DNA Sequencing DNA sequencing How we obtain the

DNA Sequencing

DNA sequencing How we obtain the sequence of nucleotides of a species …ACGTGACTGAGGACCGTG CGACTGACTGGGT CTAGACTACGTTTTA TATATACGTCGTCGT ACTGATGACTAGATTACAG ACTGATTTAGATACCTGAC TGATTTTAAAAAAATATT… CS 273 a 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! CS 273 a Lecture 3, Autumn 08, Batzoglou

Why humans are so similar N Out of Africa Heterozygosity: H H = 4 Nu/(1 + 4 Nu) u ~ 10 -8, N ~ 104 H ~ 4 10 -4 CS 273 a Lecture 3, Autumn 08, Batzoglou A small population that interbred reduced the genetic variation Out of Africa ~ 40, 000 years ago

Human population migrations • Out of Africa, Replacement § “Grandma” of all humans (Eve) ~150, 000 yr • Ancestor of all mt. DNA § “Grandpa” of all humans (Adam) ~100, 000 yr • Ancestor of all Y-chromosomes • Multiregional Evolution § Fossil records show a continuous change of morphological features § Proponents of theory doubt mt. DNA and other genetic evidence • New fossil records bury “multirigionalists” § Nice article in Economist on that http: //www. economist. com/science/displaystory. cfm? story_id=9507453 CS 273 a Lecture 3, Autumn 08, Batzoglou

DNA Sequencing – Overview • Gel electrophoresis 1975 § 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 § Microbial and environmental sequencing § Cancer genome sequencing 2015 CS 273 a 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 CS 273 a Lecture 3, Autumn 08, Batzoglou

DNA Sequencing – vectors DNA Shake DNA fragments Vector Circular genome (bacterium, plasmid) CS 273 a Lecture 3, Autumn 08, Batzoglou Known location + = (restriction site)

Different types of vectors VECTOR Size of insert Plasmid 2, 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 CS 273 a 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 CS 273 a Lecture 3, Autumn 08, Batzoglou

Method to sequence longer regions genomic segment cut many times at random (Shotgun) Get one or two reads from each segment ~900 bp CS 273 a Lecture 3, Autumn 08, Batzoglou ~900 bp

Reconstructing the Sequence (Fragment Assembly) reads Cover region with high redundancy Overlap & extend reads to reconstruct the original genomic region CS 273 a Lecture 3, Autumn 08, Batzoglou

Definition of Coverage C Length of genomic segment: Number of reads: Length of each read: G N L Definition: C=NL/G Coverage 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 nucleotides CS 273 a Lecture 3, Autumn 08, Batzoglou

Repeats Bacterial genomes: Mammals: 5% 50% Repeat types: • Low-Complexity DNA (e. g. ATATACATA…) • Microsatellite repeats • Transposons § SINE (a 1…ak)N where k ~ 3 -6 (e. g. CAGCAGTAGCAGCACCAG) (Short Interspersed Nuclear Elements) e. g. , ALU: ~300 -long, 106 copies § LINE § LTR retroposons (Long Interspersed Nuclear Elements) ~4000 -long, 200, 000 copies (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 CS 273 a Lecture 3, Autumn 08, Batzoglou

Sequencing and Fragment Assembly AGTAGCACAGA CTACGACGAGA CGATCGTGCGACGGCGTA GTGTGCTGTAC TGTCGTGTGTG TGTACTCTCCT 3 x 109 nucleotides 50% of human DNA is composed of repeats Error! Glued together two distant regions CS 273 a Lecture 3, Autumn 08, Batzoglou

What can we do about repeats? Two main approaches: • Cluster the reads • Link the reads CS 273 a Lecture 3, Autumn 08, Batzoglou

What can we do about repeats? Two main approaches: • Cluster the reads • Link the reads CS 273 a Lecture 3, Autumn 08, Batzoglou

What can we do about repeats? Two main approaches: • Cluster the reads • Link the reads CS 273 a Lecture 3, Autumn 08, Batzoglou

Sequencing and Fragment Assembly AGTAGCACAGA CTACGACGAGA CGATCGTGCGACGGCGTA GTGTGCTGTAC TGTCGTGTGTG TGTACTCTCCT 3 x 109 nucleotides A R B ARB, CRD or C CS 273 a Lecture 3, Autumn 08, Batzoglou R D ARD, CRB ?

Sequencing and Fragment Assembly AGTAGCACAGA CTACGACGAGA CGATCGTGCGACGGCGTA GTGTGCTGTAC TGTCGTGTGTG TGTACTCTCCT 3 x 109 nucleotides CS 273 a Lecture 3, Autumn 08, Batzoglou

Strategies for whole-genome sequencing 1. Hierarchical – Clone-by-clone i. iii. Break genome into many long pieces Map each long piece onto the genome Sequence each piece with shotgun Example: Yeast, Worm, Human, Rat 2. Online version of (1) – Walking i. iii. Break genome into many long pieces Start sequencing each piece with shotgun 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 CS 273 a Lecture 3, Autumn 08, Batzoglou