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 …ACGTGACTGAGGACCGTG CGACTGAGACTGACTGGGT CTAGCTAGACTACGTTTTA TATATATATACGTCGTCGT ACTGATGACTAGATTACAG ACTGATTTAGATACCTGAC TGATTTTAAAAAAATATT…

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 ~ 10 4  H ~ 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 that

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 Resequencing of humans  Microbial and environmental sequencing Microbial and environmental sequencing  Cancer genome sequencing Cancer genome sequencing

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 Vector Circular genome (bacterium, plasmid) Known location (restriction site)

CS273a Lecture 1, Autumn 10, Batzoglou Different types of vectors VECTORSize of insert Plasmid 2,000-10,000 Can control the size Cosmid40,000 BAC (Bacterial Artificial Chromosome) 70, ,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 / Image credits: 454 Life Sciences

CS273a Lecture 1, Autumn 10, Batzoglou Solexa / ABI SOLiD 12 Image credits: Illumina, Applied Biosystems

CS273a Lecture 1, Autumn 10, Batzoglou Illumina / Affymetrix genotyping 13 Image credits: Illumina, Affymetrix

CS273a Lecture 1, Autumn 10, Batzoglou CS273a Lecture 1 Human Genome Project (1990 – 2003) $3 billion3 Gbp ,000 Gbp ,000 Gbp ,000,000 Gbp Growth of DNA Sequencing Capacity

CS273a Lecture 1, Autumn 10, Batzoglou CS273a Lecture million species Each individual has different DNA Within individual, some cells have different DNA (i.e. cancer) Sequencing Applications

CS273a Lecture 1, Autumn 10, Batzoglou CS273a Lecture 1 What genes are on/off, when, and in which cells? Where do molecules bind to DNA? Sequencing Applications