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 262 Lecture 9, Win 06, 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: ~1/2, 000 Other organisms have much higher polymorphism rates CS 262 Lecture 9, Win 06, Batzoglou

CS 262 Lecture 9, Win 06, Batzoglou

Human population migrations • Out of Africa, Replacement § Single mother of all humans (Eve) ~150, 000 yr § Single father of all humans (Adam) ~70, 000 yr § Humans out of Africa ~40000 years ago replaced others (e. g. , Neandertals) § Evidence: mt. DNA • Multiregional Evolution § Fossil records show a continuous change of morphological features § Proponents of theory doubt mt. DNA and other genetic evidence CS 262 Lecture 9, Win 06, Batzoglou

Why humans are so similar Out of Africa A small population that interbred reduced the genetic variation Out of Africa ~ 40, 000 years ago CS 262 Lecture 9, Win 06, Batzoglou

Migration of human variation http: //info. med. yale. edu/genetics/kkidd/point. html CS 262 Lecture 9, Win 06, Batzoglou

Human variation in Y chromosome CS 262 Lecture 9, Win 06, Batzoglou

CS 262 Lecture 9, Win 06, 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 262 Lecture 9, Win 06, 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 ~500 letters at a time CS 262 Lecture 9, Win 06, Batzoglou

DNA Sequencing – vectors DNA Shake DNA fragments Vector Circular genome (bacterium, plasmid) CS 262 Lecture 9, Win 06, 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 262 Lecture 9, Win 06, 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 262 Lecture 9, Win 06, Batzoglou

Electrophoresis diagrams CS 262 Lecture 9, Win 06, Batzoglou

Challenging to read answer CS 262 Lecture 9, Win 06, Batzoglou

Reading an electropherogram 1. 2. 3. 4. Filtering Smoothening Correction for length compressions A method for calling the letters – PHRED – PHil’s Read EDitor (by Phil Green) Several better methods exist, but labs are reluctant to change CS 262 Lecture 9, Win 06, Batzoglou

Output of PHRED: a read A read: 500 -700 nucleotides A C G A A T C A G …A 16 18 21 23 25 15 28 30 32 … 21 Quality scores: -10 log 10 Prob(Error) Reads can be obtained from leftmost, rightmost ends of the insert Double-barreled sequencing: (1990) Both leftmost & rightmost ends are sequenced, reads are paired CS 262 Lecture 9, Win 06, Batzoglou

Method to sequence longer regions genomic segment cut many times at random (Shotgun) Get one or two reads from each segment ~500 bp CS 262 Lecture 9, Win 06, Batzoglou ~500 bp

Reconstructing the Sequence (Fragment Assembly) reads Cover region with ~7 -fold redundancy (7 X) Overlap reads and extend to reconstruct the original genomic region CS 262 Lecture 9, Win 06, Batzoglou

Definition of Coverage C Length of genomic segment: Number of reads: Length of each read: L n l Definition: C=nl/L Coverage How much coverage is enough? Lander-Waterman model: Assuming uniform distribution of reads, C=10 results in 1 gapped region /1, 000 nucleotides CS 262 Lecture 9, Win 06, 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 262 Lecture 9, Win 06, 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 262 Lecture 9, Win 06, Batzoglou

What can we do about repeats? Two main approaches: • Cluster the reads • Link the reads CS 262 Lecture 9, Win 06, Batzoglou

Sequencing and Fragment Assembly AGTAGCACAGA CTACGACGAGA CGATCGTGCGACGGCGTA GTGTGCTGTAC TGTCGTGTGTG TGTACTCTCCT 3 x 109 nucleotides A R B ARB, CRD or C CS 262 Lecture 9, Win 06, Batzoglou R D ARD, CRB ?

Sequencing and Fragment Assembly AGTAGCACAGA CTACGACGAGA CGATCGTGCGACGGCGTA GTGTGCTGTAC TGTCGTGTGTG TGTACTCTCCT 3 x 109 nucleotides CS 262 Lecture 9, Win 06, 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 262 Lecture 9, Win 06, Batzoglou

Hierarchical Sequencing

Hierarchical Sequencing Strategy a BAC clone genome 1. 2. 3. 4. 5. 6. Obtain a large collection of BAC clones Map them onto the genome (Physical Mapping) Select a minimum tiling path Sequence each clone in the path with shotgun Assemble Put everything together CS 262 Lecture 9, Win 06, Batzoglou map

Methods of physical mapping Goal: Make a map of the locations of each clone relative to one another Use the map to select a minimal set of clones to sequence Methods: • • Hybridization Digestion CS 262 Lecture 9, Win 06, Batzoglou

1. Hybridization p 1 Short words, the probes, attach to complementary words 1. 2. 3. 4. Construct many probes Treat each BAC with all probes Record which ones attach to it Same words attaching to BACS X, Y overlap CS 262 Lecture 9, Win 06, Batzoglou pn

Hybridization – Computational Challenge (i, j): 1, if pi hybridizes to Cj 0, otherwise Definition: Consecutive ones matrix 1 s are consecutive in each row & col Computational problem: Reorder the probes so that matrix is in consecutive-ones form Can be solved in O(m 3) time (m > n) CS 262 Lecture 9, Win 06, Batzoglou Cj 1 Cj 2 ………………. Cjn Matrix: m probes n clones C 1 C 2 ………………. Cn p 1 p 2 …………. pm 0 0 1 …………………. . 1 1 1 0 …………………. . 0 1…………………. . . 0 pi 1 pi 2…………. pim 1 1 1 0 0 0……………. . 0 0 1 1 1……………. . 0 0 0 1 1 1 0……………. . 0 0 0 0……… 1 1 1 0 0 0 0……… 0 1 1 1

pi 1 pi 2…………………. pim pi 1 pi 2…………. pim 1 1 1 0 0 0……………. . 0 0 1 1 1……………. . 0 0 0 1 1 1 0……………. . 0 0 0 0……… 1 1 1 0 0 0 0……… 0 1 1 1 Cj 1 Cj 2 ………………. Cjn Hybridization – Computational Challenge If we put the matrix in consecutive-ones form, then we can deduce the order of the clones & which pairs of clones overlap CS 262 Lecture 9, Win 06, Batzoglou

Hybridization – Computational Challenge A probe (short word) can hybridize in many places in the genome Computational Problem: Find the order of probes that implies the minimal probe repetition Equivalent: find the shortest string of probes such that each clone appears as a substring APX-hard Solutions: Greedy, probabilistic, lots of manual curation CS 262 Lecture 9, Win 06, Batzoglou p 1 p 2 …………. pm C 1 C 2 ………………. Cn Additional challenge: 0 0 1 …………………. . 1 1 1 0 …………………. . 0 1…………………. . . 0

2. Digestion Restriction enzymes cut DNA where specific words appear 1. Cut each clone separately with an enzyme 2. Run fragments on a gel and measure length 3. Clones Ca, Cb have fragments of length { li, lj, lk } overlap Double digestion: Cut with enzyme A, enzyme B, then enzymes A + B CS 262 Lecture 9, Win 06, Batzoglou

Online Clone-by-clone The Walking Method

The Walking Method 1. Build a very redundant library of BACs with sequenced cloneends (cheap to build) 2. Sequence some “seed” clones 3. “Walk” from seeds using clone-ends to pick library clones that extend left & right CS 262 Lecture 9, Win 06, Batzoglou

Walking: An Example CS 262 Lecture 9, Win 06, Batzoglou

Walking off a Single Seed • Low redundant sequencing • Many sequential steps CS 262 Lecture 9, Win 06, Batzoglou

Walking off a single clone is impractical Cycle time to process one clone: 1 -2 months 1. 2. 3. 4. 5. Grow clone Prepare & Shear DNA Prepare shotgun library & perform shotgun Assemble in a computer Close remaining gaps A mammalian genome would need 15, 000 walking steps ! CS 262 Lecture 9, Win 06, Batzoglou

Walking off several seeds in parallel Efficient Inefficient • Few sequential steps • Additional redundant sequencing In general, can sequence a genome in ~5 walking steps, with <20% redundant sequencing CS 262 Lecture 9, Win 06, Batzoglou

Whole-Genome Shotgun Sequencing

Whole Genome Shotgun Sequencing genome cut many times at random plasmids (2 – 10 Kbp) known dist cosmids (40 Kbp) ~500 bp CS 262 Lecture 9, Win 06, Batzoglou forward-reverse paired reads ~500 bp