DNA Sequencing

CS273a Lecture 3, Autumn 08, Batzoglou DNA sequencing How we obtain the sequence of nucleotides of a species …ACGTGACTGAGGACCGTG CGACTGAGACTGACTGGGT CTAGCTAGACTACGTTTTA TATATATATACGTCGTCGT ACTGATGACTAGATTACAG ACTGATTTAGATACCTGAC TGATTTTAAAAAAATATT…

CS273a Lecture 3, Autumn 08, 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 3, Autumn 08, 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 3, Autumn 08, 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 bury “multirigionalists”  Nice article in Economist on that

CS273a Lecture 3, Autumn 08, Batzoglou DNA Sequencing – Overview Gel electrophoresis  Predominant, old technology by F. Sanger Whole genome strategies  Physical mapping  Walking  Shotgun sequencing Computational fragment assembly The future—new sequencing technologies  Pyrosequencing, single molecule methods, …  Assembly techniques 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 3, Autumn 08, 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 3, Autumn 08, Batzoglou DNA Sequencing – vectors + = DNA Shake DNA fragments Vector Circular genome (bacterium, plasmid) Known location (restriction site)

CS273a Lecture 3, Autumn 08, 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 3, Autumn 08, 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 3, Autumn 08, Batzoglou Method to sequence longer regions cut many times at random (Shotgun) genomic segment Get one or two reads from each segment ~900 bp

CS273a Lecture 3, Autumn 08, Batzoglou Reconstructing the Sequence (Fragment Assembly) Cover region with high redundancy Overlap & extend reads to reconstruct the original genomic region reads

CS273a Lecture 3, Autumn 08, Batzoglou Definition of Coverage Length of genomic segment:G Number of reads: N Length of each read:L Definition: Coverage C = N L / G How much coverage is enough? Lander-Waterman model:Prob[ not covered bp ] = e -C Assuming uniform distribution of reads, C=10 results in 1 gapped region /1,000,000 nucleotides C

CS273a Lecture 3, Autumn 08, Batzoglou Repeats Bacterial genomes:5% Mammals:50% Repeat types: Low-Complexity DNA (e.g. ATATATATACATA…) Microsatellite repeats (a 1 …a k ) N where k ~ 3-6 (e.g. CAGCAGTAGCAGCACCAG) Transposons  SINE (Short Interspersed Nuclear Elements) e.g., ALU: ~300-long, 10 6 copies  LINE (Long Interspersed Nuclear Elements) ~4000-long, 200,000 copies  LTR retroposons (Long Terminal Repeats (~700 bp) at each end) cousins of HIV Gene Families genes duplicate & then diverge (paralogs) Recent duplications ~100,000-long, very similar copies

CS273a Lecture 3, Autumn 08, Batzoglou Sequencing and Fragment Assembly AGTAGCACAGA CTACGACGAGA CGATCGTGCGA GCGACGGCGTA GTGTGCTGTAC TGTCGTGTGTG TGTACTCTCCT 3x10 9 nucleotides 50% of human DNA is composed of repeats Error! Glued together two distant regions

CS273a Lecture 3, Autumn 08, Batzoglou What can we do about repeats? Two main approaches: Cluster the reads Link the reads

CS273a Lecture 3, Autumn 08, Batzoglou What can we do about repeats? Two main approaches: Cluster the reads Link the reads

CS273a Lecture 3, Autumn 08, Batzoglou What can we do about repeats? Two main approaches: Cluster the reads Link the reads

CS273a Lecture 3, Autumn 08, Batzoglou Sequencing and Fragment Assembly AGTAGCACAGA CTACGACGAGA CGATCGTGCGA GCGACGGCGTA GTGTGCTGTAC TGTCGTGTGTG TGTACTCTCCT 3x10 9 nucleotides C R D ARB, CRD or ARD, CRB ? ARB

CS273a Lecture 3, Autumn 08, Batzoglou Sequencing and Fragment Assembly AGTAGCACAGA CTACGACGAGA CGATCGTGCGA GCGACGGCGTA GTGTGCTGTAC TGTCGTGTGTG TGTACTCTCCT 3x10 9 nucleotides

CS273a Lecture 3, Autumn 08, Batzoglou Strategies for whole-genome sequencing 1.Hierarchical – Clone-by-clone i.Break genome into many long pieces ii.Map each long piece onto the genome iii.Sequence each piece with shotgun Example: Yeast, Worm, Human, Rat 2.Online version of (1) – Walking i.Break genome into many long pieces ii.Start sequencing each piece with shotgun iii.Construct map as you go Example: Rice genome 3.Whole genome shotgun One large shotgun pass on the whole genome Example: Drosophila, Human (Celera), Neurospora, Mouse, Rat, Dog