cs273a lecture 1, autumn 10, batzoglou dna sequencing
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CS273a Lecture 1, Autumn 10, Batzoglou
DNA Sequencing
CS273a Lecture 1, Autumn 10, Batzoglou
DNA sequencing
How we obtain the sequence of nucleotides of a species
…ACGTGACTGAGGACCGTGCGACTGAGACTGACTGGGTCTAGCTAGACTACGTTTTATATATATATACGTCGTCGTACTGATGACTAGATTACAGACTGATTTAGATACCTGACTGATTTTAAAAAAATATT…
CS273a Lecture 1, Autumn 10, Batzoglou
Which representative of the species?
Which human?
Answer one:
Answer two: it doesn’t matter
Polymorphism rate: number of letter changes between two different members of a species
Humans: ~1/1,000
Other organisms have much higher polymorphism rates Population size!
CS273a Lecture 1, Autumn 10, Batzoglou
Why humans are so similar
A small population that interbred reduced the genetic variation
Out of Africa ~ 40,000 years ago
Out of Africa
Heterozygosity: H
H = 4Nu/(1 + 4Nu)
u ~ 10-8, N ~ 104
H ~ 410-4
N
CS273a Lecture 1, Autumn 10, Batzoglou
Human population migrations
• Out of Africa, Replacement “Grandma” of all humans (Eve) ~150,000yr
• Ancestor of all mtDNA
“Grandpa” of all humans (Adam) ~100,000yr• Ancestor of all Y-chromosomes
• Multiregional Evolution Fossil records show a continuous change of
morphological features Proponents of the theory doubt mtDNA and
other genetic evidence
• New fossil records support out of Africa Good popular article in Economist on thathttp://www.economist.com/science/displaystory.cfm?story_id=9507453
CS273a Lecture 1, Autumn 10, Batzoglou
DNA Sequencing – Overview
• Gel electrophoresis Old technology by F. Sanger Newer sequencing technologies
• Whole genome strategies Physical mapping Shotgun sequencing
• Computational fragment assembly
• Future variants of sequencing Resequencing of humans Microbial and environmental sequencing Cancer genome sequencing
1975
2015
CS273a Lecture 1, Autumn 10, Batzoglou
DNA Sequencing
Goal:
Find the complete sequence of A, C, G, T’s in DNA
Challenge:
There is no machine that takes long DNA as an input, and gives the complete sequence as output
Can only sequence ~900 letters at a time
CS273a Lecture 1, Autumn 10, Batzoglou
DNA Sequencing – vectors
+ =
DNA
Shake
DNA fragments
VectorCircular genome(bacterium, plasmid)
Knownlocation
(restrictionsite)
CS273a Lecture 1, Autumn 10, Batzoglou
Different types of vectors
VECTOR Size of insert
Plasmid2,000-10,000
Can control the size
Cosmid 40,000
BAC (Bacterial Artificial Chromosome)
70,000-300,000
YAC (Yeast Artificial Chromosome)
> 300,000
Not used much recently
CS273a Lecture 1, Autumn 10, Batzoglou
DNA Sequencing – gel electrophoresis
1. Start at primer(restriction site)
2. Grow DNA chain
3. Include dideoxynucleoside (modified a, c, g, t)
4. Stops reaction at all possible points
5. Separate products with length, using gel electrophoresis
CS273a Lecture 1, Autumn 10, Batzoglou
Pyrosequencing / 454
11Image credits: 454 Life Sciences
CS273a Lecture 1, Autumn 10, Batzoglou
Solexa / ABI SOLiD
12Image credits: Illumina, Applied Biosystems
CS273a Lecture 1, Autumn 10, Batzoglou
Illumina / Affymetrix genotyping
13Image credits: Illumina, Affymetrix
CS273a Lecture 1, Autumn 10, BatzoglouCS273a Lecture 1
Human Genome Project (1990 – 2003)
$3 billion 3 Gbp
2008 100,000 Gbp
2010 500,000 Gbp
2011 5,000,000 Gbp
Growth of DNA Sequencing Capacity
CS273a Lecture 1, Autumn 10, BatzoglouCS273a Lecture 1
100 million species
Each individualhas different DNA
Within individual, some cellshave different DNA(i.e. cancer)
Sequencing Applications
CS273a Lecture 1, Autumn 10, BatzoglouCS273a Lecture 1
What genes are on/off,when, and in which cells?
Where do moleculesbind to DNA?
Sequencing Applications