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BLAST

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  1. a glimpse under the hood BLAST

  2. Prospero: Our revels now are ended. These our actors,As I foretold you, were all spirits, andAre melted into air, into thin air:And like the baseless fabric of this vision,The cloud-capp'd tow'rs, the gorgeous palaces,The solemn temples, the great globe itself,Yea, all which it inherit, shall dissolve,And, like this insubstantial pageant faded,Leave not a rack behind. We are such stuffAs dreams are made on; and our little lifeIs rounded with a sleep. find this

  3. Prospero: Our revels now are ended. These our actors,As I foretold you, were all spirits, andAre melted into air, into thin air:And like the baseless fabric of this vision,The cloud-capp'd tow'rs, the gorgeous palaces,The solemn temples, the great globe itself,Yea, all which it inherit, shall dissolve,And, like this insubstantial pageant faded,Leave not a rack behind. We are such stuffAs dreams are made on; and our little lifeIs rounded with a sleep. in here

  4. Prospero: Our revels now are ended.These our actors,As I foretold you,were all spirits, andAre melted into air, into thin air:And like the baseless fabric of this vision,The cloud-capp'd tow'rs,the gorgeous palaces,The solemn temples, the great globe itself,Yea, all which it inherit, shall dissolve,And, like this insubstantial pageant faded,Leave not a rack behind. We are such stuffAs dreams aremade on; and our little lifeIs rounded with a sleep.

  5. cloud-capp'd tow'rs cloud-capped towers cloudcapp'd towers cloud-tipped towels

  6. cloud-capped towers cloud-capp'd tow'rs Our revels now are ended. These our actors, As I foretold you, were all spirits, and Are melted into air, into thin air: And like the baseless fabric of this vision, The cloud-capped towers, the gorgeous palaces, The solemn temples, the great globe itself, Yea, all which it inherit, shall dissolve, And, like this insubstantial pageant faded,

  7. cloud-capped towers r: And like the baseless fabric of this vision, The cloud-capped towers, the gorgeous palaces, The solemn temples, the great globe itself, Yea, all which it inherit, shall dissolve, And, like this insubstantial pageant faded,

  8. vision, The cloud-capped towers, the gorgeous palaces, The r: And like the baseless fabric of this vision, The cloud-capped towers, the gorgeous palaces, The + + + + + +

  9. vision, The cloud-capped towers, the gorgeous palaces, The r: And like the baseless fabric of this vision, The cloud-capped towers, the gorgeous palaces, The solemn temples, the great globe itself, Yea, all which it inherit, shall dissolve, And, like this insubstantial pageant faded, + + + + + +

  10. The Natural scenario

  11. 1 gene in 1 Panda genome

  12. ~ 1 kilobase (kb) in ~ 2.30  gigabases (Gb)

  13. in

  14. Panda genome GenBank

  15. Learning goals [BLAST] Describe the principle Describe the steps Discuss the concepts Use BLAST!

  16. Εὑρίσκω Heuristic

  17. The algorithm in 13 steps

  18. Information reduction in uncertainty degree of surprise

  19. Prospero: Our revels now are ended.These our actors,As I foretold you,were all spirits, andAre melted into air, into thin air:And like the baseless fabric of this vision,The cloud-capp'd tow'rs,the gorgeous palaces,The solemn temples, the great globe itself,Yea, all which it inherit, shall dissolve,And, like this insubstantial pageant faded,Leave not a rack behind. We are such stuffAs dreams aremade on; and our little lifeIs rounded with a sleep.

  20. vifnknvikctgesqtgntgggqagntggdqagstggspqgstgaspqgstgaspqgstgasqpgssepsnpvssghsvstvsvsqtstssekqdtiqvksallkdymglkvtgpcnenfimflvphiyidvdtedtnielrttlkktnnaisfesnsgslekkkyvklpsngttgeqgsstgtvrgdtepisdssssssssssssssssssssssssssseslpangpdsptvkpprnlqnicetgknfklvvyikent low complexity 1.

  21. PAQILWQEDARRKGSM PAQ PAQI nt words = 11 characters aa words = 3 characters PAQIL PAQILW PAQILWQ PAQILWQE PAQILWQED… 2. build word list

  22. Biology in BLAST 3. & 4.

  23. evaluate matching words e.g. score with BLOSUM PAQ PAQI PAQIL PAQILW QVL PAQILWQ QIA PAQILWQE RIL … PAQILWQED… 3. 20 x 20 x 20 8000 possible scores

  24. cloud-capp'd tow'rs cloud-capped towers cloudcapp'd towers cloud-tipped towels QIL QVL QIA RIL …

  25. Small P G cyclic side-chain A C Aromatic S - OH T Y Amines W M H N F K E Aliphatic R D Q I V L Charged Hydrophobic Hydrophilic

  26. BLOSUM62 8000 words  ca 50 QIL 5 + 4 + 4 T = 10 QVL 5 + 3 + 4 QIA 5 + 4 - 1 RIL … 1 + 4 + 4 4. retain high-scoring words

  27. What about nucleotide words?

  28. Repeat 3. and 4. for each word listed 5. A 50n polypeptide will yield ~12,500 words

  29. create search tree QIL Q N QVL QII Q N NIL V I I QV QI NI 6. L I L L QII QIL QVL NIL

  30. plant seeds Query high-scoring words in position n n = 3 QIL,QVL … Exact matches 7. Database

  31. stop when maximal score drops by a threshold X + + + + + + 8v1 grow HSPs

  32. vision, The cloud-capped towers, the gorgeous palaces, The r: And like the baseless fabric of this vision, The cloud-capped towers, the gorgeous palaces, The + + + + + +

  33. vision, The cloud-capped towers, the gorgeous palaces, The The image of “cloud-capped towers”, unites ethereality and might - - + - - - - + - - - -

  34. use words at T  longer word list X A X X X Query sequence X X X 8v2 Database sequence grow HSPs

  35. extend ‘close’ words as one A X X 8v2 A grow HSPs

  36. filter HSPs HSP HSP HSP HSP 9. ≥ S ≤ S S cut-off score

  37. determine score significance Could 2 random sequences with the lengths of the query and database resp. score achieve the ≥ HSP score? 10.

  38. determine score significance DB S Query S*** S* 10. S**** S** S***** World of randomness S******

  39. determine score significance 10. K, λ calculated for the scoring matrix m’ effective length of database seq n’ effective length of query seq

  40. determine score significance 10.

  41. Extend ≥2 HSPs S 65 40 52 45 Poisson 40 < 45 11. Sum of scores (65+40) > (52+45)

  42. Perform Smith-Waterman (local) alignments BLAST BLAST2 12. Calculate bit score (S’) and E-value of each SW alignment

  43. Deliver results > E < E 13. E user selected cut-off score

  44. A practical example BLAST

  45. * Mutation

  46. * * Primer binding sites PCR Transposon mutagenesis

  47. * * PCR Seq 1 Seq 2

  48. Self-study BLAST