Next-Generation Sequencing (NGS)

Basics, Development and Application of

High-throughput DNA Sequencing

Muhammed Demiral

Table of Contents

Definition of gene sequencing

Basic principles of gene sequencing

Development and improvement of NGS techniques

Fields of application

Future of NGS

Definition of gene sequencing

DNA sequencing [CELL MOL] The determination of the

sequence of nucleotides in deoxyribonucleic acid (DNA)

molecules.

Sequencing: the act, or process, of determining the sequence

of proteins or nucleic acids

taken from: Allison, L. A. Fundamental Molecular Biology; Blackwell Pub.: Malden, MA, 2007.

taken from: McGraw-Hill

dictionary of bioscience, 2nd

ed; McGraw-Hill: New York,

2003.

taken from: Cammack, R., Ed.

Oxford dictionary of biochemistry

and molecular biology, 2. ed; Oxford

Univ. Press: Oxford, 2008.

Basic principles of gene sequencing

1977: publication of two different approaches to gene sequencing

Chemical cleavage method (Maxam-Gilbert method)

Principle: base-specific chemical cleavage

Chemical agents modify certain bases

Hot piperidine (90 °C) is used to cleave the modified DNA at the position of

the modified base

Electrophoresis and autoradiography

Chemical agents Cleaved base(s)

Dimethyl sulfate G

Formic acid G+A

Hydrazine C+T

Hydrazine + 1,5 M NaCl C

5‘ 32PACTGTCCGA 3‘ base-specific chemical

cleavage of the DNA strand in

four different aliquots

Cleavage at G Cleavage at G+A Cleavage at T+C Cleavage at C

32PACTG 32PACTGTCCG

32PA 32PACTG 32PACTGTCCG 32PACTGTCCGA

32PAC 32PACT 32PACTGT 32PACTGTC 32PACTGTCC

32PAC 32PACTGTC 32PACTGTCC

electrophoretic resolution/separation

and autoradiographic verification

+

-

G C T+C G+A

A

C

C

T

G

T

C

A

G

5‘

3‘

Gel runs in

this direction Read in this

direction

Dideoxy (chain-termination) method (Sanger‘s method)

Principle: in-vitro strand synthesis and chain termination

H OH

Base Base

2´-Deoxyribonucleoside-5´-Triphosphate (dNTP) 2',3-dideoxyribonucleoside triphosphate (ddNTP)

a dNTP possesses a

hydroxyl group necessary

for chain elongation

no hydroxyl group

no chain elongation

chain termination

A DNA strand is elongated by the use of a DNA polymerase

The reaction contains also necessary components für an in-vitro DNA

synthesis – including one of the four different ddNTPs

DNA polymerases can incorporate ddNTPs like dNTPs

ddNTPs have no 3‘-OH group chain is terminated after the incorporation

of a ddNTP (no formation of a phosphodiester linkage between two

nucleotides)

The DNA template is divided into four separate sequencing reactions

containing all four standard dNTPs and to each reaction is added only one

of the four ddNTPs

sequencing reaction components

Primer1

DNA polymerase

dNTPs und ddNTPs²

DNA template1

1 one of them being

radioactively labelled

² only one sort in each

of the four aliquots

taken from: Voet, D.; Voet, J. G. Biochemistry, 4th ed.; John Wiley & Sons: Hoboken, NJ, 2011, p. 178

Maxam-Gilbert method Sanger‘s method

+ The sequence within an unknown DNA

base sequence can be determined with

the help of a restriction map

+ The sequence stems from the original

DNA molecule (no enzymetically

produced copies) no errors in the

base sequence

− Only short DNA sequences can be

sequenced

− In general, the reaction runs slower and

is less reliable

− The reactions require different harmful

chemicals

+ Longer fragments can be sequenced

+ Fast and reliable method easily

automatable

+ Radioactive substances can be replaced

by fluorescent dyes faster and safer,

as the steps for autoradiography are no

longer required

− A Primer is needed; part of the sequence

must be known (solution: use of random

primers and primer design (e.g. Edman

degradation of proteins and the genetic

code))

Advantages and disadvantages of the sequencing methods

Improvement in Sanger‘s method:

radioactive labels, autoradiographic

detection and manual data interpretation

were replaced with fluorescent labels,

laser-induced fluorescence detection and

computer-based data analysis

Maxam-Gilbert Chemical fragmentation

Sanger

Chain termination method

(later: capillary (gel) electrophoresis)

Shotgun sequencing

Roche Pyrosequencing

Illumina Illumina sequencing

Life Technologies

Sequencing by Oligonucleotide Ligation and

Detection (SOLiD)

Ion semiconductor sequencing

Helicos BioSciences True single molecule sequencing (tSMS)

Pacific Biosciences Single molecule real time sequencing (SMRT)

Oxford Nanopore

Technologies

Nanopore sequencing

First-generation

sequencing

Second-generation

sequencing

Third-generation

sequencing

Next-

ge

nera

tio

n s

eq

uen

cin

g (

NG

S)

First-generation

sequencing

Second-generation

sequencing

Third-generation

sequencing

• in-vitro amplification of the template DNA

steps of molecular cloning are reduced

• simplified sample preparation (minimisation,

automation and standardisation of working

steps better reproducibility and reduction of

error sources), to some extent machines are

maintained by only one technician/lab worker

• Improvement of sequencing accuracy

• Increase in number and quantitiy of samples

• Increase in read length

• Increase in data output

• Decrease of (overall) costs

Development of costs

taken from: Wetterstrand KA. DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP).

http://www.genome.gov/sequencingcosts/ (accessed January 6, 2014)

Development of costs

taken from: Wetterstrand KA. DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP).

http://www.genome.gov/sequencingcosts/ (accessed January 6, 2014)

Fields of application

Examples:

Archaeology

Anthropology

Genetics and genomics (e.g. evolutionary genomics)

Biotechnology

Molecular biology

Biochemistry

Forensics

Key aspects:

Immunogenetics/pharmacogenomics individualised medicines and therapies (minimise side effects

and adverse events; maximise effects)

Medical genetics/human genetics (detection of mutations, genetically determined (hereditary) diseases)

Oncology/oncogenomics better prediction of the individual course of disease; better therapy selection

Epigenomics

DNA methylation: addition of a methyl group to DNA nucleotides; influence on gene expression

ChIP-seq: technique for the analysis of Proteine-DNA-interaction

Histone modification: Histone proteins package and order the DNA

Transcription factors: proteins that

bind to the DNA and regulate the

transcription process taken from: About ENCODE Data at UCSC. https://genome.ucsc.edu/ENCODE/aboutScaleup.html (accessed January 1, 2014).

Outlook and future of NGS

Ten years after the Human Genome Project: qualified views

For the future: growing importance of NGS

Improvement and development of old and new techniques respectively

New projects

It is fair to say that all of these predictions have come true,

with some caveats that offer important lessons about the

best path forward for genomics and personalized

medicine. The promise of a revolution in human health

remains quite real. Those who somehow expected

dramatic results overnight may be disappointed, but

should remember that genomics obeys the First Law of

Technology: we invariably overestimate the short-term

impacts of new technologies and underestimate their

longer-term effects.

quote taken from: Collins, F. Has the revolution arrived? Nature 2010, 464 (7289), 674–675.

References and further literature

General information on DNA sequencing

[1] Bioinformatics for High Throughput Sequencing; Springer Science+Business Media, LLC: New York, NY,

2012.

[2] Alphey, L. DNA-Sequenzierung; Spektrum, Akad. Verl.: Heidelberg ;, Berlin, 1998.

[3] Brooker, R. J. Genetics. Analysis & Principles, 4th ed.; McGraw-Hill: New York, 2012.

[4] Christen, P. Biochemie. Eine Einführung mit 40 Lerneinheiten, 1st ed.; Springer: Berlin, 2005.

[5] Hartwell, L. Genetics. From Genes to Genomes, 4th ed.; McGraw-Hill: New York, 2011.

[6] Karp, G. Cell and Molecular Biology. Concepts and Experiments, 6th ed.; John Wiley: Hoboken, NJ,

2010.

[7] Löffler, G.; Schölmerich, J. Basiswissen Biochemie. Mit Pathobiochemie; 7th ed.; Springer: Heidelberg,

2008.

[8] Metzler, D. E. Biochemistry. The chemical reactions of living cells 1; Acad. Press: New York, 2001.

[9] Mülhardt, C. Der Experimentator: Molekularbiologie, Genomics, 6th ed.; Spektrum Akad.-Verlag:

Heidelberg, 2009.

[10] Nelson, D. L.; Cox, M. M. Principles of Biochemistry, 4. ed.; Freeman: New York, NY, 2004.

[11] Pierce, B. A. Genetics. A conceptual approach, 4th ed.; W.H. Freeman: New York, 2012.

[12] Snustad, D. P.; Simmons, M. J. Principles of Genetics, 6th ed.; Wiley: Hoboken, NJ, 2012.

[13] Voet, D.; Voet, J. G. Biochemistry, 4th ed.; John Wiley & Sons: Hoboken, NJ, 2011.

[14] Walker, J. M.; Rapley, R. Molecular Biology and Biotechnology, 5th ed.; Royal Society of Chemistry:

Cambridge, 2009.

Human Genome Project

[1] International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human

genome. Nature 2001, 409 (6822), 860–921.

[2] J. C. Venter et al. The Sequence of the Human Genome. Science 2001, 291 (5507), 1304–1351.

[3] Collins, F. Has the revolution arrived? Nature 2010, 464 (7289), 674–675.

Websites on NGS projects

[1] National Human Genome Research Institute (NHGRI). Homepage. http://www.genome.gov/ (accessed

December 30, 2013).

[2] Nationales Genomforschungsnetz (NGFN). Programm der medizinischen Genomforschung.

http://www.ngfn.de/ (accessed December 31, 2013).

Sanger‘s method

[1] Sanger, F.; Coulson, A. A rapid method for determining sequences in DNA by primed synthesis with DNA

polymerase. Journal of Molecular Biology 1975, 94 (3), 441–448.

[2] Sanger, F.; Nicklen, S.; Coulson, A. R. DNA Sequencing with Chain-Terminating Inhibitors. Proceedings of

the National Academy of Sciences 1977, 74 (12), 5463–5467.

Maxam-Gilbert method

[1] Maxam, A. M.; Gilbert, W. A New Method for Sequencing DNA. Proceedings of the National Academy of

Sciences 1977, 74 (2), 560–564.

Next-Generation Sequencing

[1] Anderson, M. W.; Schrijver, I. Next Generation DNA Sequencing and the Future of Genomic Medicine.

Genes 2010, 1 (1), 38–69.

[2] Ansorge, W. J. Next-generation DNA sequencing techniques. New Biotechnology 2009, 25 (4), 195–203.

[3] Barron, A. E.; Hert, D. G.; Fredlake, C. P. Advantages and limitations of next-generation sequencing

technologies: A comparison of electrophoresis and non-electrophoresis methods. Electrophoresis 2008, 29

(23), 4618–4626.

[4] Barzon, L.; Lavezzo, E.; Militello, V.; Toppo, S.; Palù, G. Applications of Next-Generation Sequencing

Technologies to Diagnostic Virology. IJMS 2011, 12 (11), 7861–7884.

[5] Berglund, E. C.; Kiialainen, A.; Syvänen, A.-C. Next-generation sequencing technologies and applications

for human genetic history and forensics. Invest Genet 2011, 2 (1), 23.

[6] França, L. T. C.; Carrilho, E.; Kist, T. B. L. A review of DNA sequencing techniques. Quart. Rev. Biophys.

2002, 35 (02).

[7] Hadfield, J., Loman, N. Next-Generation Genomics: World Map of High-throughput Sequencers.

http://omicsmaps.com/ (accessed January 2, 2014).

[8] Kircher, M.; Kelso, J. High-throughput DNA sequencing - concepts and limitations. Bioessays 2010, 32

(6), 524–536.

[9] Lerner, H.; Fleischer, R. Prospects for the Use of Next-Generation Sequencing Methods in Ornithology.

The Auk 2010, 127 (1), 4–15.

[10] Mardis, E. R. Next-Generation DNA Sequencing Methods. Annu. Rev. Genom. Human Genet. 2008, 9

(1), 387–402.

[11] Mardis, E. R. A decade’s perspective on DNA sequencing technology. Nature 2011, 470 (7333), 198–

203.

[12] Metzker, M. L. Emerging technologies in DNA sequencing. Genome Research 2005, 15 (12), 1767–

1776.

[13] Metzker, M. L. Sequencing technologies — the next generation. Nat Rev Genet 2009, 11 (1), 31–46.

[14] Niedringhaus, T. P.; Milanova, D.; Kerby, M. B.; Snyder, M. P.; Barron, A. E. Landscape of Next-

Generation Sequencing Technologies. Anal. Chem. 2011, 83 (12), 4327–4341.

[15] Pettersson, E.; Lundeberg, J.; Ahmadian, A. Generations of sequencing technologies. Genomics 2009,

93 (2), 105–111.

[16] Quail, M.; Smith, M. E.; Coupland, P.; Otto, T. D.; Harris, S. R.; Connor, T. R.; Bertoni, A.; Swerdlow, H.

P.; Gu, Y. A tale of three next generation sequencing platforms: comparison of Ion torrent, pacific biosciences

and illumina MiSeq sequencers. BMC Genomics 2012, 13 (1), 341.

[17] Shendure, J.; Ji, H. Next-generation DNA sequencing. Nat Biotechnol 2008, 26 (10), 1135–1145.

[18] Shendure, J.; Mitra, R. D.; Varma, C.; Church, G. M. Advanced sequencing technologies: methods and

goals. Nat Rev Genet 2004, 5 (5), 335–344.

[19] Varshney, R. K.; Nayak, S. N.; May, G. D.; Jackson, S. A. Next-generation sequencing technologies and

their implications for crop genetics and breeding. Trends in Biotechnology 2009, 27 (9), 522–530.

[20] Voelkerding, K. V.; Dames, S. A.; Durtschi, J. D. Next-Generation Sequencing: From Basic Research to

Diagnostics. Clinical Chemistry 2009, 55 (4), 641–658.

[21] Welch, J. S.; Link, D. C. Genomics of AML: Clinical Applications of Next-Generation Sequencing.

Hematology 2011, 2011 (1), 30–35.

[22] Zhou, X.; Ren, L.; Meng, Q.; Li, Y.; Yu, Y.; Yu, J. The next-generation sequencing technology and

application. Protein Cell 2010, 1 (6), 520–536.

Capillary electrophoresis

[1] Karger, B. L.; Guttman, A. DNA Sequencing by Capillary Electrophoresis. ELECTROPHORESIS 2009, 30

(S1), S196.

[2] Life Technologies/Applied Biosystems. DNA Sequencing & Fragment Analysis by Capillary

Electrophoresis. http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/dna-

sequencing-fragment-analysis.html (accessed January 1, 2014).

Sequencing by Oligonucleotide Ligation and Detection (SOLiD™ technology)

[1] Applied Biosystems. Overview of SOLiD™ Sequencing Chemistry.

http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-

sequencing/next-generation-systems/solid-sequencing-chemistry.html (accessed December 30, 2012).

[2] Applied Biosystems. SOLiD™ System Accuracy.

http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_057511

.pdf (accessed December 30, 2013).

[3] Applied Biosystems. SOLiD™ System Barcoding.

http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_057554

.pdf (accessed December 30, 2013).

Ion semiconductor sequencing

[1] Rothberg, J. M.; Hinz, W.; Rearick, T. M.; Schultz, J.; Mileski, W.; Davey, M.; Leamon, J. H.; Johnson, K.;

Milgrew, M. J.; Edwards, M.; Hoon, J.; Simons, J. F.; Marran, D.; Myers, J. W.; Davidson, J. F.; Branting, A.;

Nobile, J. R.; Puc, B. P.; Light, D.; Clark, T. A.; Huber, M.; Branciforte, J. T.; Stoner, I. B.; Cawley, S. E.;

Lyons, M.; Fu, Y.; Homer, N.; Sedova, M.; Miao, X.; Reed, B.; Sabina, J.; Feierstein, E.; Schorn, M.; Alanjary,

M.; Dimalanta, E.; Dressman, D.; Kasinskas, R.; Sokolsky, T.; Fidanza, J. A.; Namsaraev, E.; McKernan, K.

J.; Williams, A.; Roth, G. T.; Bustillo, J. An integrated semiconductor device enabling non-optical genome

sequencing. Nature 2011, 475 (7356), 348–352.

VisiGen sequencing system

[1] VisiGen Biotechnologies. Real-Time DNA Sequencing.

http://www.abrf.org/Other/ABRFMeetings/ABRF2005/Hardin.pdf (accessed December 30, 2013).

Pyrosequencing

[1] 454 Life Sciences. How is genome sequencing done? http://www.454.com/downloads/news-events/how-

genome-sequencing-is-done_FINAL.pdf (accessed December 29, 2013).

[2] Ahmadian, A.; Ehn, M.; Hober, S. Pyrosequencing: History, biochemistry and future. Clinica Chimica Acta

2006, 363 (1-2), 83–94.

[3] Chowdhury, F. a. A. Pyrosequencing - An Alternative to Traditional Sanger Sequencing. American Journal

of Biochemistry and Biotechnology 2012, 8 (1), 14–20.

[4] Fakruddin, M.; Chowdhury, A.; Hossain, N.; Khanjada Shahnewaj Bin Mannan, R. M. M. Pyrosequencing

- Principles And Applications. International Journal of Life science and Pharma Reviews (IJLPR) 2012, 2 (2),

65–76.

[5] Gharizadeh, B. Method development and applications of Pyrosequencing technology: Stockholm, 2003.

[6] Gharizadeh, B.; Akhras, M.; Nourizad, N.; Ghaderi, M.; Yasuda, K.; Nyrén, P.; Pourmand, N.

Methodological improvements of pyrosequencing technology. Journal of Biotechnology 2006, 124 (3), 504–

511.

[7] Nakano, M.; Komatsu, J.; Matsuura, S.-i.; Takashima, K.; Katsura, S.; Mizuno, A. Single-molecule PCR

using water-in-oil emulsion. Journal of Biotechnology 2003, 102 (2), 117–124.

[8] Qiagen N.V. Pyrosequencing – genetic analysis for clinical research. http://www.pyrosequencing.com/

(accessed December 29, 2013).

[9] Qiagen N.V. Pyrosequencing — the synergy of sequencing and quantification.

http://www.qiagen.com/literature/render.aspx?id=200102 (accessed January 7, 2014).

[10] Ronaghi, M. Pyrosequencing Sheds Light on DNA Sequencing. Genome Research 2001, 11 (1), 3–11.

[11] Ronaghi, M.; Karamohamed, S.; Pettersson, B.; Uhlén, M.; Nyrén, P. Real-Time DNA Sequencing Using

Detection of Pyrophosphate Release. Analytical Biochemistry 1996, 242 (1), 84–89.

[12] Rothberg, J. M.; Leamon, J. H. The development and impact of 454 sequencing. Nat Biotechnol 2008,

26 (10), 1117–1124.

[13] Siqueira, J. F.; Fouad, A. F.; Rôças, I. N. Pyrosequencing as a tool for better understanding of human

microbiomes. Journal of Oral Microbiology 2012, 4 (0).

[14] Winge, M. Pyrosequencing - a new approach to DNA analysis. Innovations in Pharmaceutical

Technology 2000, 4 (0), 18–24.

Illumina sequencing

[1] Illumina Inc. An Introduction to Next-Generation Sequencing Technology.

http://www.illumina.com/Documents/products/Illumina_Sequencing_Introduction.pdf (accessed December 29,

2013).

[2] Illumina Inc. Illumina Sequencing Technology.

http://www.illumina.com/documents/products/techspotlights/techspotlight_sequencing.pdf (accessed

December 29, 2013).

True Single Molecule Sequencing (tSMS™ technology)

[1] Thompson, J. F.; Steinmann, K. E. Single Molecule Sequencing with a HeliScope Genetic Analysis

System. In Current Protocols in Molecular Biology; Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D.,

Seidman, J., Smith, J. A., Struhl, K., Eds.; John Wiley & Sons, Inc: Hoboken, NJ, USA, 2001.

[2] Bowers, J.; Mitchell, J.; Beer, E.; Buzby, P. R.; Causey, M.; Efcavitch, J. W.; Jarosz, M.; Krzymanska-

Olejnik, E.; Kung, L.; Lipson, D.; Lowman, G. M.; Marappan, S.; McInerney, P.; Platt, A.; Roy, A.; Siddiqi, S.

M.; Steinmann, K.; Thompson, J. F. Virtual terminator nucleotides for next-generation DNA sequencing. Nat

Meth 2009, 6 (8), 593–595.

[3] Helicos Biosciences. Helicos DNA Sample Preparation Manual.

http://mbcf.dfci.harvard.edu/Helicos/helicos_pdf/TR.pdf (accessed December 30, 2013).

[4] Helicos Biosciences. True Direct DNA Measurement.

http://helicosbio.com/Portals/0/Documents/Helicos%20tSMS%20Technology%20Primer.pdf (accessed

December 30, 2013).

Single molecule real time sequencing (SMRT™ technology)

[1] Pacific Biosciences. Single Molecule Real Time (SMRT) DNA Sequencing.

http://www.pacificbiosciences.com/assets/files/pacbio_technology_backgrounder.pdf (accessed December

30, 2013).

Nanopore sequencing

[1] Branton, D.; Deamer, D. W.; Marziali, A.; Bayley, H.; Benner, S. A.; Butler, T.; Di Ventra, M.; Garaj, S.;

Hibbs, A.; Huang, X.; Jovanovich, S. B.; Krstic, P. S.; Lindsay, S.; Ling, X. S.; Mastrangelo, C. H.; Meller, A.;

Oliver, J. S.; Pershin, Y. V.; Ramsey, J. M.; Riehn, R.; Soni, G. V.; Tabard-Cossa, V.; Wanunu, M.; Wiggin, M.;

Schloss, J. A. The potential and challenges of nanopore sequencing. Nat Biotechnol 2008, 26 (10), 1146–

1153.

[2] McNally, B.; Singer, A.; Yu, Z.; Sun, Y.; Weng, Z.; Meller, A. Optical Recognition of Converted DNA

Nucleotides for Single-Molecule DNA Sequencing Using Nanopore Arrays. Nano Lett. 2010, 10 (6), 2237–

2244.

[3] Noblegen Biosciences. Technology. http://www.noblegenbio.com/Technology.html (accessed December

30, 2013).

[4] Oxford Nanopore Technologies. Introduction to nanopore sensing.

http://www.nanoporetech.com/technology/introduction-to-nanopore-sensing/introduction-to-nanopore-sensing

(accessed December 29, 2013).

[5] Schneider, G. F.; Dekker, C. DNA sequencing with nanopores. Nat Biotechnol 2012, 30 (4), 326–328.

[6] Soni, G. V.; Meller, A. Progress toward Ultrafast DNA Sequencing Using Solid-State Nanopores. Clinical

Chemistry 2007, 53 (11), 1996–2001.