DNA sequencing I
Historical method – SangerN “chain termination”
Latest method – ion torrent – seq. via pH measurement
Both rely on DNA polymerase to copy template,
i.e. “sequencing by synthesis”
Old technology –
chain terminationNobel:
clone target DNA in bac.
to get ~1011 copies
needed for 4 seq rxns:
DNA template + primer
+ pol + dNTP + ddATP
(or ddCTP etc., each in
separate tube);
ddNTP’s lack 3’OH,
incorporate normally
but can’t be extended;
run gel w/4 lanes; bands
in G lane show size of
frags. ending in G, etc.
Di-dexoy NTPs
lack 3’OH group
They are incorporated
normally, but
next base can’t be
chemically attached
because it attaches
thru 3’ O
missing OH
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
*
**
*
Each sequencing run -> ~500bp of sequence
this method used for human genome project
But needed ~108 seq rxns, 107 gels
even @ 104 gels/d, $10/rxn
-> 1000 days (3yrs) and $1B
Latest method - Ion Torrent
Part A: produce ~107 copies of individual DNA fragments
on mm-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)
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
F
R’
R’
F
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)
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
F
Each droplet acts like test
tube to isolate individ. DNA
species. Because many
copies of F are on each
bead, many product strands
( ~107) starting with F get
attached to each bead
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
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 incorporated, generating ~107 H+ ions as dATP ->dAMP+PP+H+
If T is not
the next base,
no H+ will
be produced
http://upload.wikimedia.org/wikipedia/commons/1/10/DNTP_nucleotide_incorporation_reaction.svg
A run of n bases of the same type -> ~n*107 H+ ions
Flow in dATP, record H+ signal, wash
repeat with dCTP, then dTTP, then dGTP
then repeat cycle of 4 dNTP additions …
G
T
C
A
Sequence of H+ signals (1, 2, 0, 0, 1 …) tells you sequence
A CC
A…
Electrical detection of H+ ions with ISFET
H+ ions accumulating on gate induce e- carriers below,
which allows current to flow
between S and D
H+
H+
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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
-> ~106 wells/
1cm2 chip
? rationale for position of multiple FETs
inflow
Attach top,
walls and
Inflow/outflow
ports for
fluidics
Top view of
assembled
~1cm2 chip
outflow
Reader with chip clamped in place
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. ~DpH 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
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
8
2
1
Model simulations
for pH change due
to 1 to 8 base incorporations (e.g. TTTT..)
They sample pH change
in individual wells many
times during cycle, then
fit data to these curves to infer how many bases were
Incorporated; the inference of # of bases = “raw data”
Raw data for first 100 flows of dNTPs reading a sequence
Note signal from bases presumably not incorporated (<<1)
gradually increases. Why do you think signal degrades?
Phasing
Their explanation is that polymerase slips behind or
jumps ahead on some of the ~107 identical templates
on a bead, then mixing in sequence from templates “out
of phase”; slippage could be due to failure to incorporate
a base on some templates due to loss of polymerase
(pol molecules can diffuse out of well); jumping ahead
could be due to incomplete wash out of previous base:
e.g. if seq. is C-T-C-G and not all dCTP washed out after 1st
C, during T cycle a dTTP and then dCTP could be
incorporated on some templates, and these would then
be ahead by 1 base when the next base is flowed in
Can you use this information to “clean up” signal?
It allows them to model which particular sequencedependent erroneous signals might be mixed in,
and subtract them -> “corrected base calls”
Note improved uniformity and closeness to integer values
But they don’t provide enough info to evaluate procedure
“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
Even after data processing, the maximum # of bases they
can read accurately from each bead is currently ~100.
98%
100%
Histogram of
read lengths
with indicated
accuracy
They stop reads
when (not-fullydisclosed) error
checking thresholds
are exceeded
Other accuracy estimates from sequencing bacterial
DNAs which have been sequenced by other methods
100%
99%
99%
97%
Homopolymer length
Position in read
1
3
5
60
120
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?
What is “fold-coverage”?
“coverage”?
They also used this method to sequence the genome
of Gordon Moore (of Moore’s Law!)
To estimate accuracy, they compared SNPs identified
using ion torrent vs another method (SOLiD)
The good news:
they disagreed
<0.1% of the time
when both called
het. or hom. SNPS
The bad news:
they disagreed or
missed >1M out
of ~3M SNPS
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 106wells/chip x 100 bases/well
= 1011 bases = 30*(3x109) = 30x “coverage”
This is first report with ion torrent, so expect technical
improvements and cost reductions …
They claim 109 wells/chip are feasible,
so possibly 1 chip/genome… but how much can
the error rate be reduced?
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!