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Technology & Methods Seminar: “Tiling Arrays - Probing Genome and Transcriptome Structure”. On the use of Affymetrix Tiling Arrays for Comparative Genomic Hybridizations. Norman Pavelka (Rong Li lab). March 29, 2007. Background : Role of MYO1 in cytokinesis. Myo1.

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Presentation Transcript
slide1

Technology & Methods Seminar:

“Tiling Arrays - Probing Genome and Transcriptome Structure”

On the use of Affymetrix Tiling Arrays for Comparative Genomic Hybridizations

Norman Pavelka

(Rong Li lab)

March 29, 2007

slide2

Background: Role of MYO1 in cytokinesis

Myo1

  • Phenotype of yeast cells experiencing an acute loss of MYO1:
  • Severe cytokinesis defect
  • Impaired cell viability
  • Phenotype of yeast cells experiencing a chronic loss of MYO1:
  • Extremely heterogenous
  • Occasionally: full recovery of cytokinesis proficiency and of growth ability
slide3

Biological question: What genome changes occurred in e-strains?

  • Polyploidization?
  • Aneuploidization?
  • Interstitial deletions?
  • Reciprocal translocations?
  • Non-reciprocal translocations?
  • Amplifications?
  • Single-nucleotide mutations?

Albertson & Pinkel, Hum Mol Genet (2003)

slide4

Method: array-based Comparative Genomic Hybridization (aCGH)

U.C. Berkeley Division of Biostatistics Working Paper Series (2002), paper 106.

slide5

Technology: Affymetrix Yeast Tiling Arrays

~6.5 million unique probes on the chip

Gresham et al., Science (2006)

~12.5 million bp in the yeast genome

Designed to interrogate the yeast genome with a 5bp resolution:

slide6

Experimental protocol:

Strain 2b

(wt)

Ladder

75 mU DNase I

150 mU DNase I

25

500

2000

6000

Fragment length (nt)

200

1000

4000

Strain 7a-1

  • Extraction of genomic DNA with Phenol / Chloroform / Isoamylalcohol
  • “Controlled” fragmentation with DNase I (5 min at 37 °C)
  • End-labeling with TdT and biotin-dUTP
  • Hybridize on Affy chips
  • Stain with streptavidin-PE
  • Wash and scan chips
slide7

2b (low DNase I, large fragments)

7a-1 (low DNase I, large fragments)

2b (high DNase I, small fragments)

7a-1 (high DNase I, small fragments)

slide8

Limitations: What genome changes can we see by aCGH?

  • Polyploidization?
  • Aneuploidization?
  • Interstitial deletions?
  • Reciprocal translocations?
  • Non-reciprocal translocations?
  • Amplifications?
  • Single-nucleotide mutations?

Albertson & Pinkel, Hum Mol Genet (2003)

slide9

Observation #1: Deletion of the MYO1 locus

MYO1 locus

+1

0

log2(ratio)

-10

+1

0

log2(ratio)

-10

Chromosome VIII

slide10

Observation #2: “Duplication” of the TRP1 locus

TRP1

+10

log2(ratio)

0

-1

+10

log2(ratio)

0

-1

Chromosome IV

Caveat #1: No information on where the signal comes from!

slide11

Caveat #2: Highly repetitive sequences!

(aka “Saturation” effect)

+1

log2(ratio)

0

-1

+1

log2(ratio)

0

Full-length Ty1

Full-length Ty1

Ty1 LTR

-1

Chromosome II

slide12

Observation #3: Gradual loss of signal towards telomeres

+1

log2(ratio)

0

-1

+1

log2(ratio)

0

-1

Full sequence of chromosome II

slide16

Possible observation #2: Single-nucleotide changes?

+10

log2(ratio)

0

-10

35

45

0

5

10

15

20

30

40

25

Probes on the chip

Genomic DNA

35

45

0

5

10

15

20

30

40

25

Gresham et al., Science (2006)

slide17

Summary:

  • What can be seen by CGH on Tiling Arrays?
    • Anything that causes a change in the copy number of a DNA segment, e.g. aneuploidies, deletions/amplifications, non-reciprocal translocations, etc.
    • Mutations that affect the hybridization of multiple overlapping probes, i.e. single-nucleotide changes.
  • What can not be seen by CGH?
    • Anything that does not cause a change in the copy number of a DNA segment, e.g. polyploidization, reciprocal translocations etc.
    • If probes are too long and non-overlapping, single-nucleotide mutations will not be detectable.
  • What are the most common pitfalls?
    • No information about where the signal actually comes from!
    • No reliable information from probes hybridizing to highly-repetitive sequence (because of “saturation” effect)!
    • If some chromosomes are gained or lost, this will affect the log-ratios also of all other chromosomes (because of “dilution” effect)!
slide18

Acknowledgements:

  • Microarray group:
  • Karin Zueckert-Gaudenz
  • Allison Peak
  • Chris Seidel
  • Rong Li lab:
  • Giulia Rancati
  • Rong Li