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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