Erwin R Schmidt Institut fuumlr Molekulargenetik

Vorlesung 10 01 07 2014

Thema Gentechnologie

Pyrosequenzierung

The Pyrosequencingtrade technology is a relatively new DNA sequencing method originally developed here at KTH at the Department of Biotechnology The technology has been commercialized and is today marketed by Biotage AB The technique utilizes the cooperativity between four different enzymes and the phenomenon of bioluminescence to monitor the incorporation of nucleotides into the DNA A short description of the steps in the Pyrosequencing process is given below Initial step The reaction mixture consists of the four enzymes (DNA polymerase ATP sulfurylase luciferase and apyrase) different substrates needed for the reactions and the single stranded DNA to be sequenced Step 1 - Polymerase One of the four nucleotides dNTP (dATP dCTP dGTP dTTP) is added to the reaction mixture If the added nucleotide is complementary to the base in the DNA strand it is incorporated and inorganic pyrophosphate (PPi) is released Step 2 - ATP sulfurylase The PPi is converted into ATP by the enzyme ATP sulfurylase Step 3 - Luciferase The luciferase catalyzes a reaction where ATP is used to generate light The amount of light is proportional to the amount of ATP and hence also proportional to the amount of incorporated nucleotides via the PPi The light is then detected by a CCD camera Step 4 - Apyrase Remaining dNTP and ATP are degraded by the apyrase before the next nucleotide in the iterative cycle is added to the reaction mixture My research is devoted to developing a good mathematical model of the reaction system This will help us to understand the mechanisms governing the system in detail Once a satisfactory model has been developed it can be used to optimize the method with respect to substrate and enzyme concentrations as well as the choice of enzymes (kinetic parameters) As the demand for even better DNA sequencing techniques is steadily increasing as new applications arise there is a lot to gain by optimization

Pyrosequenzierung

The Pyrosequencingtrade technology is a relatively new DNA sequencing method originally developed here at KTH at the Department of Biotechnology The technology has been commercialized and is today marketed by Biotage AB The technique utilizes the cooperativity between four different enzymes and the phenomenon of bioluminescence to monitor the incorporation of nucleotides into the DNA A short description of the steps in the Pyrosequencing process is given below Initial step The reaction mixture consists of the four enzymes (DNA polymerase ATP sulfurylase luciferase and apyrase) different substrates needed for the reactions and the single stranded DNA to be sequenced Step 1 - Polymerase One of the four nucleotides dNTP (dATP dCTP dGTP dTTP) is added to the reaction mixture If the added nucleotide is complementary to the base in the DNA strand it is incorporated and inorganic pyrophosphate (PPi) is released Step 2 - ATP sulfurylase The PPi is converted into ATP by the enzyme ATP sulfurylase Step 3 - Luciferase The luciferase catalyzes a reaction where ATP is used to generate light The amount of light is proportional to the amount of ATP and hence also proportional to the amount of incorporated nucleotides via the PPi The light is then detected by a CCD camera Step 4 - Apyrase Remaining dNTP and ATP are degraded by the apyrase before the next nucleotide in the iterative cycle is added to the reaction mixture My research is devoted to developing a good mathematical model of the reaction system This will help us to understand the mechanisms governing the system in detail Once a satisfactory model has been developed it can be used to optimize the method with respect to substrate and enzyme concentrations as well as the choice of enzymes (kinetic parameters) As the demand for even better DNA sequencing techniques is steadily increasing as new applications arise there is a lot to gain by optimization

The Pyrosequencingtrade technology is a relatively new DNA sequencing method originally developed here at KTH at the Department of Biotechnology The technology has been commercialized and is today marketed by Biotage AB The technique utilizes the cooperativity between four different enzymes and the phenomenon of bioluminescence to monitor the incorporation of nucleotides into the DNA A short description of the steps in the Pyrosequencing process is given below Initial step The reaction mixture consists of the four enzymes (DNA polymerase ATP sulfurylase luciferase and apyrase) different substrates needed for the reactions and the single stranded DNA to be sequenced Step 1 - Polymerase One of the four nucleotides dNTP (dATP dCTP dGTP dTTP) is added to the reaction mixture If the added nucleotide is complementary to the base in the DNA strand it is incorporated and inorganic pyrophosphate (PPi) is released Step 2 - ATP sulfurylase The PPi is converted into ATP by the enzyme ATP sulfurylase Step 3 - Luciferase The luciferase catalyzes a reaction where ATP is used to generate light The amount of light is proportional to the amount of ATP and hence also proportional to the amount of incorporated nucleotides via the PPi The light is then detected by a CCD camera Step 4 - Apyrase Remaining dNTP and ATP are degraded by the apyrase before the next nucleotide in the iterative cycle is added to the reaction mixture My research is devoted to developing a good mathematical model of the reaction system This will help us to understand the mechanisms governing the system in detail Once a satisfactory model has been developed it can be used to optimize the method with respect to substrate and enzyme concentrations as well as the choice of enzymes (kinetic parameters) As the demand for even better DNA sequencing techniques is steadily increasing as new applications arise there is a lot to gain by optimization

bdquoNext Generationldquo Sequencing (NGS)

Erwin R Schmidt Institut fuumlr Molekulargenetik

Johannes Gutenberg Universitaumlt Mainz

DNA-Sequencing

A brief historical overview The different platforms of NGS Benchtop versus High Output Cost and Reliabilty Future technologies Summary

NGS Short History of (Nucleotide) Sequencing How many generations do we have

First Generation Sequencing

bull Nearest neighbor technology

bull Combined with sequence or base specific nuclease digestion

The first nucleotide sequence of a complete biomolecule was the Alanine tRNA of Yeast

by Robert W Holley et al in 1964 Nobel Prize in Physiology and Medicine 1968

(for 77 nt)

Generation 2 The real breakthrough

bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis

bull Nobel Prize Chemistry 1980

bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation

bull Nobel Prize Chemistry 1980

Development of the Sanger Sequencing

1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448

1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467

DNA-Sequencing

A brief historical overview The different platforms of NGS Benchtop versus High Output Cost and Reliabilty Future technologies Summary

NGS Short History of (Nucleotide) Sequencing How many generations do we have

First Generation Sequencing

bull Nearest neighbor technology

bull Combined with sequence or base specific nuclease digestion

The first nucleotide sequence of a complete biomolecule was the Alanine tRNA of Yeast

by Robert W Holley et al in 1964 Nobel Prize in Physiology and Medicine 1968

(for 77 nt)

Generation 2 The real breakthrough

bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis

bull Nobel Prize Chemistry 1980

bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation

bull Nobel Prize Chemistry 1980

Development of the Sanger Sequencing

1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448

1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467

NGS Short History of (Nucleotide) Sequencing How many generations do we have

First Generation Sequencing

bull Nearest neighbor technology

bull Combined with sequence or base specific nuclease digestion

The first nucleotide sequence of a complete biomolecule was the Alanine tRNA of Yeast

by Robert W Holley et al in 1964 Nobel Prize in Physiology and Medicine 1968

(for 77 nt)

Generation 2 The real breakthrough

bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis

bull Nobel Prize Chemistry 1980

bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation

bull Nobel Prize Chemistry 1980

Development of the Sanger Sequencing

1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448

1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467

First Generation Sequencing

bull Nearest neighbor technology

bull Combined with sequence or base specific nuclease digestion

The first nucleotide sequence of a complete biomolecule was the Alanine tRNA of Yeast

by Robert W Holley et al in 1964 Nobel Prize in Physiology and Medicine 1968

(for 77 nt)

Generation 2 The real breakthrough

bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis

bull Nobel Prize Chemistry 1980

bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation

bull Nobel Prize Chemistry 1980

Development of the Sanger Sequencing

1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448

1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467

The first nucleotide sequence of a complete biomolecule was the Alanine tRNA of Yeast

by Robert W Holley et al in 1964 Nobel Prize in Physiology and Medicine 1968

(for 77 nt)

Generation 2 The real breakthrough

bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis

bull Nobel Prize Chemistry 1980

bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation

bull Nobel Prize Chemistry 1980

Development of the Sanger Sequencing

1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448

1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467

Generation 2 The real breakthrough

bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis

bull Nobel Prize Chemistry 1980

bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation

bull Nobel Prize Chemistry 1980

Development of the Sanger Sequencing

1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448

1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467

Development of the Sanger Sequencing

1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448

1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467

The Maxam and Gilbert Method based on base specific chemical degradation of end-

labelled DNA-restriction fragments

In the same year (1977) but 10 months before Sanger published the chain terminator Method Maxam and Gilbert published their DNA sequencing method based on chemical degradation of end-labelled DNA restriction fragments A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564

Which sequencing method was superior

Maxam and Gilbert sequencing

bull A very robust method

bull Not sensitive to secondary structures

bull Shows base modification

But

bull Requires work with with

bull strong carcinogens and miliCuries of

radioisotopes

bull Is not automatable

bull Very laborious and requires long

exposition times

A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564

Which sequencing method was superior

Sanger sequencing

bull Reading the sequence easier

bull No carcinogenic chemicals involved

bull Exposure times were only a few hours

bull the sequencing reactions could be done by

acute the technician

bull but

the natural DNA-Polymerases are sensitive to

secondary structures and stretches of

homopolymeric nucleotides This changed

only when the bdquosequenasesldquo were invented

Sanger-sequencing has won the race

Maxam and Gilbert

Number of citations

- 7690 times

Sanger Nicklen and Coulson

Number of citations

ndash 62757 times

Source Google Scholar

Generation 3 on line sequencing

- number of different techniques

- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer

- automated bdquobase callingldquo

bdquoClassicalldquo on line sequencing is still in use

bull The demand is still increasing

bull Results are robust low error rate lt 11000-110000 bp

bull Up to 1500 nt readable in a row

bull Cost per sample ~ 3-5 euro (014 Cent Bpds)

bull Comprehensive service available commercially

Generation 4

Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod

of the yearldquo

bull introduces a new dimension in sequence determination

bull Several platforms exist providing different possibilities

The advent of NGS is reflected by the number of genome projects and data base entries

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

In particular bacterial genome projects boost since 2008

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24

Source httpgenomesonlineorgcgi-binGOLDindexcgi

Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi

Incomplete Genome Projects 27988 Archaeal 457

Bacterial 19494 Eukaryal 6413

Last updated 2014-01-24

Source GOLD = Genomes Online Database at the DOE Joint Genome Institute

NGS has revolutionized genome science

bull Reduction of costs

bull Reduction of time

bull Reduction of labour

bull Increase in bioinformatical challenge

The different platforms

The genome scale

bull 454Roche GenomeSequencer FLX

bull ABI SOLiD Sequencing System

bull IlluminaSolexa Hi-Seq20002500

bull Ion Torrent Proton

bull Pacific Bioscience

bull (Helicos)

The bench top scale

bull 454 GS JuniorRoche

bull Illumina MiSeq

bull Illumina NextSeq500

bull Ion Torrent PGMLife Technologies

454Roche GS FLX

The basis is Emulsion PCR and Pyrosequencing

sst-DNA single-stranded template DNA

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Which sequencing method was superior

Maxam and Gilbert sequencing

bull A very robust method

bull Not sensitive to secondary structures

bull Shows base modification

But

bull Requires work with with

bull strong carcinogens and miliCuries of

radioisotopes

bull Is not automatable

bull Very laborious and requires long

exposition times

A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564

Which sequencing method was superior

Sanger sequencing

bull Reading the sequence easier

bull No carcinogenic chemicals involved

bull Exposure times were only a few hours

bull the sequencing reactions could be done by

acute the technician

bull but

the natural DNA-Polymerases are sensitive to

secondary structures and stretches of

homopolymeric nucleotides This changed

only when the bdquosequenasesldquo were invented

Sanger-sequencing has won the race

Maxam and Gilbert

Number of citations

- 7690 times

Sanger Nicklen and Coulson

Number of citations

ndash 62757 times

Source Google Scholar

Generation 3 on line sequencing

- number of different techniques

- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer

- automated bdquobase callingldquo

bdquoClassicalldquo on line sequencing is still in use

bull The demand is still increasing

bull Results are robust low error rate lt 11000-110000 bp

bull Up to 1500 nt readable in a row

bull Cost per sample ~ 3-5 euro (014 Cent Bpds)

bull Comprehensive service available commercially

Generation 4

Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod

of the yearldquo

bull introduces a new dimension in sequence determination

bull Several platforms exist providing different possibilities

The advent of NGS is reflected by the number of genome projects and data base entries

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

In particular bacterial genome projects boost since 2008

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24

Source httpgenomesonlineorgcgi-binGOLDindexcgi

Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi

Incomplete Genome Projects 27988 Archaeal 457

Bacterial 19494 Eukaryal 6413

Last updated 2014-01-24

Source GOLD = Genomes Online Database at the DOE Joint Genome Institute

NGS has revolutionized genome science

bull Reduction of costs

bull Reduction of time

bull Reduction of labour

bull Increase in bioinformatical challenge

The different platforms

The genome scale

bull 454Roche GenomeSequencer FLX

bull ABI SOLiD Sequencing System

bull IlluminaSolexa Hi-Seq20002500

bull Ion Torrent Proton

bull Pacific Bioscience

bull (Helicos)

The bench top scale

bull 454 GS JuniorRoche

bull Illumina MiSeq

bull Illumina NextSeq500

bull Ion Torrent PGMLife Technologies

454Roche GS FLX

The basis is Emulsion PCR and Pyrosequencing

sst-DNA single-stranded template DNA

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Which sequencing method was superior

Sanger sequencing

bull Reading the sequence easier

bull No carcinogenic chemicals involved

bull Exposure times were only a few hours

bull the sequencing reactions could be done by

acute the technician

bull but

the natural DNA-Polymerases are sensitive to

secondary structures and stretches of

homopolymeric nucleotides This changed

only when the bdquosequenasesldquo were invented

Sanger-sequencing has won the race

Maxam and Gilbert

Number of citations

- 7690 times

Sanger Nicklen and Coulson

Number of citations

ndash 62757 times

Source Google Scholar

Generation 3 on line sequencing

- number of different techniques

- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer

- automated bdquobase callingldquo

bdquoClassicalldquo on line sequencing is still in use

bull The demand is still increasing

bull Results are robust low error rate lt 11000-110000 bp

bull Up to 1500 nt readable in a row

bull Cost per sample ~ 3-5 euro (014 Cent Bpds)

bull Comprehensive service available commercially

Generation 4

Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod

of the yearldquo

bull introduces a new dimension in sequence determination

bull Several platforms exist providing different possibilities

The advent of NGS is reflected by the number of genome projects and data base entries

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

In particular bacterial genome projects boost since 2008

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24

Source httpgenomesonlineorgcgi-binGOLDindexcgi

Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi

Incomplete Genome Projects 27988 Archaeal 457

Bacterial 19494 Eukaryal 6413

Last updated 2014-01-24

Source GOLD = Genomes Online Database at the DOE Joint Genome Institute

NGS has revolutionized genome science

bull Reduction of costs

bull Reduction of time

bull Reduction of labour

bull Increase in bioinformatical challenge

The different platforms

The genome scale

bull 454Roche GenomeSequencer FLX

bull ABI SOLiD Sequencing System

bull IlluminaSolexa Hi-Seq20002500

bull Ion Torrent Proton

bull Pacific Bioscience

bull (Helicos)

The bench top scale

bull 454 GS JuniorRoche

bull Illumina MiSeq

bull Illumina NextSeq500

bull Ion Torrent PGMLife Technologies

454Roche GS FLX

The basis is Emulsion PCR and Pyrosequencing

sst-DNA single-stranded template DNA

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Sanger-sequencing has won the race

Maxam and Gilbert

Number of citations

- 7690 times

Sanger Nicklen and Coulson

Number of citations

ndash 62757 times

Source Google Scholar

Generation 3 on line sequencing

- number of different techniques

- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer

- automated bdquobase callingldquo

bdquoClassicalldquo on line sequencing is still in use

bull The demand is still increasing

bull Results are robust low error rate lt 11000-110000 bp

bull Up to 1500 nt readable in a row

bull Cost per sample ~ 3-5 euro (014 Cent Bpds)

bull Comprehensive service available commercially

Generation 4

Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod

of the yearldquo

bull introduces a new dimension in sequence determination

bull Several platforms exist providing different possibilities

The advent of NGS is reflected by the number of genome projects and data base entries

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

In particular bacterial genome projects boost since 2008

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24

Source httpgenomesonlineorgcgi-binGOLDindexcgi

Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi

Incomplete Genome Projects 27988 Archaeal 457

Bacterial 19494 Eukaryal 6413

Last updated 2014-01-24

Source GOLD = Genomes Online Database at the DOE Joint Genome Institute

NGS has revolutionized genome science

bull Reduction of costs

bull Reduction of time

bull Reduction of labour

bull Increase in bioinformatical challenge

The different platforms

The genome scale

bull 454Roche GenomeSequencer FLX

bull ABI SOLiD Sequencing System

bull IlluminaSolexa Hi-Seq20002500

bull Ion Torrent Proton

bull Pacific Bioscience

bull (Helicos)

The bench top scale

bull 454 GS JuniorRoche

bull Illumina MiSeq

bull Illumina NextSeq500

bull Ion Torrent PGMLife Technologies

454Roche GS FLX

The basis is Emulsion PCR and Pyrosequencing

sst-DNA single-stranded template DNA

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Generation 3 on line sequencing

- number of different techniques

- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer

- automated bdquobase callingldquo

bdquoClassicalldquo on line sequencing is still in use

bull The demand is still increasing

bull Results are robust low error rate lt 11000-110000 bp

bull Up to 1500 nt readable in a row

bull Cost per sample ~ 3-5 euro (014 Cent Bpds)

bull Comprehensive service available commercially

Generation 4

Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod

of the yearldquo

bull introduces a new dimension in sequence determination

bull Several platforms exist providing different possibilities

The advent of NGS is reflected by the number of genome projects and data base entries

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

In particular bacterial genome projects boost since 2008

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24

Source httpgenomesonlineorgcgi-binGOLDindexcgi

Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi

Incomplete Genome Projects 27988 Archaeal 457

Bacterial 19494 Eukaryal 6413

Last updated 2014-01-24

Source GOLD = Genomes Online Database at the DOE Joint Genome Institute

NGS has revolutionized genome science

bull Reduction of costs

bull Reduction of time

bull Reduction of labour

bull Increase in bioinformatical challenge

The different platforms

The genome scale

bull 454Roche GenomeSequencer FLX

bull ABI SOLiD Sequencing System

bull IlluminaSolexa Hi-Seq20002500

bull Ion Torrent Proton

bull Pacific Bioscience

bull (Helicos)

The bench top scale

bull 454 GS JuniorRoche

bull Illumina MiSeq

bull Illumina NextSeq500

bull Ion Torrent PGMLife Technologies

454Roche GS FLX

The basis is Emulsion PCR and Pyrosequencing

sst-DNA single-stranded template DNA

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

bdquoClassicalldquo on line sequencing is still in use

bull The demand is still increasing

bull Results are robust low error rate lt 11000-110000 bp

bull Up to 1500 nt readable in a row

bull Cost per sample ~ 3-5 euro (014 Cent Bpds)

bull Comprehensive service available commercially

Generation 4

Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod

of the yearldquo

bull introduces a new dimension in sequence determination

bull Several platforms exist providing different possibilities

The advent of NGS is reflected by the number of genome projects and data base entries

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

In particular bacterial genome projects boost since 2008

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24

Source httpgenomesonlineorgcgi-binGOLDindexcgi

Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi

Incomplete Genome Projects 27988 Archaeal 457

Bacterial 19494 Eukaryal 6413

Last updated 2014-01-24

Source GOLD = Genomes Online Database at the DOE Joint Genome Institute

NGS has revolutionized genome science

bull Reduction of costs

bull Reduction of time

bull Reduction of labour

bull Increase in bioinformatical challenge

The different platforms

The genome scale

bull 454Roche GenomeSequencer FLX

bull ABI SOLiD Sequencing System

bull IlluminaSolexa Hi-Seq20002500

bull Ion Torrent Proton

bull Pacific Bioscience

bull (Helicos)

The bench top scale

bull 454 GS JuniorRoche

bull Illumina MiSeq

bull Illumina NextSeq500

bull Ion Torrent PGMLife Technologies

454Roche GS FLX

The basis is Emulsion PCR and Pyrosequencing

sst-DNA single-stranded template DNA

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Generation 4

Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod

of the yearldquo

bull introduces a new dimension in sequence determination

bull Several platforms exist providing different possibilities

The advent of NGS is reflected by the number of genome projects and data base entries

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

In particular bacterial genome projects boost since 2008

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24

Source httpgenomesonlineorgcgi-binGOLDindexcgi

Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi

Incomplete Genome Projects 27988 Archaeal 457

Bacterial 19494 Eukaryal 6413

Last updated 2014-01-24

Source GOLD = Genomes Online Database at the DOE Joint Genome Institute

NGS has revolutionized genome science

bull Reduction of costs

bull Reduction of time

bull Reduction of labour

bull Increase in bioinformatical challenge

The different platforms

The genome scale

bull 454Roche GenomeSequencer FLX

bull ABI SOLiD Sequencing System

bull IlluminaSolexa Hi-Seq20002500

bull Ion Torrent Proton

bull Pacific Bioscience

bull (Helicos)

The bench top scale

bull 454 GS JuniorRoche

bull Illumina MiSeq

bull Illumina NextSeq500

bull Ion Torrent PGMLife Technologies

454Roche GS FLX

The basis is Emulsion PCR and Pyrosequencing

sst-DNA single-stranded template DNA

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

The advent of NGS is reflected by the number of genome projects and data base entries

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

In particular bacterial genome projects boost since 2008

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24

Source httpgenomesonlineorgcgi-binGOLDindexcgi

Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi

Incomplete Genome Projects 27988 Archaeal 457

Bacterial 19494 Eukaryal 6413

Last updated 2014-01-24

Source GOLD = Genomes Online Database at the DOE Joint Genome Institute

NGS has revolutionized genome science

bull Reduction of costs

bull Reduction of time

bull Reduction of labour

bull Increase in bioinformatical challenge

The different platforms

The genome scale

bull 454Roche GenomeSequencer FLX

bull ABI SOLiD Sequencing System

bull IlluminaSolexa Hi-Seq20002500

bull Ion Torrent Proton

bull Pacific Bioscience

bull (Helicos)

The bench top scale

bull 454 GS JuniorRoche

bull Illumina MiSeq

bull Illumina NextSeq500

bull Ion Torrent PGMLife Technologies

454Roche GS FLX

The basis is Emulsion PCR and Pyrosequencing

sst-DNA single-stranded template DNA

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

In particular bacterial genome projects boost since 2008

httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics

Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24

Source httpgenomesonlineorgcgi-binGOLDindexcgi

Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi

Incomplete Genome Projects 27988 Archaeal 457

Bacterial 19494 Eukaryal 6413

Last updated 2014-01-24

Source GOLD = Genomes Online Database at the DOE Joint Genome Institute

NGS has revolutionized genome science

bull Reduction of costs

bull Reduction of time

bull Reduction of labour

bull Increase in bioinformatical challenge

The different platforms

The genome scale

bull 454Roche GenomeSequencer FLX

bull ABI SOLiD Sequencing System

bull IlluminaSolexa Hi-Seq20002500

bull Ion Torrent Proton

bull Pacific Bioscience

bull (Helicos)

The bench top scale

bull 454 GS JuniorRoche

bull Illumina MiSeq

bull Illumina NextSeq500

bull Ion Torrent PGMLife Technologies

454Roche GS FLX

The basis is Emulsion PCR and Pyrosequencing

sst-DNA single-stranded template DNA

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24

Source httpgenomesonlineorgcgi-binGOLDindexcgi

Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi

Incomplete Genome Projects 27988 Archaeal 457

Bacterial 19494 Eukaryal 6413

Last updated 2014-01-24

Source GOLD = Genomes Online Database at the DOE Joint Genome Institute

NGS has revolutionized genome science

bull Reduction of costs

bull Reduction of time

bull Reduction of labour

bull Increase in bioinformatical challenge

The different platforms

The genome scale

bull 454Roche GenomeSequencer FLX

bull ABI SOLiD Sequencing System

bull IlluminaSolexa Hi-Seq20002500

bull Ion Torrent Proton

bull Pacific Bioscience

bull (Helicos)

The bench top scale

bull 454 GS JuniorRoche

bull Illumina MiSeq

bull Illumina NextSeq500

bull Ion Torrent PGMLife Technologies

454Roche GS FLX

The basis is Emulsion PCR and Pyrosequencing

sst-DNA single-stranded template DNA

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi

Incomplete Genome Projects 27988 Archaeal 457

Bacterial 19494 Eukaryal 6413

Last updated 2014-01-24

Source GOLD = Genomes Online Database at the DOE Joint Genome Institute

NGS has revolutionized genome science

bull Reduction of costs

bull Reduction of time

bull Reduction of labour

bull Increase in bioinformatical challenge

The different platforms

The genome scale

bull 454Roche GenomeSequencer FLX

bull ABI SOLiD Sequencing System

bull IlluminaSolexa Hi-Seq20002500

bull Ion Torrent Proton

bull Pacific Bioscience

bull (Helicos)

The bench top scale

bull 454 GS JuniorRoche

bull Illumina MiSeq

bull Illumina NextSeq500

bull Ion Torrent PGMLife Technologies

454Roche GS FLX

The basis is Emulsion PCR and Pyrosequencing

sst-DNA single-stranded template DNA

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

NGS has revolutionized genome science

bull Reduction of costs

bull Reduction of time

bull Reduction of labour

bull Increase in bioinformatical challenge

The different platforms

The genome scale

bull 454Roche GenomeSequencer FLX

bull ABI SOLiD Sequencing System

bull IlluminaSolexa Hi-Seq20002500

bull Ion Torrent Proton

bull Pacific Bioscience

bull (Helicos)

The bench top scale

bull 454 GS JuniorRoche

bull Illumina MiSeq

bull Illumina NextSeq500

bull Ion Torrent PGMLife Technologies

454Roche GS FLX

The basis is Emulsion PCR and Pyrosequencing

sst-DNA single-stranded template DNA

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

The different platforms

The genome scale

bull 454Roche GenomeSequencer FLX

bull ABI SOLiD Sequencing System

bull IlluminaSolexa Hi-Seq20002500

bull Ion Torrent Proton

bull Pacific Bioscience

bull (Helicos)

The bench top scale

bull 454 GS JuniorRoche

bull Illumina MiSeq

bull Illumina NextSeq500

bull Ion Torrent PGMLife Technologies

454Roche GS FLX

The basis is Emulsion PCR and Pyrosequencing

sst-DNA single-stranded template DNA

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

454Roche GS FLX

The basis is Emulsion PCR and Pyrosequencing

sst-DNA single-stranded template DNA

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

The number of sequences is depending on the number of wells in plate

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

454Roche GS FLX

Pyrosequencing

AcircPS = Adenosinephosphosulfate

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

GS FLX+ System

Sequencing Kit New GS FLX Titanium XL+

GS FLX Titanium XLR70

Read Length Up to 1000 bp Up to 600 bp

Mode Read Length 700 bp 450 bp

Throughput Profile

- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp

- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp

Typical Throughput 700 Mb 450 Mb

Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon

Consensus Accuracy 99997 99995

Run Time 23 hours 10 hours

Sample Input gDNA or cDNA

gDNA cDNA or amplicons (PCR products)

Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions

Data from Roche http454comproductsgs-flx-system

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

454Roche GS FLX Titanium

bull Advantages

bull Long read length gt400 nt up to 1000

bull Low error rate but sensitive to homooligomers

bull Disadvantages

bull Data output lt 07 Gb

bull Cost per Gigabase is highest among all systems

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection

Template preparation Emulsion PCR Sequencing Hybridization and ligation

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

By successive rounds labelled oligonucleotide ligation to the template each base in the

template is determined twice

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Process of SOLiD Sequencing

Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Each base is sequenced twice

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Applied Biosystems SOLiDTM Sequencing

bull Advantage bull Very good data quality since every base

sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to

1536 sample per run) bull Cost effective 2000 eurohuman genome

bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases

Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

IlluminaSolexaTM-Sequencing

bull Sequencing by Synthesis

bull Modified chain terminating method

bull Bridge amplification

bull Paired end and mate pair libraries possible

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

IlluminaSolexaTM-Sequencing Clustering and sequencing

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

IlluminaSolexaTM-Sequencing

Advantages (Hi-SeqTM 20002500)

Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun

Read length PE 2x150 bases (increasing)

Cost per Gb ~ lt50euro or 1500eurohuman genome

Disadvantages

bull Hardware investment is high (~600000 euro plus periphery)

bull Medium high error rate (~05 increasing with read length)

bull High maintenance costs (service contract gt80000 euroyear)

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

bull Based on sequencing by Synthesis

bull Available since 2010

bull Emulsion PCR for library construction

bull Beads with amplified molecules are primed with an adapter

bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions

bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Annual Reviews

G A T C

Figure modified by E R Schmidt

Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Life TechnologiesIon Torrent-Sequencing by pH Monitoring

Advantages

Very cost efficient (human genome lt 1000 euro)

Read length 200 bases (increasing)

Very short running times

(~ 2-4 hrs)

Hardware investment is bdquolowldquo (~ 80000 US $)

Disadvantages

bull High error rate (gt10 increasing with read length)

bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo

bull Data output medium (depending on chip eg Proton PII = 32 Gb)

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Pacific BiosciencesSingle molecule real time (SMRT)-sequencing

bull Based on sequencing by synthesis on single molecules

bull Available since 2010 bull Special library construction leading to circular

molecules (enables multiple sequencing of the same molecule)

bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)

bull fluorescence labelled dNTP are measured in real time during incorporation

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Zero-mode waveguide

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Pacific BiosciencesSingle molecule polymerase active site monitoring

Advantages

Read length up to 10000 bases (average gt 1000 b)

Very short running times

(~ 2hrs)

Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo

Disadvantages

bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)

bull Significant investment in hardware (gt600 keuro)

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)

bull Sequencing by Synthesis with single molecules as templates

bull Modified chain terminating method

bull Bridge amplification

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Benchtop NGS Sequencing

bull Illumina Mi-SeqTM

bull Roche 454 JuniorTM

bull Ion Torrent PGMTM

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Platform List price Approximate cost per

run

Minimum throughput (read length)

Run time CostMb Mbh

454 GS Junior $108000 $1100 35 Mb (400 bases)

8 h $31 44

Ion Torrent PGM (314 chip)

$80490ab $225c 10 Mb (100 bases)

3 h $225 33

(316 chip) $425 100 Mbd (100 bases)

3 h $425 333

(318 chip) $625 1000 Mb (100 bases)

3 h $063 3333

MiSeq $125000 $750 1500 Mb (2 times 150 bases)

27 h $05 555

Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs

From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198

Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013

httpwwwgenomegovimagescontentcost_per_megabasejpg

Generation 5 Future Technology Nanopore Sequencing

bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently

When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data

In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through

httpswwwnanoporetechcomhome From

Applications of NGS

bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced

complexity sequencing bull Ribosome profiling

No summary table

Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going

Thank you very much for your attention

Benjamin Rieger bioinformatician

Nicole Naumann technical assistant

Rudolf Baader technical assistant

Steffen Rapp NGS Unit manager

httpwwwgenomegovimagescontentcost_per_megabasejpg

Generation 5 Future Technology Nanopore Sequencing

bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently

When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data

In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through

httpswwwnanoporetechcomhome From

Applications of NGS

bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced

complexity sequencing bull Ribosome profiling

No summary table

Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going

Thank you very much for your attention

Benjamin Rieger bioinformatician

Nicole Naumann technical assistant

Rudolf Baader technical assistant

Steffen Rapp NGS Unit manager

Generation 5 Future Technology Nanopore Sequencing

bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently

When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data

In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through

httpswwwnanoporetechcomhome From

Applications of NGS

bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced

complexity sequencing bull Ribosome profiling

No summary table

Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going

Thank you very much for your attention

Benjamin Rieger bioinformatician

Nicole Naumann technical assistant

Rudolf Baader technical assistant

Steffen Rapp NGS Unit manager

bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently

When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data

In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through

httpswwwnanoporetechcomhome From

Applications of NGS

bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced

complexity sequencing bull Ribosome profiling

No summary table

Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going

Thank you very much for your attention

Benjamin Rieger bioinformatician

Nicole Naumann technical assistant

Rudolf Baader technical assistant

Steffen Rapp NGS Unit manager

Applications of NGS

bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced

complexity sequencing bull Ribosome profiling

No summary table

Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going

Thank you very much for your attention

Benjamin Rieger bioinformatician

Nicole Naumann technical assistant

Rudolf Baader technical assistant

Steffen Rapp NGS Unit manager

No summary table

Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going

Thank you very much for your attention

Benjamin Rieger bioinformatician

Nicole Naumann technical assistant

Rudolf Baader technical assistant

Steffen Rapp NGS Unit manager

Benjamin Rieger bioinformatician

Nicole Naumann technical assistant

Rudolf Baader technical assistant

Steffen Rapp NGS Unit manager