1. Dynamic Programming (cont’d) CS 466 Saurabh Sinha

2. Affine Gap Penalties In nature, a series of k indels often come as a single event rather than a series of k single nucleotide events: Normal scoring would give the same score for both alignments This is more likely. This is less likely. ATA__GGC ATGATCGC ATA_G_GC ATGATCGC

3. Accounting for Gaps Gaps- contiguous sequence of spaces in one of the rows Score for a gap of length x is: -(ρ + σx) where ρ >0 is the penalty for introducing a gap: gap opening penalty ρ will be large relative to σ: gap extension penalty because you do not want to add too much of a penalty for extending the gap.

4. Affine gap penalty in DP When computing s i,j, need to look at s i,j-1, s i,j-2, s i,j-3,…. and s i-1,j, s i-2,j, … Each cell needs O(n) time for update O(n 2 ) cells Therefore, O(n 3 ) algorithm We can still do this in O(n 2 ) time

5. Affine Gap Penalty Recurrences s i,j = s i-1,j - σ max s i-1,j –(ρ+σ) s i,j = s i,j-1 - σ max s i,j-1 –(ρ+σ) s i,j = s i-1,j-1 + δ (v i, w j ) max s i,j s i,j Continue Gap in w (deletion) Start Gap in w (deletion): from middle Continue Gap in v (insertion) Start Gap in v (insertion):from middle Match or Mismatch End deletion: from top End insertion: from bottom

6. Optional Reading Section 6.10 (J & P) Multiple Alignment

7. Gene Prediction

8. Gene: A sequence of nucleotides coding for protein Gene Prediction Problem: Determine the beginning and end positions of genes in a genome Gene Prediction: Computational Challenge

10. The Genetic Code SOURCE: http://www.bioscience.org/atlases/genecode/genecode.htm

11. In 1961 Sydney Brenner and Francis Crick discovered frameshift mutations Systematically deleted nucleotides from DNA –Single and double deletions dramatically altered protein product –Effects of triple deletions were minor –Conclusion: every triplet of nucleotides, each codon, codes for exactly one amino acid in a protein Codons

12. In 1964, Charles Yanofsky and Sydney Brenner proved colinearity in the order of codons with respect to amino acids in proteins As a result, it was incorrectly assumed that the triplets encoding for amino acid sequences form contiguous strips of information. Great Discovery Provoking Wrong Assumption

13. Exons and Introns In eukaryotes, the gene is a combination of coding segments (exons) that are interrupted by non-coding segments (introns) This makes computational gene prediction in eukaryotes even more difficult Prokaryotes don’t have introns - Genes in prokaryotes are continuous

14. Splicing exon1 exon2exon3 intron1intron2 transcription translation splicing exon = coding intron = non-coding Batzoglou

15. Gene prediction More difficult in eukaryotes than in prokaryotes (due to introns). In human genome, ~3% of DNA sequence is genes Lot of “junk” DNA between genes, and even inside genes (between exons). Gene prediction must deal with this.

16. Gene prediction: broadly speaking Statistical approaches: look for features than appear frequently in genes and infrequently elsewhere Similarity based approaches: a newly sequenced gene may be similar to a known gene. –even this is not so simple. The exon structures may be different between otherwise similar genes

17. Statistical approaches

18. Let us consider gene prediction in prokaryotes (no introns) Detect potential coding regions by looking at ORFs –A region of length n is comprised of (n/3) codons –Stop codons break genome into segments between consecutive Stop codons –The subsegments of these that start from the Start codon (ATG) are ORFs Genomic Sequence Open reading frame ATGTGA Open Reading Frames (ORFs)

19. ORFs 6 reading frames in any given sequence –6 ways to map the DNA sequence to codon sequence (+1,+2,+3,-1,-2,-3) –3 on either strand Look at all 6 reading frames for ORFs

20. Long open reading frames may be a gene –At random, we should expect one stop codon every (64/3) ~= 21 codons –However, genes are usually much longer than this A basic approach is to scan for ORFs whose length exceeds certain threshold –This is naïve because some genes (e.g. some neural and immune system genes) are relatively short Long vs.Short ORFs

21. Codon usage In a given sequence (e.g., an ORF), compute frequency distribution of codons (64 element array): codon usage array Codon usage array for coding sequences is different from that for non-coding sequences If the codon usage array for an ORF is much more similar to that of coding sequences than to that of non-coding sequences, the ORF could be a gene

22. Codon usage Codons coding for “Arg” in human: –CGU: 37%, CGC: 38%, CGA: 7%, CGG: 10%, AGA: 5%, AGG: 3% –In a coding sequence, codon CGC is 12 times more likely than codon AGG –An ORF preferring CGC over AGG is likely to be a gene

23. Codon Usage in Human Genome

24. Codon usage One way to test if an ORF is a gene is to compute –Pr(ORF sequence under a coding sequence model) –Pr(ORF sequence under a non-coding model) –Ratio of the two. These methods work best in prokaryotes The exon-intron trouble is not handled yet

25. Promoter Structure in Prokaryotes (E.Coli) Transcription starts at offset 0. Pribnow Box (-10) Gilbert Box (-30) Ribosomal Binding Site (+10)

26. Ribosomal Binding Site

27. Splicing Signals: an additional statistical clue, for eukaryotes Exons are interspersed with introns and typically flanked by GT and AG

28. Splice site detection 5’ 3’ Donor site Position % From lectures by Serafim Batzoglou (Stanford)

29. Consensus splice sites

30. Statistical approaches: summary Codon usage Promoter motifs Ribosome binding site Splicing sites

31. Similarity based approaches

32. Some genomes may be very well-studied, with many genes having been experimentally verified. Closely-related organisms may have similar genes Unknown genes in one species may be compared to genes in some closely- related species

33. The basic approach Given a protein sequence, and a genomic sequence, find a set of substrings of the genomic sequence whose concatenation best fits the protein sequence Deals with the exon-intron problem First cut: Find fragments in the genomic sequence that match portions of the protein sequence (local alignment) Then find the “optimal” subset of non-overlapping fragments

34. Exon chaining Each of the fragments of the genomic sequence that somewhat match the protein (locally) is a putative exon The “goodness” of the match is the “weight” assigned to this putative exon Thus, we have a set of weighted intervals (l,r,w): for a fragment from l to r, with weight w representing how well it matches (a portion of) the protein

35. Exon Chaining Problem Input: A set of weighted intervals (l,r,w) Output: A maximum weight chain of non-overlapping intervals from this set

36. Exon Chaining Problem: Graph Representation This problem can be solved with dynamic programming in O(n) time. 21 edge from every l i to r i edge between every two successive vertices

37. Assumptions No two intervals have a common boundary point. So the (l i,r i ) define 2n distinct points, if there are n intervals

38. Exon Chaining Algorithm ExonChaining (G, n) //Graph, number of intervals for i ← to 2n s i ← 0 for i ← 1 to 2n if vertex v i in G corresponds to right end of the interval I j ← index of vertex for left end of the interval I w ← weight of the interval I s i ← max {s j + w, s i-1 } else s i ← s i-1 return s 2n

39. Not very helpful A chain is a set of non-overlapping exons in order (left to right) But the matching protein portions may not be in the same order !

40. Spliced Alignment Begins by selecting either all putative exons between potential acceptor and donor sites or by finding all substrings similar to the target protein (as in the Exon Chaining Problem). This set is further filtered in a such a way that attempt to retain all true exons, with some false ones. Then find the chain of exons such that the sequence similarity to the target protein sequence is maximized

41. Spliced Alignment Problem: Formulation Input: Genomic sequences G, target sequence T, and a set of candidate exons (blocks) B. Output: A chain of exons Γ such that the global alignment score between Γ* and T is maximized Γ* - concatenation of all exons from chain Γ

42. Dynamic programming Genomic sequence G = g 1 g 2 …g n Target sequence T = t 1 t 2 …t m As usual, we want to find the optimal alignment score of the i-prefix of G and the j-prefix of T Problem is, there are many i-prefixes possible (since multiple blocks may include position i)

43. Idea Find the optimal alignment score of the i-prefix of G and the j-prefix of T assuming that this alignment uses a particular block B at position i S(i, j, B) For every block B that includes i

44. Recurrence If i is not the starting vertex of block B: S(i, j, B) = max { S(i – 1, j, B) – indel penalty S(i, j – 1, B) – indel penalty S(i – 1, j – 1, B) + δ(g i, t j ) } If i is the starting vertex of block B: S(i, j, B) = max { S(i, j – 1, B) – indel penalty max all blocks B’ preceding block B S(end(B’), j, B’) – indel penalty max all blocks B’ preceding block B S(end(B’), j – 1, B’) + δ(g i, t j ) }