Download Powerpoint slides - Berkeley Statistics

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project

Document related concepts

Gene therapy of the human retina wikipedia , lookup

Metagenomics wikipedia , lookup

DNA damage theory of aging wikipedia , lookup

RNA silencing wikipedia , lookup

Minimal genome wikipedia , lookup

Oncogenomics wikipedia , lookup

Bisulfite sequencing wikipedia , lookup

Polycomb Group Proteins and Cancer wikipedia , lookup

History of RNA biology wikipedia , lookup

DNA supercoil wikipedia , lookup

Nucleic acid double helix wikipedia , lookup

Cancer epigenetics wikipedia , lookup

Nutriepigenomics wikipedia , lookup

Molecular cloning wikipedia , lookup

Genome (book) wikipedia , lookup

Non-coding RNA wikipedia , lookup

Epigenetics of human development wikipedia , lookup

Epitranscriptome wikipedia , lookup

Genomic library wikipedia , lookup

Human genome wikipedia , lookup

DNA vaccination wikipedia , lookup

Gene expression profiling wikipedia , lookup

Epigenomics wikipedia , lookup

Cre-Lox recombination wikipedia , lookup

No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup

Comparative genomic hybridization wikipedia , lookup

Cell-free fetal DNA wikipedia , lookup

Extrachromosomal DNA wikipedia , lookup

Genetic engineering wikipedia , lookup

Point mutation wikipedia , lookup

NEDD9 wikipedia , lookup

Gene wikipedia , lookup

Genome evolution wikipedia , lookup

Designer baby wikipedia , lookup

Genomics wikipedia , lookup

Mir-92 microRNA precursor family wikipedia , lookup

Site-specific recombinase technology wikipedia , lookup

Non-coding DNA wikipedia , lookup

Helitron (biology) wikipedia , lookup

Microevolution wikipedia , lookup

Genome editing wikipedia , lookup

Deoxyribozyme wikipedia , lookup

Nucleic acid analogue wikipedia , lookup

Therapeutic gene modulation wikipedia , lookup

Vectors in gene therapy wikipedia , lookup

Primary transcript wikipedia , lookup

History of genetic engineering wikipedia , lookup

RNA-Seq wikipedia , lookup

Artificial gene synthesis wikipedia , lookup

Transcript
Introduction to genome biology and
microarrays experiment
Statistics for Microarray Data Analysis – Lecture 1
The Fields Institute for Research in
Mathematical Sciences
May 25, 2002
1
The human genome
• The cell is the fundamental working unit of every
living organism.
• Humans: trillions of cells (metazoa);
other organisms like yeast: one cell (protozoa).
• Cells are of many different types (e.g. blood, skin,
nerve cells), but all can be traced back to a single
cell, the fertilized egg.
2
Genes
• The human genome is distributed along 23 pairs of
chromosomes.
- 22 autosomal pairs;
- the sex chromosome pair, XX for females and XY for
males.
• In each pair, one chromosome is paternally inherited, the
other maternally inherited.
• Chromosomes are made of compressed and entwined
DNA.
• A (protein-coding) gene is a segment of chromosomal
DNA that directs the synthesis of a protein.
3
Chromosomes and DNA
4
DNA
• A deoxyribonucleic acid or DNA molecule is a
double-stranded polymer composed of four
basic molecular units called nucleotides.
• Each nucleotide comprises a phosphate group,
a deoxyribose sugar, and one of four nitrogen
bases: adenine (A), guanine (G), cytosine (C),
and thymine (T).
• The two chains are held together by hydrogen
bonds between nitrogen bases.
• Base-pairing occurs according to the following
rule: G pairs with C, and A pairs with T.
5
6
Genetic and physical maps
7
Genetic and physical maps
• Physical distance: number of base pairs (bp).
• Genetic distance: expected number of
crossovers between two loci, per chromatid, per
meiosis.
• Measured in Morgans (M) or centiMorgans (cM).
• 1cM ~ 1 million bp (1Mb) in humans
8
Central dogma
• The expression of the genetic information stored in the
DNA molecule occurs in two stages:
(i) transcription, during which DNA is transcribed into
mRNA;
(ii) translation, during which mRNA is translated to
produce a protein.
DNA  mRNA  protein
• Other important aspects of regulation: methylation,
alternative splicing, etc.
• The correspondence between DNA's four-letter alphabet
and a protein's twenty-letter alphabet is specified by the
genetic code, which relates nucleotide triplets to amino
acids.
9
Idea: measure the amount of mRNA to see which genes are
being expressed in (used by) the cell.
Measuring protein might be better, but is currently harder.
10
Differential expression
• Each cell contains a complete copy of the
organism's genome.
• Cells are of many different types and states
E.g. blood, nerve, and skin cells, dividing cells,
cancerous cells, etc.
• What makes the cells different?
• Differential gene expression, i.e., when, where,
and in what quantity each gene is expressed.
• On average, 40% of our genes are expressed at
any given time.
11
RNA
• A ribonucleic acid or RNA molecule is a nucleic
acid similar to DNA, but
- single-stranded;
- ribose sugar rather than deoxyribose sugar;
- uracil (U) replaces thymine (T) as one of the
bases.
• RNA plays an important role in protein synthesis
and other chemical activities of the cell.
• Several classes of RNA molecules, including
messenger RNA (mRNA), transfer RNA (tRNA),
ribosomal RNA (rRNA), and other small RNAs.
12
Exons and introns
• Genes comprise only about 2% of the human
genome; the rest consists of non-coding regions,
whose functions may include providing
chromosomal structural integrity and regulating
when, where, and in what quantity proteins are
made (regulatory regions).
• The terms exon and intron refer to coding
(translated into a protein) and non-coding DNA,
respectively.
13
Functional genomics
• The various genome projects have yielded the
complete DNA sequences of many organisms.
E.g. human, mouse, yeast, fruitfly, etc.
• Human: 3 billion base-pairs, 30-40 thousand
genes.
• Challenge: go from sequence to function, i.e.,
define the role of each gene and understand
how the genome functions as a whole.
14
Introduction to microarrays
15
Gene expression assays
• DNA microarrays rely on the hybridization
properties of nucleic acids to monitor DNA or
RNA abundance on a genomic scale in different
types of cells.
• The main types of gene expression assays:
-
High-density nylon membrane arrays;
Serial analysis of gene expression (SAGE);
Short oligonucleotide arrays (Affymetrix);
Long oligonucleotide arrays (Agilent);
Fibre optic arrays (Illumina);
cDNA arrays (Brown/Botstein)*.
16
Applications of microarrays
•
•
•
•
•
•
•
Measuring transcript abundance;
Genotyping;
Estimating DNA copy number;
Determining identity by descent;
Measuring mRNA decay rates;
Identifying protein binding sites;
Determining sub-cellular localization of gene
products;
• ……..
17
18
The process
Building the chip:
MASSIVE PCR
PCR PURIFICATION
and PREPARATION
PREPARING SLIDES
RNA
preparation:
CELL CULTURE
AND HARVEST
PRINTING
Hybing the
chip:
POST PROCESSING
ARRAY HYBRIDIZATION
RNA ISOLATION
DATA ANALYSIS
cDNA PRODUCTION
PROBE LABELING
19
Taken from Schena & Davis
Building the chip
Arrayed Library
(96 or 384-well plates of
bacterial glycerol stocks)
PCR amplification
Directly from colonies with
SP6-T7 primers in 96-well
plates
Consolidate into
384-well plates
Spot as microarray
on glass slides
20
Ngai Lab, UC Berkeley)
Pins collect cDNA
from wells
384 well
plate
Print-tip
group 1
cDNA clones
Contains cDNA
probes
Glass Slide
Array of bound cDNA probes
4x4 blocks = 16 print-tip groups
Spotted in duplicate
Print-tip
group 6
21
Sample preparation
22
Hybridization
Binding of cDNA target samples to cDNA probes on the slide
Hybridize for
5-12 hours
23
Hybridization chamber
3XSSC
HYB CHAMBER
ARRAY
LIFTERSLIP
SLIDE
LABEL
SLIDE LABEL
• Humidity
• Temperature
• Formamide
(Lowers the Tm)
24
Expression profiling with DNA microarrays
cDNA “B”
Cy3 labeled
cDNA “A”
Cy5 labeled
Laser 1
Hybridization
Laser 2
Scanning
+
Analysis
Image Capture
25
RGB overlay of Cy3 and Cy5 images
26
Raw data
• Human cDNA arrays
-
~ 43K spots;
16–bit TIFFs: ~ 20Mb per channel;
~ 2,000 x 5,500 pixels per image;
Spot separation: ~ 136um;
For a “typical” array:
Mean = 43, med = 32, SD = 26 pixels per
spots
27
Image analysis
• The raw data from a cDNA microarray
experiment consist of pairs of image files,
16-bit TIFFs, one for each of the dyes.
• Image analysis is required to extract
measures of the red and green
fluorescence intensities for each spot on
the array.
28
Image analysis
29
GenePix
Image analysis
1. Addressing. Estimate location of
spot centers.
2. Segmentation. Classify pixels as
foreground (signal) or background.
3. Information extraction. For
each spot on the array and each
dye
• signal intensities;
• background intensities;
• quality measures.
R and G for each spot on the array.
30
Spot
• Batch automatic addressing.
• Segmentation. Seeded region growing (Adams &
Bischof 1994): adaptive segmentation method, no
restriction on the size or shape of the spots.
• Intensity extraction
- Foreground. Mean of pixel intensities within a spot.
- Background. Morphological opening: non-linear filter
which generates an image of the estimated background
intensity for the entire slide.
• Spot quality measures.
• Software package. Spot, built on the freely
available software package R.
31
Quality measures
• Spot
- One channel, R or G
- Signal/noise ratio;
- Variation in pixel intensities;
- Identification of “bad spots” (no signal), etc.
- Two channels, R/G
- Brightness: foreground/background ratio;
- Uniformity: variation in pixel intensities and ratios of intensities;
- Morphology: area, perimeter, circularity.
• Array (slide)
- Percentage of spots with no signal;
- Range of intensities;
- Distribution of spot signal area, etc.
• How to use quality measures in subsequent analyses?
32
Terminology
• Probe: DNA spotted on the array, aka. spot,
immobile substrate.
• Target: DNA hybridized to the array, mobile
substrate.
• Sector: collection of spots printed using the same
print-tip (or pin), aka. print-tip-group, pin-group,
spot matrix, grid.
• Batch: collection of slides with the same probe
layout.
• The terms slide or array are often used to refer to
the printed microarray.
33
Biological
Question
Data
Analysis &
Modelling
Microarray
Life Cycle
Sample
Preparation
Microarray
Detection
Microarray
Reaction
Taken from Schena & Davis
34
WWW resources
• Complete guide to “microarraying”
http://cmgm.stanford.edu/pbrown/mguide/
• http://www.microarrays.org
- Parts and assembly instructions for printer and
scanner;
- Protocols for sample prep;
- Software;
- Forum, etc.
• Animation:
http://www.bio.davidson.edu/courses/genomics/chip/chip.html
35
Acknowledgments
Introduction to biology: based on Bioconductor short course lecture 1
(http://www.bioconductor.org/) and Temple short course lecture 1
with pictures from http://www.accessexcellence.org/
Introduction to microarray: based on IPAM, UCLA
http://www.ipam.ucla.edu/programs/fg2000/tutorials.html#terry_speed
Thanks also to:
Natalie Thorne (WEHI)
Ingrid Lönnstedt (Uppsala)
36