naslednja (next) in tretja generacija visokozmogljivega

NASLEDNJA (NEXT) IN TRETJA GENERACIJA VISOKOZMOGLJIVEGA
SEKVENCIRANJA
Naslednja generacija (HTS-NG) – PCR amplifikacija za ojačitev signala
- Sekveniranje z amplifikacijo na kroglicah (Roche/454FLX)
- Sekvenciranje s sintezo (Illumina/Solexa Genome analyzer)
-Sekvenciranje z ligacijo (Applied Biosystems SOLID System)
Tretja generacija – sekvenciranje brez ampifikacije
-
Heliscope (tSMS – true single molecule sequencing, 2007)
-
SMRT (single molecule real time sequencer)
-
RNAP (single molecule real time sequencer)
-
Nanopore DNA sequencer
-
Ion Torrent DNA sequencer
SEKVENCIRANJE DNA
http://universe-review.ca/R11-16-DNAsequencing.htm
NASLEDNJA GENERACIJA SEKVENCIRANJA NG-HTS
Skupne lastnosti NG-HTS:
-Kompleksnost encimskih reakcij, kemije, programske in strojne opreme,
optike, itd.
-Premočrtna priprava knjižnic (vzorcev) pred sekvenciranjem.
-Priprava knjižnic fragmentov DNA s prileganjem za platformo značilnih
podaljškov (angl. Linker) in PCR amplifikacija.
-PCR amplifikacija enoverižnih fragmentov knjižnice in sekveniranje
pomnoženih fragmentov. (Roche – emulzijske kroglice; ABI – kloni kroglic; illumina
– kloni mostičkov)
General concepts for clonal-array generation and sequencing
a | Bead-chips. Genomic DNA is fragmented and adaptors are ligated to create an insert library that is
flanked by two universal priming sites. Because of the random fragmentation, the complexity of this
signature sequence library is equivalent to the genome. This library is cloned on beads using emulsion
PCR technology. A water-in-oil emulsion is created from a PCR mix that contains a limiting dilution of
DNA and beads. The emulsion creates micro-compartments with, on average, a single bead and single
DNA template each. After PCR, beads with clones are affinity selected and assembled onto a planar
substrate. A subsequent cycle-sequencing reaction is used to read out the sequence on the clones.
(Roche).
b | Sequencing by synthesis (SBS). A common anchor primer is annealed to a constant sequence
(universal priming site) that is contained within the library clones that are located on the polony (clonal
bead) array (the orientation of the immobilized target might vary depending on the platform that is used).
The sequence is read out by polymerase extension in a base-by-base fashion using either reversible
terminators or sequential nucleotide addition (pyrosequencing). After incorporation of a single base or
base type, the incorporated base is identified by fluorescence (laser) or chemiluminescence (no laser
required) (Ilumina-Solexa).
c | Sequencing by ligation. The polony array set-up is similar to SBS in which a common primer is
annealed to an arrayed polony library and used to read out the sequence through a stepwise ligation of
random oligomers. The labelled oligomers are designed to have random bases inserted at every site
except the query site. The query site has one of four base substitutions, each matched to a particular
fluorescent label on the oligonucleotide. After read-out of each ligation event, the primer and the ligated
oligomer are stripped, a new primer reannealed and the process repeated with an oligomer that contains
a query base at a different position (ABI).
Fan et al. Nature Reviews Genetics 7, 632–644 (August 2006) | doi:10.1038/nrg1901
PRIMERJAVA PLATFORM ZA NG-HTS
Splošni koncepti priprave klonalnih mrež in sekveniranje
Bead chips
Sequencing by
synthesis
Sequencing by
ligation
Fan et al. Nature Reviews Genetics 7, 632–644 (August 2006) | doi:10.1038/nrg1901
Roche/454 FLX Pyrosequencer
Library fragments are mixed with agarose
beads with oligos complementary to
adapter sequences on the library.
Each bead is associated with a single
fragment.
Each fragment-bead complex is isolated
into individual oil:water micelles with PCR
mixture.
Thermal cycling of this emulsion PCR of
the micelles produces amplified unique
sequences on the bead surface.
“En mass” sequencing of PCR products
on picotiter plates (PTP) with single beads
in each picowell.
Enzyme/substrate containing beads for
the pyrosequencing reaction are added to
wells that act as floww cells for addition of
individual pure nucleotide solutions. The
CCD camera records the light emitted at
each bead.
Mardis E.R. Annual Review of Genomics and Human Genetics 9: 387-403 (2008).
Principles of Pyrosequencing
Watsonov and Crickov izpis po pirosekveniranju
Timeline of the pyrosequencing development
October 2005 Release of the Genome Sequencer 20, the first next-generation sequencing
system on the market
October 2005 Collaboration agreement signed with Roche Diagnostics
.
January 2007 Release of the Genome Sequencer FLX System
March 2007 Roche Diagnostics completes integration with 454 Life Sciences
May 2007 Complete sequence of Jim Watson published in Nature. First genome to be
sequenced for less than $1 million.
November 2007 Announcement of the 100th peer-reviewed publication enabled by 454
Sequencing
June 2008 454 Joins the 1000 Genome Project, an international effort to build the most detailed
map to date of human genetic variation as a tool for medical research
September 2008 Announcement of the 250th peer-reviewed publication enabled by 454
Sequencing
October 2008 Release of Genome Sequencer FLX Titanium Series reagents, featuring 1
million reads at 400 base pairs in length
illumina sequencing technology
is based on arrays of randomly assembled glass (silica) beads;
the beads have oligonucleotides covalently attached to the surface;
each bead has about one million oligos on its surface;
all oligos on each bead have the same sequence
Attached DNA fragments are extended and bridge amplified to create an ultra-high density
sequencing flow cell with 80-100 million clusters, each containing ~1,000 copies of the same template.
These templates are sequenced using a robust four-color DNA sequencing-by-synthesis technology
that employs reversible terminators with removable fluorescent dyes. This novel approach ensures
high accuracy and true base-by-base sequencing, eliminating sequence-context specific errors and
enabling sequencing through homopolymers and repetitive sequences.
the beads are randomly assembled on the arrays, and the location of a particular probe is initially
unknown;a process called decoding is used to find the location of each bead;
Illumina sequencing by synthesis
Illumina sequencing by synthesis
ABI- Ligation mediated
sequencing
Mardis E.R. Annual Review of Genomics and
Human Genetics 9: 387-403 (2008).
Structure of detector oligonucleotides
First two nucleotides determine the colour of the
fluorophore. Colour table show the relationship
between dinucleotides and fluorophores.
Four different dinucleotides (256 different
oligonucleotides) correspond to each fluorophore.
If first or second nucleotide (in dinucleotide) is known,
colour is unambiguously related with the other
nucleotide.
Three next positions — degenerate nucleotides: 64
different versions for each particular dinucleotide.
When ligated to the sequencing primer, only one from
these 64 versions would fit to the position.
Detector oligonucleotides (DO) are 8-mers
fluorescently labeled on 3' end. DO's can't be too
short, otherwise T4 ligase would not recognize
them as a substrate.
Altogether, there are 1024 different detection
oligos: (dinucleotide + 3 degenerate)4=54.
Three last positions: universal bases, they are the
same for all detector oligonucleotides.
Dark oligonucleotides have the same internal
structure, but have no fluorophores.
seq.molbiol.ru/sch_seq_ligase.html
The principles of 2-base encoding/decoding
Mardis E.R. Annual Review of Genomics and Human Genetics 9: 387-403 (2008).
TRETJA GENERACIJA SEKVENCIRANJA
- Heliscope (tSMS – true single molecule sequencing, 2007)
- SMRT (single molecule real time sequencer)
- RNAP (single molecule real time sequencer)
- Nanopore DNA sequencer
- Ion Torrent DNA sequencer
TRETJA GENERACIJA SEKVENCIRANJA
tSMS (Heliscope) – PRAVO SEKVENCIRANJE POSAMEZNIH MOLEKUL
-
Priprava knjižnice z naključno
razgradnjo DNA
Dodajanje poli-A repov
Hibridizacija z oligo-dT v pretočnih
celicah
Sekvenciranje z dodajanjem
posameznih fluorescentnig
nukleotidov
Odčitavanje signala
Odčitki dolgi 55 bp, 8 dni za 28 Gb
v enem zagonu reakcije
tSMS (Heliscope) – ODČITAVANJE SIGNALA PRI SEKVENCIRANJU
POSAMEZNIH MOLEKUL
Slika, ki jo podaja HeliScope molekulski sekvenator. V povečavi je prikazana
molekula DNA, ki je v tem ciklu vključila nukleotid “G”.
http://www.helicosbio.com/Technology/TrueSingleMoleculeSequencing/tabid/64/Default.aspx
SMRT (single molecule real time sequencer)
SMRT celice, od katerih vsaka vsebuje tisoč “zero-mode waveguides” (ZMW).
Ena molekula DNA polimeraze je pripeta na dno vsakega ZMV, kar omogoča
opazovanje polimerizacije posamezne molekule DNA polimeraze.
Opazujemo sintezo novonastale molekuče DNA s fluorescentno označenimi
dNTP.
SEKVENCIRANJE Z NANOPORAMI
Ni barvnega označevanja nukleotidov in CCD detekcije.
Izrablja transokacijo DNA čez nanopore in s tem povezane odčitke električnega
signala. Nukleotid blokira ionski tok čez nanoporo – vsak nukleotid ima drugačno
časovno periodo blokiranja!
.
Nanopora: alfa-hemolizin, kovalentno vezan na molekulo ciklodekstrina
ION TORRENT TEHNOLOGIJA SEKVENCIRANJA
PostLight™ tehnologija sekvenciranjem na polprevodniških čipih.
Pri detekciji niso potrebni optični instrumenti.
Direktna povezava med kemično in digitalno informacijo.
MEHANIZEM VGRAJEVANJA NUKLEOTIDOV PRI SEKVENCIRARNJU
Z ION TORRENTOM
V naravi vgraditev vsakega nukleotida v verigo DNA vodi do sprostitev
vodikovega protona (H+).
Ion Torrent vsebuje visoko-gostotno mrežo mikroaparatur v žepkih
(luknjicah), kjer se proces vgrajevanja nukelotidov dogaja masivno in
paralelno.
Vsak žepek vsebuje svojo molekulo DNA. Pod žepkom je za ione občutljiva
plast (senzor), ki beleži pH spremembe zaradi sproščenih protonov.
Ker se nukleotidi dodajajo zaporedno, vemo, zaradi katerega nukleotida je
prišlo do spremembe pH.
BELEŽENJE SIGNALA PRI VGRAJEVANJU NUKLEOTIDOV
UPORABA NASLEDNJE GENERACIJE SEKVENCIRANJA
HTS-NG od leta 2005 dalje revolucija v raziskavah genomov (tudi rastlinskih, živalskih,
človeškega)
V letu 2003 je NHGRI (National Human Genome Research Institute napove 100-kratno
znižanje cene na bp v 5 letih in v 10 leith 10 000-kratno znižanje cene na bp, kar bi
privedlo do “1000 $ genoma”.
SNP genotyping
illumina
CNV genotyping
Fan et al. Nature Reviews Genetics 7, 632–644 (August 2006) | doi:10.1038/nrg1901
RNA-seq
-sekvencriranje cDNA z NG-HTS
- Sekvenciranje RNA direktno s tretjo generacijo
Chip-seq
Povzetek
• Nova generacija visokozmogljivostnega sekvenciranje omogoča razpoznavanje zaporedij
DNA na ravni celega genoma, z resolucijo posameznega baznega para.
• Iz vsakega vzorca se pripravi z adaptorji ligirana knjižnica, ki vsebuje vse v vzorcu
prisotne fragmente DNA ali RNA (cDNA).
• Vse platforme bazirajo na ligaciji adaptorjev in pomnoževanju, imajo pa različne pristope
sekvenciranja:
-Pirosekvenciranje (Roche-Nimblegen)
-Sekvenciranje s sintezo (Illumina-Solexa)
-Sekvenciranje z ligacijo (ABI)
•Razvile so se tudi metode, ki pred sekvenciranjem ne potrebujejo pomnoževanja.
Bionformatics
www.gwumc.edu
http://bioinformatics.ubc.ca/about/what_is_bioinformatics/images/computer.gif
Bioinformatics
Wikipedia
Making sense of the huge amounts of DNA data produced by gene sequencing
projects.
Bioinformatics and computational biology involve the use of techniques from
applied mathematics, informatics, statistics, and computer science to solve biological
problems.
Research in computational biology often overlaps with systems biology.
Major research efforts in the field include sequence alignment, gene finding, genome
assembly, protein structure alignment, protein structure prediction, prediction of gene
expression and protein-protein interactions, and the modeling.
The terms bioinformatics and computational biology are often used interchangeably,
although the former typically focuses on algorithm development and specific
computational methods, while the latter focuses more on hypothesis testing and
discovery in the biological domain.
Bioinformatics
More hypothesis-driven research in computational biology.
More technique-driven research in bioinformatics.
A common thread in projects in bioinformatics and computational biology is the
use of mathematical tools to extract useful information from noisy data
produced by high-throughput biological techniques.
A representative problem in bioinformatics is the assembly of high-quality DNA
sequences from fragmentary "shotgun" DNA sequencing.
In computational biology, a representative problem might be statistical testing of
a hypothesis of common gene regulation using data from mRNA microarrays or
mass spectrometry.