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sequencing in the 21st century:a key technology
• sequencing without previous amplification
• Archon X prize initiated research: goal: 100 genomes in 10 day for less than
10000$/genome; 1 error in 100,000 bp, with sequences accurately covering 98% of the genome
prize: $10 MILLION
SBS – „sequencing by synthesis“
• tSMS - true single molecule sequencing by Helicos
• FRET – fluorescence resonance energy transfer - based approach by Visigen
• SMRT- single molecule real time by PacBio
we use DNA polymerase
• other approach: Nanopore sequencing – different methods
• TEM (combines SBS + transmission electron microscopy) by ZSGenetics
• 3rd generation sequencing =
SINGLE MOLECULE SEQUENCING
(SMS) except “HANS” by NABsys
SBS: Helicos
• poly(dA) tailed templates bind to
poly(dT)-oligonucleotides that are anchored
to surface
• Cy5 (cyanine dye – non radioactive
fluorescent) labeled dNTP added
(only 1 out of 4 each cycle!)
• polymerase activity
• washing
• imaging
• cleaving
• cyclical addition of reagents.
Helicos
• illumination with laser
• direct RNA sequencing is possible
30bp read length,70000$ / human genome, 1gb takes 12h
SBSrt :Visigen
• FRET based approach –
Fluorescence resonance energy transfer = improvement on tSMS
• no cyclic addition of reagents
• polymerase contains donor fluorophore
• each nucleotide carries one of four differently colored acceptor fluorophores
• proximity of donor and acceptor
fluorophores results in a FRET signal • fluorescently- tagged PPi cleaved off
• human genome in 1h
SBSrt: PacBio• SBS in ZMW zero mode waveguides
• immobilized polymerase
• labeled nucleotides (fluorophore-to phosphate!)
• laser-beam-mediated illumination of a small detection volume (20 zeptoliters = 10^-21 L)
SBSrt: PacBio• detection: diffusion: microseconds incorporation: milliseconds• up to 10 bases per second
• 2013:complete high-quality sequence of 1 genome in just 15 min preparation to sequence in 12h
Read length: thousands of bpCosts: cheap 100$
nanopore sequencing
• 1989 : Deamer, Branton
DNA is driven through a pore electro-
phoretically
• measurement of ionic current
• Problem:
appropriate pore geometry
and translocation speedIf problems are solved cheapest method with unlimited read length!
nanopore sequencing
• Natural vs synthetic systems
• Staphylococcus aureus toxin,
α-hemolysin
(protein pores in a lipid bilayer)
• solid state nanopores
(less 5nm in diameter and length in silicon- based chip)
OxfordNanopore Technologies
direct label-free exonuclease sequencing
• α-hemolysin-aminocyclo-
dextrin pore in a lipid bilayer
• enzyme cuts off dNMP
• dNMP reduces electric
current to 4 different levels
(A,T,G or C)
NABsys (not SMS)
• combines nanopore + SBH (hybridization) called HANS (hybridization-assisted
nanopore sequencing; no SMS!)• template : cutted in 100000bp or longer• using a library of DNA probes (6mers) and
nanopores to detect where these probes have hybridized to double stranded DNA
• genome in less than 1 hour for < 1000$ with 25x covergage
nanopore improvement
• other approaches based on nanopore:
• combination nanopores and fluorescent labels,
• reverse DNA translocation (magnetic tweezers ); 2,000-fold reduction of speed
• combination solid state and hemolysin
• transverse electric circuit – functionalized electrode
TEM: ZS Genetics
• PCR with labeled dNTP• transmission electron
microscopy• high speed digital imaging
• natural DNA is essentially invisible to EM analysis
• but labeled nucleotides are visible: they can be
Iodated (Z = 53) Brominated (Z = 35)
20 year old, 23,000 base-pairs
comparison
Visigen: genome in 1hourPacBio: thousands of bp, 100$NABsys: genome 1h, 25 x coverageZs genetics: read length more than 10000
by Gupta
References• Articles:
• Single-molecule DNA sequencing technologies for future genomics research , Pushpendra K. Gupta, Trends in Biotechnology, Volume 26, Issue 11, 602-611, 1 November 2008
• The potential and challenges of nanopore sequencing, Branton, Deamer, Nat Biotechnol. 2008 October; 26(10): 1146–1153.
• Internet:
• http://www.zsgenetics.com• http://www.helicosbio.com/• http://visigenbio.com/• http://www.pacificbiosciences.com• http://www.nabsys.com/• http://www.nanoporetech.com/• http://www.abrf.org/Other/ABRFMeetings/ABRF2005/Hardin.pdf• http://genomics.xprize.org/
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