1. CSE182-L10 MS Spec Applications + Gene Finding + Projects

2. Relative abundance computation Once we have features matched across runs, we have data identical to microarrays. Features can be ‘identified’ in separate MS2 experiments run feature intensity

3. Structural genomics via MS

4. Cross-linking Cross-links are ‘fixed’ length that bind to amino-acids. How can they help predict structure? Protocol –Cross-link native protein –Denature, digest –MS/MS (identify cross-linked peptides) Potentially valuable, but not widely used

5. Identifying Cross-linked peptides Identify all peptide pairs, whose mass explains the parent mass. Given a list of peptide pairs, find the pair, and the linked position that best explains the MS2 data. What is the number of possible candidate pairs. Fragmentation in the presence of linkers is poorly understood How do you separate cross-linked peptides from singly linked, and non-cross-linked peptides?

6. Identifying cross-linked peptides Use isotopically labeled cross-linking agents. Cross-linked peptides will show up as pairs separated by a small mass. Non cross-linked peptides appear at one position only.

7. MS application: Protein-protein interaction Proteins combine to form functional complexes. An antibody is a special kind of protein that can recognize a specific protein Use an antibody to recognize a protein in a complex. Isolate & Purify the complex that binds to the antibody. Identify all the proteins in the complex via mass spectrometry.

8. Mass Spectrometry: conclusion Mass Spectrometry can be used to identify peptides, modifications, quantitation, protein structure, protein-protein interaction (complex formation) Each of these poses significant computational challenges.

9. Proteomic Databases/Tools

10. Eukaryotic Gene Prediction

11. Eukaryotic gene structure

12. Translation

13. Gene Features ATG 5’ UTR intron exon 3’ UTR Acceptor Donor splice site Transcription start Translation start

14. Gene identification Eukaryotic gene definitions: –Location that codes for a protein –The transcript sequence(s) that encodes the protein –The protein sequence(s) Suppose you want to know all of the genes in an organism. This was a major problem in the 70s. PhDs, and careers were spent isolating a single gene sequence. All of that changed with the development of high throughput methods like EST sequencing

15. EST Sequencing Suppose we could collect all of the mRNA. However, mRNA is unstable An enzyme called reverse transcriptase is used to make a DNA copy of the RNA. Use DNA polymerase to get a complementary DNA strand. Sequence the (stable) cDNA from both ends. This leads to a collection of transcripts/expressed sequences (ESTs). Many might be from the same gene AAAA TTTT AAAA TTTT

16. EST Sequencing Often, reverse transcriptase breaks off early. Why is this a good thing? The 3’ end may not have a much coding sequence. We can assemble the 5’ end to get more of the coding sequence

17. Project 2 EST assembly Given a collection of EST (3’) sequences, your goal is to cluster all ESTs from the same gene, and produce a consensus. How would you do it if we also had 5’ EST sequences?

18. Project 1 Goal: Look for signals in the UTR. The UTR is not boring. It often folds into a 2 D structure and subsequently affects transcription/translation of genes. What are Riboswitches? miRNA?

19. Project 3 Goal is to predict expressed genes using ESTs/proteins and mass spectrometry.

20. Project guidelines 4 Checkpoints. The first is mainly to identify a project, project partners, and answer a few simple questions to get started. Deadline 11/3/05.

21. Gene Finding: The 1st generation Given genomic DNA, does it contain a gene (or not)? Key idea: The distributions of nucleotides is different in coding (translated exons) and non- coding regions. Therefore, a statistical test can be used to discriminate between coding and non-coding regions.

22. Coding versus Non-coding You are given a collection of exons, and a collection of intergenic sequence. Count the number of occurrences of ATGATG in Introns and Exons. –Suppose 1% of the hexamers in Exons are ATGATG –Only 0.01% of the hexamers in Intons are ATGATG How can you use this idea to find genes?

23. Generalizing AAAAAA AAAAAC AAAAAG AAAAAT IE Compute a frequency count for all hexamers. Use this to decide whether a sequence is an exon/intron

24. Coding versus non-coding Fickett and Tung (1992) compared various measures Measures that preserve the triplet frame are the most successful. Genscan: 5th order Markov Model Conservation across species

25. Coding vs. non-coding regions Compute average coding score (per base) of exons and introns, and take the difference. If the measure is good, the difference must be biased away from 0.

26. Coding differential for 380 genes

27. Other Signals GT ATG AG Coding

28. Coding region can be detected Coding Plot the coding score using a sliding window of fixed length. The (large) exons will show up reliably. Not enough to predict gene boundaries reliably

29. Other Signals GT ATG AG Coding Signals at exon boundaries are precise but not specific. Coding signals are specific but not precise. When combined they can be effective

30. The second generation of Gene finding Ex: Grail II. Used statistical techniques to combine various signals into a coherent gene structure. It was not easy to train on many parameters. Guigo & Bursett test revealed that accuracy was still very low. Problem with multiple genes in a genomic region

32. HMMs and gene finding HMMs allow for a systematic approach to merging many signals. They can model multiple genes, partial genes in a genomic region, as also genes on both strands.

33. The Viterbi Algorithm

34. HMMs and gene finding The Viterbi algorithm (and backtracking) allows us to parse a string through the states of an HMM Can we describe Eukaryotic gene structure by the states of an HMM? This could be a solution to the GF problem.

35. An HMM for Gene structure

36. Generalized HMMs, and other refinements A probabilistic model for each of the states (ex: Exon, Splice site) needs to be described In standard HMMs, there is an exponential distribution on the duration of time spent in a state. This is violated by many states of the gene structure HMM. Solution is to model these using generalized HMMs.

37. Length distributions of Introns & Exons

38. Generalized HMM for gene finding Each state also emits a ‘duration’ for which it will cycle in the same state. The time is generated according to a random process that depends on the state.

39. Forward algorithm for gene finding ji qkqk Emission Prob.: Probability that you emitted X i..X j in state q k (given by the 5th order markov model) Forward Prob: Probability that you emitted I symbols and ended up in state q k Duration Prob.: Probability that you stayed in state q k for j-i+1 steps

40. HMMs and Gene finding Generalized HMMs are an attractive model for computational gene finding –Allow incorporation of various signals –Quality of gene finding depends upon quality of signals.

41. DNA Signals Coding versus non-coding Splice Signals Translation start

42. Splice signals GT is a Donor signal, and AG is the acceptor signal GTAG

43. PWMs Fixed length for the splice signal. Each position is generated independently according to a distribution Figure shows data from > 1200 donor sites 321123456AAGGTGAGTCCGGTAAGTGAGGTGAGGTAGGTAAGG

44. MDD PWMs do not capture correlations between positions Many position pairs in the Donor signal are correlated

45. Choose the position which has the highest correlation score. Split sequences into two: those which have the consensus at position I, and the remaining. Recurse until

46. MDD for Donor sites

47. De novo Gene prediction: Sumary Various signals distinguish coding regions from non-coding HMMs are a reasonable model for Gene structures, and provide a uniform method for combining various signals. Further improvement may come from improved signal detection

48. How many genes do we have? Nature Science

49. Alternative splicing

50. Comparative methods Gene prediction is harder with alternative splicing. One approach might be to use comparative methods to detect genes Given a similar mRNA/protein (from another species, perhaps?), can you find the best parse of a genomic sequence that matches that target sequence Yes, with a variant on alignment algorithms that penalize separately for introns, versus other gaps.

51. Comparative gene finding tools Procrustes/Sim4: mRNA vs. genomic Genewise: proteins versus genomic CEM: genomic versus genomic Twinscan: Combines comparative and de novo approach.