Towards DNA chip technology as a standard analytical tool for the identification of marine organisms...
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Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni- bremen.de Fish and Chips: microarray-based DNA-barcoding of European Marine Fishes Kochzius M 1 , Antoniou A 2 , Botla S 1 , Campo Falgueras D 3 , Garcia Vazquez E 3 , Hauschild J 1 , Hervet C 4 , Hjörleifsdottir S 5 , Hreggvidsson G 5 , Kappel K 1 , Landi M 6 , Magoulas A 2 , Marteinsson V 5 , Nölte M 7 , Planes S 4 , Seidel C 1 , Silkenbeumer N 1 , Tinti F 6 , Turan C 8 , Venugopal MN 9 , Weber H 1 , Blohm D 1 1 Centre for Applied Gene Sensor Technology (CAG), University of Bremen, Germany 2 Institute of Marine Biology and Genetics, Hellenic Centre for Marine Research, Greece 3 Universidad de Oviedo, Spain 4 Université de Perpignan, France
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Transcript of Towards DNA chip technology as a standard analytical tool for the identification of marine organisms...
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Fish and Chips: microarray-based DNA-barcoding 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, Germany2Institute of Marine Biology and Genetics, Hellenic Centre for Marine Research, Greece3Universidad de Oviedo, Spain4Université de Perpignan, France5Prokaria, Iceland6University of Bologna, Italy7Zentrum für Technomathematik (ZeTeM), University of Bremen, Germany8Mustafa Kemal University, Turkey9College of Fisheries, Mangalore, India
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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
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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.
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The principle of DNA chips (microarrays)
CATGT
CATGT
CATGT
GAGTA
GAGTA
GAGTA
AGCTG
AGCTG
AGCTG
Surface of the DNA Chip
C ATGATTACATCGAC
Probes
C ATGATTACATCGAC
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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
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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
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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 capture-
molecules
• Avoidance of cross hybridisation
• Hybridisation efficiency
• Secondary structure of target molecules
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Layout of the microarry
16S (46)
COI (150)
cyt b (123)
Blo
ck
Arr
ay
Mic
roar
ray
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Hybridisation experiments
Psetta maxima (16S) Psetta maxima (COI) Psetta maxima (cyt b)
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COI(150)
Pro
bes
cyt b(123)
16S(46)
Targets
16S (34) COI (42) cyt b (42)
DNA microarray experiments experiments
true positive
Signals
missingtrue positive
false positive
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Flu
ores
cenc
e si
gnal
(ar
brita
ry u
nits
)
16SCOI
cyt bprobes
cyt b
COI16S
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
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DNA microarray experiments (16S)
Boops b
oo
ps
(2)
Engra
ulis
encra
sic
olu
s (
2)
Helic
ole
nus
dact
ylopte
rus
(2)
Mela
nogra
mm
us
aegle
finus
(4)
Loph
ius b
ud
egass
a (
2)
Pagellu
s aca
rne (
2)
Sco
ph
thalm
us
rho
mbu
s (1
)S
com
ber
sco
mbru
s (
2)
Serr
anu
s cabrilla
(2
)S
paru
s aura
ta (
2)
Tra
churu
s tr
ach
uru
s (1
)Trigla
lyra
(2)
Dente
x dente
x (6
)D
iplo
dus
vulg
aris
(3)
Gadus
morh
ua (
2)
Merlangiu
s m
erlangu
s (6
)M
erlucc
ius m
erlucci
us
(1)
Mic
rom
esi
stiu
s po
tasso
u (
3)
Mullu
s s
urm
ule
tus (
2)
Polla
chiu
s polla
chiu
s (
2)
Polla
chiu
s vi
rens
(4)
Pse
tta
maxi
ma (
3)
Serr
anu
s he
patu
s (1
)
Tra
churu
s m
editerr
aneus (
1)
Tra
churu
s p
ictu
ratu
s (
2) Booboo_315 ( Boops boops)
Engenc_231 ( )Engraulis encrasicolusHeldac_317 ( )Helicolenus dactylopterusLopbud_312 ( )Lophius budegassaPagaca_317 ( )Pagellus acarneScorho_322 ( )Scophthalmus rhombusScosco_321 ( )Scomber scombrusSercab_313 ( )Serranus cabrillaSpaaur_201 ( )Sparus aurataTratra_333 ( )Trachurus trachurusTrilyr_232 ( )Trigla lyra
25,804 10,628
15
20 2040 27
40
21
30
34
40
21
2035
3540
40
4040
119
120
120120
120
120
60
60
6060 40
4080
80
6040
40
40
80
8080
8080
80
8060
60
15
20
20
20
20
4040
4040
40
40
60
40
80
40
Kochzius et al., submitted
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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
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Srujana Chitipothu et al. (poster) Towards microarray-based DNA-barcoding of marine invertebrates
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AcknowledgementThe “Fish & Chips” project is a Specific Targeted Research Project (STREP)
funded by the European Commission under the contract no. 505491
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Thank you very much for your attention!
