Transcript of DNA sequencing I Historical method – Sanger N “chain termination” Latest method – ion...
- Slide 1
- 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
- Slide 2
- 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
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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!