1. CS273a Lecture 2, Autumn 10, Batzoglou DNA Sequencing (cont.)

2. CS273a Lecture 2, Autumn 10, Batzoglou CS273a Lecture 1 100 million species Each individual has different DNA Within individual, some cells have different DNA (i.e. cancer) Sequencing Applications

3. CS273a Lecture 2, Autumn 10, Batzoglou CS273a Lecture 1 What genes are on/off, when, and in which cells? Where do molecules bind to DNA? Sequencing Applications

4. CS273a Lecture 2, Autumn 10, Batzoglou Method to sequence longer regions cut many times at random (Shotgun) genomic segment Get one or two reads from each segment ~900 bp

5. CS273a Lecture 2, Autumn 10, Batzoglou Reconstructing the Sequence (Fragment Assembly) Cover region with high redundancy Overlap & extend reads to reconstruct the original genomic region reads

6. CS273a Lecture 2, Autumn 10, 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

7. CS273a Lecture 2, Autumn 10, 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

8. CS273a Lecture 2, Autumn 10, 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

9. CS273a Lecture 2, Autumn 10, Batzoglou What can we do about repeats? Two main approaches: Cluster the reads Link the reads

10. CS273a Lecture 2, Autumn 10, Batzoglou What can we do about repeats? Two main approaches: Cluster the reads Link the reads

11. CS273a Lecture 2, Autumn 10, Batzoglou What can we do about repeats? Two main approaches: Cluster the reads Link the reads

12. CS273a Lecture 2, Autumn 10, Batzoglou Sequencing and Fragment Assembly AGTAGCACAGA CTACGACGAGA CGATCGTGCGA GCGACGGCGTA GTGTGCTGTAC TGTCGTGTGTG TGTACTCTCCT 3x10 9 nucleotides C R D ARB, CRD or ARD, CRB ? ARB

13. CS273a Lecture 2, Autumn 10, Batzoglou Sequencing and Fragment Assembly AGTAGCACAGA CTACGACGAGA CGATCGTGCGA GCGACGGCGTA GTGTGCTGTAC TGTCGTGTGTG TGTACTCTCCT 3x10 9 nucleotides

14. CS273a Lecture 2, Autumn 10, 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

15. CS273a Lecture 2, Autumn 10, Batzoglou Hierarchical Sequencing

16. CS273a Lecture 2, Autumn 10, Batzoglou Hierarchical Sequencing Strategy 1.Obtain a large collection of BAC clones 2.Map them onto the genome (Physical Mapping) 3.Select a minimum tiling path 4.Sequence each clone in the path with shotgun 5.Assemble 6.Put everything together a BAC clone map genome

17. CS273a Lecture 2, Autumn 10, Batzoglou Hierarchical Sequencing Strategy 1.Obtain a large collection of BAC clones 2.Map them onto the genome (Physical Mapping) 3.Select a minimum tiling path 4.Sequence each clone in the path with shotgun 5.Assemble 6.Put everything together a BAC clone map genome

18. CS273a Lecture 2, Autumn 10, Batzoglou 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

19. CS273a Lecture 2, Autumn 10, Batzoglou 1. Hybridization Short words, the probes, attach to complementary words 1.Construct many probes 2.Treat each BAC with all probes 3.Record which ones attach to it 4.Same words attaching to BACS X, Y  overlap p1p1 pnpn

20. CS273a Lecture 2, Autumn 10, Batzoglou 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 C a, C b have fragments of length { l i, l j, l k }  overlap Double digestion: Cut with enzyme A, enzyme B, then enzymes A + B

21. CS273a Lecture 2, Autumn 10, Batzoglou Some Terminology insert a fragment that was incorporated in a circular genome, and can be copied (cloned) vector the circular genome (host) that incorporated the fragment BAC Bacterial Artificial Chromosome, a type of insert–vector combination, typically of length 100-200 kb read a 500-900 long word that comes out of a sequencing machine coverage the average number of reads (or inserts) that cover a position in the target DNA piece shotgun the process of obtaining many reads sequencing from random locations in DNA, to detect overlaps and assemble

22. CS273a Lecture 2, Autumn 10, Batzoglou Whole Genome Shotgun Sequencing cut many times at random genome forward-reverse paired reads plasmids (2 – 10 Kbp) cosmids (40 Kbp) known dist ~800 bp