Download Folie 1 - Indentifying Species with DNA Barcoding

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Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
Fish and Chips: microarray-based DNAbarcoding of European Marine Fishes
Kochzius M1, Antoniou A2, Botla S1, Campo Falgueras D3, Garcia Vazquez E3,
Hauschild J1, Hervet C4, Hjörleifsdottir S5, Hreggvidsson G5, Kappel K1, Landi
M6, Magoulas A2, Marteinsson V5, Nölte M7, Planes S4, Seidel C1,
Silkenbeumer N1, Tinti F6, Turan C8, Venugopal MN9, Weber H1, Blohm D1
1Centre
for Applied Gene Sensor Technology (CAG), University of Bremen, Germany
2Institute of Marine Biology and Genetics, Hellenic Centre for Marine Research, Greece
3Universidad de Oviedo, Spain
4Université de Perpignan, France
5Prokaria, Iceland
6University of Bologna, Italy
7Zentrum für Technomathematik (ZeTeM), University of Bremen, Germany
8Mustafa Kemal University, Turkey
9College of Fisheries, Mangalore, India
www.fish-and-chips.uni-bremen.de
Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
Environmental problems, biodiversity, and ecosystem
functioning in European Seas
• Biodiversity and ecosystems of European Seas are under
anthropogenic induced pressure, such as pollution, eutrophication,
coastal construction, and fishery overexploitation
• Compared to terrestrial ecosystems very little is known about marine
biodiversity and changes in species richness and ecosystem
function
• This is mainly due to sampling difficulties and problems in taxonomy
• There are only few scientific specialists for several groups of marine
organisms, including phytoplankton, invertebrates, as well as eggs
and larvae of fishes
www.fish-and-chips.uni-bremen.de
Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
DNA chips as a new tool for marine species
identification in biodiversity and ecosystem research
• DNA-based identification methods are established as powerful
tools and the following marine animals have been investigated:
(1) eggs, larvae and adults of fishes
(2) planktonic copepods
(3) invertebrate larvae
(4) prey in gut content or faeces of penguins, whales, and fishes
• most of these methods allow to handle only single or a few species
at the same time
• DNA microarrays are believed to have the potential of identifying
hundreds of species in parallel and to differentiate them against an
even larger number of related species.
www.fish-and-chips.uni-bremen.de
Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
The principle of DNA chips (microarrays)
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Probes A
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Surface of the DNA Chip
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www.fish-and-chips.uni-bremen.de
Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
The principle of DNA chips (microarrays)
Microarray (glass slide): hundreds of spots with probes
Spots of probes with different
signal intensities after scanning
with a fluorescence scanner
www.fish-and-chips.uni-bremen.de
Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
Sampling, sequencing, and probe design
• Sampling: all European Seas
• Sequencing of partial mtDNA genes:
COI: 532 sequences, 66 species
cyt b: 434 sequences, 41 species
16S: 479 sequences, 79 species
• Probe design:
COI: 455 bp, 470 sequences, 47 species
cyt b: 404 bp, 281 Sequences, 43 species
16S: 418-452 bp, 404 sequences, 46 species
• In silico testing against several hundred “background” sequences
from sequence data bases
www.fish-and-chips.uni-bremen.de
Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
Probe design (www.miconet.uni-bremen.de)
Considered parameters in the design of
oligonucleotide libraries:
• Standard features, such as length, Tm, GC
content
• Sensitivity and specificity
• Secondary structure of the DNA capturemolecules
• Avoidance of cross hybridisation
• Hybridisation efficiency
• Secondary structure of target molecules
Array
16S (46)
Block
COI (150)
Microarray
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Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
Layout of the microarry
cyt b (123)
www.fish-and-chips.uni-bremen.de
Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
Hybridisation experiments
Psetta maxima (16S)
Psetta maxima (COI)
Psetta maxima (cyt b)
Probes
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Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
DNA microarray experiments experiments
Targets
16S (34) COI (42) cyt b (42)
16S
(46)
COI
(150)
cyt b
(123)
Signals
true positive
missing
true positive
false positive
16S
COI
probes
cyt b
cyt b
COI
16S
tar
ge
ts
Fluorescence signal (arbritary units)
www.fish-and-chips.uni-bremen.de
Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
DNA microarray experiments
• 16S: strong true-positive signals; only a few false-positive signals
• COI: strong true-positive signals; many false-positive signals
• cyt b: weak true-positive signals, many missing; false-positive signals
Boops boops (2)
Engraulis encrasicolus (2)
Helicolenus dactylopterus (2)
Lophius budegassa (2)
Pagellus acarne (2)
Scophthalmus rhombus (1)
Scomber scombrus (2)
Serranus cabrilla (2)
Sparus aurata (2)
Trachurus trachurus (1)
Trigla lyra (2)
Dentex dentex (6)
Diplodus vulgaris (3)
Gadus morhua (2)
Melanogrammus aeglefinus (4)
Merlangius merlangus (6)
Merluccius merluccius (1)
Micromesistius potassou (3)
Mullus surmuletus (2)
Pollachius pollachius (2)
Pollachius virens (4)
Psetta maxima (3)
Serranus hepatus (1)
Trachurus mediterraneus (1)
Trachurus picturatus (2)
signal (ar
Fluorescence
25,804
35
21 20
20
40
34
35 119
20
40
30 40
40 40
40
15
120
40
27
60
120
21
120
60
120
60
40
120 60 40 80
80
Ta r ge t
s pe c ie
40
80
60 40
60
40
40
No n -ta
r g et s p
ec ie
80 60
80
80
s
60
80
80
80
80
Trilyr_232 ( Trigla lyra)
20 40 Tratra_333 ( Trachurus trachurus)
Spaaur_201 ( Sparus aurata)
15
40 Sercab_313 ( Serranus cabrilla)
Scosco_321 (Scomber scombrus )
20
Scorho_322 ( Scophthalmus rhombus)
40
40
Pagaca_317
( Pagellus acarne)
20
Lopbud_312 ( Lophius budegassa)
40
Heldac_317 ( Helicolenus dactylopterus)
20
40 Engenc_231 ( Engraulis encrasicolus)
Booboo_315 ( Boops boops)
Prob
es
bitrary units)
www.fish-and-chips.uni-bremen.de
Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
DNA microarray experiments (16S)
10,628
40
s
Kochzius et al., submitted
www.fish-and-chips.uni-bremen.de
Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
Conclusions
• A single marker system seems to be not sufficient to design
oligonucleotide probes for DNA microarrays
• Therefore, it is recommended to utilise several markers for the
genetic identification of fishes with DNA microarrays
• Nevertheless, identification of fishes is possible with DNA
microarrays, but probes have to be tested intensively in
hybridisation experiments
www.fish-and-chips.uni-bremen.de
Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
Srujana Chitipothu et al. (poster) Towards microarraybased DNA-barcoding of marine invertebrates
www.fish-and-chips.uni-bremen.de
Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
Acknowledgement
The “Fish & Chips” project is a Specific Targeted Research Project (STREP)
funded by the European Commission under the contract no. 505491
www.fish-and-chips.uni-bremen.de
Towards DNA chip technology as a standard analytical tool for the
identification of marine organisms in biodiversity and ecosystem research
Thank you very much for your
attention!