Ngs introduction

NEXT GENERATION SEQUENCING Introduction

Alagar S2014419002M.Tech/Computational Biology

1953

DNA Structurediscovery

1977

Sanger DNA sequencing bychain-terminating inhibitors

Epstein-Barrvirus

(170 Kb)

1984

1987

Abi370Sequencer

1995

Homosapien

s(3.0

Gb)

454

Solexa

Solid

2001

2005

2007

Ion Torrent

PacBio

Haemophilusinfluenzae (1.83 Mb)

2011

2012

2013

Sequencing over the Ages

Illumina

Illumina Hiseq X

454

Pinustaeda

(24 Gb)

Sequencing: from DNA to GenomesSanger chain termination (1977) Hierarchical and

Shotgun sequencing (1996)

History:

• The first DNA sequences were obtained in the early 1970s by academic researchers using laborious methods based on two-dimensional chromatography. Following the development of fluorescence-based sequencing methods with automated analysis.

• Several notable advancements in DNA sequencing were made during the 1970s. Frederick Sanger developed rapid DNA sequencing methods at the MRC Centre, Cambridge, UK and published a method for "DNA sequencing with chain-terminating inhibitors" in 1977.

• Walter Gilbert and Allan Maxam at Harvard also developed sequencing methods, including one for "DNA sequencing by chemical degradation"

Contd.• The first full DNA genome to be sequenced was that

of bacteriophage φX174 in 1977. Medical Research Council scientists deciphered the complete DNA sequence of the Epstein-Barr virus in 1984, finding it to be 170 thousand base-pairs long.

• Leroy E. Hood's laboratory at the California Institute of Technology and Smith announced the first semi-automated DNA sequencing machine in 1986.

• Followed by Applied Biosystems' marketing of the first fully automated sequencing machine, the ABI 370, in 1987.

• By 1990, the U.S. NIH had begun large-scale sequencing trials on Mycoplasma capricolum ,Escherichia coli, Caenorhabditis elegans, and Saccharomyces cerevisiae at a cost of US$0.75 per base.

Several new methods for DNA sequencing were developed in the mid to late 1990s. These techniques comprise the first of the "next-generation" sequencing methods.

In 1996, Pål Nyrén and his student Mostafa Ronaghi at the Royal Institute of Technology in Stockholm published their method of pyrosequencing.Lynx Therapeutics published and marketed "Massively parallel signature sequencing", or MPSS, in 2000. This method incorporated a parallelized, adapter/ligation-mediated, bead-based sequencing technology and served as the first commercially available "next-generation" sequencing method, though no DNA sequencers were sold to independent laboratories

Next Generation Sequencing• Employs micro and nanotechnologies to reduce the size of

sample components, reducing reagent costs and enabling massively parallel sequencing reactions.

• Highly multiplexed, allowing simultaneous sequencing and analysis of millions of samples.

• Became commercially available from 2005.• The first using Solexa sequencing technologies.• Several different sequencing methods have been developed,

all of which are continually being developed at astonishing rates.

NGS technologies

Introduction to NGShttp://ueb.ir.vhebron.net/NGS

Next-generation DNA sequencing

Sanger sequencing Cyclic-array sequencing

http://ueb.ir.vhebron.net/NGS



Sanger sequencing Next-generation sequencing

Advantages of NGS- Construction of a sequencing library € clonal amplification to generate sequencing features





Advantages:-Construction

of asequencinglibrary € clonal amplification

to generate sequencing features

✓No in vivo cloning, transformation, colony picking...









- Array-based sequencing









- Array-based sequencing

✓Higher degree of parallelism than capillary-based sequencing


NGS means high sequencing capacity

GS FLX 454 (ROCHE)

HiSeq 2000 (ILLUMINA)

5500xl SOLiD (ABI)

GS Junior

Ion TORRENT

The sequencing process, in detail

DNAfragmentation and invitro adaptor ligation

11 Library preparation



emulsion PCR

1

2

12

Library preparationClonal amplification




emulsion PCR

bridge PCR

Pyrosequencing

1

2

3

123 Cyclic array

sequencing


454 sequencing





bridge PCR

Pyrosequencing

Sequencing-by-ligation

1

emulsion PCR

2

3

123

454 sequencing

SOLiD platform

Cyclic array sequencing






bridge PCR

Pyrosequencing

Sequencing-by-ligation

Sequencing-by-synthesis

1

emulsion PCR

2

3

123

454 sequencing

SOLiD platform

Solexa technology

Cyclic array sequencing



Third Generation Sequencing• PacBio RS• Single Molecule Realtime

Sequencing – instead of sequencing clonally amplified templates from beads (Pyro) or clusters (Illumina) DNA synthesis is detected on a single DNA strand.

• Zero-mode waveguide (ZMW)• DNA polymerase is affixed to the

bottom of a tiny hole (~70nm). • Only the bottom portion of the

hole is illuminated allowing for detection of incorporation of dye-labeled nucleotide.

Third Generation Sequencing

MethodSingle-molecule

real time sequencing

Ion semiconductor

Pyrosequencing (454)

Sequencing by synthesis (Illumina)

Sequencing by ligation (SOLiD

sequencing)

Chain termination

(Sanger sequencing)

Read length 2900 bp average[ 200 bp 700 bp 50 to 250 bp 50+35 or 50+50 bp 400 to 900 bp

Accuracy87% (read length mode), 99% (accuracy mode)

98% 99.9% 98% 99.9% 99.9%

Reads per run 35–75 thousand up to 5 million 1 million up to 3 billion 1.2 to 1.4 billion N/A

Time per run 30 minutes to 2 hours 2 hours 24 hours

1 to 10 days, depending upon sequencer and specified read length

1 to 2 weeks 20 minutes to 3 hours

Cost per 1 million bases $2 $1 $10 $0.05 to $0.15 $0.13 $2400

AdvantagesLongest read length. Fast. Detects 4mC, 5mC, 6mA.

Less expensive equipment. Fast.

Long read size. Fast.

Potential for high sequence yield, depending upon sequencer model

Low cost per base.

Long individual reads. Useful for many applications.

Disadvantages

Low yield at high accuracy. Equipment can be very expensive.

Homopolymer errors.

Runs are expensive. Homopolymer errors.

Equipment can be very expensive.

Slower than other methods.

More expensive and impractical for larger sequencing projects.

References

• Sequences, sequences, and sequences. Sanger, F. s.l. : Annu Rev Biochem, 1988, Vol. 57, pp. 1-28.• Nucleotide sequence of bacteriophage phi X174 DNA. Sanger, F, Air, GM and Barrell, BG.1977,

Nature, Vol. 265, pp. 687-695.• DNA Sequencing with chain-terminating inhibitors. Snager, F, Nicklen, S and Coulson, AR. s.l. :

Proc NatI Acad Sci USA, Vol. 74, pp. 5463-5467.• Overview of DNA sequencing strategies. Shendure, JA, Porreca, GJ and Church, GM.Chapter 7,

s.l. : John Wiley & Sons, 2011.• Energy transfer primers: a new fluoresence labeling paradigm for DNA sequencing and

analysis. Ju, J, Glazer, AN and Mathies, RA. 2, s.l. : Nat Med, 1996, pp. 998-999.• 454 Sequencing. [Online] 2015. [Cited: 6 2, 2015.] http://www.454.com/.• illumina. [Online] 2015. [Cited: 6 2, 2015.] http://www.illumina.com/.• SOLiD. Applied Biosystems. [Online] 2015. [Cited: 6 2, 2015.]

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

• Ion Torrent. Applied Biosystems. [Online] 2015. [Cited: 6 2, 2015.] http://www.lifetechnologies.com/ca/en/home/brands/ion-torrent.html.

Ngs introduction

Science

Transcript of Ngs introduction