Class 13 Two sequencing methods that aim to sequence single DNA molecules Pacific Biosciences...
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Transcript of Class 13 Two sequencing methods that aim to sequence single DNA molecules Pacific Biosciences...
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Class 13 Two sequencing methods that aim to sequencesingle DNA molecules
Pacific Biosciences “zero mode” wave guide
Bayley group nanopore method
What steps in sequencing methods we considered so farwould ability to sequence single molecules avoid?
What technical problem would be eliminated?
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Pacific Bioscience seq. strategy – single-molecule, real-time
immobilize DNA pol on glasssurface at bottom of very small wells (~100nm radius); aim for1 pol molecule/well
add DNA template + primer
use dNTPs with fluor attached to terminal P so that it is cleaved off during incorporation(how different from previous fluor-dNTPS we discussed?)collect sequence in real time (enz can go ~1-100b/s)
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How long should pulses last?Why would you expect to see only 1 dye at a time?
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What steps in previous methods would enzyme-removal of fluor during synthesis eliminate?
How fast were previous methods (in terms of bases sequenced/s) given need for chemical steps and washes between base additions?
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Challenge for detecting single fluor molecules is usuallynot sensitivity, but reducing background
“Zero mode” waveguide (ZMW)– for well diameters << l w/metallic walls, propagating waves blocked; evanescent waves of exciting and emitted light decay exponentially. Detection depth (volume) ~30nm (10-19 liters) for 100nm holes in aluminum. At mM dye conc., <1 dye/detection vol on average, and any such molecule diffuses out of detection vol in ~100ms (verify: t ~x2/2D, D= 10-12m2/s) whereas dye on dNTP being incorporated by DNA pol expected to be retained by DNA pol. for ms.
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E-beam lithography makes array of <100nm diam. holes in ~ 100nm thick aluminum filmon silica slide
For well diameter << l, I(z) ~ e-kz ; excitation of fluor also inhibited by wall proximity; effectiveilluminated height theoretically~ 30nm (~ 10x smaller than TIRF)vol ~ 10-19 liters
Science 299:683, 2003
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optical set-up to excite and read fluorescence from each well
holographic wave plate divides input laser beams into array of beamlets, 1/well? -> more light/well thanIf whole field illuminated
prism diffracts emitted lightto collect diff dye-dNTPsignals in different pixels
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http://www.sciencemag.org/content/vol0/issue2008/images/data/1162986/DC1/1162986s1.mov
93 rows (1mm spacing) x 33 columns (4mm spacing)Light from each well diffracted laterally for diff. detectors
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Why do some wells stay illuminated?
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Why do you want high (~mM) dNTP conc.?
Binding rate = kon [dNTP]
Rate (#/s) at which dNTPs bind each polymerase molecule,per M conc of dNTP; if kon = 107/Ms, what conc. of dNTP do you need for pol to synthesize 10b/s?
If you use TIRF, min. vol. of illuminated spot ~ pl2*hatt
~ (p 500nm)2 100nm = 10-19m3 = 10-16liter
How many dNTPs in this vol. at 1mM? Need <1
ZMW gets you to <1 by reducing illum. vol ~1000-fold!
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Base-labeled nucleotide
Phosphate-labeled nucleotide
Idea hinges on using dNTPswith dye labels on phosphate(diff colors for each nt)so dye will be cleaved offduring incorporation (don’tneed separate chemical step) -> real-time sequencing
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Actual dNTPs used have big dyes + 6 phosphates – They “invented” these dye-NTs and then had to engineer (mutate) DNA pol to use them efficiently
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Emission spectra - would be easiest to distinguish C from G, harder to distinguish 1 from 2, and 3 from 4
(affects “substitution” error rates)
C A T G
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Start with “simple” ss-template – 150 bases withalternating regions with multiple G’s or C’s, synthesizeusing dGTP-yellow, dCTP-blue, other bases unlabeled
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They at least can pick out the G’s and C’s separated by0-2 other bases in this regionof template, but note variabilityin peak duration
Segment from previous trace
Same data in graphical form
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Now try a circular 75b ss template Their pol enzyme has “strand displacing” activity What advantages might a circular template have?
Should you see a pattern repeating every 75 b?What disadvantages would there be to having to use circular DNA templates?
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Now try all 4 bases on 150b ss template
Note variability in pulse widths
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Error rates
Single run on 150 b template ~30% of reads incomplete
12 insertions, 8 deletions, 7 mismatches (~20%)
What causes apparent insertions? what if base sticks to pol long enough to be detected but falls off before being incorporated, then
sticks again and gets incorporated?
what could you do about this? – try to engineerenzyme that binds bases tighter (might not work)
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What causes apparent deletions?
what if base were photobleached before detection?
what if enzyme incorporates a base very quickly?
If enzyme has constant average rate of incorporation of bound base/unit time, expect Poisson distribution of “hold times”, with largest number of shortest durations
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They have problem detecting the shortest hold timesbecause of photon counting statistics and noise: dyes produce ~5000 photons/sec -> 50/10ms
Expected Poisson distribution of hold times
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What could you do about this problem?
try to slow down incorporation rate chemically(they say pH change has a small effect)
try to engineer enzyme that incorporates basesmore slowly (might not work)
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Substitution errors = misreading dyes due to incomplete spectral separation, esp. hard distinguish in short pulses
what could you do? – try to improve dyes
More immediate solution to high error rates
read each DNA template multiple timesto generate a consensus sequence
(circular templates would be useful…)
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They used sequence info from 449 reads of same150 base template in different wells. Generate aconsensus sequence based on random samples of datain which ? each position appears in >15 sequences. Repeat 100x to generate 100 consensus sequences.Error rate in consensus sequences ~2-3%.
If they had 3000 wells,why did they only use449 reads? Suggests theyare getting fewer usefulwells than they say…
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Over-sequencing makes sense if the errors are random but what if the error rate depends on sequence?
Summary
Many technical hurdles have been overcome, but errorrate remains very high
Even if they got reliable sequence in real time at rate of 3b/s in each of 3000 wells, would need > 15
days to do human genome 15-fold redundantly,which is competitors’ claimed current rate; 2 yrsago they said a 1,000,000-well device was coming…
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Nanopore sensor method – Bayley group
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a-hemolysin: heptameric membrane pore-forming protein (bacterial toxin that punches holes in red blood cells)
Spontaneously forms 7-mer and inserts into lipid membrane
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When inserted in membrane, in electrolyte solution, creates channel that allows ions to cross membrane
Can easily detect single channels in artificial membrane as they cause step-like changes in current …how many ions go thru pore/sec?
How much current do you expect from 0.7nm radius pore10nm long in 1M KCl at 100mV, if conductivity s = 14S/m?I = V/R = VG = VsA/L = .1*14*p*(.7*10-9)2/10-8 = 200pA
pA0-20
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How can you make membranes, introduce pores?
25 µm
1 cm
_+
2 nm
Cl-
K+
teflon barrierwith~50mmhole
Add lipid tosolution, raiseand lower meniscus overhole, 5nm lipidbilayer formsspontaneously (!)
Add a-hemolysin protein to 1 chamber – it inserts itself!
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b-cyclodextrin: heptameric ring of sugars
spontaneously inserts inside a-hemolysin porestabilized by coordination with 7 identical sites,
one in each a-hemolysin monomer
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b-CD insertion lowers conductivity
As dNMPs go through pore, they further decrease conductivity; can b-CD be modified so that different bases will -> different decreases in conductivity?
W
Why might you wantto reduce pore diameter?
Would you expectcharges in pore toinfluence current?
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Bayley’s group have made extensive mutationsin a-HL and tested many b-CD derivatives to try to makepores that distinguish DNA bases by extent of decr. cond.
Here, devise covalent S-S linkage between a-HL and b-CD based on single cys in a-HL and S in modified b-CDin order to have stable small diam. pore
How do they get single cys in heptameric a-HL? Mix 2 types of a-HL, 1 with 1 cys and tail of 8-charged (asp) aa’s; other w/no cys or tail; they form different hetero-7mers;select desired 7mer by electrophoresis
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b-CD without S reversibly enters a-HL; with S, it inserts stably but can be removed by reducing -S-S- with DTT
b-CD with -SH group stably associates w/cys-modified a-HL
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aHL with stably inserted b-CD senses mixture of bases
Residual pore currents come in 4 types – why?
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More data, with higher conc bases
note variable dwell times
note minimal overlapin residual pore currenthistograms – good fordistinguishing bases butrequires extensive data smoothing
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Dwell times have wide distribution, but averagesdiffer for different bases. What does this suggest?
Scatter plot of dwell time vs residual pore current
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Channel can also distinguish methyl-dCMP, a variant ofC associated with silenced gene expression: this system can detect such “epigenetic” changes more easily thanother sequencing methods
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Idea for sequencing – use exonuclease to degrade template to dNMPs and read them going thru pore in the order they are produced
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Problem #1: exonuclease doesn’t work in high salt, whichis requiredfor good base discrim-ination; theylower salt onside w/exo
Test mixed salt system on simpler templates with
No T’s No A’s shows they candegrade temp-lates with exo’aseand read basesproduced
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Technical challenges
Reducing [KCL]cis to 200mM allowed exonuclease to work, but decreased ability to distinguish A’s and T’s to ~90%, not good enough for sequencing; they might be able to select exo’s that can work in high salt (e.g. brine bacteria)
<tdwell> ~10ms, but Poisson distribution => most dwells are short; for short dwells it is harder to distinguish different bases; very short dwells may -> “deletions”
Ability to distinguish 4 bases enhanced by +charge onlinker arm; may help to trap bases electrostatically;? more chemical modifications might improvediscrimination
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No data yet that exonuclease can be held near enough to pore (e.g. via aHL-exo fusion protein) that that chewed off bases can be read sequentially
Will path of some bases (drift + diffusion) be such that they are read out-of-order, or not read at all?
Can pore formation be automated and multiplexed?
Nevertheless, extraordinary accomplishment in terms of chemical adaptation of nanopore to make real-time, label-free, single-molecule detector that distinguishes 5 bases
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Some similarities between solid state FETs and ion channels:
charge on channel walls regulates rate charged objects(ions, DNA molecules, peptides) go thru pore
if pore is small enough compared to transported object, changes in charge -> exponential changes in transport
chips – use simple geometries, Coulomb interactions,engineered structures ~ mm x 30nm laterally,a few nm vertically
biology – more complex geometries, Coulomb + chemicalinteractions (H-bonding, covalent bonds), greatercontrol of nanoscale positioning via protein & DNA engineering but less control at mm scale
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Some big ideas from course:
Biology at nano-scale is not very different fromchemistry, physics at similar scale
Biological macromolecules (DNA, protein) allownew forms of engineering and controlover nanoscale phenomena
Some tools are novel to biology – e.g. replication via pcr
Biology provides new things we want to sense (like DNA) and new tools to sense them
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At nano-level, single things become detectable
Detecting single-molecule events can provide info abt.molecular structure not obtainable in bulk measurements – e.g. when replicatemolecules cannot be kept in same state forbulk measurements (e.g. phasing problem in DNA sequencing)
Aim for detailed and quantitative understanding:how many molecules per sec, unit area, vol;what do they stick to; for how long; how issignal generated; how many photons, etc
Think critically as you learn!
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Suggestions for student presentations
Go over paper with me beforehand if at all possible!
Stick to a few main points – you have only ~20 minutes
Try to teach us something interesting you learned