Post on 18-Mar-2020
Nieuwe ontwikkeling voor het bepalen van de biodiversiteit en ecologische kwaliteit.
Marco Jaspers
Holger Cremer
Timme Donders
Frank Schuren
2
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
3
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
8
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
24
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
29
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
30
31
32
Van researchchip naar eerste praktijkchip
33
DNA bindingspatroon (Legionella chip)
Toekomst: Opschaling in aantal samples mogelijk
Comparison current and new approach
Frank Schuren
37
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
38
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