Introduction to Array analysis and Genespring workgroup at NASC

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Introduction to Array analysis and Genespring
workgroup at NASC

• Neil Graham

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NASC Affymetrix service(http//affymetrix.arabido
psis.info)

• Affymetrix service started in 2002
• All Affymetrix arrays supported (all species,
  tiling, exon, SNP etc)
• Arabidopsis data automatically added to
  NASCarrays database and GEO, arrayexpress
• 350 (including ATH1, tomato), 450 (including
  Human, mouse)

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NASCarrays database

• MIAME compliant database
• Contains gt3000 hybridisations
• Associated analysis tools spot history, gene
  swinger, digital northern

How do you fully analyse the data?
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Analysis

• Analysis will depend on what you are trying to
  get out of your experiment?
• What file types have you got?
• Number of replicates
• Up / down regulated genes
• Genes with similar expression patterns

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Normalisation

• Reduces variation to enable comparison between
  slides
• Variation between hybridisation of probes
• Variation across slide
• Variation between slides

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Normalisation of probe values

• 11 probe sets per gene need method to produce
  single value
• Variety of methods
• MAS 5 (affymetrix software)
• RMA (Genespring)
• MBEI (dCHip)

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MAS 5

• Subtracts background reading form each probe
• Uses both PM and MM probes

Siganl TukeyBiweightlog(PM IM)
For a probe set Tukeys Biweight is an average
that minimises the effect of outliers. IM is the
ideal mismatch. This is the same as the MM
intensity, except in the case where the MM is
greater than the PM, in which case a new MM
values is calculated based on other probes nearby
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Signal normailsation

• To try to eliminate chip-to-chip variability.
• Sort the signal values and remove the top and
  bottom 2
• Calculate a scaling factor to adjust this middle
  96s mean to 100 (configurable, and variable)
• Multiply all signal values by the scaling factor

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RMA

• Robust Multichip Average
• Uses a set of arrays
• Only uses PM values to produce signal values
• Background adjustment (based on all MM)
• Qauntile normalisation adjusts intensities to
  produce identical intensities

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Clustering

• Mathematical method of grouping together genes
  with similar expression patterns
• Not based of signal intensity
• Hierarchical
• K means
• Self organising maps

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Hierarchical

• All genes compared to each other
• Produces a tree showing distances between genes

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K means

• Select how many clusters (K)
• Genes randomly assigned to these clusters
• Genes compared to mean of each cluster
• If gene closer to mean of other cluster them
  moved
• Means of clusters recalculated and process
  repeated

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Excel Analysis

• Files downloaded from NASCarrays
• Already normalised- MAS
• Replicate variation
• Up / down regulated genes
• Further analysis, ontologies, promoter motifs

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Genespring

• Powerful package
• Statistical tests
• Clustering
• Pathway analysis
• Promoter analysis

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Genespring workgroup at NASC

• Allows easy access and analysis of all data in
  NASCarrays including .CEL files
• Whole experiments or sets of .CEL files can be
  downloaded and analysed
• Workgroup can be accessed in 3 ways

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Web based viewer(http//genespring.arabidopsis.in
fo)

• Used to view and download data in workgroup

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Web based viewer(http//genespring.arabidopsis.in
fo)

• View experiments, gene lists in a range of ways

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Web based viewer(http//genespring.arabidopsis.in
fo)

• Download experiments, gene lists etc

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Genespring viewer

• Stand alone application free to download
• Cut down version of Genespring GX
• Use to view data and perform simple analysis

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Genespring viewer

• Download installer from web viewer page

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Genespring viewer

• Multiple ways to view experiments

Box plot
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Genespring viewer

• Generate Venn diagrams of gene lists

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Genespring viewer

• Find similar genes

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Genespring GX

• Extensive visualisation and analysis tools

• Download any sample from workgroup and create new
  experiments

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Genespring GX

• Find differentially expressed genes

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Genespring GX

• Clustering (K means, gene trees, condition trees
  etc)

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Access to workgroup

• Access requires username and password
• Web viewer available at http//genespring.arabid
  opsis.info
• Genespring viewer installer can be downloaded
  from web page
• Genespring GX either using your own licence or
  NASC licence (5 concurrent licences available)

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More information

• General information can be found at
  http//affymetrix.arabidopsis.info/workgroup.html
• Web viewer available from http//genespring.arabi
  dopsis.info
• For inquires and information about usernames
  contact genespring_at_arabidopsis.onfo

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Practical

• Demonstration of NASCarrays
• Analysis of data using excel
• Viewing data set in Genespring
• After lunch hands-on training on using Genespring
  loading data, creating experiments, basic
  analysis techniques