Transcript HighThroughput

The Biology, Technology and
Statistical Modeling of Highthroughput Genomics Data
Naomi Altman
Dept. of Statistics
Penn State U.
May 25, 2010
1
DNA 100
A Statistician’s Simplification
Every cell has the same
genetic material, stored in
the double helix of DNA.
The "backbone" is the
support of the ladder.
The rungs are "base pairs" .
Each pair consists of 2
bound codons which are
designed C, G, A, T. These
are called base pairs.
C binds only to G.
A binds only to T.
http://www.accessexcellence.org/
RC/VL/GG/chromosome.html
http://www.bioteach.ubc.ca/MolecularBiology/
AMonksFlourishingGarden/
In a diploid population, most
cells have 2 copies of each
2
chromosome.
DNA replication
When the cell divides, the DNA is
replicated by breaking the double bond
between the base pairs, and rebuilding
the double helix by creating a new
backbone and new pairs on each
strand.
The molecules making up the backbone
are asymmetric creating a direction
along the backbone. One end is called
the "3' end". The other end is the "5'
end". Duplication always goes from the
5' end to the 3' end.
http://oak.cats.ohiou.edu
/~ballardh/pbio475/Heredity
/Heredity.htm
A complex suite of proteins is involved3
in duplication.
Transcription and Translation
To make a protein:
•The DNA unzips
•mRNA binds to the exposed
codons on the coding (sense)
strand - the matching strand is
the anti-sense strand
•mRNA goes to the ribosome
where it binds to amino acids
brought to the ribosome by the
tRNA
•Each set of 3 codons encodes
1 amino acid
•The complete linear set of
amino acids defines the protein
http://www.bioteach.ubc.ca/MolecularBiology/
AMonksFlourishingGarden/
•The protein folds into its
active shape
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Transcription
•transcription factors bind to the
promoter and bind RNA polymerase
•DNA strands separate and
transcription is initiated
•transcription continues in the 3'-5'
direction until the stop codons are
reached
•The completed RNA strand is
released for post-processing
www.csu.edu.au/faculty/health/biomed/subjects/molbol/basic.htm
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Introns and Exons
promoter
Chromosome
In "higher" organisms, the gene contains
noncoding regions, called introns, and
coding regions called exons.
The introns are spliced out of the mRNA
before translation into protein.
"Splicing variants" can be formed by the cell
selecting combinations of the exons.
The resulting spliced strand is the mRNA.
We can "predict" exons using statistical
algorithms, but the gold standard is that
only exons match mRNA sequences
http://biology.unm.edu/ccouncil/Bi
ology_124/Summaries/T&T.html
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DNA 100
A Statistician’s Simplification
DNA is complicated stuff.
Protein-coding regions are called genes.
There are also other functional parts to the DNA, some of which
code for RNA and some of which are regulatory regions - i.e. they
help control how the coding regions are used - e.g. promoters
The supercoiling of the DNA may also control how the coding
regions are used.
As well, there is a lot of DNA which appears to be "junk" - i.e. to
date no function is known. But we keep making new discoveries e.g. some of the "junk" codes for small RNA pieces that are
functional.
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DNA 100
A Statistician’s Simplification
An allele is a variant of a gene
- e.g. "blood type" A, B, O in humans.
If a gene has 2 or more alleles, it is said to be polymorphic.
A Single Nucleotide Polymorphism (SNP) means that 2 individuals
from the same species have a difference in one nucleotide at
some location in their DNA. (e.g. a C in one person, and a T in
the other).
SNPs are very useful for determining the genotype of an organism
and for tracing evolution of proteins.
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DNA 100
A Statistician’s Simplification
A key step in microarray technology is
reverse transcription: going from mRNA
to DNA with the introns excised. This is
called cDNA.
At the 5' and 3' ends of the cDNA are
the regulatory regions called the
"UnTranslated Regions" or UTRs.
The 5' UTR is functional and evolves
very slowly.
The 3' UTR is less functional and hence
evolves more rapidly. It can be used to
distinguish closely related genes.
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DNA 100
A Statistician’s Simplification
DNA persists in the cell, and is the cell's memory device.
mRNA and proteins do not persist in the cell and are degraded with components
recycled.
Degradation is part of cell regulation. Cells degrade both imperfect compounds and
those no longer needed.
Understanding cellular processes is complicated by our inability to follow the synthesis
and degradation processes in single cells - so we are actually seeing the average over
many cells which may be at somewhat different stages.
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DNA 100
A Statistician’s Simplification
The function of each cell is determined by which proteins it produces.
Our objective will be to measure either proteins are produced (directly, by
measuring and identifying proteins or indirectly by measuring mRNA).
It is easier to measure mRNA than protein, but due to degradation, the
correlation between mRNA levels and protein levels is imperfect. In fact, in
some cases, the mRNA may not actually produce any protein.
In some cases we will measure the genomic DNA directly - usually to look for
differences among alleles.
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PCR
Polymerase Chain Reaction
PCR allows us to
greatly amplify
any selected
piece of DNA.
Selection is done
by choice of
primer.
DNA
This allows us to
detect small
quantities of DNA.
Labels can be added
to the new strands
by attaching
chemicals to the free
C,G,A,T
primer
new strand
denature (by high
temperature)
primer
new strand
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Electrophoresis
The PCR product is put
through an electrophoresis gel
to determine
presence/absence of the
DNAs targeted by the primer
Maryam Ahmed Khan
February 14, 2001
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PCR
Polymerase Chain Reaction
PCR is used directly to amplify genes. It is mainly used to
detect alleles i.e. variants of a gene that can be used to
e.g. identify individuals (e.g. DNA fingerprinting)
identify subpopulations (e.g. tracking ivory poaching)
determine which variants are associated with a condition
(e.g. drug efficacy)
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RT-PCR
Reverse Transcription PCR
in the cell
primer
cDNA
cDNA
cDNA
DNA
mRNA
mRNA mRNA
primer cDNA
RT-PCR is used to identify genes which
EXPRESS in the tissue
or create a cDNA library
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cDNA Library and ESTs
A cDNA library is a means of storing specific
genes or gene fragments.
The library is actually a set of "wells" containing
living cells with plasmids containing the cDNA.
Often a cDNA library for a tissue is partially
sequenced, to obtain Expressed Sequence
Tags (ESTs), short pieces of sequenced DNA
which can be used to identify which genes are
expressed in the tissue. (There is a lot of
computation involved in compiling ESTs into
gene sequences, which is called assembly.)
fig.cox.miami.edu/~cmallery/150/gene/sf16x5.jpg
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PCR Methods for Measuring
Gene Expression
PCR is considered the gold standard for detecting and
measuring gene expression.
Detection is "simple". A label (radioactive or dye) can
be added during the PCR reaction. After several
cycles, if the label is bound, then the PCR target
must be present.
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Quantitative PCR Methods for
Measuring Gene Expression
Because each cycle of PCR requires the denaturization step the number of
PCR cycles is under experimental control.
Hence, the quantity of PCR product at the end of some number of cycles can
be used to estimate the initial quantity. The estimate is usually improved by
also amplifying a "control" product with "known" initial quantity.
Quantitative PCR uses only the measured quantity at the final step of a preset
number of cycles.
Real time PCR uses a label that binds only to double stranded DNA, and
measures the quantity at the end of each cycle. This provides a curve giving
the label intensity versus the number of cycles, which can be extrapolated back
to the initial point. This method is more accurate but much more expensive.
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Real Time RT-PCR
(from the PSU Nucleic Acid Facility)
• A probe is designed to anneal to the target sequence
between mRNA and cDNA primers.
• The probe is labeled at the 5' end with a reporter
fluorochrome and a quencher fluorochrome added at
any T position or at the 3' end.
• The amount of fluorescence released during the
amplification cycle is proportional to the amount of
product generated in each cycle.
• The software calculates the threshold cycle (CT) for
each reaction with which there is a linear relationship to
the amount of starting DNA or RNA.
• Up to 96 samples are run simultaneously, so the relative
fluorescence corresponds to the relative quantity of
mRNA initially present
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Northern Blot
This is another "1-at-atime" RNA detection
method amenable to
quantification - the
"old" gold standard.
http://www.columbia.edu/cu/biology/courses/c2005/handouts/northernforweb.gif
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Microarrays
A microarray is a glass or plastic slide on which
are printed 1000's of single strands of cDNA.
RT is used to create single strand labeled cDNA
from the mRNA of a tissue.
The cDNA binds only to the complementary
strand on the slide.
Dye intensity for each "spot" is proportional to the
concentration of matching cDNA.
The intensity is summarized by a scanning
microscope, which detects the "spots".
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What is a microarray probe?
A probe is a spot on an array representing a
gene or part of a gene
On “cDNA” arrays, the probes are actual
pieces of cDNA originally extracted from a
cell.
We may not know the genetic sequence of a
cDNA.
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What is a microarray probe?
If we know the genetic sequence of the
cDNA, we can artificially synthesize a
strand of DNA with the same sequence.
This is called an oligo(nucleotide).
Oligos may be “spotted” on the array like
cDNA or may be synthesized on the array
by one of several technologies.
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cDNA versus Oligos
cDNAs have different hybridization
properties due to their biochemistry
Oligos may be chosen to have similar
hybridization properties
- and to represent maximally unique parts of
genes
- or to represent common domains
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cDNA versus Oligos
cDNAs are maintained in cDNA libraries
which are expensive to maintain and may
be mislabeled or contaminated.
Oligos are synthesized from genomic
sequence information which can be
subject to error.
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Spotted 2-Channel Array
Spotted arrays are
printed on
coated
microscope
slides.
2 RNA samples
are converted
to cDNA. Each
is labelled with
a different dye.
http://www.anst.uu.se/frgra677/bilder/micro_method_large.jpg
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"Spotted" arrays
The spot material may be a cDNA, or an oligo generally 50-70 codons long.
Some commercial arrays use only a single dye.
"Spotted" refers to the print technology. Arrays
with similar format may have oligos
synthesized directly on the array surface.
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Affymetrix Array
•Each gene is represented by a “probe set”
•Each “probe set” is 16-20 pairs of oligos
•Each oligo is 25 nucleotides
•A PM (perfect match) probe matches a
strand of cDNA
•The corresponding MM (mismatch) probe
differs from the PM by a change in the central
nucleotide
•The probe pairs are spatially dispersed
•Control probes are printed
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Format of an Affymetrix Array
http://cnx.rice.edu/content/m12388/latest/figE.JPG
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Heuristics for “Probe Sets”
MM probe is supposed to control for:
•Variation in chemical composition
•Abundance of cross-hybridizing fragments from
other genes
By combining PM and MM information from many
probes, gene to gene differences should be
minimized.
These arrays are more quantitative than other
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types of microarrays.
Microarrays for Gene Expression
Whichever technology is used, an intensity
value is obtained for every probe from every
sample.
Generally values are comparative - i.e. does
this probe express more highly in melanoma
than in a normal skin cell.
The data are very noisy. A lot of effort has gone
into data-cleaning methods which are generally
called "normalization".
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Microarrays for Gene Expression
Microarrays are "genomic" - 6000 - 40,000 genes may
be on a single array.
Microarrays have other uses
- e.g. tiling arrays cover the entire genome
- SNP arrays have 2 variants of many SNPs
- promoter arrays have upstream sequence
We will focus on gene expression arrays but most of
what we discuss will be useful for all "omic" level data.
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Measuring Gene Expression
Microarrays are very expensive (on a per
array basis) and somewhat noisy, have
broad coverage (which makes them cheap
on a per gene basis).
Real time PCR and Northern blots are more
accurate (maybe) but are "single gene"
methods.
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Measuring Gene Expression
Microarrays are used to obtain broad
coverage of the genome.
Real time PCR or Northern Blots are often
used to verify the results for a few genes,
or for some low-expression genes.
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