1 / 70

Sequencing Sequence Alignment

2. Objectives. Understand how DNA sequence data is collected and preparedBe aware of the importance of sequence searching and sequence alignment in biology and medicineBe familiar with the different algorithms and scoring schemes used in sequence searching and sequence alignment. 3. High Throughput DNA Sequencing.

corbin
Télécharger la présentation

Sequencing Sequence Alignment

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

    1. 1 Sequencing & Sequence Alignment

    2. 2 Objectives Understand how DNA sequence data is collected and prepared Be aware of the importance of sequence searching and sequence alignment in biology and medicine Be familiar with the different algorithms and scoring schemes used in sequence searching and sequence alignment

    3. 3 High Throughput DNA Sequencing

    4. 4

    5. 5 Shotgun Sequencing

    6. 6 Principles of DNA Sequencing

    7. 7 The Secret to Sanger Sequencing

    8. 8 Principles of DNA Sequencing

    9. 9 Principles of DNA Sequencing

    10. 10 Capillary Electrophoresis

    11. 11 Multiplexed CE with Fluorescent detection

    12. 12 Shotgun Sequencing

    13. 13 Shotgun Sequencing Very efficient process for small-scale (~10 kb) sequencing (preferred method) First applied to whole genome sequencing in 1995 (H. influenzae) Now standard for all prokaryotic genome sequencing projects Successfully applied to D. melanogaster Moderately successful for H. sapiens

    14. 14 The Finished Product

    15. 15 Sequencing Successes

    16. 16 Sequencing Successes

    17. 17 So what do we do with all this sequence data?

    18. 18 Sequence Alignment

    19. 19 Alignments tell us about... Function or activity of a new gene/protein Structure or shape of a new protein Location or preferred location of a protein Stability of a gene or protein Origin of a gene or protein Origin or phylogeny of an organelle Origin or phylogeny of an organism

    20. 20 Factoid:

    21. 21 Similarity versus Homology Similarity refers to the likeness or % identity between 2 sequences Similarity means sharing a statistically significant number of bases or amino acids Similarity does not imply homology Homology refers to shared ancestry Two sequences are homologous is they are derived from a common ancestral sequence Homology usually implies similarity

    22. 22 Similarity versus Homology Similarity can be quantified It is correct to say that two sequences are X% identical It is correct to say that two sequences have a similarity score of Z It is generally incorrect to say that two sequences are X% similar

    23. 23 Homology cannot be quantified If two sequences have a high % identity it is OK to say they are homologous It is incorrect to say two sequences have a homology score of Z It is incorrect to say two sequences are X% homologous Similarity versus Homology

    24. 24 Sequence Complexity

    25. 25 Assessing Sequence Similarity

    26. 26 Assessing Sequence Similarity

    27. 27 Is This Alignment Significant?

    28. 28 Some Simple Rules If two sequence are > 100 residues and > 25% identical, they are likely related If two sequences are 15-25% identical they may be related, but more tests are needed If two sequences are < 15% identical they are probably not related If you need more than 1 gap for every 20 residues the alignment is suspicious

    29. 29 Doolittles Rules of Thumb

    30. 30 Sequence Alignment - Methods Dot Plots Dynamic Programming Heuristic (Fast) Local Alignment Multiple Sequence Alignment Contig Assembly

    31. 31 Dot Plots

    32. 32 Dot Plots Invented in 1970 by Gibbs & McIntyre Good for quick graphical overview Simplest method for sequence comparison Inter-sequence comparison Intra-sequence comparison Identifies internal repeats Identifies domains or modules

    33. 33 Dot Plots & Internal Repeats

    34. 34 Dot Plot Algorithm Take two sequences (A & B), write sequence A out as a row (length=m) and sequence B as a column (length =n) Create a table or matrix of m columns and n rows Compare each letter of sequence A with every letter in sequence B. If theres a match mark it with a dot, if not, leave blank

    35. 35 Dot Plot Algorithm

    36. 36 Dot Plots Most commercial programs offer pretty good dot plot programs including: GCG/Omiga (Pharmacopeia) PepTool (BioTools Inc.) LaserGene (DNAStar) Popular freeware package is Dotter www.cgr.ki.se/cgr/groups/sonnhammer/Dotter.html Dotlet http://www.isrec.isb-sib.ch/java/dotlet/Dotlet.html JDotter http://athena.bioc.uvic.ca/sars/jdotter/main.php

    37. 37 Dynamic Programming

    38. 38 Dynamic Programming Developed by Needleman & Wunsch (1970) Refined by Smith & Waterman (1981) Ideal for quantitative assessment Guaranteed to be mathematically optimal Slow N2 algorithm Performed in 2 stages Prepare a scoring matrix using recursive function Scan matrix diagonally using traceback protocol

    39. 39 The Recursive Function

    40. 40 Identity Scoring Matrix (Sij)

    41. 41 A Simple Example...

    42. 42 A Simple Example...

    43. 43 Could We Do Better? Key to the performance of Dynamic Programming is the scoring function Dynamic Programming always gives the mathematically correct answer Dynamic Programming does not always give the biologically correct answer The weakest link -- The Scoring Matrix

    44. 44 Scoring Matrices An empirical model of evolution, biology and chemistry all wrapped up in a 20 X 20 table of integers Structurally or chemically similar residues should ideally have high diagonal or off-diagonal numbers Structurally or chemically dissimilar residues should ideally have low diagonal or off-diagonal numbers

    45. 45 A Better Matrix - PAM250

    46. 46 Using PAM250...

    47. 47 Using PAM250...

    48. 48 PAM Matrices Developed by M.O. Dayhoff (1978) PAM = Point Accepted Mutation Matrix assembled by looking at patterns of substitutions in closely related proteins 1 PAM corresponds to 1 amino acid change per 100 residues 1 PAM = 1% divergence or 1 million years in evolutionary history

    49. 49 Dynamic Programming Great for doing pairwise global alignments Produces a quantitative alignment score Problems if one tries to do alignments with very large sequences (memory requirement grows as N2 or as N x M) Serious problems if one tries to align one sequence against a database (10s of hours) Need an alternative..

    50. 50 Fast Local Alignment Methods

    51. 51

    52. 52 Fast Alignment Algorithm

    53. 53

    54. 54 Fast Alignment Algorithm

    55. 55

    56. 56 FASTA Developed in 1985 and 1988 (W. Pearson) Looks for clusters of nearby or locally dense identical k-tuples init1 score = score for first set of k-tuples initn score = score for gapped k-tuples opt score = optimized alignment score Z-score = number of S.D. above random expect = expected # of random matches

    57. 57 FASTA

    58. 58 Multiple Sequence Alignment

    59. 59 Multiple Alignment Algorithm Take all n sequences and perform all possible pairwise (n/2(n-1)) alignments Identify highest scoring pair, perform an alignment & create a consensus sequence Select next most similar sequence and align it to the initial consensus, regenerate a second consensus Repeat step 3 until finished

    60. 60 Multiple Sequence Alignment Developed and refined by many (Doolittle, Barton, Corpet) through the 1980s Used extensively for extracting hidden phylogenetic relationships and identifying sequence families Powerful tool for extracting new sequence motifs and signature sequences

    61. 61 Multiple Alignment Most commercial vendors offer good multiple alignment programs including: GCG (Accelerys) PepTool/GeneTool (BioTools Inc.) LaserGene (DNAStar) Popular web servers include T-COFFEE, MULTALIN and CLUSTALW Popular freeware includes PHYLIP & PAUP

    62. 62 Mutli-Align Websites Match-Box http://www.fundp.ac.be/sciences/biologie/bms/matchbox_submit.shtml MUSCA http://cbcsrv.watson.ibm.com/Tmsa.html T-Coffee http://www.ch.embnet.org/software/TCoffee.html MULTALIN http://www.toulouse.inra.fr/multalin.html CLUSTALW http://www.ebi.ac.uk/clustalw/

    63. 63 Multi-alignment & Contig Assembly

    64. 64 Contig Assembly Read, edit & trim DNA chromatograms Remove overlaps & ambiguous calls Read in all sequence files (10-10,000) Reverse complement all sequences (doubles # of sequences to align) Remove vector sequences (vector trim) Remove regions of low complexity Perform multiple sequence alignment

    65. 65 Chromatogram Editing

    66. 66 Sequence Loading

    67. 67 Sequence Alignment

    68. 68 Contig Alignment - Process

    69. 69 Sequence Assembly Programs Phred - base calling program that does detailed statistical analysis (UNIX) http://www.phrap.org/ Phrap - sequence assembly program (UNIX) http://www.phrap.org/ TIGR Assembler - microbial genomes (UNIX) http://www.tigr.org/softlab/assembler/ The Staden Package (UNIX) http://www.mrc-lmb.cam.ac.uk/pubseq/ GeneTool/ChromaTool/Sequencher (PC/Mac)

    70. 70 http://bio.ifom-firc.it/ASSEMBLY/assemble.html

    71. 71 Conclusions Sequence alignments and database searching are key to all of bioinformatics There are four different methods for doing sequence comparisons 1) Dot Plots; 2) Dynamic Programming; 3) Fast Alignment; and 4) Multiple Alignment Understanding the significance of alignments requires an understanding of statistics and distributions

More Related