HW1 Due This Fri 10/21.

1. HW1 Due This Fri 10/21. http://cs273a.stanford.edu [Bejerano Fall11/12]

2. TA Q&A Office Hours: • Preferred. Infinitely higher bandwidth. E-mail: • Please reserve for (short) technicalities. • Discouraged questions: • Is my answer correct? Am I on the right track? • You are experiencing genomic research. No one tells us whether our answers are right or wrong. We QA ourselves. A lot. Do it too. Spot check elements in the UCSC Browser! • Please debug this UNIX statement / output file for me. http://cs273a.stanford.edu [Bejerano Fall11/12]

3. Lecture 7 Chains & Nets cont’d Genome Reconstruction Genomic Conservation & Function Sneak peek at other functional classes http://cs273a.stanford.edu [Bejerano Fall11/12]

4. Mutations Chromosomal Mutations: Deletion Inversion Translocation Duplication (Nondisjunction) http://cs273a.stanford.edu [Bejerano Fall11/12]

5. Gene tree Speciation Duplication Loss Gene Families Orthologs : Genes related via speciation (e.g. C,M,H3) Paralogs: Genes related through duplication (e.g. H1,H2,H3) Homologs: Genes that share a common origin (e.g. C,M,H1,H2,H3) single ancestral gene Species tree http://cs273a.stanford.edu [Bejerano Fall11/12]

6. Chaining (Paralogs) Protease Regulatory Subunit 3 http://cs273a.stanford.edu [Bejerano Fall11/12]

7. Netting (Ortholog) http://cs273a.stanford.edu [Bejerano Fall11/12]

8. Convert / LiftOver "LiftOver chains" are actually chains extracted from nets, or chains filtered by the netting process. LiftOver – batch utility http://cs273a.stanford.edu [Bejerano Fall11/12]

9. Net highlights rearrangements A large gap in the top level of the net is filled by an inversion containing two genes. Numerous smaller gaps are filled in by local duplications and processed pseudo-genes. http://cs273a.stanford.edu [Bejerano Fall11/12]

10. Drawbacks • Nets relentlessly try to fill in gaps True dog ortholog just hasn’t been sequenced Heuristic guess of best chain!

11. Retroposed Genes and Pseudogenes http://cs273a.stanford.edu [Bejerano Fall11/12]

12. And Retrogenes http://cs273a.stanford.edu [Bejerano Fall11/12]

13. Conservation Track Documentation http://cs273a.stanford.edu [Bejerano Fall11/12]

14. Useful in finding pseudogenes gene pred. Ensembl and Fgenesh++ automatic gene predictions confounded by numerous processed pseudogenes. Domain structure of resulting predicted protein must be interesting! http://cs273a.stanford.edu [Bejerano Fall11/12]

15. Cautionary Note 2 http://cs273a.stanford.edu [Bejerano Fall11/12]

16. Same Region… same in all the other fish http://cs273a.stanford.edu [Bejerano Fall11/12]

17. A Rearrangement Hot Spot Rearrangements are not evenly distributed. Roughly 5% of the genome is in hot spots of rearrangements such as this one. This 350,000 base region is between two very long chains on chromosome 7. http://cs273a.stanford.edu [Bejerano Fall11/12]

18. Drawbacks • Inversions not handled optimally Chains > > > > chr1 > > > > > > > chr1 > > > < < < < chr5 < < < < < < < < chr1 < < < < Nets > > > > chr1 > > > > > > > chr1 > > > < < < < chr5 < < < <

19. Drawbacks • High copy number genes can break orthology

20. Self Chain http://cs273a.stanford.edu [Bejerano Fall11/12]

21. From pairwise to multiple alignments http://cs273a.stanford.edu [Bejerano Fall11/12]

22. Multidimensional DP • Example: in 3D (three sequences): • 7 neighbors/cell F(i,j,k) = max{ F(i-1,j-1,k-1)+S(xi, xj, xk), F(i-1,j-1,k )+S(xi, xj, - ), F(i-1,j ,k-1)+S(xi, -, xk), F(i-1,j ,k )+S(xi, -, - ), F(i ,j-1,k-1)+S( -, xj, xk), F(i ,j-1,k )+S( -, xj, xk), F(i ,j ,k-1)+S( -, -, xk) }

23. Progressive Alignment x pxy y • When evolutionary tree is known: • Align closest first, in the order of the tree • In each step, align two sequences x, y, or profiles px, py, to generate a new alignment with associated profile presult z pxyzw pzw w E.g: Blastz – Multiz shown in UCSC browser

24. Anchor based alignment Example: http://cs273a.stanford.edu [Bejerano Fall11/12]

25. Anchor based alignment E.g: Enredo - Pecan shown in ENSEMBL browser http://cs273a.stanford.edu [Bejerano Fall11/12]

26. Reconstruct the Boreoeutherian ancestor

27. Ancestral Genome Reconstruction • Given: - Genomic sequences of several mammals • - Phylogenetic tree • Find: The genomic sequence of all their ancestors ARMADILLO TGCTACTAATATTTAGTACATAGAGCCCAGGGGTGCTGCTGAAAGTCTTAAAATGCACAGTGTAGCCCCTCCTCC COW GCCTCTCTTTCTGCCCTGCAGGCTAGAATGTATCACTTAGATGTTCCAAATCAGAAAGTGTTCAGCCATTTCCATACC HORSE GTCACAATTTAGGAAGTGCCACTGGCCTCTAGAGGGTAGAAGACAGGGATGCTAATAATCATCCCACGTCATCCTACAGTGCTCAGAACAGCACCCCTACCCTCACCCC CAT GTCACAGTTTAGGGGGTACTACTGGCATCTATCGGGTGGAGGATAGGGATACTGATAATCATTCTACAGTGCACAGGACAGTACCCCTACTTTCACCCC DOG GTCACAATTTGGGGGATACTACTGGCATCTAATGGGTAGAGGACAGGGATACTGATAATTGCTTTACAGTGCACAGGACAGCACCCTTATCTTCACCCC HEDGEHOG GTCATAGTTTGATTATATGGGCTTCTTAGTAGACAAAGAAAAAGATGTTCTGGTAGTCATTCTGCTTTCCATATGATAGCACTCCCATCTTCACTTC MOUSE GTCACAGTTTGGAGGATGTTACTGACATCTAGAGAGTAGACTTTAAAGATACTGATAGTCACCCCATTGTGCACCTCC RAT GTCACAATTTGGAGGATGTTACTGGCATCTAGAGAGTAGACTTTAAGGACACTGATAATCATACTATGCTGCACTTCC RABBIT ATCACAATTTGGGGAACACCACTGGCATCTCGGGTAGCAGGCCAGGCATGCTGGTAATTATACTACAGTGCACAGTACAGTTCCCCACATCCCGCACC LEMUR ATCACAATTGGGGGTGCCACGGTCCTCCAGTGGGTAGAGAACAGGGAGGCTGATAACCACCCTGCAGTGCACAGGGCAGTGCCCCACTCCCACCAC MOUSE-LEMUR ATCACAGTTGGGGGATGCCACTGGCCTCAAGTGGGTAGAGAACAGGGAGGCTGAAAACCACCCTGCAGAGCACGGGGCAGTGCCTTCACCACCACTCC VERVET GTCAGAATTTGGGGGATGCTTCTGGCTCTACTTGGGTAGAGAAACAGGGATGCTTATAATCATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCC MACAQUE GTCAGAATTTGGGGGATGCTTCTGGCTCTACTTGGGTAGAGAAACAGGAATGCTTATAATCATCCTACAGTGCACAGGTCAGTACCCCCACCCACACTCC BABOON GTCAGAATTTGGGGGATGCTTCTGGCTCTACTTGGGTAGAAAAACAGGGATGCTTATAATCATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCC ORANGUTAN GTCACGATTTGGGAGATGCTTCTGGCTCGACTTGGGTAGAGAAGCGGGGATGCTTATAATCATCCAACAGTGCACAGGACAGTACCCCCACCCACACTCC GORILLA GTCACGATTTGGGGGATGCTTCTGGCTCAACTTGGGTAGAGAAGTGGGGATGCTTATACTCATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCC CHIMP GTCACGATTTGGGGGATGCTTCTGGCTCAACTTGGGTAGAGAAGCGGGGATGCTTATAATCATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCC HUMAN GTCACGATTTGGGGGATGCTTCTGGCTCAACTTGGGTAGAGAAGCGGGGATGCTTATAATCATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCC All of it: Functional, non-functional, introns, intergenic, repeats, everything*! • Mutational operations • Small-scale : Substitutions, deletions, insertions (inc. transposons) • Large scale: Genome rearrangement, segmental/tandem duplications • (*): Heterochromatin non-included

28. Reconstruction algorithm • Identify orthologous regions in each species

29. Reconstruction algorithm • 2) Compute multiple genome alignment ARMADILLO ----------------TGCTACTAATAT-----T-TAGTA-CATAGAG-CC-CAGGGGTGCTGCTGAAA----------GTCTTAAAATGCACAGTGTAGCCCCTCCTCC------------ACAAAGAATTAACTAGCCCAGAATGTCAGGA--------GT--A-CCAAG COW GCCTCTCTTT-----------CTGCCCTGCAGGC-TAGAA-TGTATCA-CT-TAGATGTTCCAA---------------ATCAGAAAGTGTTCAG----------CCATTTCCATACCACC----AGGAGCTA-CAATGTTGGGCTGCAGCTA--------TTTGGATCAAA HORSE GTCACAATTTAGGAAGTGCCACTGGCCT-----C-TAGAG-GGTAGAA-GA-CAGGGATGCTAATAATCATCCCACGTCATCCTACAGTGCTCAGAACAGCACCCCTACCCTCACCCCATCAACAAAGAATTATCCAGCCCAAAATGCCAATA--------GT--GCCCAGA CAT GTCACAGTTTAGGGGGTACTACTGGCAT-----C-TATCG-GGTGGAG-GA-TAGGGATACTGATAATC----------ATTCTACAGTGCACAGGACAGTACCCCTACTTTCACCCCACAA-CAAAGAATTATCCAGCCCAAAATGCCAACA--------GT--GCTCAGA DOG GTCACAATTTGGGGGATACTACTGGCAT-----C-TAATG-GGTAGAG-GA-CAGGGATACTGATAATT----------GCTTTACAGTGCACAGGACAGCACCCTTATCTTCACCCCAAAAGCAAAGTATTATCCAGCCCCAAATGCCAATG--------GT--GCTCAGA HEDGEHOG GTCATAGTTT----GATTATATGGGCTT-----CTTAGTA-GACAAAGAAA-AAGATGTTCTGGTAGTC----------ATTCTGCTTTCCATATGATAGCACTCCCATCTTCACTTCCAAAATTAAGAGTCATCATACTCAGTGTGCCAATA--------TG--GCCCAGA MOUSE GTCACAGTTTGGAGGATGTTACTGACAT-----C-TAGAG-AGTAGAC-TT-TAAAGATACTGATAGTC----------ACCCCATTGTGCAC---------------------CTCCAACAATAATGGCTCATCGAAACCTAAATGCCAATCTGCCAATTAT--GTCCATG RAT GTCACAATTTGGAGGATGTTACTGGCAT-----C-TAGAG-AGTAGAC-TT-TAAGGACACTGATAATC----------ATACTATGCTGCAC---------------------TTCCAACAATAATGGCTCATCTAGACCTAAATACCAATCTGCCAATTAT--ATCCATG RABBIT ATCACAATTTGGGGAACACCACTGGCAT-----C-TCGGGTAGCAGGC----CAGGCATGCTGGTAATT----------ATACTACAGTGCACAGTACAGTTCCCCACATCCCGCACCAACAACA--GGTTTATGCTGCCCAAAGTGCCAGTGTGC-----------CCACG LEMUR ATCACAA-TTGGGGG-TGCCACGGTCCT-----C-CAGTG-GGTAGAG-AA-CAGGGAGGCTGATAACC----------ACCCTGCAGTGCACAGGGCAGTGCC-CCACTCCCACCACAACAATGGAGAATTATTGGGCCCCAAATGCCAATA--------GT--GCCCAAG MOUSELEMUR ATCACAG-TTGGGGGATGCCACTGGCCT-----C-AAGTG-GGTAGAG-AA-CAGGGAGGCTGAAAACC----------ACCCTGCAGAGCACGGGGCAGTGCCTTCACCACCACTCCAACAACGGAGAATTATTGGGTCCCAAATGCCAATA--------GT—-GCCCAGG VERVET GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAG-AAACAGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGAACCCAAAATGTTAATA--------GT--GTCCAGG MACAQUE GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAG-AAACAGGAATGCTTATAATC----------ATCCTACAGTGCACAGGTCAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGGACCCAAAATGCTAATG--------GT--GTCCAGG BABOON GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAA-AAACAGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGGACCCAAAATGTTAATG--------GT--GTCCAGG ORANGUTAN GTCACGATTTGGGAGATGCTTCTGGCTC-----G-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCAACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCACTGGACCCAAAATGTTAATG--------GT--GTCCAGG GORILLA GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGTGGGGATGCTTATACTC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCGAAAATGTTAATG--------GT--GTCCAGG CHIMP GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCGAAAATGTTAATG--------GT--GTCCAGA HUMAN GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCTAAAATGTTAATG--------GT--GTCCAGG • Goal: Phylogenetic correctness • Two nucleotides are aligned if and only if they have a common ancestor.

30. Reconstruction algorithm • 3) Reconstruct insertion/deletion history • Find most likely explanation for gaps observed ARMADILLO ----------------TGCTACTAATAT-----T-TAGTA-CATAGAG-CC-CAGGGGTGCTGCTGAAA----------GTCTTAAAATGCACAGTGTAGCCCCTCCTCC------------ACAAAGAATTAACTAGCCCAGAATGTCAGGA--------GT--A-CCAAG COW GCCTCTCTTT-----------CTGCCCTGCAGGC-TAGAA-TGTATCA-CT-TAGATGTTCCAA---------------ATCAGAAAGTGTTCAG----------CCATTTCCATACCACC----AGGAGCTA-CAATGTTGGGCTGCAGCTA--------TTTGGATCAAA HORSE GTCACAATTTAGGAAGTGCCACTGGCCT-----C-TAGAG-GGTAGAA-GA-CAGGGATGCTAATAATCATCCCACGTCATCCTACAGTGCTCAGAACAGCACCCCTACCCTCACCCCATCAACAAAGAATTATCCAGCCCAAAATGCCAATA--------GT--GCCCAGA CAT GTCACAGTTTAGGGGGTACTACTGGCAT-----C-TATCG-GGTGGAG-GA-TAGGGATACTGATAATC----------ATTCTACAGTGCACAGGACAGTACCCCTACTTTCACCCCACAA-CAAAGAATTATCCAGCCCAAAATGCCAACA--------GT--GCTCAGA DOG GTCACAATTTGGGGGATACTACTGGCAT-----C-TAATG-GGTAGAG-GA-CAGGGATACTGATAATT----------GCTTTACAGTGCACAGGACAGCACCCTTATCTTCACCCCAAAAGCAAAGTATTATCCAGCCCCAAATGCCAATG--------GT--GCTCAGA HEDGEHOG GTCATAGTTT----GATTATATGGGCTT-----CTTAGTA-GACAAAGAAA-AAGATGTTCTGGTAGTC----------ATTCTGCTTTCCATATGATAGCACTCCCATCTTCACTTCCAAAATTAAGAGTCATCATACTCAGTGTGCCAATA--------TG--GCCCAGA MOUSE GTCACAGTTTGGAGGATGTTACTGACAT-----C-TAGAG-AGTAGAC-TT-TAAAGATACTGATAGTC----------ACCCCATTGTGCAC---------------------CTCCAACAATAATGGCTCATCGAAACCTAAATGCCAATCTGCCAATTAT--GTCCATG RAT GTCACAATTTGGAGGATGTTACTGGCAT-----C-TAGAG-AGTAGAC-TT-TAAGGACACTGATAATC----------ATACTATGCTGCAC---------------------TTCCAACAATAATGGCTCATCTAGACCTAAATACCAATCTGCCAATTAT--ATCCATG RABBIT ATCACAATTTGGGGAACACCACTGGCAT-----C-TCGGGTAGCAGGC----CAGGCATGCTGGTAATT----------ATACTACAGTGCACAGTACAGTTCCCCACATCCCGCACCAACAACA--GGTTTATGCTGCCCAAAGTGCCAGTGTGC-----------CCACG LEMUR ATCACAA-TTGGGGG-TGCCACGGTCCT-----C-CAGTG-GGTAGAG-AA-CAGGGAGGCTGATAACC----------ACCCTGCAGTGCACAGGGCAGTGCC-CCACTCCCACCACAACAATGGAGAATTATTGGGCCCCAAATGCCAATA--------GT--GCCCAAG MOUSELEMUR ATCACAG-TTGGGGGATGCCACTGGCCT-----C-AAGTG-GGTAGAG-AA-CAGGGAGGCTGAAAACC----------ACCCTGCAGAGCACGGGGCAGTGCCTTCACCACCACTCCAACAACGGAGAATTATTGGGTCCCAAATGCCAATA--------GT—-GCCCAGG VERVET GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAG-AAACAGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGAACCCAAAATGTTAATA--------GT--GTCCAGG MACAQUE GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAG-AAACAGGAATGCTTATAATC----------ATCCTACAGTGCACAGGTCAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGGACCCAAAATGCTAATG--------GT--GTCCAGG BABOON GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAA-AAACAGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGGACCCAAAATGTTAATG--------GT--GTCCAGG ORANGUTAN GTCACGATTTGGGAGATGCTTCTGGCTC-----G-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCAACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCACTGGACCCAAAATGTTAATG--------GT--GTCCAGG GORILLA GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGTGGGGATGCTTATACTC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCGAAAATGTTAATG--------GT--GTCCAGG CHIMP GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCGAAAATGTTAATG--------GT--GTCCAGA HUMAN GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCTAAAATGTTAATG--------GT--GTCCAGG

31. Reconstruction algorithm • 3) Reconstruct insertion/deletion history • Find most likely explanation for gaps observed ARMADILLO ----------------TGCTACTAATAT-----T-TAGTA-CATAGAG-CC-CAGGGGTGCTGCTGAAA----------GTCTTAAAATGCACAGTGTAGCCCCTCCTCC------------ACAAAGAATTAACTAGCCCAGAATGTCAGGA--------GT--A-CCAAG COW GCCTCTCTTT-----------CTGCCCTGCAGGC-TAGAA-TGTATCA-CT-TAGATGTTCCAA---------------ATCAGAAAGTGTTCAG----------CCATTTCCATACCACC----AGGAGCTA-CAATGTTGGGCTGCAGCTA--------TTTGGATCAAA HORSE GTCACAATTTAGGAAGTGCCACTGGCCT-----C-TAGAG-GGTAGAA-GA-CAGGGATGCTAATAATCATCCCACGTCATCCTACAGTGCTCAGAACAGCACCCCTACCCTCACCCCATCAACAAAGAATTATCCAGCCCAAAATGCCAATA--------GT--GCCCAGA CAT GTCACAGTTTAGGGGGTACTACTGGCAT-----C-TATCG-GGTGGAG-GA-TAGGGATACTGATAATC----------ATTCTACAGTGCACAGGACAGTACCCCTACTTTCACCCCACAA-CAAAGAATTATCCAGCCCAAAATGCCAACA--------GT--GCTCAGA DOG GTCACAATTTGGGGGATACTACTGGCAT-----C-TAATG-GGTAGAG-GA-CAGGGATACTGATAATT----------GCTTTACAGTGCACAGGACAGCACCCTTATCTTCACCCCAAAAGCAAAGTATTATCCAGCCCCAAATGCCAATG--------GT--GCTCAGA HEDGEHOG GTCATAGTTT----GATTATATGGGCTT-----CTTAGTA-GACAAAGAAA-AAGATGTTCTGGTAGTC----------ATTCTGCTTTCCATATGATAGCACTCCCATCTTCACTTCCAAAATTAAGAGTCATCATACTCAGTGTGCCAATA--------TG--GCCCAGA MOUSE GTCACAGTTTGGAGGATGTTACTGACAT-----C-TAGAG-AGTAGAC-TT-TAAAGATACTGATAGTC----------ACCCCATTGTGCAC---------------------CTCCAACAATAATGGCTCATCGAAACCTAAATGCCAATCTGCCAATTAT--GTCCATG RAT GTCACAATTTGGAGGATGTTACTGGCAT-----C-TAGAG-AGTAGAC-TT-TAAGGACACTGATAATC----------ATACTATGCTGCAC---------------------TTCCAACAATAATGGCTCATCTAGACCTAAATACCAATCTGCCAATTAT--ATCCATG RABBIT ATCACAATTTGGGGAACACCACTGGCAT-----C-TCGGGTAGCAGGC----CAGGCATGCTGGTAATT----------ATACTACAGTGCACAGTACAGTTCCCCACATCCCGCACCAACAACA--GGTTTATGCTGCCCAAAGTGCCAGTGTGC-----------CCACG LEMUR ATCACAA-TTGGGGG-TGCCACGGTCCT-----C-CAGTG-GGTAGAG-AA-CAGGGAGGCTGATAACC----------ACCCTGCAGTGCACAGGGCAGTGCC-CCACTCCCACCACAACAATGGAGAATTATTGGGCCCCAAATGCCAATA--------GT--GCCCAAG MOUSELEMUR ATCACAG-TTGGGGGATGCCACTGGCCT-----C-AAGTG-GGTAGAG-AA-CAGGGAGGCTGAAAACC----------ACCCTGCAGAGCACGGGGCAGTGCCTTCACCACCACTCCAACAACGGAGAATTATTGGGTCCCAAATGCCAATA--------GT—-GCCCAGG VERVET GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAG-AAACAGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGAACCCAAAATGTTAATA--------GT--GTCCAGG MACAQUE GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAG-AAACAGGAATGCTTATAATC----------ATCCTACAGTGCACAGGTCAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGGACCCAAAATGCTAATG--------GT--GTCCAGG BABOON GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAA-AAACAGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGGACCCAAAATGTTAATG--------GT--GTCCAGG ORANGUTAN GTCACGATTTGGGAGATGCTTCTGGCTC-----G-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCAACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCACTGGACCCAAAATGTTAATG--------GT--GTCCAGG GORILLA GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGTGGGGATGCTTATACTC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCGAAAATGTTAATG--------GT--GTCCAGG CHIMP GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCGAAAATGTTAATG--------GT--GTCCAGA HUMAN GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCTAAAATGTTAATG--------GT--GTCCAGG

32. Reconstruction algorithm • 3) Reconstruct insertion/deletion history • Find most likely explanation for gaps observed • This defines the presence/absence of a base at each position of each ancestor ARMADILLO ----------------TGCTACTAATAT-----T-TAGTA-CATAGAG-CC-CAGGGGTGCTGCTGAAA----------GTCTTAAAATGCACAGTGTAGCCCCTCCTCC------------ACAAAGAATTAACTAGCCCAGAATGTCAGGA--------GT--A-CCAAG COW GCCTCTCTTT-----------CTGCCCTGCAGGC-TAGAA-TGTATCA-CT-TAGATGTTCCAA---------------ATCAGAAAGTGTTCAG----------CCATTTCCATACCACC----AGGAGCTA-CAATGTTGGGCTGCAGCTA--------TTTGGATCAAA HORSE GTCACAATTTAGGAAGTGCCACTGGCCT-----C-TAGAG-GGTAGAA-GA-CAGGGATGCTAATAATCATCCCACGTCATCCTACAGTGCTCAGAACAGCACCCCTACCCTCACCCCATCAACAAAGAATTATCCAGCCCAAAATGCCAATA--------GT--GCCCAGA CAT GTCACAGTTTAGGGGGTACTACTGGCAT-----C-TATCG-GGTGGAG-GA-TAGGGATACTGATAATC----------ATTCTACAGTGCACAGGACAGTACCCCTACTTTCACCCCACAA-CAAAGAATTATCCAGCCCAAAATGCCAACA--------GT--GCTCAGA DOG GTCACAATTTGGGGGATACTACTGGCAT-----C-TAATG-GGTAGAG-GA-CAGGGATACTGATAATT----------GCTTTACAGTGCACAGGACAGCACCCTTATCTTCACCCCAAAAGCAAAGTATTATCCAGCCCCAAATGCCAATG--------GT--GCTCAGA HEDGEHOG GTCATAGTTT----GATTATATGGGCTT-----CTTAGTA-GACAAAGAAA-AAGATGTTCTGGTAGTC----------ATTCTGCTTTCCATATGATAGCACTCCCATCTTCACTTCCAAAATTAAGAGTCATCATACTCAGTGTGCCAATA--------TG--GCCCAGA MOUSE GTCACAGTTTGGAGGATGTTACTGACAT-----C-TAGAG-AGTAGAC-TT-TAAAGATACTGATAGTC----------ACCCCATTGTGCAC---------------------CTCCAACAATAATGGCTCATCGAAACCTAAATGCCAATCTGCCAATTAT--GTCCATG RAT GTCACAATTTGGAGGATGTTACTGGCAT-----C-TAGAG-AGTAGAC-TT-TAAGGACACTGATAATC----------ATACTATGCTGCAC---------------------TTCCAACAATAATGGCTCATCTAGACCTAAATACCAATCTGCCAATTAT--ATCCATG RABBIT ATCACAATTTGGGGAACACCACTGGCAT-----C-TCGGGTAGCAGGC----CAGGCATGCTGGTAATT----------ATACTACAGTGCACAGTACAGTTCCCCACATCCCGCACCAACAACA--GGTTTATGCTGCCCAAAGTGCCAGTGTGC-----------CCACG LEMUR ATCACAA-TTGGGGG-TGCCACGGTCCT-----C-CAGTG-GGTAGAG-AA-CAGGGAGGCTGATAACC----------ACCCTGCAGTGCACAGGGCAGTGCC-CCACTCCCACCACAACAATGGAGAATTATTGGGCCCCAAATGCCAATA--------GT--GCCCAAG MOUSELEMUR ATCACAG-TTGGGGGATGCCACTGGCCT-----C-AAGTG-GGTAGAG-AA-CAGGGAGGCTGAAAACC----------ACCCTGCAGAGCACGGGGCAGTGCCTTCACCACCACTCCAACAACGGAGAATTATTGGGTCCCAAATGCCAATA--------GT—-GCCCAGG VERVET GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAG-AAACAGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGAACCCAAAATGTTAATA--------GT--GTCCAGG MACAQUE GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAG-AAACAGGAATGCTTATAATC----------ATCCTACAGTGCACAGGTCAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGGACCCAAAATGCTAATG--------GT--GTCCAGG BABOON GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAA-AAACAGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGGACCCAAAATGTTAATG--------GT--GTCCAGG ORANGUTAN GTCACGATTTGGGAGATGCTTCTGGCTC-----G-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCAACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCACTGGACCCAAAATGTTAATG--------GT--GTCCAGG GORILLA GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGTGGGGATGCTTATACTC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCGAAAATGTTAATG--------GT--GTCCAGG CHIMP GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCGAAAATGTTAATG--------GT--GTCCAGA HUMAN GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCTAAAATGTTAATG--------GT--GTCCAGG NNNNNNNNNNNNNNNNNNNNNNNNNNNN-----N-NNNNN-NNNNNNN-NN-NNNNNNNNNNNNNNNNN----------NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

33. Reconstruction algorithm • 4) Infer max.-like. nucleotide at each position • Ancestral sequences are inferred! ARMADILLO ----------------TGCTACTAATAT-----T-TAGTA-CATAGAG-CC-CAGGGGTGCTGCTGAAA----------GTCTTAAAATGCACAGTGTAGCCCCTCCTCC------------ACAAAGAATTAACTAGCCCAGAATGTCAGGA--------GT--A-CCAAG COW GCCTCTCTTT-----------CTGCCCTGCAGGC-TAGAA-TGTATCA-CT-TAGATGTTCCAA---------------ATCAGAAAGTGTTCAG----------CCATTTCCATACCACC----AGGAGCTA-CAATGTTGGGCTGCAGCTA--------TTTGGATCAAA HORSE GTCACAATTTAGGAAGTGCCACTGGCCT-----C-TAGAG-GGTAGAA-GA-CAGGGATGCTAATAATCATCCCACGTCATCCTACAGTGCTCAGAACAGCACCCCTACCCTCACCCCATCAACAAAGAATTATCCAGCCCAAAATGCCAATA--------GT--GCCCAGA CAT GTCACAGTTTAGGGGGTACTACTGGCAT-----C-TATCG-GGTGGAG-GA-TAGGGATACTGATAATC----------ATTCTACAGTGCACAGGACAGTACCCCTACTTTCACCCCACAA-CAAAGAATTATCCAGCCCAAAATGCCAACA--------GT--GCTCAGA DOG GTCACAATTTGGGGGATACTACTGGCAT-----C-TAATG-GGTAGAG-GA-CAGGGATACTGATAATT----------GCTTTACAGTGCACAGGACAGCACCCTTATCTTCACCCCAAAAGCAAAGTATTATCCAGCCCCAAATGCCAATG--------GT--GCTCAGA HEDGEHOG GTCATAGTTT----GATTATATGGGCTT-----CTTAGTA-GACAAAGAAA-AAGATGTTCTGGTAGTC----------ATTCTGCTTTCCATATGATAGCACTCCCATCTTCACTTCCAAAATTAAGAGTCATCATACTCAGTGTGCCAATA--------TG--GCCCAGA MOUSE GTCACAGTTTGGAGGATGTTACTGACAT-----C-TAGAG-AGTAGAC-TT-TAAAGATACTGATAGTC----------ACCCCATTGTGCAC---------------------CTCCAACAATAATGGCTCATCGAAACCTAAATGCCAATCTGCCAATTAT--GTCCATG RAT GTCACAATTTGGAGGATGTTACTGGCAT-----C-TAGAG-AGTAGAC-TT-TAAGGACACTGATAATC----------ATACTATGCTGCAC---------------------TTCCAACAATAATGGCTCATCTAGACCTAAATACCAATCTGCCAATTAT--ATCCATG RABBIT ATCACAATTTGGGGAACACCACTGGCAT-----C-TCGGGTAGCAGGC----CAGGCATGCTGGTAATT----------ATACTACAGTGCACAGTACAGTTCCCCACATCCCGCACCAACAACA--GGTTTATGCTGCCCAAAGTGCCAGTGTGC-----------CCACG LEMUR ATCACAA-TTGGGGG-TGCCACGGTCCT-----C-CAGTG-GGTAGAG-AA-CAGGGAGGCTGATAACC----------ACCCTGCAGTGCACAGGGCAGTGCC-CCACTCCCACCACAACAATGGAGAATTATTGGGCCCCAAATGCCAATA--------GT--GCCCAAG MOUSELEMUR ATCACAG-TTGGGGGATGCCACTGGCCT-----C-AAGTG-GGTAGAG-AA-CAGGGAGGCTGAAAACC----------ACCCTGCAGAGCACGGGGCAGTGCCTTCACCACCACTCCAACAACGGAGAATTATTGGGTCCCAAATGCCAATA--------GT—-GCCCAGG VERVET GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAG-AAACAGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGAACCCAAAATGTTAATA--------GT--GTCCAGG MACAQUE GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAG-AAACAGGAATGCTTATAATC----------ATCCTACAGTGCACAGGTCAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGGACCCAAAATGCTAATG--------GT--GTCCAGG BABOON GTCAGAATTTGGGGGATGCTTCTGGCTC-----T-ACTTG-GGTAGAA-AAACAGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTATCGAAGAATCATTGGACCCAAAATGTTAATG--------GT--GTCCAGG ORANGUTAN GTCACGATTTGGGAGATGCTTCTGGCTC-----G-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCAACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCACTGGACCCAAAATGTTAATG--------GT--GTCCAGG GORILLA GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGTGGGGATGCTTATACTC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCGAAAATGTTAATG--------GT--GTCCAGG CHIMP GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCGAAAATGTTAATG--------GT--GTCCAGA HUMAN GTCACGATTTGGGGGATGCTTCTGGCTC-----A-ACTTG-GGTAGAG-AAGCGGGGATGCTTATAATC----------ATCCTACAGTGCACAGGACAGTACCCCCACCCACACTCCAGTAATGAAGAATCATTAGACCTAAAATGTTAATG--------GT--GTCCAGG GTCACAATTTGGGGGATGCTACTGGCAT-----C-TAGTG-GGTAGAG-AA-CAGGGATGCTGATAATC----------ATCCTACAGTGCACAGGACAGTGCCCCCACCCCCACTCCAACAACAAAGAATTATCCGGCCCAAAATGCCAATA--------GT--GCCCAGG

34. Reconstructing Cancer Genomes • Change gene structure and regulatory “wiring” of the genome. • Create “bad” novel fusion genes and break “good” old genes. • Example: translocation in leukemia. • GleevecTM (Novartis 2001) targets BCR-ABL oncogene. Chromosome 9 promoter ABL gene Chromosome 22 promoter BCR gene promoter BCR-ABL oncogene

35. Complex Tumor Genomes • What are detailed architectures of tumor genomes? • What rearrangements/duplications produce these architectures and what is the order of these events? • What are the novel fusion genes and old “broken” genes?

36. Tumor genome 2 Tumor genome 3 Tumor genome 4 Tumor Genomes Projects Mutation, selection Human genome Tumor genome • Identify recurrent aberrations • Identify temporal sequence of aberrations • Use these data for tumor diagnostics and therapeutics

37. Meet Your Genome contd. [Human Molecular Genetics, 3rd Edition] http://cs273a.stanford.edu [Bejerano Fall11/12]

38. Sequence Conservation implies Function • (but which function/s?...) Comparative Genomics of Distantly related species: functional region! human ...CTTTGCGA-TGAGTAGCATCTACTATTT... common ancestor ...ACGTGGGACTGACTA-CATCGACTACGA... anotherspecies Note: the inverse “no conservation  no function”is a much weaker statement given current knowledge http://cs273a.stanford.edu [Bejerano Fall11/12]

39. Vertebrates: what to sequence? , Stickleback Which species to compare to? Too close and purifying selection will be largely indistinguishable from the neutral rate. Too far and many functional orthologs will diverge beyond our ability to accurately align them. , Lizard , Opossum too far sweet spot too close  you are here [Human Molecular Genetics, 3rd Edition] http://cs273a.stanford.edu [Bejerano Fall11/12]

40. The Dawn of Whole Genome Comparative Genomics 2001 2002 40% DNA alignable 95% coding genes shared http://cs273a.stanford.edu [Bejerano Fall11/12]

41. More Species Have Joined Since http://cs273a.stanford.edu [Bejerano Fall11/12]

42. How They Measured all human-mouse alignments human-mouse ancestral repeats alignment Difference: 5% of Human Genome [Mouse consortium, Nature 2002] http://cs273a.stanford.edu [Bejerano Fall11/12]

43. Conserved elements in the Human Genome all human-mouse alignments human-mouse ancestral repeats alignment human-mouse ancestral repeats alignment election Difference: 5% of Human Genome Ultraconservation 85%id on average [Mouse consortium, Nature 2002] http://cs273a.stanford.edu [Bejerano Fall11/12]

44. Ultraconserved Elements fish 481 elements perfectly conserved (100%id) over 200bp or more between human, mouse and rat. [Bejerano et al., Science 2004] http://cs273a.stanford.edu [Bejerano Fall11/12]

45. * * * * * Ultraconserved Elements: Why? Hundreds of long substrings identical between amniotes they must have rejected many different changes. But... all functions we understand in our genome are encoded using redundant codes. Coding: 3 DNA letters → 1 Protein letter. E.g. Protein Coding Genes: DNA – 108 letters over alphabet of 4. Protein – 102 letters over alphabet of 20. [Bejerano et al., Science 2004] http://cs273a.stanford.edu [Bejerano Fall11/12]

46. * * * * * No known function requires this much conservation ? CDS ncRNA TFBS seq. http://cs273a.stanford.edu [Bejerano Fall11/12]

47. PhastCons Conserved Elements http://cs273a.stanford.edu [Bejerano Fall11/12]

48. Other Ways to Measure: indel resistance, rejected subst [Lunter et al, 2006] [Cooper et al., 2005] http://cs273a.stanford.edu [Bejerano Fall11/12]

49. Phylogenetic Shadowing , Stickleback , Lizard , Opossum “too close” can actually be a boon if you have enough closely related genomes too close  you are here [Human Molecular Genetics, 3rd Edition] http://cs273a.stanford.edu [Bejerano Fall11/12]

50. What They Found Human Genome: 3*109 letters 1.5% known function compare to other species >50% junk >5% human genome functional 3x more functional DNA than known! ~106 regions do not code for protein What do they do then? [Science 2004 Breakthrough of the Year, 5th runner up] http://cs273a.stanford.edu [Bejerano Fall11/12]