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DNA sequencing I Historical method Sanger N chain termination Latest method ion torrent seq. via pH measurement Both rely on DNA polymerase to copy template, i.e. sequencing by synthesis

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Old technology chain termination Nobel : clone target DNA in bac. to get 10 11 copies needed for 4 seq rxns: DNA template + primer + pol + dNTP + ddATP (or ddCTP etc., each in separate tube); ddNTPs lack 3OH, incorporate normally but cant be extended; run gel w/4 lanes; bands in G lane show size of frags. ending in G, etc.

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Di-dexoy NTPs lack 3OH group They are incorporated normally, but next base cant be chemically attached because it attaches thru 3 O missing OH

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More elegant later method: label each ddNTP with a diff. colored fluor run electrophoresis products in single lane camera records color of products as they run off the bottom of the gel * * * * *

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Each sequencing run -> 500bp of sequence this method used for human genome project But needed 10 8 seq rxns, 10 7 gels even @ 10 4 gels/d, $10/rxn -> 1000 days (3yrs) and $1B

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Latest method - Ion Torrent Part A: produce 10 7 copies of individual DNA fragments on m-sized beads because sequencing method requires multiple identical target molecules/bead Part B: read sequence by primer extension synthesis, 1 base at a time, detecting pH change when dNTPs are incorporated in individual wells containing single beads, using array of ion-sensitive field effect transistors (ISFETs)

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Part A - method to put many copies of single short piece of DNA on micron-size bead; diff. DNAs on diff. beads Shear target DNA; select pieces 200 bp in length (how?) Ligate forked adapter oligos to ends of sheared DNA Note this allows all pieces to be amplified with oligos F and R (the reverse complement of R = F) (without fork, F and F would be at 5 and 3 ends and their annealing on single templates would impede pcr) F R F /

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Make water-in-oil emulsion containing: 1) pcr reagents to amplify DNA using primers F and R 2) hydrophilic micron-size beads with lots of oligos F attached via their 5 ends 3) bead and DNA concentration adjusted such that 1 DNA fragment and 1 bead/water droplet Each droplet acts like test tube to isolate individ. DNA species. Because many copies of F are on each bead, many product strands ( 10 7 ) starting with F get attached to each bead F

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Break emulsion with soap, spin down beads, melt off non-covalently attached strand, spin down beads - most now have single-stranded DNA starting with F and ending with R Enrich for beads that have such templates by capturing them on paramagnetic beads with oligo R on them, collecting with magnet, and then melting them off Centrifuge enriched beads into wells just big enough to hold a single bead

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http://upload.wikimedia.org/wikipedia/commons/1/10/DNTP_nucleotide_incorporation_reaction.svg Part B: to get sequence, add primer R, DNA pol and a single dNTP, e.g. dATP; if T is next base on template, A will be in- corporated, generating 10 7 H + ions as dATP ->dAMP+PP+H + If T is not the next base, no H + will be produced

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A run of n bases of the same type -> n*10 7 H + ions

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Flow in dATP, record H + signal, wash repeat with dCTP, then dTTP, then dGTP then repeat cycle of 4 dNTP additions Sequence of H + signals (1, 2, 0, 0, 1 ) tells you sequence A CC A A C T G

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Electrical detection of H + ions with ISFET http://www.google.com/imgres?q=ion+sensitive+field+effect+transistor&um=1&hl=en&biw=1410 &bih=773&tbm=isch&tbnid=w8xp90Qj4QYOHM:&imgrefurl=http://www.wtec.org/loyola/mcc/me ms_eu/Pages/Chapter- 5.html&docid=ja485HSUF4DTXM&imgurl=http://www.wtec.org/loyola/mcc/mems_eu/Media/5_4. jpe&w=504&h=196&ei=CCW3TvfKNuTi2QWIzZTQDQ&zoom=1&iact=hc&vpx=280&vpy=281&dur=6 335&hovh=140&hovw=360&tx=150&ty=81&sig=114362777222024808894&page=1&tbnh=70&tb nw=181&start=0&ndsp=22&ved=1t:429,r:1,s:0 H+H+ H+H+ - H + ions accumulating on gate induce e - carriers below, which allows current to flow between S and D

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SEM image of cross section of chip with wells on top and sets of S and D electrodes below small size of wells -> 10 6 wells/ 1cm 2 chip ? rationale for position of multiple FETs

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Attach top, walls and Inflow/outflow ports for fluidics Top view of assembled 1cm 2 chip inflow outflow

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Reader with chip clamped in place

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Position of inflow and outflow -> only central ovoid of sensors exposed to sample Histogram pH readings from wells exposed to same solution shows sensor uniformity with s.d. pH from single base incorporation ( 0.02) Unclear if this is very important since you can check each sensor w/ known bases at start of run

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Blue = time course of pH change in 1 well due to single base incorporation Red = not fully disclosed model of pH change expected as a result of dNTP flowing by, diffusing into well, DNA pol incorporating base, H + produced and diffusing out

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Model simulations for pH change due to 1 to 8 base incor- porations (e.g. TTTT..) They sample pH change in individual wells many times during cycle, then 1 2 8 fit data to these curves to infer how many bases were Incorporated; the inference of # of bases = raw data

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Raw data for first 100 flows of dNTPs reading a sequence Note signal from bases presumably not incorporated (

It allows them to model which particular sequence- dependent erroneous signals might be mixed in, and subtract them -> corrected base calls Note improved uniformity and closeness to integer values But they dont provide enough info to evaluate procedure

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Phasing problem is inherent to all methods that rely on coordinating state of many molecules that go through cyclic changes What tends to keep DNA synthesis in phase on different templates in their system? If a sensor could sense the state of single-molecules, would phasing-type problems disappear? Keep this in mind wrt future methods

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Histogram of read lengths with indicated accuracy 100% 98% Even after data processing, the maximum # of bases they can read accurately from each bead is currently 100. They stop reads when (not-fully- disclosed) error checking thresholds are exceeded

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Other accuracy estimates from sequencing bacterial DNAs which have been sequenced by other methods Position in read 99% 60 120 Homopolymer length 1 3 5 97% 99% 100% E Coli: 4.7M bases: consensus seq. with 11-fold coverage has 1228 errors (.03%), 1171 (95%) of which are deletions How many would this predict in a human genome?

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What is fold-coverage? coverage?

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They also used this method to sequence the genome of Gordon Moore (of Moores Law!) To estimate accuracy, they compared SNPs identified using ion torrent vs another method (SOLiD) The good news: they disagreed 1M out of 3M SNPS

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Cost estimates: They sell the Ion Torrent reader without chips (fluidics and computer??) for $50,000 They used 1000 chips for Gordon Moore: @ $100 -> $100,000/human genome sequence Note 1000 chips x 10 6 wells/chip x 100 bases/well = 10 11 bases = 30*(3x10 9 ) = 30x coverage This is first report with ion torrent, so expect technical improvements and cost reductions They claim 10 9 wells/chip are feasible, so possibly 1 chip/genome but how much can the error rate be reduced?

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Main points Appreciate cleverness of emulsion pcr to put many copies of individual sequences on beads. If they are limited by sensitivity of detection of H +, they may not be able to use much smaller beads (# H + ions bead area) Major new advance is the method of electrical detection of base incorporation, which allows them to get away from specialized biochemistry and expensive optical detection methods used in competing methods next week!