MicroArray Image Analysis

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MicroArray Image Analysis

• Robin Liechti (robin.liechti_at_ie-bpv.unil.ch)
• www.ch.embnet.org/CoursEMBnet/CHIP02/.../Liechti02
  _images.ppt
• statwww.epfl.ch/davison/teaching/Microarrays/lec/w
  eek04.ppt
• Mark Reimers (National Cancer Institute)
• www.ims.nus.edu.sg/Programs/microarray/files/MReim
  ersTut1.ppt

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Microarray analysis

• Array construction, hybridisation, scanning
• Quantitation of fluorescence signals
• Data visualisation
• Meta-analysis (clustering)
• More visualisation

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Technical
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Affymetrix Gene Chip
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Images from scanner

• Resolution
• standard 10?m currently, max 5?m
• 100?m spot on chip 10 pixels in diameter
• Image format
• TIFF (tagged image file format) 16 bit (65536
  levels of grey)
• 1cm x 1cm image at 16 bit 2Mb (uncompressed)
• other formats exist e.g.. SCN (used at Stanford
  University)
• Separate image for each fluorescent sample
• channel 1, channel 2, etc.

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Images 2 color
Spot color Signal strength Gene expression
yellow Control perturbed unchanged
red Control lt perturbed induced
green Control gt perturbed repressed
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Images 1 color
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Processing of images

• Addressing or gridding
• Assigning coordinates to each of the spots
• Segmentation
• Classification of pixels either as foreground or
  as background
• Intensity extraction (for each spot)
• Foreground fluorescence intensity pairs (R, G)
• Background intensities
• Quality measures

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Affymetrix Image Reading

• About 100 pixels per probe cell
• Selects 16-25 brightest contiguous pixels
• Take average of selected pixels
• Variability in best pixels 5-20

Image courtesy of Affymetrix
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Probe Variation

• Probes vary by two orders of magnitude on each
  chip

Signal from 16 probes for the GAPDH gene on one
chip

• Individual probes dont agree on fold changes
• across chips
• Bright probes more often, but not always, more
  reliable

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Addressing
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Addressing (I)

• The basic structure of the images is known
  (determined by the arrayer)

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Addressing (II)

• The measurement process depends on the addressing
  procedure
• Addressing efficiency can be enhanced by allowing
  user intervention (slow!)
• Most software systems now provide for both manual
  and automatic gridding procedures

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Example from GenePix software
http//transcriptome.ens.fr/sgdb/tools/download/im
age_analysis_en.pdf
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Segmentation
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Segmentation (I)

• Classification of pixels as foreground or
  background -gt fluorescence intensities are
  calculated for each spot as measure of transcript
  abundance
• Production of a spot mask set of foreground
  pixels for each spot

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Segmentation (II)

• Segmentation methods
• Fixed circle segmentation
• Adaptive circle segmentation
• Adaptive shape segmentation
• Histogram segmentation

Fixed circle ScanAlyze, GenePix, QuantArray
Adaptive circle GenePix, Dapple
Adaptive shape Spot, region growing and watershed
Histogram method ImaGene, QuantArraym DeArray and adaptive thresholding
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Fixed circle segmentation

• Fits a circle with a constant diameter to all
  spots in the image
• Easy to implement
• The spots need to be of the same shape and size

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Adaptive circle segmentation

• The circle diameter is estimated separately for
  each spot

• Problematic if spot exhibits oval shapes

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Adaptive shape segmentation

• Specification of starting points or seeds

• Regions grow outwards from the seed points
  preferentially according to the difference
  between a pixels value and the running mean of
  values in an adjoining region.

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Histogram segmentation

• Uses a target mask chosen to be larger than any
  other spot
• Foreground and background intensity are
  determined from the histogram of pixel values for
  pixels within the masked area
• Example QuantArray
• Background mean between 5th and 20th percentile
• Foreground mean between 80th and 95th
  percentile
• Unstable when a large target mask is set to
  compensate for variation in spot size

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Example from GenePix software
http//transcriptome.ens.fr/sgdb/tools/download/im
age_analysis_en.pdf
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Information extraction
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Spot intensity

• The total amount of hybridization for a spot is
  proportional to the total fluorescence at the
  spot
• Spot intensity sum of pixel intensities within
  the spot mask
• Since later calculations are based on ratios
  between cy5 and cy3, we compute the average
  pixel value over the spot mask
• alternative use ratios of medians instead of
  means

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Background intensity

• Motivation spots measured intensity includes a
  contribution of non-specific hybridization and
  other chemicals on the glass
• Fluorescence from regions not occupied by DNA
  should by different from regions occupied by DNA
  -gt could be interesting to use local negative
  controls (spotted DNA that should not hybridize)
• Different background methods Local background,
  morphological opening, constant background, no
  adjustment

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Local background

• Focusing on small regions surrounding the spot
  mask.
• Median of pixel values in this region
• Most software package implement such an approach

• By not considering the pixels immediately
  surrounding the spots, the background estimate is
  less sensitive to the performance of the
  segmentation procedure

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Constant background

• Global method which subtracts a constant
  background for all spots
• Some findings suggests that the binding of
  fluorescent dyes to negative control spots is
  lower than the binding to the glass slide
• -gt More meaningful to estimate background based
  on a set of negative control spots
• If no negative control spots approximation of
  the average background third percentile of all
  the spot foreground values

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No adjustment

• Do not consider the background

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(No Transcript)
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References

• Yang, Y. H., Buckley, M. J., Dudoit, S. and
  Speed, T. P. (2001), Comparisons of methods for
  image analysis on cDNA microarray data.
  Technical report 584, Department of Statistics,
  University of California, Berkeley.
• Yang, Y. H., Buckley, M. J. and Speed, T. P.
  (2001), Analysis of cDNA microarray images.
  Briefings in bioinformatics, 2 (4), 341-349.

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Next time

• Data formats/files for Affymetrix microarrays
• CEL and CDF
• Intro to R
• Reading in microarray data
• Exploring array data
• Assignment
• For the gene, Pbx1, determine the probe design on
  either the mouse Affymetrix 1.0 ST MoGene array
  or the Zebrafish genome array
• ? What is the difference between a probe and a
  probeset?
• You should be able to use resources at
  www.affymetrix.com but you might need to register
  to get access to data files.

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For Pbx1, How many probes? What are the sequences
of the probes? Where are the probes placed along
the gene structure for Pbx1?
Google Affymetrix web site