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1 Developing GUI Microarray Analysis Tools Keith Satterley Bioinformatics, WEHI, Nov. 15 2005 Keith Satterley Bioinformatics, WEHI, Nov. 15 2005.

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Presentation on theme: "1 Developing GUI Microarray Analysis Tools Keith Satterley Bioinformatics, WEHI, Nov. 15 2005 Keith Satterley Bioinformatics, WEHI, Nov. 15 2005."— Presentation transcript:

1 1 Developing GUI Microarray Analysis Tools Keith Satterley Bioinformatics, WEHI, Nov Keith Satterley Bioinformatics, WEHI, Nov

2 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research2 1. R, Environment, tools & resources 2. Graphical tools. 3. LimmaGUI and AffylmGUI. 4. Example Analysis. Overview.Overview. 5. Resources available. 6. Future Developments.

3 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research3 R is language and environment for statistical computing and graphics. R is released under the GNU license. R is a free software environment for statistical computing and graphics. It compiles and runs on a wide variety of platforms including Unix variants, Windows and MacOS. S was developed by by John Chambers and colleagues at Bell Labs. R can be considered as a different implementation of S. R was initially written by Robert Gentleman and Ross Ihaka of the Statistics Department of the University of Auckland. Since mid-1997 a large group of individuals have contributed to R by sending code and bug reports. The R url is The R Project for Statistical Computing

4 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research4 R has an effective data handling and storage facility, A suite of operators for calculations on arrays, in particular matrices, Provides a vast number of useful statistical tools, many of which have been painstakingly tested, R produces publication-quality graphics in a variety of formats, including JPEG, postscript, eps, pdf, and bmp, A well-developed, simple and effective programming language. The R Project for Statistical Computing

5 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research5 R allows users to add additional functionality by defining new functions. C, C++ and Fortran code can be linked and called at run time. R can be extended (easily) via packages. There are about eight packages supplied with the R distribution and many more are available through the CRAN family of Internet sites The R Project for Statistical Computing

6 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research6 Frequently Asked Questions: Archives - CRAN see next. Mailing Lists Bug-tracking System: Resources for R

7 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research7 CRAN = Comprehensive R Archive Network. CRAN is a network of ftp and web servers around the world that store identical, up- to-date, versions of code and documentation for R. Resources for R

8 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research8 Australia –http://cran.au.r-project.org/ PlanetMirror, Brisbane University of Melbournehttp://cran.au.r-project.org/ Austria –http://cran.at.r-project.org/ Technische Universitaet Wienhttp://cran.at.r-project.org/ Brasil –http://cran.br.r-project.org/ Universidade Federal do Parana?? Federal University of Vicosa Oswaldo Cruz Foundation, Rio de Janeiro University of Sao Paulo, Piracicaba University of Sao Paulo, Sao Paulohttp://cran.br.r-project.org/ Canada –http://cran.stat.sfu.ca/ Simon Fraser University, Burnaby University of Torontohttp://cran.stat.sfu.ca/ China –http://www.lmbe.seu.edu.cn/CRAN/ Southeast University, Nanjinghttp://www.lmbe.seu.edu.cn/CRAN/ Denmark –http://cran.dk.r-project.org/ dotsrc.org, Aalborghttp://cran.dk.r-project.org/ France –http://cran.fr.r-project.org/ CICT, Toulouse Dept. of Biometry & Evol. Biology, University of Lyon Boese Internet, Parishttp://cran.fr.r-project.org/ Germany –http://cran.r-mirror.de/ Stefan Drees, Berlin Pangora GmbH, Hamburg Miscellaneousdata.de, Koeln mainz.de/CRAN/ University of Mainz mirrorplus.org, Muenchenhttp://cran.r-mirror.de/ mainz.de/CRAN/ Hungary –http://cran.hu.r-project.org/ Semmelweis Universityhttp://cran.hu.r-project.org/ Italy –http://cran.arsmachinandi.it/ Ars Machinandi, Arezzo Universita di Ferrara Garr Mirror, Milano Universita degli Studi di Palermohttp://cran.arsmachinandi.it/ Israel –http://cran.active.co.il/ Activetech Ltd, Tel-Avivhttp://cran.active.co.il/ Japan –ftp://ftp.u-aizu.ac.jp/pub/lang/R/CRAN University of Aizu University of Tsukubaftp://ftp.u-aizu.ac.jp/pub/lang/R/CRAN Korea –http://bibscvs.snu.ac.kr/R/ Seoul National Universityhttp://bibscvs.snu.ac.kr/R/ Netherlands –http://cran.nedmirror.nl/ Nedmirror, Amsterdamhttp://cran.nedmirror.nl/ Poland –http://novum.am.lublin.pl/CRAN/ Skubiszewski Medical University, Lublin University of Wroclawhttp://novum.am.lublin.pl/CRAN/ Portugal –http://cran.pt.r-project.org/ Universidade do Portohttp://cran.pt.r-project.org/ Slovenia –http://www.fastmirrors.org/cran/ Fastmirrors.org, Besnica Wsection.com, Ljubljanahttp://www.fastmirrors.org/cran/ South Africa –http://cbio.uct.ac.za/CRAN/ University of Cape Town Rhodes Universityhttp://cbio.uct.ac.za/CRAN/ Spain –http://cran.es.r-project.org/ Spanish National Research Network, Madridhttp://cran.es.r-project.org/ Switzerland –http://cran.ch.r-project.org/ ETH Zuerich Universitaet Bern Prokmu Hosting, Bernhttp://cran.ch.r-project.org/ Turkey –http://godel.cs.bilgi.edu.tr/mirror/cran/ Istanbul Bilgi Universityhttp://godel.cs.bilgi.edu.tr/mirror/cran/ Taiwan –http://cran.cs.pu.edu.tw/ Providence University, Taichung National Taiwan University, Taipeihttp://cran.cs.pu.edu.tw/ UK –http://cran.uk.r-project.org/ University of Bristol Sourcekeg, Londonhttp://cran.uk.r-project.org/ USA –http://cran.cnr.Berkeley.edu University of California, Berkeley, CA University of California, Los Angeles, CA University of California, Davis, CA Iowa State University, Ames, IA Michigan Technological University, Houghton, MI Washington University, St. Louis, MO University of North Carolina, Chapel Hill, NC Pair Networks, Pittsburgh, PA Statlib, Carnegie Mellon University, Pittsburgh, PA Hosting Zero, Dallas, TX Fred Hutchinson Cancer Research Center, Seattle, WAhttp://cran.cnr.Berkeley.edu

9 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research9 CRAN Mirrors – 475 packages

10 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research10 Features of R. –Graphical abilities. –Package System. –Objects in R. Resources for R

11 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research11 Graphical Capabilities in R On unix(inc. Mac OS X) X11 is used. On MS Windows it uses the MS windows system commands. This is not a GUI, but a graphics device for plotting and drawing. There are high level, low level and interactive plotting commands. plot(x) is a high level command. –If x is a time series, this produces a time-series plot. –If x is a numeric vector, it produces a plot of the values in the vector against their index in the vector. –If x is a complex vector, it produces a plot of imaginary versus real parts of the vector elements.

12 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research12 Graphical Capabilities in R Low-level plotting commands can be used to add extra information (such as points, lines or text) to the current plot. abline(a, b) –Adds a line of slope b and intercept a to the current plot. title(main, sub) –Adds a title main to the top of the current plot

13 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research13 An R command line Example library(limma) setwd("C:/aaa-R/swirl/") getwd() list.files() targets <- readTargets("SwirlTargetsFile.txt") targets RG <- read.maimages(targets$FileName, source="spot") RG par(fg="yellow",bg="green") plot(RG$R,lwd=3) abline(2000,1,lwd=5,col ="black")

14 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research14 R Graphics

15 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research15 R Graphics (cont.)

16 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research16 Bioconductor Graphics

17 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research17 R Packages Packages provide a mechanism for loading code and attached documentation. Packaging automatically checks and creates various documentation files from one source Creates distributable win.binary(.zip), mac.binary(.tgz) or source files(.tar.gz). Packages can specify dependent or suggested packages

18 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research18 install.packages() can install a package and all its dependencies (and their dependencies…), either the essential ones and/or the suggested ones (which maybe needed for examples etc.) R Packages(cont.)

19 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research19 Objects in R The entities R operates on are technically known as objects. The class of an object determines how it will be treated by what are known as generic functions. For example print, plot or summary will react according to what sort of object they are called to work on.

20 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research20 Bioconductor Url is Bioconductor is an open source and open development software project for the analysis and comprehension of genomic data. The Bioconductor core team is based primarily at the Fred Hutchinson Cancer Research Center.core teamFred Hutchinson Cancer Research Center. Aims to promote high-quality documentation and reproducible research.documentation Aims to provide access to a wide range of powerful statistical and graphical methods for the analysis of genomic data.

21 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research21 Bioconductor R and the R package system are the main vehicles for designing and releasing software. Bioconductor has a commitment to full open source discipline, All contributions are expected to exist under an open source license such as GPL2 or BSD.

22 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research22 Features of the Bioconductor site. –Packages – code –Packages – metadata –Version management system Bioconductor

23 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research23 Bioconductor Packages 140 code packages listed aCGH Classes and functions for Array Comparative Genomic Hybridization data. affxparser Affymetrix File Parsing SDK affy Methods for Affymetrix Oligonucleotide Arrays affycomp Graphics Toolbox for Assessment of Affymetrix Expression Measures affydata Affymetrix Data for Demonstration Purpose affylmGUI GUI for affy analysis using limma package affypdnn Probe Dependent Nearest Neighbours (PDNN) for the affy package affyPLM Methods for fitting probe-level models affyQCReport QC Report Generation for affyBatch objects altcdfenvs alternative cdfenvs ~~~~~~ limma Linear Models for Microarray Data limmaGUI GUI for limma package ~~~~~~ vsn Variance stabilization and calibration for microarray data webbioc Bioconductor Web Interface widgetInvoke Evaluation widgets for functions widgetTools Creates an interactive tcltk widget xcms LC/MS and GC/MS Data Analysis PLUS 250 metadata packages From: ag Affymetrix Arabidopsis Genome Array Annotation Data (ag) agahomology A data package containing annotation data for agahomology To: zebrafishcdf zebrafishcdf zebrafishprobe Probe sequence data for microarrays of type zebrafish

24 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research24 Bioconductor – use the Subversion version mgt. system Subversion! Subversion is a free/open-source version control system. (replaces CVS). That is, Subversion manages files and directories over time. Subversion clients can access their repository across networks, which allows the version repository to be accessed by many users simultaneously.

25 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research25 Subversion uses a Copy-Modify-Merge solution, rather than a Lock-Modify-Unlock procedure. it remembers every change ever written to it: A client can ask historical questions like, “What did this directory contain last Wednesday?” or “Who was the last person to change this file, and what changes did they make?” Bioconductor – Version management system

26 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research26 Graphical User Interfaces These items are known as widgets. Tcl/Tk is a tool for creating and interacting with widgets. Tcl/Tk runs on unix, Windows and Mac OS X.

27 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research27 Tcl/Tk Tcl/Tk needs to be installed on the computer as well as R. There are prewritten librarys of Tcl/Tk tools- - for eg. TkTable. The R package tcltk needs to be installed in R. The tcltk R package is an interface between the R language and Tcl/Tk commands.

28 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research28 GUI Programs On Windows Tcl/Tk talks to the MS Windows graphical window system. On Unix(&Mac), Tcl/Tk talks to the X Windows system, hence X11 must be started first. 1. Run X11 on Unix & Mac 2. load the R package tcltk using: library(tcltk) library(affylmGUI) for example, ( actually affylmGUI will automatically load tcltk )

29 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research29 R tcltk example This can be used to test if tcltk (or Tcl/Tk) is working correctly: >library(tcltk) >tt <- tktoplevel() >lbl <- tklabel(tt, text="Hello, World!") >tkpack(lbl) >but <- tkbutton(tt, text="OK") >tkpack(but)

30 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research30 R tcltk testing tools To check the path that Tcl/Tk uses to find libraries – >tclvalue(“auto_path”) – [1] "{C:\\R\\rw2020\\R-2.2.0/Tcl/lib/tcl8.4} – C:/R/rw2020/R-2.2.0/Tcl/lib./lib – C:/R/rw2020/R-2.2.0/Tcl/lib/tk8.4 – C:/R/rw2020/R-2.2.0/library/tcltk/exec“ To add an extra path to search, use: – >addTclPath(“C:/bin”) – >tclvalue(“auto_path”) – [1] "{C:\\R\\rw2020\\R-2.2.0/Tcl/lib/tcl8.4} – C:/R/rw2020/R-2.2.0/Tcl/lib./lib – C:/R/rw2020/R-2.2.0/Tcl/lib/tk8.4 – C:/R/rw2020/R-2.2.0/library/tcltk/exec C:/bin“ –For a list of package commands: – >ls(package:tcltk)

31 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research31 Help Commands in R help(mean) #help window on mean function ?mean #same as help(mean) help.search(“regression”) #Help files with alias or concept or title matching 'regression' using fuzzy matching: help.start() #Browser into R docs The Browser shows links into the R Language Definition, Installation & Administration of R, Package writing, Package documentation FAQ’s etc.

32 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research32 Some Useful R Commands for the GUI user! getwd() #Get working directory. setwd() #Set working Directory. list.files() #list files in working directory. ls() #list objects in workspace. rm(list=ls()) #Remove all objects (recommended at start of a session). savehistory(file=“History.txt”) source(file="C:/path/to/filename/file.R", echo=T) #reads commands from file.R and executes them. installed.packages() #detailed info on all packages installed. summary(RG) #displays basic data about object RG. library(limmaGUI) #loads limmaGUI package.

33 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research33 Cross Platform Issues Installation issues are varied MS Windows – able to be installed in C:\R by ordinary user Unix – can be installed by user, but duplications if multiple users do so. Mac OS X – special procedures necessary

34 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research34 LimmaGUI limmaGUI is a Graphical User Interface (GUI) based on R-Tcl/Tk for the exploration and linear modelling of data from two-colour spotted microarray experiments, especially the assessment of differential expression in complex experiments. Swirl Example Analysis.

35 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research35 AffylmGUI AffylmGUI enables the user to perform quality assessment, low-level analysis and linear modeling of data from Affymetrix GeneChips®, with the ultimate goal of identifying differentially expressed genes. Estrogen Example Analysis

36 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research36 WEHI website Resources WEHI Bioinformatics home page Microarray Data Analysis LIMMA:Linear Models for Microarray Data limmaGUI: affylmGUI: James Wettenhall's Bioinformatics Home Page: R-Tcl/Tk Examples, Worked Examples for limma/affylmGUI at

37 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research37 Future Directions for AffylmGUI additional plots to aid in quality assessment of a set of chips, including RNA degradation plots; calculation and display of QC parameters recommended by Affymetrix (Affymetrix, 2004), such as percent present, ratios of 3’/5’ expression for hybridization controls and the like; fitting of mixed linear models where there is technical replication; support for other single-channel platforms.

38 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research38 Future Directions for LimmaGUI additional plots to aid in quality assessment of a set of chips; fitting of mixed linear models where there is technical replication; fitting of mixed linear models where there is biological replication; ?

39 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research39 Aknowledgments James Wettenhall Gordon Smyth Ken Simpson Terry Speed Bioinformatics – many seminars on microarrays!

40 Developing GUI Microarray Analysis Tools The Walter and Eliza Hall Institute of Medical Research40


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