Genome-wide Copy Number Analysi Genome-wide Copy Number Analysi s s Qunyuan Zhang,Ph.D. Division of Statistical Genomics Department of Genetics & Center for Genome Sciences Washington University School of Medicine 02 - 08 – 2006 Course: M 21-621 Computational Statistical Genetics
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Genome-wide Copy Number Analysis Qunyuan Zhang,Ph.D. Division of Statistical Genomics Department of Genetics & Center for Genome Sciences Washington University.
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Genome-wide Copy Number AnalysisGenome-wide Copy Number Analysis
Qunyuan Zhang,Ph.D.
Division of Statistical Genomics
Department of Genetics & Center for Genome Sciences
Washington University School of Medicine
02 - 08 – 2006
Course: M 21-621 Computational Statistical Genetics
Four QuestionsFour Questions
What is Copy Number ?What is Copy Number ?
What can Copy Number tell us?What can Copy Number tell us?
How to measure/quantify Copy Number?How to measure/quantify Copy Number?
How to analyze Copy Number?How to analyze Copy Number?
What is Copy Number ?What is Copy Number ?
Gene Copy Number
The gene copy number (also "copy number variants" or CNVs) is the amount of copies of a particular gene in the genotype of an individual. Recent evidence shows that the gene copy number can be elevated in cancer cells. For instance, the EGFR copy number can be higher than normal in Non-small cell lung cancer. …Elevating the gene copy number of a particular gene can increase the expression of the protein that it encodes.
From Wikipedia www.wikipedia.org
DNA Copy Number A Copy Number Variant (CNV) represents a copy number change involving a D
NA fragment that is ~1 kilobases or larger. From Nature Reviews Genetics, Feuk et al. 2006
DNA Copy Number ≠ DNA Tandem Repeat Number (e.g. micro satellites) <10 bases
DNA Copy Number ≠ RNA Copy Number RNA Copy Number = Gene Expression Level
DNA transcription mRNA
Copy Number is the amount of copies of a particular fragment of nucleic acid molecular chain. It refers to DNA Copy Number in most publications.
What can Copy Number tell us?What can Copy Number tell us?
Genetic Diversity/Polymorphisms
- restriction fragment length polymorphism (RFLP)- amplified fragment length polymorphism (AFLP)- random amplification of polymorphic DNA (RAPD)- variable number of tandem repeat (VNTR; e.g., mini- and
microsatellite)- single nucleotide polymorphism (SNP)- presence/absence of transportable elements…- structural alterations (e.g., deletions, duplications, inversions … )- DNA copy number variant (CNV)
Association with phenotypes/diseases genes/genetic factors
Genetic Alterations in Tumor Cells (DNA Copy Number Changes)
Homologous repeats
Segmental duplications
Chromosomal rearrangements
Duplicative transpositions
Non-allelic recombinations
……
Normal cell
Tumor cells
deletion amplification
CN=0 CN=1 CN=2 CN=3 CN=4
CN=2
How to measure/quantify Copy Number?How to measure/quantify Copy Number?
Quantitative Polymerase Chain Reaction (Q-PCR) : DNA Amplification
(dNTPs, primers, Taq polymerase, fluorescent dye)
PCR
less CN amplification less DNA low fluorescent intensity
more CN amplification more DNA high fluorescent intensity
(one fragment each time)
Microarray : DNA Hybridization
(dNTPs, primers, Taq polymerase, fluorescent dye)
PCR
less CN amplification less DNA arrayed probes low intensities
more CN amplification more DNA arrayed probes high intensities
(multiple/different fragments, mixed pool)
Hybridization
Microarray: From Image to Copy Number
Tumor NormalAffymetrix Mapping
250K Sty-I chip
~250K probe sets
~250K SNPs
CN=1
CN=0
CN>2
CN=2
CN=2
CN=2
probe set (24 probes)
Deletion
Deletion
Amplification
more DNA copy number more DNA hybridization higher intensity
~400 cancer patients
Normal tissue & tumor tissue (~400 pairs, ~800 DNA samples)
Affymetrix 250K Sty-I Human Mapping SNP Array
DNA hybridization signals (intensities on chip images)
Genotype calling
SNP genotypes
LOH analysis DNA copy number analysis (genotypic changes) (DNA copy number changes)
How to Analyze Copy Number?How to Analyze Copy Number?
?
A Real Example
General Procedures for Copy Number Analysis
Finished chips (scanner) Raw image data [.DAT files] (experiment info [ .EXP]) (image processing software)