3rd generation sequencing

„next-next generation single molecule sequencing“

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/

Thanks

for your attention!