Next Generation Sequencing

Next Generation Sequencing (NGS)
Fernando Alvarez
Sección Biomatemática,
Facultad de Ciencias, UdelaR
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Uruguay
Montevide
o
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TANGO
World Champ
1930
1950 (Maraca
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Next Generation Sequencing
module
Next Generation Sequencing technologies. Monday
morning
Genome Sequencing and Assembling. Monday
afternoom
Assembling Practical Monday afternoom
RNA seq Theoretical and Practical. Tuesday
morning
Chiptser (Data analysis platform for microarray,
proteomics and NGS data) Tuesday afternoom
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Agenda
1- Sanger Sequencing
2- Next Generation Sequencing – Deep sequencing
Sanger sequencing Technologies
Genome sequencers- Ultra high throughput sequencers
- 454 Roche
- Solexa illumina
Description of the different technologies, comparison of
their throughput, limitations
3- Next-next generation sequencers (single molecule
sequencers)
-Pacific Biosciences
-Helicos
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DNA Sequencing
1977 Sanger enzymatic method
dd(nucleotides)
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DNA Sequencing
Requirements:
Template (to be sequenced)
Primer
dNTP’s
ddNTP’s
4 independent reactions
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DNA Sequencing
Result:
Fragments of different size
according to where the
ddNTP has been
incorporated
Gel
denaturing
Gel
separate the fragmentS
to
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DNA Sequencing
Sequenced
segment:
4040-3920 =
bases
120
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Parallelization in the lab
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Fluorometry and
Sequencing
1986:
contribution of fluorometry.
Use of ddNTP’s linked to fluorescent labels
Requeriments:
Template
Primer
dNTP’s
ddNTP’s (each one linked to a
different Dye)
Only 1 reaction
Automatic Sequencing
Nature 321, 674-679 (12 June 1986)
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Last 90
Applied Biosystems
ABI 3730XL
96 capilaries
800 nt per read
1 Mb / days
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Capillary sequencers (Sanger
Centre, 2000)
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Agenda
1- Sanger Sequencing
2- Next Generation Sequencing – Deep sequencing
Sanger sequencing Technologies
Genome sequencers- Ultra high throughput sequencers
- 454 Roche
- Solexa illumina
Description of the different technologies, comparison of
their throughput, limitations
3- Next-next generation sequencers (single molecule
sequencers)
-Pacific Biosciences
-Helicos
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ROCHE 454
454 Sequencing is a massively-parallel
pyrosequencing system capable of
sequencing roughly 20 megabases of
raw DNA per 7-hour (GS20). The GSFLX is
able to sequence 100 Mb. The system relies
on fixing nebulized and adapter-ligated DNA
fragments to small DNA-capture beads in a
water-in-oil emulsion. The DNA fixed to these
beads is then amplified by PCR. Finally, each
DNA-bound bead is placed into a ~44 μm well
on a PicoTiterPlate, a fiber optic chip. A mix of
enzymes such as polymerase, ATP
sulfurylase, and luciferase are also packed
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454 sequencer ROCHE
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Amplification
emPCR (Emulsion PCR)
1 DNA fragment  1 bead  1
microreactor
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One Fragment = One Bead (limiting concentration)
1,6 millones de
secuenciadores
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Pyrosequencing
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Colección de Datos
http://www.youtube.com/watch?v=nFfgWGFe0aA&feature=related
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Pyrogram
1st cycle 2nd cycle
AGGGG TGGC
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Some Data
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Some Data
2011:
Genome Sequencer FLX System
GS Junior System
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Some throughput Data
Genome Sequencer FLX System
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Solexa Technology (200 Gb sequencer)
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Deep sequencing
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250 millions of clusters,
1000 copies each
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http://seq.molbiol.ru/
Cesped de
primers
http://seq.molbiol.ru/
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Illumina
2011:
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Illumina HiSeq1000-2000
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Illumina miSeq
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Comparison of the technologies
Technology
Read
Length
Number of
Reads
Total
throughput
Capillary
(Sanger)
800 nt
96
<80 kb
400
1.6 millions
Roche
(from 300 to
500)
Solexa
Solid
76 (100)
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600
Mb
Aprox
100 millions 10 Gb (x2)
(x2)
(today 200
GB )
500 millions
16 GB
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Agenda
1- Sanger Sequencing
2- Next Generation Sequencing – Deep sequencing
Sanger sequencing Technologies
Genome sequencers- Ultra high throughput sequencers
- 454 Roche
- Solexa illumina
Description of the different technologies, comparison of
their throughput, limitations
3- Next-next generation sequencers (single molecule
sequencers)
-Pacific Biosciences
-Helicos
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Next-Next Generation Sequencers
Sequencing without amplification
Oxford Nanopore
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PACBIO
HELICOS
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Secuenciación
2010-2011:
J. Rothberg
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Ion Torrent
2011 PGM:
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Ion Torrent
2011 PGM:
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Secuenciación
2011 PGM:
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Oxford Nanopore Tech:
Oxford Nanopore Tech:
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Oxford Nanopore
α-hemolysin Staphylococcus aureus
exonuclease
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Huma sequencing project (finished)
13 years, 3 dólares
2008
3 months, 60 k dolar,
Today, 1 week 10 k dolars
In the neext few years
Few hours or minutes, less than 1000 dolars
-Genome sequencing will become a routine clinical technique
This is allowing and will allow even more,
to pose questions that were unthinkable few years ago
-Eg. what is the genetic basis of longevity, hypertension,
or whatever trait with a very complex genetic basis
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Many things can be done, and many more will
be possible in the future,
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