ngs introduction
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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
Introduction to NGShttp://ueb.ir.vhebron.net/NGS
Next-generation DNA sequencing
Sanger sequencing Next-generation sequencing
Advantages of NGS- Construction of a sequencing library € clonal amplification to generate sequencing features
Introduction to NGShttp://ueb.ir.vhebron.net/NGS
Next-generation DNA sequencing
Sanger sequencing Next-generation sequencing
Advantages:-Construction
of asequencinglibrary € clonal amplification
to generate sequencing features
✓No in vivo cloning, transformation, colony picking...
Introduction to NGShttp://ueb.ir.vhebron.net/NGS
Next-generation DNA sequencing
Sanger sequencing Next-generation sequencing
Advantages:-Construction
of asequencinglibrary € clonal amplification
to generate sequencing features
✓No in vivo cloning, transformation, colony picking...
- Array-based sequencing
Introduction to NGShttp://ueb.ir.vhebron.net/NGS
Next-generation DNA sequencing
Sanger sequencing Next-generation sequencing
Advantages:-Construction
of asequencinglibrary € clonal amplification
to generate sequencing features
✓No in vivo cloning, transformation, colony picking...
- 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
Next-generation DNA sequencing
DNAfragmentation and invitro adaptor ligation
emulsion PCR
1
2
12
Library preparationClonal amplification
Introduction to NGShttp://ueb.ir.vhebron.net/NGS
Next-generation DNA sequencing
DNAfragmentation and invitro adaptor ligation
emulsion PCR
bridge PCR
Pyrosequencing
1
2
3
123 Cyclic array
sequencing
Library preparationClonal amplification
454 sequencing
Introduction to NGShttp://ueb.ir.vhebron.net/NGS
Next-generation DNA sequencing
DNAfragmentation and invitro adaptor ligation
bridge PCR
Pyrosequencing
Sequencing-by-ligation
1
emulsion PCR
2
3
123
454 sequencing
SOLiD platform
Cyclic array sequencing
Library preparationClonal amplification
Introduction to NGShttp://ueb.ir.vhebron.net/NGS
Next-generation DNA sequencing
DNAfragmentation and invitro adaptor ligation
bridge PCR
Pyrosequencing
Sequencing-by-ligation
Sequencing-by-synthesis
1
emulsion PCR
2
3
123
454 sequencing
SOLiD platform
Solexa technology
Cyclic array sequencing
Library preparationClonal amplification
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
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.
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