!&
The next generation
sequencing
Métodos Atuais
Sequenciamento de próxima geração
1 - melhor custo-benefício para projetos de alta demanda de dados;
2 – custo por pb muito menor;
3 – sequenciamento muito mais rápido e eficiente (>40 Gbase/corrida).
'&
The next generation technologies
Roche 454
Solid
Illumina/Solexa
Ion Torrent
PacBio
Nanopore
Sequenciamento de
segunda geração
Sequenciamento de
terceira geração
Sequenciamento de
quarta geração
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http://genome.gov/splash.htm
CUSTO DO SEQUENCIAMENTO POR BASE
,&
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enoma humano – U$ 2 bi; ~3 bilhões bp; 11 anos; ~10X
Genoma – U$ 3.000,00; ~3 bilhões bp; 2 dias; ~100X
CUSTO DO SEQUENCIAMENTO POR GENOMA
http://genome.gov/splash.htm
Roche 454
Illumina
Solid
Mardis, ER. A decade’s perspective on DNA sequencing technology. Nature, 2011.
A evolução do
sequenciamento de DNA
Mardis. Science 2011
"&
Kahn, SD. On the future of Genomic Data. Science, 2011
A evolução do
sequenciamento de DNA
Kahn SD. Science 2011
Objetivo – rastrear as variações genéticas em populações humanas
Objetivo – gerar conhecimento sobre o genoma de câncer com o intuito de
gerar tratamentos e diagnósticos
(&
Objetivo – analisar o genoma de 10 mil espécies de vertebrados
Hoje …
Objetivo – analisar o genoma de 5 mil espécies de insetos e outros
artrópodos
Maiores problemas dos
sequenciamentos em
larga escala!!!
B&
Sequenciamento de
segunda geração
Roche 454
Solid
Illumina/Solexa
Ion Torrent
Amplificação e
síntese
2004-2005
Roche – 454 Life science
Utiliza tecnologia de pirosequenciamento - 1986
Roche 454 – 2
nd
generation
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Análise genômica 2013
Princípio
Mecanismo do
Pirosequenciamento
ATP-sulfurilase – Conversão PPi ! ATP
Luciferase – Usa ATP p/ converter luciferina ! oxyluciferina =
LUZ
Apirase – degrada os ATPs e nucleotideos livres
the
order
of
the
signal
peaks
the
DNA
sequence
can
be
determined
[
14
].
Free
ATP
and
nucleotides
are
degraded
by
the
apyrase.
This
disables
light
emission
and
regen-erates
the
solution.
The
complete
enzyme
process
can
be
performed
in
a
single
well,
offering
a
fast
reaction
time
of
approximately
20
min
per
96-well
plate
[
9
].
A
lot
of
sequencing
platforms
are
available
today.
All
of
them
use
short
fragments,
so-called
reads,
to
investigate
genome
sequences
[
15
],
chromatin
immunoprecipitation
(ChIP)
or
mapping
of
DNA
methylation.
The
two
most
common
variants
based
on
conventional
pyrosequencing
principle
are
the
454
Sequencing,
and
the
PyroMark
ID
system.
The
second
developmental
stage
of
pyrosequen-cing
was
the
invention
of
surface-based
systems.
These
systems
also
follow
the
principle
to
detect
every
single
nucleotide
incorporation
step,
but
enzymatically
induced
lightnings
are
not
necessary
anymore.
This
recent
gener-ation
provides
options
for
high-throughput
functional
transcriptomics.
Various
capabilities
like
discovery
of
transcription
factor
bindings
or
non-coding
RNA
expres-sion
profiling
could
have
been
established
by
now
[
6
].
Here,
the
Genome
analyzer
and
SOLiD
are
widely
spread
variants.
All
these
methods
have
various
advantageous
and
drawbacks
and
access
or
local
facilities
will
influence
the
choice
of
the
according
technology
[
16
].
These
four
systems
are
presented
in
detail
next.
The
first
large-scale
adaption
of
the
pyrosequencing
technique,
invented
by
454
Life
Sciences
[
17
]
and
later
commercialized
by
Roche,
is
a
high-throughput
system
[
18
].
It
can
be
described
as
pyrosequencing
in
high-density
picoliter
reactors.
Fragmentized
DNA
is
attached
to
streptavidin
beads
that
are
consequently
captured
into
aqueous
droplets
in
an
oil
solution.
This
so-called
emul-sion
PCR
separates
DNA
molecules
along
with
the
pri-mer-coated
beads.
Thus,
the
droplets
form
small
amplification
reactors
[
19
].
Every
bead
is
transferred
to
a
picoliter
plate
and
analyzed
by
normal
pyrosequencing.
454
instruments
are
sequencing
up
to
500
million
bases
within
ten
hours.
The
read
length
(250
nt)
is
shorter
than
with
Sanger
technology
(600
nt)
because
it
is
limited
by
the
used
pyrosequencing
chemistry.
As
a
result
of
decrease
in
apyrase’s
efficiency
in
degrading
excess
2
Analytical
Biotechnology
COBIOT-1093;
NO.
OF
PAGES
9
Please
cite
this
article
in
press
as:
Mutz
K-O,
et
al.:
Transcriptome
analysis
using
next-generation
sequencing,
Curr
Opin
Biotechnol
(2012),
http://dx.doi.org/10.1016/j.copbio.2012.09.004
Figure
1
5′
3′
3
′
5′
polymerase
dNTP
PPi
ATP
sulfurylase
luciferase
apyrase
ATP
dNTP
dNDP
dNMP
ADP
AMP
time
light
N
3′
5′
5′
3′
polymerase
primer
sulfurylase
luciferase
APS
PPi
ATP
luciferin
oxyluciferin
(a)
(c)
(b)
(d)
pyrosequencing
454 sequencing
Solexa
laser detector
SOLiD
polymerization
emulsion PCR
primer
adapter
Current Opinion in Biotechnology
Basic
principles
of
NGS
techniques.
(a)
pyrosequencing:
the
incorporation
of
a
new
nucleotide
generates
detectable
light.
(b)
454
sequencing:
nucleotide
incorporation
is
associated
with
the
release
of
pyrophosphate
resulting
in
a
light
signal.
(c)
Solexa:
DNA
fragments
build
double-stranded
bridges
and
after
the
addition
of
the
labeled
terminators
the
sequencing
cycle
starts.
(d)
SOLiD:
if
the
adapters
are
bound,
emulsion
PCR
is
carried
out
to
generate
so-called
bead
clones.
C&
Figure 1. The GS FLX system working principle. (A) Prepare adapter ligated ssDNA library (A-[insert]-B). (B) Emulsion based clonal amplification. (C) Depositing DNA beads into the PicoTiter™ plate. (D) Sequencing and base calling. (http://www.454.com)
© Higher Education Press and Springer-V erlag Berlin Heidelberg 2010 523 The next-generation sequencing technology and application
Protein
&
Cell
Zhou et al., 2010. Protein Cell
http://www.youtube.com/watch?v=bFNjxKHP8Jc
http://www.youtube.com/watch?v=JNqXgLKOzKU
Biotin tag
*&
!$&
SOLiD
Life Technologies
!"#$%&'()*+,)
13
2 Overview of Sequencing Technology Platforms
which the sequencing reactions begin. These high-throughput sequencing systems,
with the exception of PacBio RS, require amplifi cation of the sequencing library
DNA to form spatially distinct and detectable sequencing features (Fig.
2.3
).
Amplifi cation can be performed in situ, in emulsion or in solution to generate
clus-ters of clonal DNA copies. Sequencing is performed using either DNA polymerase
synthesis for fl uorescent nucleotides or the ligation of fl uorescent oligonucleotides
(Fig.
2.4
).
The high-throughput sequencing platforms integrate a variety of fl uidic and optic
technologies to perform and monitor the molecular sequencing reactions. The fl uidics
systems that enable the parallelization of the sequencing reaction form the core of the
high-throughput sequencing platform. Micro-liter scale fl uidic devices support the
DNA immobilization and sequencing using automated liquid dispensing
mecha-nisms. These instruments enable the automated fl ow of reagents onto the immobilized
Fig. 2.3 Generation of sequencing features. High-throughput sequencing systems have taken
different approaches in the generation of the detectable sequencing features. ( a ) Emulsion PCR is
applied in the GS FLX and SOLiD systems. Single enrichment bead and sequencing library fragment
are emulsifi ed inside an aqueous reaction bubble. PCR is then applied to populate the surface of
the bead by clonal copies of the template. Beads with immobilized clonal DNA collections are
deposited onto a Picotiter plate (GS FLX) or on a glass slide (SOLiD). ( b ) Bridge-PCR is used
to generate the in situ clusters of amplifi ed sequencing library fragments on a solid support.
Immobilized amplifi cation primers are used in the process. ( c ) Rolling circle amplifi cation is used
to generate long stretches of DNA that fold into nanoballs that are arrayed in the CGA technology.
( d ) Biotinylated DNA polymerase binds to bubble adapted template in the PacBio RS system.
Polymerase/template complex is immobilized on the bottom of a zero mode wave guide (ZMW)
*-
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Sequencing by Oligonucleotide Ligation and Detection
Life Technologies
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Amplific
ação
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e câmeras
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• 1 dNTP de cada vez
• ñ utiliza nucleotídeos modificados e cascatas enzimáticas
• ñ utiliza detecção óptica (fluorescência e
quimioluminescência)
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PacBio RS sequencer
Sequenciamento de terceira geração
Amplificação e
síntese
''&
Detecção de modificações epigenéticas
Custos
1Gb of data
'"&
Mecanismo
Constituintes Principais
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Commendations for Nature News & Comment in the 2012 Online Media Awards
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Related stories
NATURE | NEWS
Nanopore genome sequencer makes its debut
Technique promises it will produce a human genome in 15 minutes.
17 February 2012
Technology that its parent company says will sequence a human genome in just 15 minutes opened its
first data run to scrutiny today.
Oxford Nanopore Technologies, based in Oxford, UK, revealed the initial results from its GridION system
at the Advances in Genome Biology and Technology meeting in Marco Island, Florida. The firm expects
to start selling its new machine in the second half of this year and also plans to launch the world’s first
miniaturized, disposable sequencer — the MinION — which will retail for less than US$900.
Given its flexibility, scalability and low entry price, “this
technology could have a seriously disruptive effect on
the sequencing industry,” says Daniel MacArthur, a
geneticist who
blogs about the genomics industry.
That industry is already seeing significant jockeying for
position with Swiss drug giant Roche last month
launching a takeover bid for the manufacturer of the
sector’s dominant technology: Illumina of San Diego,
California (see
Roche takeover bid poses challenge to
Illumina
). In the same month, up-and-coming company
Ion Torrent Systems of Guilford, Connecticut, vowed to
begin selling a machine by the end of the year that can
sequence an entire human genome in a day for less
than $1,000 per sequence.And last April, Pacific Biosciences of Menlo Park, California, launched its own
sequencing technology.
Oxford Nanopore’s system uses nanopore sequencing to rapidly read DNA sequences. A strand of DNA
is fed through a biological pore and the various bases are identfied by measuring the difference in their
electrical conductivity as they pass through the pore (see
Personal genomes: Standard and pores
).
The launch of the nanopore machines marks the end of a
decades-long wait. Nanopore technology was first mooted in the early 1990s,
Fast track: nanopore sequencing identifies
individual bases as a strand of DNA is passed
through a pore.
IEMEDIA SOLUTIONS
