Nieuwe ontwikkeling voor het bepalen van de biodiversiteit en ecologische kwaliteit.

Marco Jaspers

Holger Cremer

Timme Donders

Frank Schuren

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Noodzaak: Europese Kaderrichtlijn Water

Monitoring, onderhoud en verbetering van de

ecologische kwaliteit van oppervlaktewateren verplicht

Behoefte aan een krachtig en betrouwbaar instrument

Gebaseerd op de samenstelling van indicator

organismen

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Doel van het Hydrochip project

Ontwikkeling van een op DNA gebaseerd meetsysteem waarmee snel de samenstelling van de diatomeeën populatie kan worden vastgesteld om op die manier de ecologische kwaliteit te kunnen vaststellen.

DNA Chip: - Generiek - Inzicht in Populatie

DNA EXTRACTIE

Markergen (b.v. toxine, 16S)

GENERIEKE PCR

Chip

ENKELSTRENGS MAKEN

HYBRIDISATIE (BINDING)

MASS SEQUENCING

Mass Sequencing: Generiek Inzicht in Populatie

Detectie van Waterorganismen via DNA

Voorbeeld van een research chip

Op zoek naar Markers: Open approach

Onbevooroordeeld , niet gebaseerd op bestaande kennis.

Focus op biodiversiteit en nieuwe markers

Monsters geselecteerd op basis van diversiteit

DNA isolatie gevolgd door mass sequencing

18s analysis for eukaryotes

16s analysis for prokaryotes

Frank Schuren

DNA targets

Eukaryoten (waaronder diatomeeën en meeste algen):

18s rRNA gen: aanwezig in alle eukaryoten, sterk

geconserveerd, veel gebruikt voor taxonomie

Prokaryoten (bacteriën):

16s rRNA gen: aanwezig in alle bacteriën, sterk

geconserveerd, veel gebruikt voor taxonomie,

interessant voor blauwalgen

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Flora analysis by 454 pyrosequencing

Characteristics Next generation sequencing technique

105-106 individual sequence runs in 10 hours!

Short sequence tags: 16s rDNA hypervariable regions

Advantages:

Provides very good view on composition complex flora

Probes based on relevant sequence data

Frank Schuren

Sampling diverse locations

Frank Schuren

Biodiversity at phylum level

8 4 0 6

1 5 1 4

1 2 9 9

9 0 1

7 8 9

2 6 21 9 2

1 6 21 4 91 3 09 36 85 74 23 73 53 33 23 13 03 02 72 42 01 41 41 39887766555544

Bacillar iophyta(1 0 0 ) Total D ikarya(1 0 0 ) Total

D inophyceae(1 0 0 ) Total Chrysophyceae(1 0 0 ) Total

O om ycetes (1 0 0 ) Total PX_clade(9 9 ) Total

Cryptom onadales (1 0 0 ) Total G lom erom ycota(1 0 0 ) Total

Raphidophyceae(1 0 0 ) Total Synurophy ceae(1 0 0 ) Total

Eustigm atophyceae(1 0 0 ) Total Chrysophyceae(9 6 ) Total

Cnidaria(1 0 0 ) Total D ikarya(9 8 ) Total

P inguiophyceae(9 8 ) Total Chrysophyceae(9 5 ) Total

D ikarya(9 7 ) Total Porifera(1 0 0 ) Total

PX_clade(1 0 0 ) Total Ciliophora(1 0 0 ) Total

Porifera(8 7 ) Total Raphidophyceae(9 3 ) Total

Synurophyceae(9 6 ) Total G lom erom ycota(9 5 ) Total

Apicom plexa(1 0 0 ) Total G lom erom ycota(9 3 ) Total

Perk insea(1 0 0 ) Total Chrysophyceae(8 9 ) Total

Porifera(6 3 ) Total Porifera(8 8 ) Total

Chrysophyceae(8 6 ) Total Raphidophyceae(7 2 ) Total

Arthropoda(1 0 0 ) Total Ciliophora(8 4 ) Total

Bacillar iophyta(6 1 ) Total Perk insea(8 0 ) Total

Streptophyta(1 0 0 ) Total Synurophy ceae(6 1 ) Total

Eustigm atophyceae(7 5 ) Total Synurophy ceae(7 5 ) Total

Frank Schuren

Most abundant diatom sequences

Organism blast Watertype M31 M08 R06 M12 R04 M01 M27 M27 M27 M07b

x Waterlocatie HC-DEK- HC-ETE- HC-GAL- HC-GER- HC-HEI- HC-TEW- HC-TPN- HC-TPZ- HC-TPZ- HC-VEC-

x sample ID 1_A4_HC-DEK-01.4

1_A6_HC-ETE-01.1

1_A9_HC-GAL-02.4

1_A11_HC-GER-01.1

1_B4_HC-HEI-01.1

1_C2_HC-TEW-02.6

1_C6_HC-TPN-02.4

1_C8_HC-TPZ-02.4

1_C9_HC-TPZ-03.4

1_C10_HC-VEC-03.4

x clusterseqs o4391 o4392 o4393 o4394 o4396 o4397 o4398 o4399 o4400 o4401

Grand Total 15683 240 324 361 32 454 588 645 64 92 1373

Synedra ulna Total 4068 7 2 2 9 402 44 16 3 0 2

Gomphonema affine Total 2112 3 253 35 0 8 6 13 5 0 22

Cocconeis pediculus Total 1435 0 0 15 0 0 0 4 0 0 1274

Stephanodiscus hantzschii Total 1389 0 18 2 0 0 13 0 0 6 0

Cyclotella meneghiniana Total 1276 0 12 0 3 4 1 0 3 0 0

Cyclotella cf. scaldensis G1W11 Total 1042 0 0 0 0 0 0 0 0 0 1

Navicula radiosa Total 924 0 0 2 4 0 337 515 27 34 3

uncultured eukaryote Total 535 3 1 60 5 0 2 4 0 5 6

Peridinium foliaceum endosymbiont Total 298 0 0 0 0 0 49 0 0 14 2

Nitzschia thermalis Total 223 177 0 0 0 0 0 0 0 0 0

Thalassiosira pseudonana Total 222 12 1 1 0 0 1 0 0 0 0

Achnanthidium minutissimum Total 166 0 0 6 0 0 3 38 0 5 3

Thalassiosira guillardii Total 137 0 0 2 0 0 0 0 3 4 1

Encyonema minutum Total 123 7 0 0 0 0 2 0 0 0 0

Synedra acus Total 123 0 0 0 0 2 14 0 0 3 2

Nitzschia palea Total 100 0 8 0 0 0 4 0 0 4 0

Cyclotella sp. CCMP331 Total 92 0 0 0 0 0 0 0 0 0 0

uncultured diatom Total 83 0 0 26 0 5 3 11 0 0 21

Navicula cryptocephala Total 77 0 0 1 1 2 7 0 0 3 0

Planothidium lanceolatum Total 73 0 0 55 1 0 0 0 0 0 0

Cymatopleura elliptica Total 61 0 0 0 0 0 0 0 0 0 0

Eunotia implicata Total 58 0 0 0 0 0 0 0 0 0 0

Nitzschia sigma Total 47 0 0 1 0 0 1 0 0 0 0

Frank Schuren

Samenvatting Open Approach

• Biodiversiteit kan door sequencing in kaart worden gebracht

• Vooral op phylum niveau

• Geen makkelijke link naar soorten

• Heel veel nieuwe sequenties: hoe hier mee om te gaan?

• De technologie is relatief duur en arbeidsintensief: nog geen

routine

Frank Schuren

Searching for Markers: Closed approach

To be able to compare results from molecular and

microscopical analyses linking both datasets is essential

For most indicator organisms currently no reliable DNA

sequences available in databases!

Unfortunately collecting living diatoms is not a standard

approach as it is for many other living organisms.

Therefore alternative approaches have been evaluated

(culturing, single-cell analysis)

Frank Schuren

Attempts into experimental hydrobiology

Culturing diatoms is not trivial

Little expertise available, nearly no

publications/books

Expertise not present in NL, but is present in Gent

Collaboration started with Wim Vijvermans and

Pieter van Ormelingen, UGent

technician TNO visited Gent for few days

After some initial problems culturing started to work

Frank Schuren

Experiences culturing diatoms

Culturing diatoms is feasible

Protocols are available for certain well-studied genera

Growth is slow

In our experience obtaining a diverse collection of species is

very difficult: some species are encountered all the time,

whereas many others get easily lost

Considering the amount of time and effort in relation to the

outcome we decided to quit this path

Frank Schuren

Micromanipulator for single-cell analysis

Frank Schuren

Some examples

Frank Schuren

Approach used

• Pick single diatoms with micromanipulator

• Put cell in eppendorf cup and perform PCR amplification of

specific gene fragment

• Perform a second nested PCR reaction to obtain sufficient

material for sequencing

• Sequence analysis through Sanger sequencing

• Collect diatom from samples

• Embed single diatom

• Perform species determination through microscopy

• Link DNA-sequence to species name

Frank Schuren

Results single-cell analysis

• The good news is: it worked!

• The bad news is: it is very laborious and success rate is low

• Complex protocol with failures at each step:

• Capillary size/diatom size

• Getting diatom out of capillary into eppendorf cup

• Obtaining PCR product

• Retrieving diatoms

• Orientation embedded diatoms

• Ability to determinate single valves

• In total 600 DNA –sequences were determined!

Frank Schuren

Wish list Herman van Dam: 150 most abundant species in NL

Nr

%monster

s

gemiddelde

abundantie (%)

cumulatieve

abundantie (%) Taxon zout

1 67,33 15,25 15,25 Achnanthidium minutissimum zoet

2 64,11 9,73 24,98 Cocconeis placentula s.l. zeer licht brak

3 70,95 4,84 29,81 Gomphonema parvulum zoet

4 46,92 2,62 32,44 Rhoicosphenia abbreviata zeer licht brak

5 35,39 2,62 35,06 Eunotia bilunaris zeer zoet

6 37,55 2,18 37,24 Nitzschia paleacea zoet

7 50,73 1,81 39,05 Nitzschia palea zoet

8 38,07 1,78 40,83 Gomphonema olivaceum zoet

9 42,39 1,64 42,47 Navicula gregaria zeer licht brak

10 38,07 1,56 44,03 Eolimna minima zoet

11 8,91 1,48 45,51 Eunotia exigua [1] zeer zoet

12 32,92 1,41 46,92 Amphora pediculus zoet

13 24,60 1,17 48,09 Melosira varians zoet

14 27,28 1,14 49,23 Nitzschia frustulum zeer licht brak

15 23,39 1,14 50,36 Diatoma tenuis zeer licht brak

16 19,17 0,95 51,32 Fragilaria famelica zeer licht brak

17 29,85 0,93 52,25 Tabularia tabulata-groep licht brak

18 20,09 0,88 53,13 Fragilaria capucina s.l. zoet

19 10,05 0,88 54,01 Tabellaria flocculosa zeer zoet

20 34,71 0,86 54,87 Navicula cryptotenella zoet

Frank Schuren

Hydrochip analyses

• In 2010 and 2011 several hundreds of samples were

analyzed by microscopy and Hydrochip analysis

• Because of differences in species coverage direct comparison

of results was not yet possible

• Molecular analysis shows species not detected by microscopy

and vice versa

• To enable comparison of data we turned to an ecological

parameter comparison: trophy calculation

Frank Schuren

Comparison results after translation to eutrophy level

phytoplankton phytobenthos

Frank Schuren

Current situation

Translation of data to ecological parameters works reasonably

well (except for oligotrophic samples for which indicator

species sequences are lacking)

Current research chip is translated to routine chip containing

about 150 reliable probes

Implementation of technology will be part of new EU-Life

project which most likely will start later this year

In this project we want to make the translation from prototype

to implementation (Vitens)

Frank Schuren

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Van Diatomee naar Ecologische Kwaliteit

Trofiegraad

Sample preparation

DNA Isolatie

PCR,

DNA labelling

Binding op chip

Data Analyse

Vertaalslag

HYDROCHIP

Frank Schuren

Toekomst: Opschaling naar verschillende type indicatororganismen mogelijk

Naast diatomeeën Waterplanten Macro evertebraten Blauwalgen Meerdere indicatoren geven mogelijk een gedetailleerder beeld van de ecologische kwaliteit

Verdere toekomst: Lab on a Chip Automatisering en miniaturisering van: - monstervoorbewerking - DNA isolatie en labelling - DNA binding op chip en detectie - Analyse en interpretatie Mogelijkheid tot online monitoring van waterkwaliteit

Voordelen Hydrochip in de toekomst:

Objectiever want gestandaardiseerde en op termijn

geautomatiseerde methode

Analyse van groot aantal relevante organismen in

ecosysteem

Snel resultaat: dus snel sturing op basis van analyse

resultaten mogelijk

Intensievere analyses mogelijk (meer tijdstippen,

meer locaties etc)

Goedkoper dan huidige analyses

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Partners:

STOWA: Bas vd Wal, Tessa vd Wijngaart TNO: Frank Schuren, Holger Cremer, Timme Donders, Jordy Coolen,

Jolanda Kool, Heleen Koolmees, Marco Jaspers

Vitens: Bendert de Graaf, Mark Kiestra, Adrie Atsma, Wouter v Delft

Hoogheemraadschap Hollands Noorderkwartier Gert van Ee

Waternet Ron vd Oost

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31

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Van researchchip naar eerste praktijkchip

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DNA bindingspatroon (Legionella chip)

Toekomst: Opschaling in aantal samples mogelijk

Comparison current and new approach

Frank Schuren

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Workflow

Microbial flora

analysis by 454

sequencing

microtiter format

DNA isolation and

labeling

multi-well chip printing

multi-well hybridization

scan results for

data interpretation

Automated selection

microarray probes

Samples array development and analysis

DNA isolation DNA isolation array development sample analysis

Data analysis and interpretation

analysis array development

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0,0

1,0

2,0

3,0

4,0

5,0

6,0

0,0 1,0 2,0 3,0 4,0 5,0 6,0

Hab

itat

typ

e h

yd

roch

ip

Habitat type microscopy

Additional results for free: Cyanobacteria

Frank Schuren

Cyanobacteria abundance during the season

Frank Schuren