment services will return an alignment if provided
3363 with RNA or DNA sequences, with varying reliability.}
3364 \label{nucleomsatools}
3367 \subsection{Translate cDNA}
3369 The {\sl Calculate $\Rightarrow$ Translate cDNA} function in the alignment
3370 window is only available when working with a nucleic acid alignment. It uses the standard codon translation table given in the online help documentation to translate a nucleotide alignment, or the currently selected region, into a set of aligned peptide sequences. Any features or annotation present on the nucleotide alignment will also be translated, allowing DNA alignment analysis results to be transferred on to peptide products for further investigation.
3372 \subsection{Linked DNA and Protein Views}
3375 Views of alignments involving DNA sequences are linked to views of alignments containing their peptide products in a similar way to views of protein sequences and views of their associated structures. Peptides translated from cDNA that have been fetched from EMBL records for DNA contigs are linked to their `parent' coding regions. Mousing over a region of the peptide highlights codons in views showing the original coding region.
3378 %\begin{figure}[htbp]
3380 \includegraphics[width=2.8in]{images/cdnatranslinkedwin.pdf}
3382 %\caption{{\bf Linked DNA and Protein Views.} }
3388 \subsection{Coding regions from EMBL records}
3390 Many EMBL records that can be retrieved with the sequence fetcher contain exons.
3391 Coding regions will be marked as features on the EMBL nucleotide sequence, and
3392 Uniprot database cross references will be listed in the tooltip displayed when
3393 the mouse hovers over the sequence ID. Uniprot database cross references
3394 extracted from EMBL records are sequence cross references, and associate a
3395 Uniprot sequence's coordinate system with the coding regions annotated on the
3396 EMBL sequence. Jalview utilises cross-reference information in two ways.
3397 \subsubsection{Retrieval of Protein or DNA Cross References}
3398 The {\sl Calculate $\Rightarrow$ Get Cross References } function is only available when Jalview recognises that there are protein/DNA cross-references present on sequences in the alignment. When selected, it retrieves the cross references from the alignment's dataset (a set of sequence and annotation metadata shared between alignments) or using the sequence database fetcher. This function can be used for EMBL sequences containing coding regions to open the Uniprot protein products in a new alignment window. The new alignment window that is opened to show the protein products will also allow dynamic highlighting of codon positions in the EMBL record for each residue in the protein product(s).
3400 \subsubsection{Retrieval of protein DAS features on coding regions}
3402 The Uniprot cross-references derived from EMBL records can be used by Jalview to visualize protein sequence features directly on nucleotide alignments. This is because the database cross references include the sequence coordinate mapping information to correspond regions on the protein sequence with that of the nucleotide contig. Jalview will use the Uniprot accession numbers associated with the sequence to retrieve features, and then map them onto the nucleotide sequence's coordinate system using the coding region location.
3404 \begin{figure}[htbp]
3406 \label{dnadasfeatures}
3407 \includegraphics[width=5in]{images/dnadasfeatures.pdf}
3409 \caption{Uniprot and PDB sum features retrieved {\sl via} DAS and mapped onto
3410 coding regions of EMBL record V00488 (an earlier version of Jalview is shown
3416 \exercise{Visualizing protein features on coding regions}
3418 \exstep{Use the sequence fetcher to retrieve EMBL record V00488.}
3419 \exstep{Ensure that {\sl View $\Rightarrow$ Show Sequence Features} is checked and change the alignment view format to Wrapped mode so the distinct exons can be seen.}
3420 \exstep{Open the {\sl DAS Settings} tab in the {\sl Sequence Feature Settings\ldots} window and fetch features for V00488 from the Uniprot reference server, and any additional servers that work with the Uniprot coordinate system.}
3421 \exstep{Mouse over the features retrieved, note that they have been mapped onto the coding regions, and in some cases broken into several parts to cover the distinct exons.}
3422 \exstep{Open a new alignment view containing the Uniprot protein product with {\sl Calculate $\Rightarrow$ Get Cross References $\Rightarrow$ Uniprot } and examine the database references and sequence features. Experiment with the interactive highlighting of codon position for each residue.
3425 % \section{Working with RNA}
3426 % \label{workingwithrna}
3428 % \subsection{RNA specific alignment colourschemes}
3429 % \label{rnacolschemes}
3431 % \subsection{Exploring RNA secondary structure with VARNA}
3434 % \subsection{RNA Secondary structure annotation}
3435 % \subsubsection{Interactive creation of RNA secondary structure annotation}
3436 % \label{rnasecstrediting}
3438 % \subsubsection{Import and export of RNA secondary structure annotation}
3439 % \label{rnasecstrio}
3442 % \chapter{Advanced Jalview}
3444 % \section{Customising Jalview}
3445 % \subsection{Setting preferences}
3447 % The Jalview Desktop stores configuration and history information in a file stored in the users home directory, called `.jalview\_properties'. Many of the options stored in this file are presented in the {\sl Desktop $\Rightarrow$ Tools $\Rightarrow$ Preferences\ldots} dialog. These preferences include default settings for : {\bf Visual} layout settings for alignment views and controlling the display of the default alignment, {\bf Connection} preferences such as the standard set of URL paths that are available from the links menu and the URL which is opened when a Sequence's ID is double clicked, {\bf Editing} settings like {\sl Pad Gaps} and autocalculation of consensus, {\bf Output} settings control the degree of meta-information written in alignment files and mode of EPS Figuregeneration, and finally the {\bf Das Settings} which allows the default DAS sources to be configured.
3449 % \subsection{Adding your own URL links}
3451 % \subsection{Working with Databases and Database Cross References}
3452 % \label{getcrossrefs}
3454 % {\sl Calculations $\Rightarrow$ Get Cross References }
3456 % \section{Jalview IO Interface}
3457 % \subsection{Multiple views}
3458 % \subsection{Annotation files}
3459 % \subsection{Feature files}
3460 % \subsection{Moving sequence associated annotation}
3461 % \subsection{Propagating features}
3462 % \section{Structures}
3463 % \subsection{Working with Modeller files}
3464 % \subsection{Using local PDB files}
3465 % \section{Pairwise alignments}