[{"text": "The following\ncontent is provided", "start": 0.58, "duration": 1.86}, {"text": "by MIT OpenCourseWare under\na Creative Commons license.", "start": 2.44, "duration": 3.9}, {"text": "Additional information\nabout our license", "start": 6.34, "duration": 2.16}, {"text": "and MIT OpenCourseWare\nin general", "start": 8.5, "duration": 2.37}, {"text": "is available at ocw.mit.edu.", "start": 10.87, "duration": 1.86}, {"text": "GEORGE CHURCH: OK.", "start": 22.81, "duration": 0.75}, {"text": "Welcome back.", "start": 23.56, "duration": 1.35}, {"text": "We're going to go through a very\nspecific example of association", "start": 24.91, "duration": 6.27}, {"text": "studying, illustrating this\nextremely important statistic,", "start": 31.18, "duration": 3.36}, {"text": "the chi-square statistic\nin the simplest case.", "start": 34.54, "duration": 4.62}, {"text": "That simple case will be a\nsingle allele combination,", "start": 39.16, "duration": 6.93}, {"text": "two alleles and two possible\nphenotypic outcomes,", "start": 46.09, "duration": 6.3}, {"text": "HIV resistance and\nHIV non-resistance.", "start": 52.39, "duration": 4.92}, {"text": "So to set up this\nassociation study, which", "start": 57.31, "duration": 2.97}, {"text": "would be computational,\nlet's talk a little bit", "start": 60.28, "duration": 3.51}, {"text": "about the biology here.", "start": 63.79, "duration": 1.199}, {"text": "All viruses need to get\ninto your cells somehow.", "start": 68.89, "duration": 3.66}, {"text": "HIV has a number of\nproteins to which it binds", "start": 72.55, "duration": 4.89}, {"text": "on the surface of your cell.", "start": 77.44, "duration": 1.17}, {"text": "These proteins are not-- they're\nnot designed to bind HIV.", "start": 78.61, "duration": 3.6}, {"text": "They do something else.", "start": 82.21, "duration": 1.27}, {"text": "This is in a family of\nchemokine receptors.", "start": 83.48, "duration": 2.33}, {"text": "This is involved in\nintracellular signaling,", "start": 85.81, "duration": 3.15}, {"text": "and so it's a receptor\nnormally for these chemokines.", "start": 88.96, "duration": 5.4}, {"text": "But it is also a\nreceptor for the virus.", "start": 94.36, "duration": 3.72}, {"text": "And in the human population,\nas we alluded to earlier,", "start": 98.08, "duration": 6.43}, {"text": "there are at least two\nalleles, two common alleles.", "start": 104.51, "duration": 3.32}, {"text": "One of them, the top\none, capital CCR5,", "start": 107.83, "duration": 4.89}, {"text": "has this long,\nopen reading frame.", "start": 112.72, "duration": 1.5}, {"text": "And this you could translate\nby your pearl strip", "start": 114.22, "duration": 2.97}, {"text": "that you wrote for\nthe problem set.", "start": 117.19, "duration": 1.89}, {"text": "And the DNA sequences\nin the middle here", "start": 119.08, "duration": 3.37}, {"text": "and the protein sequence derived\nfrom it is on the top strand.", "start": 122.45, "duration": 5.46}, {"text": "And that's the capital CCR5.", "start": 127.91, "duration": 1.879}, {"text": "And the little delta or\ndeletion Ccr5 is below it.", "start": 129.789, "duration": 4.111}, {"text": "You've locked out 32\nbase pairs, which is not", "start": 133.9, "duration": 3.33}, {"text": "an interval multiple of three.", "start": 137.23, "duration": 1.47}, {"text": "And as you know\nfrom last lecture,", "start": 138.7, "duration": 2.97}, {"text": "that means it's going to be\na different genetic code.", "start": 141.67, "duration": 2.31}, {"text": "At every point downstream, this\nis going to be frame-shifted,", "start": 143.98, "duration": 3.21}, {"text": "and so you get a whole\nnew set of amino acids", "start": 147.19, "duration": 2.29}, {"text": "for the entirety of the rest\nof the carboxy terminus.", "start": 149.48, "duration": 4.92}, {"text": "So this in black\nhere-- so we're showing", "start": 154.4, "duration": 3.23}, {"text": "here in the single-letter code\na somewhat realistic folding", "start": 157.63, "duration": 5.1}, {"text": "of the-- schematic folding\nof the protein, showing here", "start": 162.73, "duration": 3.18}, {"text": "disulfide between two cysteines.", "start": 165.91, "duration": 2.43}, {"text": "Transmembrane region, we\nhave these hydrophobic alpha", "start": 168.34, "duration": 3.66}, {"text": "helices.", "start": 172.0, "duration": 0.84}, {"text": "And finally, in the\nblack amino acids,", "start": 172.84, "duration": 2.94}, {"text": "the C terminus, the\nend of the protein", "start": 175.78, "duration": 2.34}, {"text": "are all substituted in\nthis deletion mutant.", "start": 178.12, "duration": 3.45}, {"text": "Now that is sufficient to\ncause resistance to the virus.", "start": 181.57, "duration": 5.31}, {"text": "It probably has some\neffect on its ability", "start": 186.88, "duration": 2.49}, {"text": "to bind chemokines or react\nto them in some other way.", "start": 189.37, "duration": 4.12}, {"text": "And presumably, this is not--", "start": 193.49, "duration": 3.68}, {"text": "there is some deleterious\neffect we do not know about.", "start": 197.17, "duration": 2.97}, {"text": "But in any case,\nwe can assess this.", "start": 200.14, "duration": 3.075}, {"text": "We can make a genetic\nassay for this.", "start": 203.215, "duration": 2.235}, {"text": "And we can look in\nhuman populations", "start": 205.45, "duration": 4.62}, {"text": "for their resistance to HIV and\nfor the presence of either two", "start": 210.07, "duration": 4.8}, {"text": "alleles of the resistance or\ntwo alleles of the susceptible.", "start": 214.87, "duration": 6.57}, {"text": "Now I'm kind of biasing\nyou here in this.", "start": 221.44, "duration": 2.25}, {"text": "We haven't done the\nassociation study.", "start": 223.69, "duration": 1.633}, {"text": "I really shouldn't be referring\nto it as resistant or not,", "start": 225.323, "duration": 2.417}, {"text": "but I think it helps\nyou visualize it.", "start": 227.74, "duration": 2.61}, {"text": "We'll be rigorous enough\nin the next slide.", "start": 230.35, "duration": 2.11}, {"text": "So here is, if you will,\nthe big allele, right?", "start": 232.46, "duration": 4.85}, {"text": "It's the original.", "start": 237.31, "duration": 1.28}, {"text": "It's the non-deleted allele.", "start": 238.59, "duration": 1.54}, {"text": "So when you do a PCR\nassay where you're", "start": 240.13, "duration": 2.43}, {"text": "amplifying with two primers\nacross the region that", "start": 242.56, "duration": 3.54}, {"text": "could be deleted\nor not, you'll tend", "start": 246.1, "duration": 2.1}, {"text": "to get a large\namplification product.", "start": 248.2, "duration": 2.16}, {"text": "You prime synthesis\nuntil you get something", "start": 250.36, "duration": 3.81}, {"text": "which has 403 base pairs.", "start": 254.17, "duration": 2.185}, {"text": "And there's enough of it\nthat you can display it", "start": 256.355, "duration": 2.0}, {"text": "on electrophoretic gel,\nand it migrates slowly", "start": 258.355, "duration": 2.745}, {"text": "because it's large.", "start": 261.1, "duration": 2.22}, {"text": "If you have a homozygote\nfor the deletion allele--", "start": 263.32, "duration": 2.79}, {"text": "we'll not call it the HIV\nresistance allele just yet--", "start": 266.11, "duration": 4.29}, {"text": "then you'll get a\n371-base-pair PCR product.", "start": 270.4, "duration": 4.8}, {"text": "And if you have\nthe heterozygote,", "start": 275.2, "duration": 1.5}, {"text": "you'll get both, the large and\nthe small allele represented", "start": 276.7, "duration": 3.39}, {"text": "on this electrophoretic assay.", "start": 280.09, "duration": 1.47}, {"text": "A very simple, very robust\nDNA PCR-based assay.", "start": 281.56, "duration": 4.26}, {"text": "So now let's ask whether\none allele or the other", "start": 289.32, "duration": 6.11}, {"text": "is more abundant in people\nwhich are observed to be", "start": 295.43, "duration": 5.25}, {"text": "seropositive or seronegative.", "start": 300.68, "duration": 1.98}, {"text": "Seropositive means\nthat they have", "start": 302.66, "duration": 2.04}, {"text": "circulating antibodies\nin their serum", "start": 304.7, "duration": 2.82}, {"text": "which react positively\nto the HIV virus.", "start": 307.52, "duration": 4.53}, {"text": "And so that's not\nnecessarily, but is commonly", "start": 312.05, "duration": 5.11}, {"text": "associated with being heavily\nexposed to the HIV virus,", "start": 317.16, "duration": 3.26}, {"text": "and therefore\nbasically infected.", "start": 320.42, "duration": 2.28}, {"text": "Seronegative means\nthat you are--", "start": 322.7, "duration": 4.62}, {"text": "it's an indication\nthat you're resistant.", "start": 327.32, "duration": 2.19}, {"text": "Now we could do this as\na two-by-two matrix here", "start": 329.51, "duration": 4.125}, {"text": "of alleles versus\noutcomes, or we", "start": 333.635, "duration": 3.555}, {"text": "could do it as a\nthree-by-two where we", "start": 337.19, "duration": 2.25}, {"text": "have genotypes versus outcomes.", "start": 339.44, "duration": 2.94}, {"text": "The three genotypes are\nbig-big, little-little,", "start": 342.38, "duration": 4.05}, {"text": "and big-little heterozygote.", "start": 346.43, "duration": 1.635}, {"text": "But let's just do it-- just keep\nit simple in terms of alleles.", "start": 348.065, "duration": 2.625}, {"text": "If there is a selective advan--", "start": 350.69, "duration": 2.19}, {"text": "or there's a\nperceivable association", "start": 352.88, "duration": 4.98}, {"text": "between the seropositive,\nseronegative, and the allele,", "start": 357.86, "duration": 3.93}, {"text": "you should be able to see it\nin both the genotype or just", "start": 361.79, "duration": 3.565}, {"text": "the allele.", "start": 365.355, "duration": 0.5}, {"text": "The allele is simpler.", "start": 365.855, "duration": 1.155}, {"text": "It's a two-by-two matrix.", "start": 367.01, "duration": 1.53}, {"text": "And so these are the data,\njust these four boxes here.", "start": 368.54, "duration": 4.68}, {"text": "The big allele is 1,278 observed\nnegative and 1,368 observed", "start": 373.22, "duration": 7.53}, {"text": "positive for a total of 2,646.", "start": 380.75, "duration": 3.06}, {"text": "You can see that\nthere are fewer total", "start": 383.81, "duration": 2.67}, {"text": "in the population surveyed\nof the deletion mutant.", "start": 386.48, "duration": 3.577}, {"text": "This is consistent with the\nclaim that I made all along.", "start": 390.057, "duration": 2.333}, {"text": "It's about 9% in general\nhuman population,", "start": 392.39, "duration": 2.85}, {"text": "and it's 9% of this population.", "start": 395.24, "duration": 1.995}, {"text": "That's good.", "start": 397.235, "duration": 2.005}, {"text": "But now you have to\ncorrect for that.", "start": 399.24, "duration": 1.64}, {"text": "You need to-- you\ncan't just ask,", "start": 400.88, "duration": 3.24}, {"text": "is it more frequent here or not?", "start": 404.12, "duration": 1.47}, {"text": "You have to correct for\nthe actual frequencies", "start": 405.59, "duration": 2.52}, {"text": "in the population.", "start": 408.11, "duration": 0.78}, {"text": "And the way that's done is you\ncalculate another table, which", "start": 408.89, "duration": 6.33}, {"text": "is the expected number of each\nof these combinations of big", "start": 415.22, "duration": 6.6}, {"text": "with seronegative,\nbig with seropositive,", "start": 421.82, "duration": 1.86}, {"text": "and so forth, under\nthe assumption", "start": 423.68, "duration": 2.19}, {"text": "that it's completely random.", "start": 425.87, "duration": 1.965}, {"text": "You know the allele\nfrequencies in the population,", "start": 427.835, "duration": 2.385}, {"text": "but say that they just randomly\nassociated with whether it's", "start": 430.22, "duration": 3.405}, {"text": "seropositive or seronegative.", "start": 433.625, "duration": 1.545}, {"text": "You use these totals\nhere and the frequencies", "start": 435.17, "duration": 2.61}, {"text": "in the population to\ngenerate this expected end", "start": 437.78, "duration": 3.75}, {"text": "of the random assumption.", "start": 441.53, "duration": 1.17}, {"text": "And then you look for\ndeviation of the real ones", "start": 442.7, "duration": 3.09}, {"text": "of the observed\nversus the expected.", "start": 445.79, "duration": 1.715}, {"text": "So any deviation between the\nexpected and the observed", "start": 454.99, "duration": 4.32}, {"text": "is going to be\nsignificant in this.", "start": 459.31, "duration": 1.59}, {"text": "So you want to do is take the\ndifference between the observed", "start": 463.49, "duration": 3.23}, {"text": "and expected.", "start": 466.72, "duration": 0.71}, {"text": "So we're working on\na statistic here,", "start": 467.43, "duration": 2.41}, {"text": "a type of measure\nthat will determine,", "start": 469.84, "duration": 2.28}, {"text": "how far from expectation\nare the observations", "start": 472.12, "duration": 3.36}, {"text": "on the left-hand side\nin this two-by-two?", "start": 475.48, "duration": 3.27}, {"text": "And so for every square\nin the two-by-two,", "start": 478.75, "duration": 3.39}, {"text": "you find a corresponding\nexpected number", "start": 482.14, "duration": 2.64}, {"text": "and you subtract it.", "start": 484.78, "duration": 1.14}, {"text": "That's the starting point.", "start": 485.92, "duration": 1.29}, {"text": "But you don't care whether\nit's negative or positive.", "start": 487.21, "duration": 3.66}, {"text": "You want to make it positive.", "start": 490.87, "duration": 1.51}, {"text": "So the trick we'll often use\nis to take the square of that.", "start": 492.38, "duration": 2.598}, {"text": "So you could take\nthe absolute value.", "start": 494.978, "duration": 1.542}, {"text": "You take the square for the\nchi-square, oddly enough.", "start": 496.52, "duration": 3.11}, {"text": "And then you want to put it\non some kind of standard scale", "start": 499.63, "duration": 3.84}, {"text": "so that when you do a chi-square\nfor any kind of phenomenon,", "start": 503.47, "duration": 3.57}, {"text": "you'll be able to compare.", "start": 507.04, "duration": 1.2}, {"text": "And the way you do that is you\ndivide by the expected number.", "start": 508.24, "duration": 3.63}, {"text": "Or as this puts it on, if\nthese numbers were really huge,", "start": 511.87, "duration": 4.53}, {"text": "this would bring them down.", "start": 516.4, "duration": 3.13}, {"text": "Very small, it'll\nbring the same point.", "start": 519.53, "duration": 2.34}, {"text": "And so you take the sum over\nall the squares in here,", "start": 521.87, "duration": 3.45}, {"text": "and that's 15.6.", "start": 525.32, "duration": 1.94}, {"text": "And that's saying that it\ndeviates from the expectation", "start": 527.26, "duration": 5.89}, {"text": "by this amount normalized as\na fraction of the expected.", "start": 533.15, "duration": 5.49}, {"text": "Now to determine just a little\nsidelight on the chi-square,", "start": 538.64, "duration": 5.34}, {"text": "in order to determine\nthe probability of 15.6", "start": 543.98, "duration": 3.42}, {"text": "being significant,\nwhat does that", "start": 547.4, "duration": 1.41}, {"text": "mean, looking at this at 15.6?", "start": 548.81, "duration": 1.952}, {"text": "You want to turn this\ninto a probability,", "start": 550.762, "duration": 1.708}, {"text": "because probabilities are the\ncommon language that we can all", "start": 552.47, "duration": 2.67}, {"text": "share our surprise at this\nbeing different from expected,", "start": 555.14, "duration": 4.29}, {"text": "the null hypothesis that\nthis is the same as expected.", "start": 559.43, "duration": 2.825}, {"text": "And so in order\nto evaluate that,", "start": 562.255, "duration": 1.375}, {"text": "you have to ask how\nmuch freedom there--", "start": 563.63, "duration": 2.22}, {"text": "degrees of freedom.", "start": 565.85, "duration": 1.18}, {"text": "This is jargon for,\nhow many different ways", "start": 567.03, "duration": 5.26}, {"text": "can these two numbers vary?", "start": 572.29, "duration": 1.72}, {"text": "Well, since we know\nthe total number,", "start": 574.01, "duration": 2.96}, {"text": "when we observe the number of\nCCR5, that fixes in the columns", "start": 576.97, "duration": 7.15}, {"text": "the number of deletions, right?", "start": 584.12, "duration": 1.68}, {"text": "Because it's just going to be\nthe number of big alleles taken", "start": 585.8, "duration": 3.45}, {"text": "from the total.", "start": 589.25, "duration": 1.48}, {"text": "So in a way, the degrees\nof freedom is just one.", "start": 590.73, "duration": 2.18}, {"text": "You fixed this one number, and\nthe other one is now known.", "start": 592.91, "duration": 4.44}, {"text": "And the same thing's true\nfor rows and columns.", "start": 597.35, "duration": 2.08}, {"text": "So the rows minus--", "start": 599.43, "duration": 1.19}, {"text": "it's just one and\nthe columns is one.", "start": 600.62, "duration": 2.49}, {"text": "And so the whole\ndegrees of freedom", "start": 603.11, "duration": 1.83}, {"text": "is the product that rows\nand columns, which is one.", "start": 604.94, "duration": 2.97}, {"text": "So you plug this in.", "start": 607.91, "duration": 0.93}, {"text": "If you look in your\nstandard statistical--", "start": 608.84, "duration": 1.92}, {"text": "or your favorite statistical\nbook or software package, Excel", "start": 610.76, "duration": 3.84}, {"text": "or whatever, you plug\nin this chi-square value", "start": 614.6, "duration": 2.82}, {"text": "and this degrees of freedom,\nand you get this probability,", "start": 617.42, "duration": 2.95}, {"text": "which is very significant.", "start": 620.37, "duration": 1.52}, {"text": "Generally better than 5%\nmeans that you will only", "start": 621.89, "duration": 5.22}, {"text": "be wrong 20% of the time.", "start": 627.11, "duration": 2.32}, {"text": "Some people would prefer to--", "start": 629.43, "duration": 2.69}, {"text": "this means you'll be wrong eight\ntimes in 10,000 or something", "start": 632.12, "duration": 4.21}, {"text": "like that.", "start": 636.33, "duration": 0.5}, {"text": "Very, very infrequently.", "start": 636.83, "duration": 2.64}, {"text": "OK.", "start": 639.47, "duration": 1.17}, {"text": "Now this is great.", "start": 640.64, "duration": 1.84}, {"text": "This is a two-by-two matrix.", "start": 642.48, "duration": 1.25}, {"text": "We found an association.", "start": 643.73, "duration": 1.56}, {"text": "We have a plausible\nmolecular mechanism", "start": 645.29, "duration": 2.79}, {"text": "in the previous slide.", "start": 648.08, "duration": 1.56}, {"text": "But how was it that\nwe pulled CCR5 out", "start": 649.64, "duration": 3.69}, {"text": "of-- this rabbit out of the hat?", "start": 653.33, "duration": 1.5}, {"text": "I mean, why CCR5?", "start": 654.83, "duration": 2.43}, {"text": "There are 40,000-some\ngenes in the human genome.", "start": 657.26, "duration": 3.09}, {"text": "Yes?", "start": 660.35, "duration": 0.57}, {"text": "AUDIENCE: What is it?", "start": 660.92, "duration": 0.875}, {"text": "I was curious as to,\nwhat population is it?", "start": 661.795, "duration": 4.165}, {"text": "I mean, in which there's\nslightly more seropositive", "start": 665.96, "duration": 5.272}, {"text": "or seronegative people.", "start": 671.232, "duration": 0.958}, {"text": "This must be a particular--", "start": 672.19, "duration": 2.754}, {"text": "GEORGE CHURCH: Oh, yeah.", "start": 674.944, "duration": 2.286}, {"text": "This is a case\ncontrol study where", "start": 677.23, "duration": 1.89}, {"text": "you try to get a roughly\nequal number of negatives", "start": 679.12, "duration": 2.88}, {"text": "and positives that are matched\nfor socioeconomic group", "start": 682.0, "duration": 3.45}, {"text": "and race and gender\nand things like that.", "start": 685.45, "duration": 2.37}, {"text": "So that's a setup.", "start": 687.82, "duration": 1.51}, {"text": "But then the-- if there's not\na huge risk ratio for the two", "start": 689.33, "duration": 6.29}, {"text": "alleles, then you'll expect them\nto be close to the population", "start": 695.62, "duration": 2.66}, {"text": "frequency, which means\nthat you can't just", "start": 698.28, "duration": 1.75}, {"text": "look at it on inspection\nand say, oh, yeah.", "start": 700.03, "duration": 2.46}, {"text": "All the seropositive\nare the big allele.", "start": 702.49, "duration": 2.79}, {"text": "We wouldn't need a\nchi-square at that point.", "start": 705.28, "duration": 2.88}, {"text": "But this is pretty close to\nthe population frequency,", "start": 708.16, "duration": 3.79}, {"text": "and so that's why we\nhad to do a chi-square.", "start": 711.95, "duration": 4.0}, {"text": "OK.", "start": 715.95, "duration": 1.5}, {"text": "But the question\nthat we're taking now", "start": 717.45, "duration": 5.73}, {"text": "is, how did we pick CCR5?", "start": 723.18, "duration": 1.45}, {"text": "And I introduced this as\nbeing a putative receptor", "start": 724.63, "duration": 3.56}, {"text": "for the virus, but one of\nthe first pieces of evidence", "start": 728.19, "duration": 2.842}, {"text": "that this was a putative\nreceptor for the virus", "start": 731.032, "duration": 1.958}, {"text": "was this association study.", "start": 732.99, "duration": 2.71}, {"text": "So how was it that it hit\nthe chemokine receptor?", "start": 735.7, "duration": 3.632}, {"text": "Well, you could say, well,\nbecause some kind of hunch", "start": 739.332, "duration": 2.208}, {"text": "about chemokines being\ninvolved in immunology,", "start": 741.54, "duration": 2.49}, {"text": "and immunology being\nimportant in fighting viruses,", "start": 744.03, "duration": 3.69}, {"text": "but that wouldn't suggest\nthat it's an actual receptor.", "start": 747.72, "duration": 2.64}, {"text": "I think these are--", "start": 753.51, "duration": 1.64}, {"text": "there's all kinds of\ninspired guesswork.", "start": 755.15, "duration": 1.93}, {"text": "There's biochemistry happening\nbehind the scene, and so on.", "start": 757.08, "duration": 2.925}, {"text": "But let's just put that\naside for the moment", "start": 760.005, "duration": 2.17}, {"text": "and take the more\ngeneral case that you", "start": 762.175, "duration": 1.625}, {"text": "wanted to test not just\nthis one hypothesis,", "start": 763.8, "duration": 2.64}, {"text": "that CCR5 is involved,\nbut you've implicitly", "start": 766.44, "duration": 2.67}, {"text": "then tested or explicitly\ntested every gene.", "start": 769.11, "duration": 3.812}, {"text": "You've gone through\nevery gene and you've", "start": 772.922, "duration": 1.708}, {"text": "taken either the most\ncommon alleles or you've", "start": 774.63, "duration": 3.12}, {"text": "sequenced your own\ngenome and you've", "start": 777.75, "duration": 1.59}, {"text": "found the alleles that you have,\nwhether they're common or rare.", "start": 779.34, "duration": 3.275}, {"text": "You don't care.", "start": 782.615, "duration": 0.625}, {"text": "And you want to ask,\nwhat's the association?", "start": 783.24, "duration": 2.82}, {"text": "Let's consider the upper\nright-hand panel here", "start": 790.85, "duration": 4.35}, {"text": "where we have some risk ratio,\na fairly subtle one, 1.5.", "start": 795.2, "duration": 5.46}, {"text": "Remember, we were talking\nabout risk ratios of 75", "start": 800.66, "duration": 2.64}, {"text": "in the case of autism.", "start": 803.3, "duration": 1.05}, {"text": "This is just 1.5.", "start": 804.35, "duration": 1.02}, {"text": "Very subtle risk ratio, just\nlike I think in the last one", "start": 805.37, "duration": 4.02}, {"text": "could have been a\nsubtle risk ratio.", "start": 809.39, "duration": 1.86}, {"text": "And the x-axis is going to\nbe the number of alleles", "start": 813.93, "duration": 4.535}, {"text": "that you're\nhypothetically testing.", "start": 818.465, "duration": 2.115}, {"text": "This could be related\nto the number of genes", "start": 820.58, "duration": 1.92}, {"text": "that you're testing,\nor it could be", "start": 822.5, "duration": 1.98}, {"text": "more than the number of\ngenes that you're testing,", "start": 824.48, "duration": 1.56}, {"text": "because you can have more\nthan one allele per gene", "start": 826.04, "duration": 2.04}, {"text": "that you might want to test.", "start": 828.08, "duration": 1.167}, {"text": "Is this allele of this gene\nimportant in this disease,", "start": 829.247, "duration": 3.153}, {"text": "or is this other one?", "start": 832.4, "duration": 1.245}, {"text": "For example, sickle\ncell is important,", "start": 833.645, "duration": 2.085}, {"text": "but various other hemoglobin\nmutations are not.", "start": 835.73, "duration": 3.51}, {"text": "This chemokine deletion is\nimportant and maybe another one", "start": 839.24, "duration": 2.67}, {"text": "isn't.", "start": 841.91, "duration": 0.72}, {"text": "Many chemokine mutations\nwill be neutral.", "start": 842.63, "duration": 3.11}, {"text": "OK.", "start": 845.74, "duration": 0.5}, {"text": "So as you increase the number\nof genes and alleles from--", "start": 846.24, "duration": 3.77}, {"text": "here's 10 to the fourth on up--", "start": 850.01, "duration": 2.67}, {"text": "then the number of sib pairs--", "start": 852.68, "duration": 3.24}, {"text": "so this is a simulation covering\nall kinds of experiments", "start": 855.92, "duration": 4.65}, {"text": "that you could do\nwhere you can use", "start": 860.57, "duration": 3.03}, {"text": "computational\nmethods to help guide", "start": 863.6, "duration": 1.86}, {"text": "the design of experiments.", "start": 865.46, "duration": 2.4}, {"text": "If you did an\nexpensive experiment", "start": 867.86, "duration": 1.59}, {"text": "and you just happened to\nuse too few patients and too", "start": 869.45, "duration": 3.87}, {"text": "many alleles, then you may\nhave misused your resources.", "start": 873.32, "duration": 3.255}, {"text": "You should have maybe done\nfewer alleles and more patients", "start": 876.575, "duration": 2.535}, {"text": "or something like that.", "start": 879.11, "duration": 2.68}, {"text": "And this provides\nsome guidance here.", "start": 881.79, "duration": 1.82}, {"text": "But you can see\nthat in order to get", "start": 883.61, "duration": 1.68}, {"text": "to a very large\nnumber of alleles,", "start": 885.29, "duration": 1.98}, {"text": "you need a fairly\nmodest increase", "start": 887.27, "duration": 1.68}, {"text": "in the number of patients.", "start": 888.95, "duration": 1.09}, {"text": "And that's due to\nthe exponential term", "start": 890.04, "duration": 4.43}, {"text": "that you have in these\nprobability distributions.", "start": 894.47, "duration": 4.69}, {"text": "So you actually--\nbut nevertheless,", "start": 899.16, "duration": 1.97}, {"text": "it's a big deal cost-wise\ngoing up from, say,", "start": 901.13, "duration": 5.49}, {"text": "400 patients to 1,600.", "start": 906.62, "duration": 4.5}, {"text": "Nevertheless, it's\nonly linear in patients", "start": 911.12, "duration": 2.85}, {"text": "while it's exponential in the\nnumber of alleles you can test,", "start": 913.97, "duration": 4.47}, {"text": "so that's the good news.", "start": 918.44, "duration": 2.03}, {"text": "And you can see some\nof these other panels", "start": 920.47, "duration": 1.75}, {"text": "show the effect of\nvarying other parameters.", "start": 922.22, "duration": 4.35}, {"text": "For example, here\non the left you've", "start": 926.57, "duration": 1.92}, {"text": "got varying the population\nfrequency of the allele.", "start": 928.49, "duration": 3.99}, {"text": "As you get to very,\nvery rare alleles", "start": 932.48, "duration": 3.15}, {"text": "on the right hand of the\nhorizontal axis here, then", "start": 935.63, "duration": 4.26}, {"text": "the number of sib pairs--", "start": 939.89, "duration": 4.32}, {"text": "brothers-sisters,\nbrother-brothers", "start": 944.21, "duration": 1.8}, {"text": "that you need-- starts\nto go through the roof.", "start": 946.01, "duration": 2.19}, {"text": "And now it's exponential\non both axes,", "start": 948.2, "duration": 4.86}, {"text": "or that is to say that\nit's a direct relationship.", "start": 953.06, "duration": 2.31}, {"text": "Question?", "start": 955.37, "duration": 0.54}, {"text": "AUDIENCE: What's that\nz value over there?", "start": 955.91, "duration": 1.718}, {"text": "GEORGE CHURCH: Hmm?", "start": 957.628, "duration": 0.792}, {"text": "AUDIENCE: What's that\nz value of over there?", "start": 958.42, "duration": 2.53}, {"text": "GEORGE CHURCH: Oh, that's\nthe population frequency that", "start": 960.95, "duration": 4.345}, {"text": "you're--", "start": 965.295, "duration": 0.5}, {"text": "AUDIENCE: On the bottom?", "start": 965.795, "duration": 1.199}, {"text": "GEORGE CHURCH: Hmm?", "start": 966.994, "duration": 0.946}, {"text": "Well, on-- on the one we were\njust talking about on the left,", "start": 967.94, "duration": 4.86}, {"text": "the population frequency\nis the horizontal axis,", "start": 972.8, "duration": 2.52}, {"text": "and it varies from near unity\nto 10 to the minus nine.", "start": 975.32, "duration": 5.34}, {"text": "It's a very rare\nallele frequency.", "start": 980.66, "duration": 3.3}, {"text": "On the right, the one we\nstarted with, the top right,", "start": 983.96, "duration": 3.78}, {"text": "we just picked 0.5,\nwhich would be very--", "start": 987.74, "duration": 3.24}, {"text": "be around here on the left-hand\nend of the left-hand quadrant.", "start": 990.98, "duration": 4.84}, {"text": "So you pick one of these\nallele frequencies, a fairly--", "start": 995.82, "duration": 2.66}, {"text": "where it's equal\nfrequency of the two", "start": 998.48, "duration": 1.95}, {"text": "alleles in the\npopulation as where you", "start": 1000.43, "duration": 2.62}, {"text": "do the rest of the simulation.", "start": 1003.05, "duration": 1.25}, {"text": "You can think of\nall these panels", "start": 1004.3, "duration": 2.43}, {"text": "could be some\nmulti-dimensional display,", "start": 1006.73, "duration": 1.74}, {"text": "but I wanted to take\nthem out one at a time.", "start": 1008.47, "duration": 2.76}, {"text": "And actually, I did\nall these in Excel", "start": 1011.23, "duration": 3.48}, {"text": "using the equations that are\npresent in this reference", "start": 1014.71, "duration": 3.92}, {"text": "that's given in the slides here.", "start": 1018.63, "duration": 2.44}, {"text": "A good sign of a\nwell-written paper", "start": 1024.53, "duration": 1.77}, {"text": "is for a sort of\naverage individual", "start": 1026.3, "duration": 2.309}, {"text": "to be able to reproduce it.", "start": 1028.609, "duration": 2.891}, {"text": "So we have how many--\nso we've been talking", "start": 1031.5, "duration": 5.2}, {"text": "about new polymorphisms.", "start": 1036.7, "duration": 2.34}, {"text": "In fact, the question that\ncame up during the break", "start": 1039.04, "duration": 2.25}, {"text": "was, if selection and drift\ncause allele frequencies to fix", "start": 1041.29, "duration": 6.779}, {"text": "fairly rapidly, drift\nin small populations,", "start": 1048.069, "duration": 3.511}, {"text": "normal-sized\npopulations will drift", "start": 1051.58, "duration": 1.95}, {"text": "in fairly short evolutionary\ntimes and selection", "start": 1053.53, "duration": 4.303}, {"text": "if you have a high selection\ncoefficient of cost fixation.", "start": 1057.833, "duration": 2.417}, {"text": "Why do we have any allele\nfrequencies at all?", "start": 1060.25, "duration": 1.89}, {"text": "Why isn't everything fixed at\n100% for one particular allele?", "start": 1062.14, "duration": 4.5}, {"text": "And the answer is mutation.", "start": 1066.64, "duration": 1.53}, {"text": "And where all these new\nmutations or polymorphisms", "start": 1068.17, "duration": 2.408}, {"text": "come from-- actually,\nthese should probably", "start": 1070.578, "duration": 1.792}, {"text": "be called mutations,\nbecause most of them", "start": 1072.37, "duration": 2.1}, {"text": "have frequencies less than 1%.", "start": 1074.47, "duration": 2.31}, {"text": "Well, here's a specific model\nwhich doesn't follow all", "start": 1076.78, "duration": 3.103}, {"text": "the assumptions we had before,\nbut we'll use a different--", "start": 1079.883, "duration": 2.417}, {"text": "we'll use a back-of-the-envelope\ncalculation to give you", "start": 1082.3, "duration": 2.333}, {"text": "a feeling for what\nis true for the--", "start": 1084.633, "duration": 3.517}, {"text": "what is likely to be the case\nfor the human population.", "start": 1088.15, "duration": 2.77}, {"text": "So the human population,\nthere's a little bit", "start": 1090.92, "duration": 2.39}, {"text": "of unknown in some\nof these parameters,", "start": 1093.31, "duration": 2.99}, {"text": "so you should take these all\nwith a grain of salt. Actually,", "start": 1096.3, "duration": 2.5}, {"text": "everything I say\nyou should take.", "start": 1098.8, "duration": 1.42}, {"text": "But the number of\npopulations that we've", "start": 1100.22, "duration": 4.25}, {"text": "had since a bottleneck\nin the human population--", "start": 1104.47, "duration": 3.75}, {"text": "maybe there were as few\nas 10 to the fourth humans", "start": 1108.22, "duration": 3.12}, {"text": "at some point or another.", "start": 1111.34, "duration": 2.28}, {"text": "Maybe less.", "start": 1113.62, "duration": 1.68}, {"text": "But since that time, there have\nbeen about 5,000 generations.", "start": 1115.3, "duration": 2.91}, {"text": "And during that\ntime, our population", "start": 1118.21, "duration": 1.68}, {"text": "has now grown to\nsix billion people.", "start": 1119.89, "duration": 2.25}, {"text": "That's the n prime nn.", "start": 1122.14, "duration": 3.57}, {"text": "And the mutation rate, as\nmentioned earlier in response", "start": 1125.71, "duration": 6.073}, {"text": "to a question, is\naround 10 to the minus", "start": 1131.783, "duration": 1.667}, {"text": "eighth per base pair per\ngenome per generation.", "start": 1133.45, "duration": 6.13}, {"text": "And so the genome size\nis about six billion.", "start": 1139.58, "duration": 2.69}, {"text": "Coincidentally, the same\nas the population size.", "start": 1142.27, "duration": 2.16}, {"text": "Total coincidence.", "start": 1144.43, "duration": 1.95}, {"text": "Then 10 to the minus eight\ntimes that number of base pairs", "start": 1146.38, "duration": 3.45}, {"text": "means you have\nabout 60 mutations", "start": 1149.83, "duration": 2.49}, {"text": "or so occurring in a generation.", "start": 1152.32, "duration": 4.99}, {"text": "So you've got a steady\nflow of new mutations.", "start": 1157.31, "duration": 2.74}, {"text": "Now if you take\nthat-- just roughly", "start": 1160.05, "duration": 1.57}, {"text": "speaking, if you take that\nover 5,000 generations,", "start": 1161.62, "duration": 2.31}, {"text": "you've got 60 times 5,000.", "start": 1163.93, "duration": 2.7}, {"text": "You've got a very large number--", "start": 1166.63, "duration": 1.86}, {"text": "on the order of 10 to\nthe fourth mutations", "start": 1168.49, "duration": 3.21}, {"text": "that have accumulated\nin our population.", "start": 1171.7, "duration": 1.93}, {"text": "Assuming relatively\nlittle drift because", "start": 1173.63, "duration": 3.26}, {"text": "of this exponentially growing\npopulation and relatively", "start": 1176.89, "duration": 2.67}, {"text": "little selection, then\nthis is the number", "start": 1179.56, "duration": 3.27}, {"text": "that might have accumulated.", "start": 1182.83, "duration": 1.41}, {"text": "You can do subtle corrections\nfor this exponential growth.", "start": 1184.24, "duration": 4.68}, {"text": "The number of mutations\nyou got at the beginning", "start": 1188.92, "duration": 3.073}, {"text": "will have higher frequency,\nbut there'll be fewer of them,", "start": 1191.993, "duration": 2.417}, {"text": "because population was smaller.", "start": 1194.41, "duration": 1.332}, {"text": "But anyway, you\nget the picture, is", "start": 1195.742, "duration": 1.458}, {"text": "that the total\nnumber of mutations", "start": 1197.2, "duration": 1.89}, {"text": "in any one of this that\nare new since that new--", "start": 1199.09, "duration": 3.27}, {"text": "since 5,000 generations ago\nwill be on the order of 10", "start": 1202.36, "duration": 4.03}, {"text": "to the fourth in your body, not\nall of them doing good for you.", "start": 1206.39, "duration": 5.99}, {"text": "And for each of those\nrare mutations--", "start": 1212.38, "duration": 2.717}, {"text": "they might have a frequency of\nabout 10 to the minus five--", "start": 1215.097, "duration": 2.458}, {"text": "there will be about 10 to\nthe fourth people on Earth", "start": 1221.67, "duration": 4.348}, {"text": "that will share that very\nrare mutation with you.", "start": 1226.018, "duration": 2.042}, {"text": "10 to the minus 5\nsounds very rare,", "start": 1228.06, "duration": 2.782}, {"text": "but when you multiply it\nby six billion people,", "start": 1230.842, "duration": 1.958}, {"text": "there are a lot of people\nwho share that with you.", "start": 1232.8, "duration": 2.34}, {"text": "It's a new mutation.", "start": 1235.14, "duration": 1.85}, {"text": "OK.", "start": 1236.99, "duration": 0.5}, {"text": "So the take-home is--", "start": 1237.49, "duration": 1.49}, {"text": "and this is from\nthis reference here.", "start": 1238.98, "duration": 3.36}, {"text": "High genomic\ndeleterious mutations", "start": 1242.34, "duration": 3.36}, {"text": "accumulate over these 4,000--", "start": 1245.7, "duration": 3.48}, {"text": "some large number\nof generations.", "start": 1249.18, "duration": 1.92}, {"text": "And they would confound\nlinkage methods", "start": 1251.1, "duration": 2.7}, {"text": "and they would\nconfound assumptions", "start": 1253.8, "duration": 2.04}, {"text": "that say that the\ncommon alleles--", "start": 1255.84, "duration": 2.28}, {"text": "common alleles are causative.", "start": 1258.12, "duration": 4.07}, {"text": "OK.", "start": 1262.19, "duration": 0.51}, {"text": "So let's say we've done\nan association study.", "start": 1262.7, "duration": 2.25}, {"text": "Either we've done it\non one gene, picked", "start": 1264.95, "duration": 2.25}, {"text": "one out of the hat\nlike CCR5, or we've", "start": 1267.2, "duration": 2.28}, {"text": "done it on a full genome\nsurvey, 40,000 genes,", "start": 1269.48, "duration": 2.58}, {"text": "all the alleles we know of.", "start": 1272.06, "duration": 3.06}, {"text": "In order to do the latter,\nwe had to have a big patient", "start": 1275.12, "duration": 3.06}, {"text": "population.", "start": 1278.18, "duration": 1.69}, {"text": "But let's say we've done that.", "start": 1279.87, "duration": 1.25}, {"text": "Now we want to prove that\nthat association is--", "start": 1281.12, "duration": 3.9}, {"text": "that great statistic\nthat we got out,", "start": 1285.02, "duration": 1.83}, {"text": "if we have a large enough\npatient population,", "start": 1286.85, "duration": 1.89}, {"text": "is still just a statistic.", "start": 1288.74, "duration": 2.25}, {"text": "What will constitute proof is\nafter we find the association,", "start": 1290.99, "duration": 4.05}, {"text": "we make a copy of that\nmutation, isolated away", "start": 1295.04, "duration": 3.51}, {"text": "from the three million\nother polymorphisms that", "start": 1298.55, "duration": 2.55}, {"text": "are floating around\nin your body,", "start": 1301.1, "duration": 2.77}, {"text": "and we do some kind of test.", "start": 1303.87, "duration": 1.52}, {"text": "Ideally, we would\nmake an isogenic pair", "start": 1305.39, "duration": 3.12}, {"text": "of humans that only differ\nby this one mutation.", "start": 1308.51, "duration": 2.94}, {"text": "That is not generally considered\nmedically feasible or ethical.", "start": 1311.45, "duration": 7.53}, {"text": "But somehow or another, you\ncan do it on human cells", "start": 1318.98, "duration": 2.61}, {"text": "or you can do it in a\nmammalian model system.", "start": 1321.59, "duration": 5.52}, {"text": "But you have to make\nsomething that's", "start": 1327.11, "duration": 1.77}, {"text": "close to isogenic, a\ncopy of this mutation,", "start": 1328.88, "duration": 2.7}, {"text": "and show that it has a\nphenotype that makes sense.", "start": 1331.58, "duration": 3.22}, {"text": "And then, just to make\nsure you haven't introduced", "start": 1334.8, "duration": 2.27}, {"text": "other mutations, the\nreally careful scientists", "start": 1337.07, "duration": 3.24}, {"text": "will then revert that one\npolymorphism and show now", "start": 1340.31, "duration": 3.3}, {"text": "you no longer have\nthat phenotype.", "start": 1343.61, "duration": 2.46}, {"text": "OK.", "start": 1346.07, "duration": 0.96}, {"text": "So let's walk\nthrough an example.", "start": 1347.03, "duration": 4.45}, {"text": "Here's the third\nexample in this lecture", "start": 1351.48, "duration": 4.51}, {"text": "of a very specific\nallele where we've", "start": 1355.99, "duration": 2.8}, {"text": "shown the molecular\nbasis of it, and it's", "start": 1358.79, "duration": 2.22}, {"text": "the second example\nwhere it's not coding,", "start": 1361.01, "duration": 2.148}, {"text": "and it's the second example\nwhere it's going to repeat.", "start": 1363.158, "duration": 2.292}, {"text": "So this is just to--", "start": 1365.45, "duration": 1.71}, {"text": "I'm not giving you a\nrandom sample here.", "start": 1367.16, "duration": 1.71}, {"text": "I'm specifically biasing this\ntowards very interesting genes,", "start": 1368.87, "duration": 4.77}, {"text": "very interesting traits\nthat are associated", "start": 1373.64, "duration": 3.6}, {"text": "with non-coding\nrepetitive alleles.", "start": 1377.24, "duration": 2.91}, {"text": "In this case, the trait\nthat got the attention", "start": 1380.15, "duration": 3.6}, {"text": "of these researchers-- it\nisn't necessarily proven", "start": 1383.75, "duration": 3.12}, {"text": "that this is the relationship.", "start": 1386.87, "duration": 2.37}, {"text": "But the association\nthat was studied", "start": 1389.24, "duration": 1.65}, {"text": "was between anxiety-related\ntraits, anxiety,", "start": 1390.89, "duration": 4.35}, {"text": "and a polymorphism in the\nserotonin transporter.", "start": 1395.24, "duration": 3.6}, {"text": "This is a science paper.", "start": 1398.84, "duration": 3.3}, {"text": "Now the next step\nin this is to--", "start": 1402.14, "duration": 2.4}, {"text": "OK, that was found associated.", "start": 1404.54, "duration": 2.28}, {"text": "There were lots of other alleles\nrandomly being moved around,", "start": 1406.82, "duration": 6.51}, {"text": "because these are humans.", "start": 1413.33, "duration": 1.74}, {"text": "We have no control over who\nmates with who, or very little.", "start": 1415.07, "duration": 4.362}, {"text": "So you get what you're given.", "start": 1422.21, "duration": 1.98}, {"text": "You can do the survey\nas best you can.", "start": 1424.19, "duration": 1.83}, {"text": "But now in order to move it\ntowards a mechanistic basis", "start": 1426.02, "duration": 4.59}, {"text": "and a proof of introducing\njust this one point mutation.", "start": 1430.61, "duration": 3.69}, {"text": "Now what is the mutation\nfound in this case of anxiety", "start": 1434.3, "duration": 3.75}, {"text": "relationship in this\nserotonin transporter?", "start": 1438.05, "duration": 2.74}, {"text": "It's upstream of the first\nRNA encoding axon one.", "start": 1440.79, "duration": 4.34}, {"text": "It is a 44 base pair deletion\nin a repetitive region,", "start": 1445.13, "duration": 3.95}, {"text": "in a region which might be\nresponsible for promoting", "start": 1449.08, "duration": 4.03}, {"text": "transcription.", "start": 1453.11, "duration": 1.65}, {"text": "You have the short and the long\nalleles, just like in the CCR5.", "start": 1454.76, "duration": 3.93}, {"text": "In this case, it's in a\nputative promoter element.", "start": 1458.69, "duration": 2.76}, {"text": "And when you make this\nconstruct and you hook it up", "start": 1461.45, "duration": 3.57}, {"text": "to a luciferase enzymatic\nactivity in vitro,", "start": 1465.02, "duration": 5.95}, {"text": "in cultured cells so\nyou don't actually", "start": 1470.97, "duration": 2.12}, {"text": "have to construct\na mutant human,", "start": 1473.09, "duration": 3.21}, {"text": "you can now see the\nlong allele always", "start": 1476.3, "duration": 2.19}, {"text": "produces higher\nlevels of expression", "start": 1478.49, "duration": 3.33}, {"text": "than the short allele.", "start": 1481.82, "duration": 1.05}, {"text": "And these little error bars\non top of each of the measures", "start": 1482.87, "duration": 5.22}, {"text": "show your statistical measure\nof standard deviation,", "start": 1488.09, "duration": 3.63}, {"text": "as we did in the first lecture.", "start": 1491.72, "duration": 3.195}, {"text": "And if the black mean, which\nis the height of the bar,", "start": 1494.915, "duration": 5.235}, {"text": "is different from the white\nmean by more than a couple", "start": 1500.15, "duration": 3.143}, {"text": "of these standard deviations,\nwhich is the root mean", "start": 1503.293, "duration": 2.167}, {"text": "square of the standard\ndeviations of each", "start": 1505.46, "duration": 3.24}, {"text": "of the measures, then you call\nit statistically significant.", "start": 1508.7, "duration": 4.09}, {"text": "And that's what these\ntriple stars mean.", "start": 1512.79, "duration": 2.0}, {"text": "It's the statistical\nshorthand for this", "start": 1514.79, "duration": 2.37}, {"text": "is statistically significant\nat the cutoff that we're using.", "start": 1517.16, "duration": 3.06}, {"text": "Say, 5%.", "start": 1520.22, "duration": 1.68}, {"text": "Now in this case, it's\nkind of showing off", "start": 1521.9, "duration": 1.86}, {"text": "because every one of\nthem is statistically", "start": 1523.76, "duration": 1.75}, {"text": "significant by two different--", "start": 1525.51, "duration": 4.04}, {"text": "completely different\nassays here.", "start": 1529.55, "duration": 1.56}, {"text": "But the point is\nthis does not prove", "start": 1531.11, "duration": 2.16}, {"text": "that anxiety is meaningfully\nassociated with this.", "start": 1533.27, "duration": 6.27}, {"text": "But it does prove that this\nrepetitive polymorphism", "start": 1539.54, "duration": 5.04}, {"text": "is causally capable of\ndifferential transcription", "start": 1544.58, "duration": 8.25}, {"text": "levels for a reporter gene.", "start": 1552.83, "duration": 2.82}, {"text": "So by introducing it into\na clean cellular system,", "start": 1555.65, "duration": 7.65}, {"text": "you can test at least\npart of the mechanism that", "start": 1563.3, "duration": 3.12}, {"text": "might be involved in the\nassociation that originally", "start": 1566.42, "duration": 2.37}, {"text": "got your attention.", "start": 1568.79, "duration": 2.73}, {"text": "Now as you can\nsee, we're getting", "start": 1571.52, "duration": 2.97}, {"text": "into a fairly mature and fairly\nnew phase of human genetics.", "start": 1574.49, "duration": 6.66}, {"text": "Some of the concepts\nthat are used here", "start": 1581.15, "duration": 1.71}, {"text": "will be useful in a\nvariety of other systems", "start": 1582.86, "duration": 2.58}, {"text": "where we have relatively little\ncontrol over the genetics.", "start": 1585.44, "duration": 3.42}, {"text": "But certainly in humans,\nthere's a very large need.", "start": 1588.86, "duration": 4.65}, {"text": "And where this\nhappened historically", "start": 1593.51, "duration": 2.73}, {"text": "is with Mendelian linkage,\nwhich would involve", "start": 1596.24, "duration": 2.85}, {"text": "very large families with\na complete pedigree,", "start": 1599.09, "duration": 4.86}, {"text": "multi-generation,\ngreat-grandfathers and mothers,", "start": 1603.95, "duration": 3.21}, {"text": "all the way down, hundreds\nof people in a family.", "start": 1607.16, "duration": 3.615}, {"text": "And that's the problem, because\nthere aren't that many diseases", "start": 1610.775, "duration": 2.625}, {"text": "for which you have\nlarge families", "start": 1613.4, "duration": 1.375}, {"text": "with simple Mendelian\ninheritance.", "start": 1614.775, "duration": 1.895}, {"text": "Common diseases tend to\nbe much more complicated,", "start": 1616.67, "duration": 2.34}, {"text": "involving multiple\ngenes simultaneously", "start": 1619.01, "duration": 2.91}, {"text": "and small families.", "start": 1621.92, "duration": 3.36}, {"text": "Then there's linkage\ndisequilibrium,", "start": 1625.28, "duration": 3.72}, {"text": "which depends on\nthese common alleles", "start": 1629.0, "duration": 5.633}, {"text": "where the population has\ngone through a particularly", "start": 1634.633, "duration": 2.167}, {"text": "small bottleneck and where\nyour common allele is", "start": 1636.8, "duration": 3.84}, {"text": "a pretty good distance away\nfrom the causative one.", "start": 1640.64, "duration": 2.76}, {"text": "But it allows you to\nmap it into a ballpark,", "start": 1643.4, "duration": 2.58}, {"text": "and then you hunker down and\ndo the more expensive testing", "start": 1645.98, "duration": 5.22}, {"text": "of hypotheses for all and\nsequencing and looking", "start": 1651.2, "duration": 2.76}, {"text": "for potentially\ncausative alleles.", "start": 1653.96, "duration": 1.728}, {"text": "Once you find a potentially\ncausative allele, something", "start": 1655.688, "duration": 2.292}, {"text": "that looks suspicious,\nmaybe something", "start": 1657.98, "duration": 2.02}, {"text": "that's in a coding region\nthat it's conserved--", "start": 1660.0, "duration": 2.33}, {"text": "that's the first priority,\ndespite all the counterexamples", "start": 1662.33, "duration": 3.18}, {"text": "I've given.", "start": 1665.51, "duration": 1.27}, {"text": "And then you go ahead.", "start": 1666.78, "duration": 1.23}, {"text": "But the problem is that\nyou're at the mercy", "start": 1668.01, "duration": 1.792}, {"text": "of the recombination\nthat might have occurred", "start": 1669.802, "duration": 1.875}, {"text": "in that population, but you get\nto very interesting populations", "start": 1671.677, "duration": 2.803}, {"text": "like people of African\nAmerican descent", "start": 1674.48, "duration": 4.29}, {"text": "where the population\nis very old, hasn't", "start": 1678.77, "duration": 2.88}, {"text": "passed through a\npopulation bottleneck.", "start": 1681.65, "duration": 2.7}, {"text": "The useful linkage\ndisequilibrium", "start": 1684.35, "duration": 2.52}, {"text": "is on a couple of kilobases\nrather than hundreds", "start": 1686.87, "duration": 3.45}, {"text": "of kilobases, so it\nactually is hard to find", "start": 1690.32, "duration": 2.19}, {"text": "things that are linked.", "start": 1692.51, "duration": 1.32}, {"text": "That is to say, insists\nbut not causatives.", "start": 1693.83, "duration": 2.58}, {"text": "Insists to the causative allele.", "start": 1696.41, "duration": 2.97}, {"text": "Instead, you have to look\ndirectly for causative alleles.", "start": 1699.38, "duration": 2.64}, {"text": "And that's where-- now you look\nfor common causative alleles.", "start": 1702.02, "duration": 2.82}, {"text": "This has the problem that\nwe've been talking about,", "start": 1707.765, "duration": 2.125}, {"text": "which is that maybe common\nalleles aren't often causative.", "start": 1709.89, "duration": 4.97}, {"text": "They've been selected out.", "start": 1714.86, "duration": 1.47}, {"text": "So then you get into\nthe scenario, what if we", "start": 1716.33, "duration": 2.07}, {"text": "wanted to look at all alleles?", "start": 1718.4, "duration": 1.53}, {"text": "Well, theoretically, there's\nnothing wrong with that.", "start": 1719.93, "duration": 3.84}, {"text": "It'd be great.", "start": 1723.77, "duration": 1.68}, {"text": "We could do the\nassociation studies.", "start": 1725.45, "duration": 1.62}, {"text": "We'd have to have fairly\nlarge populations.", "start": 1727.07, "duration": 3.24}, {"text": "But you saw it was linear\nwith an exponential increase", "start": 1730.31, "duration": 2.34}, {"text": "in the number of hypotheses, and\nyou could rank those hypotheses", "start": 1732.65, "duration": 3.06}, {"text": "by a priori likelihood and\nso forth, but it's expensive.", "start": 1735.71, "duration": 3.57}, {"text": "And so the discussion\nnow has to include", "start": 1739.28, "duration": 3.54}, {"text": "a little bit of discussion\nof the new technologies that", "start": 1742.82, "duration": 4.29}, {"text": "might make this less expensive,\nand a discussion of how we got", "start": 1747.11, "duration": 3.36}, {"text": "the first genome and how\nthe new technologies might", "start": 1750.47, "duration": 2.43}, {"text": "be a little different.", "start": 1752.9, "duration": 1.68}, {"text": "But we're going to do this\nin the context of computation", "start": 1754.58, "duration": 2.49}, {"text": "in the sense of, how do we\ndeal with random and systematic", "start": 1757.07, "duration": 2.74}, {"text": "errors?", "start": 1759.81, "duration": 0.5}, {"text": "As we plan our\nstrategy for getting", "start": 1760.31, "duration": 3.06}, {"text": "each of our genomes, personal\ngenomes sequence for $1,000", "start": 1763.37, "duration": 3.27}, {"text": "or so, how do we choose\nwhich technology to pursue?", "start": 1766.64, "duration": 4.21}, {"text": "Which ones have the\nlowest intrinsic, random,", "start": 1770.85, "duration": 3.2}, {"text": "and systematic errors?", "start": 1774.05, "duration": 1.3}, {"text": "So we needed to study\nthe first genome", "start": 1775.35, "duration": 1.76}, {"text": "to see, what would we mean by\nrandom and systematic errors?", "start": 1777.11, "duration": 4.08}, {"text": "How many people here know what\nthe difference is already?", "start": 1781.19, "duration": 3.6}, {"text": "A few.", "start": 1787.37, "duration": 0.52}, {"text": "OK.", "start": 1787.89, "duration": 0.5}, {"text": "But anyway, whether you do or\nnot, it's good for the soul", "start": 1788.39, "duration": 3.33}, {"text": "to go through some\nreal examples of this.", "start": 1791.72, "duration": 2.7}, {"text": "A random mutation\nis something which", "start": 1794.42, "duration": 1.74}, {"text": "occurs every time you\ndo the experiment.", "start": 1796.16, "duration": 2.88}, {"text": "You get a different error.", "start": 1799.04, "duration": 2.64}, {"text": "And a systematic\none is something", "start": 1801.68, "duration": 4.56}, {"text": "where if you do it over\nand over the same way,", "start": 1806.24, "duration": 2.32}, {"text": "you'll get the same\nerror or a simple class", "start": 1808.56, "duration": 2.36}, {"text": "of errors occurring more\nthan once, over and over.", "start": 1810.92, "duration": 4.59}, {"text": "So for sequencing, the process\ninvolves picking something", "start": 1815.51, "duration": 4.535}, {"text": "that you want to\nsequence-- we'll", "start": 1820.045, "duration": 1.375}, {"text": "call it a clone or a template.", "start": 1821.42, "duration": 1.53}, {"text": "Then actually doing\nthe sequencing", "start": 1822.95, "duration": 1.86}, {"text": "and then assembling\nthis into a meaningful", "start": 1824.81, "duration": 3.09}, {"text": "interpreted sequence.", "start": 1827.9, "duration": 1.79}, {"text": "And this is an\nexample where we might", "start": 1829.69, "duration": 4.81}, {"text": "have chosen these big clones.", "start": 1834.5, "duration": 6.87}, {"text": "In other words, you might\nfragment the genome up randomly", "start": 1841.37, "duration": 2.55}, {"text": "into large clones of\n100 kilobases called", "start": 1843.92, "duration": 2.22}, {"text": "bacterial artificial\nchromosomes, for example.", "start": 1846.14, "duration": 2.55}, {"text": "And then that's all\nrandom, and so there's", "start": 1848.69, "duration": 2.46}, {"text": "a shotgun of that scale.", "start": 1851.15, "duration": 1.77}, {"text": "And then we break it up\nrandomly into smaller pieces,", "start": 1852.92, "duration": 2.92}, {"text": "which provide us with\nlittle sequences.", "start": 1855.84, "duration": 1.64}, {"text": "And then in the computer,\nwe assemble these by methods", "start": 1857.48, "duration": 2.46}, {"text": "that we'll discuss in\nthe next lecture, which", "start": 1859.94, "duration": 4.47}, {"text": "can take even fairly different\nsequences than assemble them.", "start": 1864.41, "duration": 3.19}, {"text": "In this case, we're talking\nabout very similar sequences.", "start": 1867.6, "duration": 3.44}, {"text": "And then you assemble the little\nsequences into big sequences,", "start": 1871.04, "duration": 2.783}, {"text": "and you assemble\nthe big sequences", "start": 1873.823, "duration": 1.417}, {"text": "into even bigger sequences, and\nthen you have the whole thing.", "start": 1875.24, "duration": 2.67}, {"text": "But you can see there's a lot\nof opportunity for error here.", "start": 1877.91, "duration": 3.468}, {"text": "We make it look\nsimple in this slide,", "start": 1881.378, "duration": 1.542}, {"text": "but we're going to talk about\nrandom and systematic errors", "start": 1882.92, "duration": 3.06}, {"text": "in a moment.", "start": 1885.98, "duration": 2.04}, {"text": "Where did we get\nthose sequences,", "start": 1888.02, "duration": 1.59}, {"text": "those little sequences\nthat we want to assemble?", "start": 1889.61, "duration": 2.73}, {"text": "We're going to go through\na few methods in a moment,", "start": 1892.34, "duration": 2.26}, {"text": "but the most common\none by far I think", "start": 1894.6, "duration": 2.54}, {"text": "that generates over\n90% of the human genome", "start": 1897.14, "duration": 3.24}, {"text": "sequence in the\nlast couple of years", "start": 1900.38, "duration": 2.04}, {"text": "is capillary electrophoresis\nof fluorescently labeled", "start": 1902.42, "duration": 4.71}, {"text": "polymerase terminated products.", "start": 1907.13, "duration": 2.19}, {"text": "When you electrophoresis\nthings, you're", "start": 1909.32, "duration": 2.07}, {"text": "separating DNA fragments,\nwhich are n nucleotides long,", "start": 1911.39, "duration": 5.985}, {"text": "from a DNA fragment which is\nn plus one nucleotides long.", "start": 1917.375, "duration": 3.255}, {"text": "That's a very subtle\ndifference as n gets big.", "start": 1920.63, "duration": 2.4}, {"text": "As n gets big, it\ngets harder and harder", "start": 1923.03, "duration": 2.25}, {"text": "to separate n from n plus one.", "start": 1925.28, "duration": 2.115}, {"text": "As it does so, you start\ngetting all kinds of errors.", "start": 1927.395, "duration": 2.925}, {"text": "The total number of errors here\nin the lower left-hand corner", "start": 1930.32, "duration": 3.99}, {"text": "is the number of insertions\nplus the number of deletions", "start": 1934.31, "duration": 2.43}, {"text": "plus the number of\nsubstitutions plus n, which", "start": 1936.74, "duration": 3.57}, {"text": "is an abbreviation for no call.", "start": 1940.31, "duration": 3.33}, {"text": "It means the software felt\nthat it was so close that it", "start": 1943.64, "duration": 3.36}, {"text": "couldn't call it at all.", "start": 1947.0, "duration": 1.0}, {"text": "It just said it's n.", "start": 1948.0, "duration": 0.83}, {"text": "I don't know whether\nit's an A, C-- it", "start": 1948.83, "duration": 1.583}, {"text": "doesn't know whether it's an A,\nC, G, or T. Calls it a no call.", "start": 1950.413, "duration": 3.977}, {"text": "And so if you look\nthrough this table,", "start": 1954.39, "duration": 2.27}, {"text": "it shows on the vertical axis\nfor each of these six bar", "start": 1956.66, "duration": 7.02}, {"text": "charts is as you go up\non the vertical axis,", "start": 1963.68, "duration": 4.14}, {"text": "you go from very\nshort reads where", "start": 1967.82, "duration": 2.43}, {"text": "it's easy to separate n from\nn plus one electrophoretically", "start": 1970.25, "duration": 2.7}, {"text": "to very long ones where\nyou start accumulating", "start": 1972.95, "duration": 2.55}, {"text": "insertions deletion\nerrors, insertion deletion", "start": 1975.5, "duration": 3.18}, {"text": "substitution, and n.", "start": 1978.68, "duration": 1.56}, {"text": "They all go up with length.", "start": 1980.24, "duration": 1.93}, {"text": "And so you can think of that\nas a random error superimposed", "start": 1982.17, "duration": 4.22}, {"text": "on the systematic error of if\nyou always do the experiments,", "start": 1986.39, "duration": 3.99}, {"text": "the same base pair is always\nat the end of your run,", "start": 1990.38, "duration": 3.24}, {"text": "it's going to always have\na higher random error rate.", "start": 1993.62, "duration": 2.64}, {"text": "So this is kind of a combination\nof random and systematic error.", "start": 1996.26, "duration": 3.04}, {"text": "So let's go through\nsome examples here.", "start": 1999.3, "duration": 3.38}, {"text": "We have just\nisolating the template", "start": 2002.68, "duration": 3.09}, {"text": "to prepare it for sequencing,\nthere are systematic errors.", "start": 2005.77, "duration": 5.58}, {"text": "If you have certain\nkinds of repeats,", "start": 2011.35, "duration": 1.92}, {"text": "long, inverted repeats, or\ncertain kinds of restriction", "start": 2013.27, "duration": 3.09}, {"text": "elements that the bacteria\ndoesn't like or likes", "start": 2016.36, "duration": 2.79}, {"text": "to chew up, then you\nwon't get the clone.", "start": 2019.15, "duration": 3.81}, {"text": "And that would plague the early\npart of the Genome Project,", "start": 2022.96, "duration": 2.55}, {"text": "is you'd keep trying again\nand again the same way,", "start": 2025.51, "duration": 2.612}, {"text": "and you just wouldn't\nget certain clones.", "start": 2028.122, "duration": 1.708}, {"text": "It's as if there\nwas a hole there.", "start": 2029.83, "duration": 1.53}, {"text": "You know that there's\nsomething there,", "start": 2031.36, "duration": 1.583}, {"text": "but you couldn't clone it.", "start": 2032.943, "duration": 2.077}, {"text": "Sequencing-- hairpins can\nform in the single strand", "start": 2035.02, "duration": 3.54}, {"text": "of nucleic acids\nwhere you're trying", "start": 2038.56, "duration": 1.5}, {"text": "to separate n from n plus one.", "start": 2040.06, "duration": 2.04}, {"text": "And those hairpins then take\nout the gel electrophoresis", "start": 2042.1, "duration": 4.89}, {"text": "and make it seem like it's much\nsmaller than it actually is.", "start": 2046.99, "duration": 3.81}, {"text": "Tandem repeats cause\na problem in all three", "start": 2050.8, "duration": 2.76}, {"text": "of these stages in sequencing.", "start": 2053.56, "duration": 3.42}, {"text": "You get some\npolymerase stuttering", "start": 2056.98, "duration": 2.55}, {"text": "and you get little artifacts.", "start": 2059.53, "duration": 2.189}, {"text": "In assembly repeats,\nyou're assembling", "start": 2061.719, "duration": 3.091}, {"text": "by sequence alignment.", "start": 2064.81, "duration": 2.5}, {"text": "If you have a\nrepeat, the repeat's", "start": 2067.31, "duration": 1.46}, {"text": "going to align just\nas well because it's", "start": 2068.77, "duration": 2.34}, {"text": "a repeat within the\ngenome as a repeat", "start": 2071.11, "duration": 2.105}, {"text": "due to experimental repeating,\nand so the alignment", "start": 2073.215, "duration": 2.125}, {"text": "can be off.", "start": 2075.34, "duration": 1.56}, {"text": "The errors that you got\nfrom these earlier steps", "start": 2076.9, "duration": 2.25}, {"text": "make it hard to assemble.", "start": 2079.15, "duration": 1.71}, {"text": "Polymorphisms look like errors.", "start": 2080.86, "duration": 1.71}, {"text": "Chimeric clones means\nyou've got things", "start": 2082.57, "duration": 2.01}, {"text": "that have been misassembled\nearly on here, and so on.", "start": 2084.58, "duration": 7.43}, {"text": "OK.", "start": 2092.01, "duration": 2.66}, {"text": "When we do this random\nselection of big clones", "start": 2094.67, "duration": 5.232}, {"text": "and random selection of\nlittle clones for sequencing,", "start": 2099.902, "duration": 2.208}, {"text": "we want to know when to quit.", "start": 2102.11, "duration": 1.95}, {"text": "Now you could say,\nwell, we're going", "start": 2104.06, "duration": 1.83}, {"text": "to quit once we get\nit all assembled,", "start": 2105.89, "duration": 1.56}, {"text": "but you need to accumulate\na certain amount of data", "start": 2107.45, "duration": 2.67}, {"text": "in advance before you\ntry to assemble it.", "start": 2110.12, "duration": 1.95}, {"text": "And so there are\nvarious calculations", "start": 2112.07, "duration": 3.75}, {"text": "as to when to quit.", "start": 2115.82, "duration": 1.29}, {"text": "And this is related to\nthe Poisson distribution,", "start": 2117.11, "duration": 2.47}, {"text": "but it's not exactly.", "start": 2119.58, "duration": 1.07}, {"text": "And in fact, one of the\nstudies done in 1988", "start": 2120.65, "duration": 4.11}, {"text": "made some poor\nassumptions about--", "start": 2124.76, "duration": 4.23}, {"text": "remember, we mentioned\nin the first lecture", "start": 2128.99, "duration": 3.33}, {"text": "the assumptions of the\nPoisson distribution", "start": 2132.32, "duration": 3.99}, {"text": "that make it an\napproximation-- when", "start": 2136.31, "duration": 1.86}, {"text": "it becomes an approximation\nto a more formal distribution", "start": 2138.17, "duration": 4.14}, {"text": "like the binomial.", "start": 2142.31, "duration": 0.87}, {"text": "Anyway, that approximation,\nyou should be getting,", "start": 2143.18, "duration": 3.24}, {"text": "as you get higher\nand higher coverage,", "start": 2146.42, "duration": 1.71}, {"text": "meaning more and more\nexperimental repeats,", "start": 2148.13, "duration": 4.11}, {"text": "you'll eventually\nfill all the gaps.", "start": 2152.24, "duration": 2.22}, {"text": "And that means you'll get\n100% complete coverage,", "start": 2154.46, "duration": 4.17}, {"text": "and it should asymptotically\napproach that.", "start": 2158.63, "duration": 2.28}, {"text": "Well, if you use the\nPoisson incorrectly,", "start": 2160.91, "duration": 2.94}, {"text": "as authors did in 1988, you\nbasically go off to infinity.", "start": 2163.85, "duration": 4.23}, {"text": "You get 200% coverage, which\nis physically impossible.", "start": 2168.08, "duration": 4.72}, {"text": "But both an earlier\nstudy, which they ignored,", "start": 2172.8, "duration": 2.6}, {"text": "and a more recent\nstudy got it right.", "start": 2175.4, "duration": 2.43}, {"text": "And these are slightly\ndifferent measures,", "start": 2177.83, "duration": 3.18}, {"text": "but they both converge on 100%.", "start": 2181.01, "duration": 3.24}, {"text": "And I urge you to\nlook at that if you're", "start": 2184.25, "duration": 1.98}, {"text": "designing an experiment\nwith simple formula.", "start": 2186.23, "duration": 3.33}, {"text": "On the other hand, if you want\nto design the experiment more", "start": 2189.56, "duration": 2.97}, {"text": "explicit, you eventually\nget to a point", "start": 2192.53, "duration": 2.55}, {"text": "where a simple analytic\nformula won't do,", "start": 2195.08, "duration": 2.73}, {"text": "and you have to do Monte Carlo.", "start": 2197.81, "duration": 1.35}, {"text": "We treated this\nin the first class", "start": 2199.16, "duration": 2.13}, {"text": "of analytic versus numeric\ndifferentiable equations", "start": 2201.29, "duration": 3.54}, {"text": "and then many other cases.", "start": 2204.83, "duration": 1.51}, {"text": "The simulation you\nwant to do is beyond--", "start": 2206.34, "duration": 3.04}, {"text": "is too hard to do analytically.", "start": 2209.38, "duration": 2.9}, {"text": "So Gene Myers set up-- just\nlisted all the things he", "start": 2212.28, "duration": 3.74}, {"text": "thought would be--", "start": 2216.02, "duration": 1.77}, {"text": "could affect the ability to\nassemble a genome sequence.", "start": 2217.79, "duration": 4.68}, {"text": "The read length and types of\nrepeats, and all this stuff", "start": 2222.47, "duration": 5.52}, {"text": "was simulated.", "start": 2227.99, "duration": 1.17}, {"text": "And he cranked the simulation on\nreal human genome size projects", "start": 2229.16, "duration": 4.35}, {"text": "and came to the conclusion\nthat you could do a shotgun", "start": 2233.51, "duration": 2.7}, {"text": "assembly of a mammalian genome.", "start": 2236.21, "duration": 2.25}, {"text": "A company called\nCelera was formed.", "start": 2238.46, "duration": 1.86}, {"text": "Gene Myers was hired\nas the computer guru.", "start": 2240.32, "duration": 4.2}, {"text": "They put together a\nlarge stable of computers", "start": 2244.52, "duration": 2.73}, {"text": "and they started doing this on\nthe human and the mouse genome.", "start": 2247.25, "duration": 3.81}, {"text": "The human genome was in the\nend not done by this method,", "start": 2251.06, "duration": 2.58}, {"text": "but the mouse genome was, and\nit was a pretty good assembly.", "start": 2253.64, "duration": 2.91}, {"text": "So the Drosophila genome\nmissing the Drosophila repeats", "start": 2256.55, "duration": 3.12}, {"text": "was also done by this method.", "start": 2259.67, "duration": 1.69}, {"text": "So this is scaled\nvery gracefully", "start": 2261.36, "duration": 2.51}, {"text": "from the first shotgun sequence,\nwhich was on a four kV plasmid", "start": 2263.87, "duration": 5.61}, {"text": "to mammalian size genomes.", "start": 2269.48, "duration": 3.03}, {"text": "No mammalian genome is\ncompletely sequenced,", "start": 2272.51, "duration": 2.16}, {"text": "so we can't really\ndeclare victory.", "start": 2274.67, "duration": 1.77}, {"text": "But the sort of simulation\nthat he did here", "start": 2276.44, "duration": 3.51}, {"text": "has played out very nicely.", "start": 2279.95, "duration": 1.155}, {"text": "Right here in Boston,\nthere will be--", "start": 2283.76, "duration": 2.22}, {"text": "when we start\nthinking of the future", "start": 2285.98, "duration": 1.5}, {"text": "of sequencing\ntechnology, we would all", "start": 2287.48, "duration": 1.68}, {"text": "like-- this is almost\nuncontroversial as", "start": 2289.16, "duration": 2.88}, {"text": "to how much we desire that the\ngenome not cost $3 billion,", "start": 2292.04, "duration": 4.05}, {"text": "but it costs $1,000.", "start": 2296.09, "duration": 1.83}, {"text": "And now there are\na number of people", "start": 2297.92, "duration": 2.19}, {"text": "who are taking very definite\nsteps to get us to that point.", "start": 2300.11, "duration": 6.87}, {"text": "To understand systematic\nand random errors that", "start": 2306.98, "duration": 3.06}, {"text": "can occur in these\nsteps, let's take", "start": 2310.04, "duration": 2.04}, {"text": "this dideoxy gel electrophoresis\nwe've been talking about here.", "start": 2312.08, "duration": 3.48}, {"text": "You have four different\ncolored terminators.", "start": 2315.56, "duration": 2.1}, {"text": "These is where the polymerase\ncan't go any further", "start": 2317.66, "duration": 3.09}, {"text": "because there's a\nblocking group here,", "start": 2320.75, "duration": 2.782}, {"text": "which is fluorescent labeled.", "start": 2323.532, "duration": 1.208}, {"text": "And if the template\non the far left", "start": 2324.74, "duration": 1.98}, {"text": "here is ready to accept\nan A, you'll get an A.", "start": 2326.72, "duration": 3.6}, {"text": "And this n and n minus\none will separate", "start": 2330.32, "duration": 3.75}, {"text": "on this electrophoresis and\nthe four colors will give you", "start": 2334.07, "duration": 2.97}, {"text": "this four-colored pattern\nwhere the intensity reflects", "start": 2337.04, "duration": 3.66}, {"text": "good termination\nat that position,", "start": 2340.7, "duration": 2.97}, {"text": "and you can basically\ngo along reading it.", "start": 2343.67, "duration": 2.16}, {"text": "G, C, G, G, A, T.", "start": 2345.83, "duration": 2.16}, {"text": "Now this is in the\nwell-behaved part.", "start": 2347.99, "duration": 1.77}, {"text": "An example of a systematic error\nthat you get in this extension", "start": 2349.76, "duration": 3.75}, {"text": "occurs here in the upper\nright-hand corner of slide 40.", "start": 2353.51, "duration": 3.81}, {"text": "Now you've got-- because\nof one of these hairpins", "start": 2357.32, "duration": 2.19}, {"text": "that I mentioned\nbefore, you've got", "start": 2359.51, "duration": 1.458}, {"text": "a pile-up of seven nucleotides\nall at the same position.", "start": 2360.968, "duration": 3.732}, {"text": "A completely\nalternative method that", "start": 2364.7, "duration": 1.542}, {"text": "doesn't involve\ngel electrophoresis", "start": 2366.242, "duration": 1.458}, {"text": "is called pirA\nsequencing, and this", "start": 2367.7, "duration": 2.22}, {"text": "is an equivalent that can be\ndone with fluorescent addition.", "start": 2369.92, "duration": 4.56}, {"text": "Instead of separating\nthem in time", "start": 2374.48, "duration": 2.28}, {"text": "by their size in\nelectrophoresis,", "start": 2376.76, "duration": 2.79}, {"text": "in time you ask serially,\ndo you want an A here?", "start": 2379.55, "duration": 3.57}, {"text": "If yes, then you\nget a little green--", "start": 2383.12, "duration": 2.28}, {"text": "you get a little\npeek of representing", "start": 2385.4, "duration": 3.67}, {"text": "the release of pyrophosphate\nor the incorporation", "start": 2389.07, "duration": 2.67}, {"text": "of fluorescent A.", "start": 2391.74, "duration": 1.315}, {"text": "And then you ask,\ndo you want a T,", "start": 2393.055, "duration": 2.075}, {"text": "and so forth, and you go along.", "start": 2395.13, "duration": 1.32}, {"text": "And each signal means\nyes to the answer", "start": 2396.45, "duration": 3.78}, {"text": "that it's ready for\nthat particular base.", "start": 2400.23, "duration": 2.32}, {"text": "And you can see this\ndoesn't have any problems", "start": 2402.55, "duration": 1.94}, {"text": "with this hairpin region.", "start": 2404.49, "duration": 1.56}, {"text": "So when you have a\nsystematic error,", "start": 2408.69, "duration": 1.5}, {"text": "you have to change\nthe method that you're", "start": 2410.19, "duration": 1.708}, {"text": "using fairly radically.", "start": 2411.898, "duration": 1.772}, {"text": "The opposite strand or a\ncompletely different method.", "start": 2413.67, "duration": 3.765}, {"text": "If you have-- early on,\nwe had huge differences", "start": 2420.23, "duration": 4.05}, {"text": "in intensity of the\nfluorophores and new enzymes", "start": 2424.28, "duration": 3.48}, {"text": "and new fluorophores were\ndeveloped by Tabor and Matthews", "start": 2427.76, "duration": 3.39}, {"text": "and Glaser, and so forth.", "start": 2431.15, "duration": 1.3}, {"text": "And this was a huge advance\nin making everything uniform", "start": 2432.45, "duration": 4.13}, {"text": "and eliminating\nsystematic errors.", "start": 2436.58, "duration": 2.74}, {"text": "So the sort of things that are\non the horizon that hopefully", "start": 2439.32, "duration": 2.63}, {"text": "will be discussed\ntomorrow in Boston,", "start": 2441.95, "duration": 3.09}, {"text": "we've talked about this short\nnumber of base extensions", "start": 2445.04, "duration": 6.06}, {"text": "like power\nsequencing, the longer", "start": 2451.1, "duration": 1.98}, {"text": "extensions in capillary\narrays, which, capillary arrays", "start": 2453.08, "duration": 3.9}, {"text": "are changing from\nlow-end capillaries", "start": 2456.98, "duration": 1.71}, {"text": "into microfabricated chips.", "start": 2458.69, "duration": 3.09}, {"text": "I'll show an example of a\nmass spec in just a moment.", "start": 2461.78, "duration": 2.235}, {"text": "Sequencing by\nhybridization on arrays", "start": 2467.15, "duration": 1.83}, {"text": "will illustrate as a prelude,\nas an example of Affymetrix", "start": 2468.98, "duration": 4.05}, {"text": "technology in this slide.", "start": 2473.03, "duration": 4.83}, {"text": "OK.", "start": 2477.86, "duration": 0.51}, {"text": "So the idea here, this is\nmainly for re-sequencing.", "start": 2478.37, "duration": 4.38}, {"text": "Some of the ways of reducing\nthe cost of sequencing", "start": 2482.75, "duration": 3.03}, {"text": "the human genome will not\napply to sequencing brand", "start": 2485.78, "duration": 2.7}, {"text": "new genomes.", "start": 2488.48, "duration": 0.78}, {"text": "But still, we need\nto pursue them,", "start": 2489.26, "duration": 1.71}, {"text": "because this may be the way that\nwe get the $1,000 human genome,", "start": 2490.97, "duration": 2.91}, {"text": "even if we don't get other ones.", "start": 2493.88, "duration": 2.38}, {"text": "And here, you know the\nsequence, except there's", "start": 2496.26, "duration": 3.47}, {"text": "a possibility that there could\nbe a polymorphism at any base.", "start": 2499.73, "duration": 2.81}, {"text": "Could be any single base\nsubstitution at any base.", "start": 2502.54, "duration": 3.1}, {"text": "And so you don't\nnecessarily know", "start": 2505.64, "duration": 1.695}, {"text": "in advance what the common\nones are or the rare ones,", "start": 2507.335, "duration": 2.355}, {"text": "but you do know the\ncanonical sequence.", "start": 2509.69, "duration": 2.73}, {"text": "And so at every position, you'll\nmake a 25-mer oligonucleotide", "start": 2512.42, "duration": 6.33}, {"text": "which will bind to a\nfluorescently labeled", "start": 2518.75, "duration": 1.77}, {"text": "version of your genome,\nor a piece of your genome.", "start": 2520.52, "duration": 3.39}, {"text": "And this is actually developed\nfor HIV re-sequencing", "start": 2523.91, "duration": 2.85}, {"text": "by Affymetrix.", "start": 2526.76, "duration": 1.41}, {"text": "And at this middle\nposition, you'll", "start": 2528.17, "duration": 3.45}, {"text": "put in all four possible\nsubstitutions, T, G, C, or A.", "start": 2531.62, "duration": 7.165}, {"text": "And you'll consider\neach possible template.", "start": 2538.785, "duration": 6.085}, {"text": "So if you have the\ntemplate, which is-- let's", "start": 2544.87, "duration": 1.88}, {"text": "say these are the\ntwo alleles that", "start": 2546.75, "duration": 1.417}, {"text": "occur in a human population\nor in your sample.", "start": 2548.167, "duration": 3.653}, {"text": "You can have this sequence and\nthen all the variations on it,", "start": 2551.82, "duration": 3.36}, {"text": "stepping along, changing T,\nG, C, A for the first base,", "start": 2555.18, "duration": 4.273}, {"text": "the second base, the\nthird base, and so forth,", "start": 2559.453, "duration": 1.917}, {"text": "till you hit this one.", "start": 2561.37, "duration": 0.917}, {"text": "And this is where the\nreal polymorphism occurs.", "start": 2562.287, "duration": 2.253}, {"text": "And you could have either\nthis as the context", "start": 2564.54, "duration": 2.49}, {"text": "or this as the context.", "start": 2567.03, "duration": 2.28}, {"text": "And this is schematic And\nthis is the real data.", "start": 2569.31, "duration": 3.0}, {"text": "These are real data\ndown here where", "start": 2572.31, "duration": 1.86}, {"text": "you have the context of\nthe A allele or the context", "start": 2574.17, "duration": 2.97}, {"text": "of the C allele.", "start": 2577.14, "duration": 1.05}, {"text": "You can have the homozygotes\nor the heterozygotes.", "start": 2578.19, "duration": 3.45}, {"text": "And in the homozygous A,\nyou can see it lights up", "start": 2581.64, "duration": 3.063}, {"text": "the best, the best\nhybridization when you have", "start": 2584.703, "duration": 1.917}, {"text": "the A in the middle position.", "start": 2586.62, "duration": 1.208}, {"text": "The middle position\nis most sensitive", "start": 2587.828, "duration": 1.552}, {"text": "to hybridization changes.", "start": 2589.38, "duration": 1.87}, {"text": "And for the C, you have\nit in the C row here.", "start": 2591.25, "duration": 5.03}, {"text": "And remember, you're\nchanging all the bases", "start": 2596.28, "duration": 2.13}, {"text": "in every position.", "start": 2598.41, "duration": 0.75}, {"text": "One, two, three, four.", "start": 2599.16, "duration": 0.917}, {"text": "This is the one where the middle\nbase of the middle position", "start": 2600.077, "duration": 3.073}, {"text": "is in the right context.", "start": 2603.15, "duration": 1.63}, {"text": "So this is in the A\ncontext and the C context.", "start": 2604.78, "duration": 2.33}, {"text": "And in the heterozygote, you\nget both the A and the C.", "start": 2607.11, "duration": 3.36}, {"text": "And so this has\nbeen done on HIV.", "start": 2610.47, "duration": 2.25}, {"text": "It's been done on\nBRCA1, on mitochondria.", "start": 2612.72, "duration": 2.01}, {"text": "And now they've applied\nit with whole wafers", "start": 2614.73, "duration": 2.91}, {"text": "to the entire human genome.", "start": 2617.64, "duration": 1.99}, {"text": "And this probably costs on\nthe order of $3 million or so.", "start": 2619.63, "duration": 5.0}, {"text": "Mass spectrometry is\nanother way that's", "start": 2624.63, "duration": 1.77}, {"text": "used, probably not for\nsequencing the whole genome,", "start": 2626.4, "duration": 2.125}, {"text": "but it's used for single\nnucleotide polymorphisms.", "start": 2628.525, "duration": 3.065}, {"text": "This costs on the order\nof $0.50 per polymorphism.", "start": 2631.59, "duration": 5.07}, {"text": "If there's three million\nof them in your genome,", "start": 2636.66, "duration": 3.99}, {"text": "that's a lot of $0.50.", "start": 2640.65, "duration": 3.093}, {"text": "[LAUGHTER]", "start": 2643.743, "duration": 3.387}, {"text": "And here's what it looks\nlike when you read it out.", "start": 2647.13, "duration": 2.55}, {"text": "It's really just\nlike electrophoresis.", "start": 2649.68, "duration": 2.01}, {"text": "You can now separate\nan addition of an A", "start": 2651.69, "duration": 5.43}, {"text": "from an addition of a G. And the\ndifference in mass between an A", "start": 2657.12, "duration": 5.415}, {"text": "and a G, this is\neven more subtle", "start": 2662.535, "duration": 1.395}, {"text": "than the difference of\nan n and an n plus one.", "start": 2663.93, "duration": 1.917}, {"text": "This is just a difference\nbetween an A and a C.", "start": 2665.847, "duration": 1.958}, {"text": "It's detectable in this.", "start": 2667.805, "duration": 1.105}, {"text": "In fact, it's detectable\nand quantitative enough", "start": 2668.91, "duration": 2.67}, {"text": "that you can pool samples.", "start": 2671.58, "duration": 1.332}, {"text": "This is a bit of a stunt,\nbut it's an important stunt", "start": 2672.912, "duration": 2.208}, {"text": "to show that this is really\na very precise method, albeit", "start": 2675.12, "duration": 4.98}, {"text": "still fairly expensive.", "start": 2680.1, "duration": 2.43}, {"text": "Now just in closing,\nI want to give you", "start": 2682.53, "duration": 2.79}, {"text": "the simplest possible\nexample of how we", "start": 2685.32, "duration": 4.26}, {"text": "can search through sequences.", "start": 2689.58, "duration": 1.44}, {"text": "Next week, we'll give you\na much more rigorous way", "start": 2691.02, "duration": 2.083}, {"text": "that you can look\nthrough the sequences", "start": 2693.103, "duration": 1.625}, {"text": "with very extensive differences\nbetween the sequences.", "start": 2694.728, "duration": 2.362}, {"text": "But here, the theme\nof today's talk", "start": 2697.09, "duration": 3.02}, {"text": "is the subtle\npolymorphism differences", "start": 2700.11, "duration": 2.73}, {"text": "that occur between you and me.", "start": 2702.84, "duration": 1.53}, {"text": "And here, one different--\nso generally, you're", "start": 2704.37, "duration": 2.79}, {"text": "looking for exact matches.", "start": 2707.16, "duration": 1.32}, {"text": "And a good way to look\nfor exact matches is--", "start": 2708.48, "duration": 2.97}, {"text": "good ways are hashing, suffix\narrays, and suffix trees", "start": 2711.45, "duration": 3.12}, {"text": "where basically\nin each of these,", "start": 2714.57, "duration": 1.93}, {"text": "you're looking for using\na word, a word either", "start": 2716.5, "duration": 4.61}, {"text": "that's built up and stored\nfrom the end, the suffix,", "start": 2721.11, "duration": 3.72}, {"text": "one letter at a\ntime, or it's a chunk", "start": 2724.83, "duration": 2.7}, {"text": "that you might have as a hash.", "start": 2727.53, "duration": 1.62}, {"text": "And you make up a lookup table.", "start": 2729.15, "duration": 2.142}, {"text": "And the size of\nthat lookup table--", "start": 2731.292, "duration": 1.458}, {"text": "it's a trade-off between\nspeed of searching", "start": 2732.75, "duration": 1.792}, {"text": "and the size of the table.", "start": 2734.542, "duration": 1.268}, {"text": "The size is going to be-- if\nthe word is n nucleotides long,", "start": 2735.81, "duration": 3.568}, {"text": "it's going to be four to the n.", "start": 2739.378, "duration": 1.292}, {"text": "It's going to be the\nstorage space you", "start": 2740.67, "duration": 1.583}, {"text": "have to put on disk\nor RAM, RAM if you", "start": 2742.253, "duration": 3.307}, {"text": "want it to be a fast search.", "start": 2745.56, "duration": 1.35}, {"text": "And so 16 is the magic number,\nsort of in the ballpark", "start": 2746.91, "duration": 3.48}, {"text": "for a human genome,\nbecause four to the 16th", "start": 2750.39, "duration": 2.1}, {"text": "is four billion sequences\nyou can represent.", "start": 2752.49, "duration": 2.91}, {"text": "But it's a huge table.", "start": 2755.4, "duration": 1.27}, {"text": "You have to have a table of\nfour billion times however", "start": 2756.67, "duration": 2.66}, {"text": "many bytes you need to\nstore the positions.", "start": 2759.33, "duration": 4.8}, {"text": "Typically, about four\nbytes of storage.", "start": 2764.13, "duration": 2.34}, {"text": "If you cut back on\nthis a little bit,", "start": 2766.47, "duration": 1.59}, {"text": "you'll end up with\ncollisions where", "start": 2768.06, "duration": 1.458}, {"text": "you'll have two things that\nhave the same hash or suffix.", "start": 2769.518, "duration": 3.792}, {"text": "If you make it--", "start": 2773.31, "duration": 1.62}, {"text": "and that's if you\nmake it smaller.", "start": 2774.93, "duration": 1.423}, {"text": "It'll take less space.", "start": 2776.353, "duration": 0.917}, {"text": "If you make it\nbigger, it'll take", "start": 2777.27, "duration": 1.41}, {"text": "a ridiculous amount of RAM.", "start": 2778.68, "duration": 2.34}, {"text": "And then here's a\nkind of whimsical--", "start": 2781.02, "duration": 4.05}, {"text": "another example of pearl\nwhere you not only want", "start": 2785.07, "duration": 2.43}, {"text": "to find all the mutations\nhere at a very high density--", "start": 2787.5, "duration": 2.61}, {"text": "a ridiculously high density,\nnot one particular base", "start": 2790.11, "duration": 2.97}, {"text": "for every few base pairs.", "start": 2793.08, "duration": 1.538}, {"text": "You not only want to find them,\nbut you want to correct them.", "start": 2794.618, "duration": 2.542}, {"text": "And here, the pearl\ndoes the substitution.", "start": 2797.16, "duration": 2.97}, {"text": "And after gene therapy,\neverybody walks out happy.", "start": 2800.13, "duration": 3.635}, {"text": "OK.", "start": 2803.765, "duration": 0.5}, {"text": "[LAUGHTER]", "start": 2804.265, "duration": 2.095}]