Sequencing technologies – the next generation · Sequencing technologies – the next generation...
Transcript of Sequencing technologies – the next generation · Sequencing technologies – the next generation...
Sequencing technologies – the next generation
1 July 2015
Ann-Kathrin Wagner
Overview
1. Introduction and repetition
2. Next generation sequencing based on fluorescence dyes
3. Comparison of methods used by different instruments
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Introduction
• US$ 1000 Human Genome Project by 2014
• Personalized genomics for medical purposes
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Repetition: DNA
Sequence: order of nucleotides within DNA
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http://www.rsc.org/images/Arsenic-DNA-chemical-structure_410_tcm18-214805.jpg
Repetition: DNA
Sequence: order of nucleotides within DNA
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http://www.ucl.ac.uk/~sjjgsca/PointMutations.gif
Repetition: DNA
Sequence: order of nucleotides within DNA
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http://www.ucl.ac.uk/~sjjgsca/PointMutations.gif
http://biobook.nerinxhs.org/bb/genetics/dna/ 1000px-Aminoacids_table.png
Addition to Sanger sequencing
è use of fluorophores instead of radiophosphates
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https://en.wikipedia.org/wiki/DNA_sequencing
NGS – fluorescence based
1. Fluorophore attachment 2. Template prep 3. Sequencing
4. Data analysis
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Fluorescence signal – base linked fluorescent nucleotides
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3´-O-azidomethyl *
3´-O-allyl * **
* * Terminating functional group
** inhibitory function Metzker, M.; Nature Reviews (2010)
Fluorescence signal - phospholinked
è no terminating group
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Metzker, M.; Nature Reviews (2010)
Template prep – clonally amplified
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Metzker, M.; Nature Reviews (2010)
Template prep – single molecule
C: hybridization to primer D: attachment to solid support E: polymerases attachment
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Metzker, M.; Nature Reviews (2010)
Sequencing – cyclic reversible termination
4-color 1-color at cyclic a time reversible termination
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Metzker, M.; Nature Reviews (2010)
Sequencing – sequencing by ligation
SoLiD:
èdecode DNA sequence
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Metzker, M.; Nature Reviews (2010)
Sequencing - SMRT
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Metzker, M.; Nature Reviews (2010)
Zero Mode Waveguides (ZMW)
è subwavelength holes in metal film
è studies at µM concentration & µs temporal resolution
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Levene, M.; Science (2003)
ZMW
è circular polarized light
eff. observation profile
eff. observation volume
è limits Veff to first 10-20 nm
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S(z) = I(z)p(z) p(z)p(z)+C
Veff =πd 2
4
S(z)dz∫( )2
S2 (z)dz∫
Levene, M.; Science (2003)
Signal detection & data analysis
532nm 643nm
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Eid, J.; Science (2009)
Signal detection & data analysis
ssDNA 2-base artificial sequence pattern (CG)
è sensitive to pH and concentration change
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Eid, J.; Science (2009)
Signal detection & data analysis
Long-read DNA sequencing for 2 bases
è suggesting two polymerization rates
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Eid, J.; Science (2009)
Signal detection & data analysis
Four-color sequencing
interpulse duration:
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Eid, J.; Science (2009)
Summary of commercially available techniques
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Metzker, M.; Nature Reviews (2010)
Summary of commercially available techniques
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Metzker, M.; Nature Reviews (2010)
Summary of commercially available techniques
ion torrent
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en.wikipedia.org/wiki/Ion_semiconductor_sequencing
Metzker, M.; Nature Reviews (2010)
Comparison
è PacBio max. read length Illumina max. throughput Ion torrent fastest sequencing
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van Dijk, E.; Elsevier (2014)
US$ 1000 HGP
è Illumina`s HiSeq X Ten system
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National Human Genome Research Institute
Summary
• 3 major improvements of NGS methods:
1. Prep in cell free environment è no cloning 2. Lots of parallel measurements 3. Direct detection è no electrophoresis
• 2 types of fluorophore attachment
• ZMW
è Reduce of cost and time & increase of throughput
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Literature
• Metzker, M.; Sequencing technologies – the next generation; Nature Reviews (2010) • Levene, M.; Zero-mode waveguides for single-molecule analysis at high concentrations; Science (2003) • Eid, J.; Real-time DNA sequencing from single polymerase molecules; Science (2009) • van Dijk, E.; Ten years of next-generation sequencing technology; Elsevier (2014) • Pushkarev, D.; Single-molecule sequencing of an individual human genome; Nature Biotechnology (2009) • Metzker, M.; Emerging technologies in DNA sequencing; Cold Spring Harbor Laboratory Press (2005) • wikipedia/DNA-sequencing • datasheet-hiseq-x-ten from Illumina • PacBio_RS_II_Brochure from Pscific Biotechologies • Seo, T.; Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides;
PNAS (2005)
• http://biobook.nerinxhs.org/bb/genetics/dna/1000px-Aminoacids_table.png • http://www.ucl.ac.uk/~sjjgsca/PointMutations.gif • http://www.rsc.org/images/Arsenic-DNA-chemical-structure_410_tcm18-214805.jpg
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Thank you for
your attention!
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