1. CS273a Lecture 5, Win07, Batzoglou Quality of assemblies—mouse N50 contig length Terminology: N50 contig length If we sort contigs from largest to smallest, and start Covering the genome in that order, N50 is the length Of the contig that just covers the 50 th percentile. 7.7X sequence coverage

2. CS273a Lecture 5, Win07, Batzoglou Quality of assemblies—dog 7.5X sequence coverage

3. CS273a Lecture 5, Win07, Batzoglou Quality of assemblies—chimp 3.6X sequence Coverage Assisted Assembly

4. CS273a Lecture 5, Win07, Batzoglou History of WGA 1982: -virus, 48,502 bp 1995: h-influenzae, 1 Mbp 2000: fly, 100 Mbp 2001 – present  human (3Gbp), mouse (2.5Gbp), rat *, chicken, dog, chimpanzee, several fungal genomes Gene Myers Let’s sequence the human genome with the shotgun strategy That is impossible, and a bad idea anyway Phil Green 1997

5. CS273a Lecture 5, Win07, Batzoglou Some new sequencing technologies

6. CS273a Lecture 5, Win07, Batzoglou Molecular Inversion Probes

7. CS273a Lecture 5, Win07, Batzoglou Illumina Genotype Arrays

8. CS273a Lecture 5, Win07, Batzoglou Single Molecule Array for Genotyping—Solexa

9. CS273a Lecture 5, Win07, Batzoglou Nanopore Sequencing http://www.mcb.harvard.edu/branton/index.htm

10. CS273a Lecture 5, Win07, Batzoglou Pyrosequencing on a chip Mostafa Ronaghi, Stanford Genome Technologies Center 454 Life Sciences

11. CS273a Lecture 5, Win07, Batzoglou Polony Sequencing

12. CS273a Lecture 5, Win07, Batzoglou Short read sequencing protocol Random, high-coverage clone library (Cov G = 7 – 10x) Low-coverage of clone by reads (Cov R = 1 – 2x)

13. CS273a Lecture 5, Win07, Batzoglou Short read sequencing protocol

14. CS273a Lecture 5, Win07, Batzoglou Ordering clones into clone contigs

15. CS273a Lecture 5, Win07, Batzoglou Contig assembly

16. CS273a Lecture 5, Win07, Batzoglou Contig assembly

17. CS273a Lecture 5, Win07, Batzoglou Assembly quality SequenceCoverage Contig N50 (Kb) Base quality (Q) Misassemblie s (#/Mb) Small indels (#/Mb) D. Melanogaster (118 Mb) 94.2%160.238.42.51.6 Human chr21 (34 Mb) 97.5%79.035.61.92.3 Human chr11 (131 Mb) 96.3%57.434.42.81.9 Human chr1 (223 Mb) 96.2%63.034.43.02.0 Read length = 200 bp, Error rate = 1%, Net coverage = 20.0x

18. CS273a Lecture 5, Win07, Batzoglou Some future directions for sequencing 1.Personalized genome sequencing Find your ~3,000,000 single nucleotide polymorphisms (SNPs) Find your rearrangements Goals: Link genome with phenotype Provide personalized diet and medicine (???) designer babies, big-brother insurance companies Timeline: Inexpensive sequencing:2010-2015 Genotype–phenotype association:2010-??? Personalized drugs:2015-???

19. CS273a Lecture 5, Win07, Batzoglou Some future directions for sequencing 2.Environmental sequencing Find your flora: organisms living in your body External organs: skin, mucous membranes Gut, mouth, etc. Normal flora: >200 species, >trillions of individuals Flora–disease, flora–non-optimal health associations Timeline: Inexpensive research sequencing:today Research & associationswithin next 10 years Personalized sequencing2015+ Find diversity of organisms living in different environments Hard to isolate Assembly of all organisms at once

20. CS273a Lecture 5, Win07, Batzoglou Some future directions for sequencing 3.Organism sequencing Sequence a large fraction of all organisms Deduce ancestors Reconstruct ancestral genomes Synthesize ancestral genomes Clone—Jurassic park! Study evolution of function Find functional elements within a genome How those evolved in different organisms Find how modules/machines composed of many genes evolved

21. CS273a Lecture 5, Win07, Batzoglou Multiple Sequence Alignment

22. CS273a Lecture 5, Win07, Batzoglou

23. Genome Evolution – Macro Events Inversions Deletions Duplications

24. CS273a Lecture 5, Win07, Batzoglou Synteny maps Comparison of human and mouse