sarah stephenson csiro
TRANSCRIPT
LAND AND WATER
Sarah Stephenson| Research Project Officer
Metagenetic examinations of benthic eukaryl estuarine communitiesSMBE - Eukaryotic Omics 2013
Australian benthic eukaryl estuarine communities
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson2 |
• Diverse and complex.
• 85% Australian’s live on the coast.
• Ecosystem services: Fishing, aquaculture, water purification.
• Multiple stressors: Mining, agriculture, urbanization, waste water treatment
plants, climate change.
Correlative and experimental studies:
Identify OTUs that are:
• Strongly correlated to the environmental variables of interest.
• Weakly correlated. Pick up subtle differences in biotic structure. • Whose presence is random across space and time, or which are derived from exogenous sources.
copper
sand (%)
salinity
turbidity
copper
sand (%)
salinity
turbidity
How can we link ecosystem function and structure to disturbance models?
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson2
Ecological processes:RedundancyKeystoneIdiosyncratic
Limitations of using macrobenthic communities for environmental monitoring 25-40 relatively large
metazoan taxa
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson4
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson
What are we sequencing?• Complete analysis of the genomic constituents of a complex biological sample is
impractical for routine monitoring.
Chariton et al. (2010)Bik et al. (2012)
• Targeted DNA fragments• Ribosomal genes (rDNA) are required for translation (essential and universal). • 18S rDNA universal (many copies) in eukaryotes.• Sequences easily downloaded into sequence manipulation software.• Varying levels of taxonomic resolution. • Broad coverage. • Numerous approaches, with all DNA fragments having limitations.
Adaptor A Adaptor BBar-code Bar-codePrimer Primer
Forward Fusion primer Reverse Fusion primer
18S amplicon
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AGGTTCTTCAATCGGACCCCATTCTTCAATCGGACC
TTCTTCTTCAATCGGACC
CTTCACTTCAATCGGACCz
PCR to produce a pool of 18S rDNA fragments
DNA is extracted & purified
Pooled samples are sequenced
Chemical separation: samples assigned a unique barcode and pooled prior to analysis.
Gasket A
Gasket B
Post analysis assignment of sequences into a priori defined samples.
Treatment A
Treatment B
Treatment B rep1
Treatment A rep1
Treatment A rep2
Treatment A rep3
Treatment A rep4
Treatment B rep2
Treatment B rep3
Treatment B rep4
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson6 |
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson
South East Queensland Study
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Australian population growth projections
Area contains 3.05 million people.Projection is 8.7 million by 2056.
South East Queensland Estuaries
• Aim: Use high throughput sequencing to examine the benthic composition along five estuaries of varying ecological integrity.
• Examine whether molecular biodiversity assessments can be discriminated among estuaries.
• Identify whether molecular derived biotic composition is correlated with nutrients and other physico-chemical variables.
• Utilise estuaries currently being monitored under the EHMP’s Healthy Waterways project.
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson8 |
No Biodiversity indicators
9 | Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson
www.healthywaterways.org
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson
Estuary Score (2010)
Noosa B+
Maroochydore C
Pine C-
Currumbin C
Logan F
2010 catchment & estuary report card
C-
C
B+
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C
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson
Experimental design
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site 1 site 2 site 3 site 4 site 5
5 Sample grabsa b c d e
1. Noosa 3. Currumbin 4. Pine2. Maroochydore 5. Logan
EHMP sites sampled in 2010Physico-chemistry
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson
Bioinformatics and statistical method
Pissodes castaneus AJ850007 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAAATAG GCG------- -T---TTTTG ACATCCTAAA GGCCCGCCGA CGG------- G--------- --GG------ ---------- ---------- Chrysomela scripta AF267436 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGTG ATTTGTCTGG TTAATTCCGA TAACGAACGA GATTCTAGCC TGCTAACTAG GCGT------ -T---TTTCG ACATCCCAAA GGCCCGCCGG TTG------- A--------- --GG------ ---------- ---------- Plegaderus cf. nitidus AY02833 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAAATAG GCGT------ TA---ATTCG ACATCTCAAA GGCCCGCCGG CCC------- G--------- --GC------ ---------- ---------- Rhynchophorus cruentatus AF389 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGC------ -T---TTTTG ACATCCTAAA GGCCCGCCGG CCG------- G--------- --CG------ ---------- ---------- Ocypus opthalmicus AJ810739 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGTG ATCTGTCAGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGT-----A CT----TTCG ACATCTCAAA GGTCG----- ---------- ---------- ---------- ---------- ---------- Galerucella nympheae AF267443 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGT------ -A---TTTCG ACATCCCAAA GGCCCGTCGG TAT------- G--------- --GA------ ---------- ---------- Microsporus sp. AF427599 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGTG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGTTTGAGA ATTCAATCCG GTATCTATGA GGTGCGTGCA CGCGATCAGC TATGGGCTGA GAGAGCGCCG TCGGTGGCAT GGACCGTTGT Apotomus rufithorax AF012497 --TGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGATTG ATTTGTCTGC TTAATTGCGA TAACGAACGA GATTCTAGCC TGCTAAATAG GCGTT----- ACTTA--CCG GTATCTCGAA GATTCGCATC TCC------- --TGGTCGTT CAGCATGTTG TTCGTTGCAT GTGTGGGCGT Carabus nemoralis AF012507 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGT-----A CT---TTCCG GTATCTCGAA GGCCGACGTC TCT------- C--------- --GGGTTTCC GTTTG----- -----GCGCG Oregus aereus AF012500 GGTGGGTGGT GGTGCATG-C CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGTA----A CT---TACCG GTATCTCGAA GGCTCGCGTC TCT------- C--------- --GGTTCCCG CGT-----GT TGCGTTTGCG Lancetes varius AJ318684 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAAATAG GCGTT----A CT---TTCCG GCATCCCGAA GGCTCGCGTC ACC------- C--------- --GTTCGCC- ---------- -------GGT Hydraena iberica AJ810730 GGTGCGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGATCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGTT----T CT-----CCG ACATCTCAAA GGCCA----- ---------- ---------- ---------- ---------- ---------- Trechus sp. nr. chalybeus AF00 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGACCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGTT----A CT---TACCG GTATCCTGAA GGCTC--GTG TCT------- T--------- --TCGTATTC TGGCGTGCAT T-TGTCG--- Dalyat mirabilis AY926478 Cara ---GGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGTTAACTAG GCGTT----A CT---TACCG GTATCTCGAA GGCTCGCGTC TCT------- C--------- --GTATTCG- ---------- ---------- Diaprepes abbreviatus AY157729 GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGTA----A AA---TTTTG ACATCTTAAA GGCCCGCCGG CCG------- A--------- --CGCGGTGT GTGGCGTGCG TGCGCGGCGC Pachnaeus litus AY157730 Curcu GGTGGGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGTA----A AA---TTTTG ACATCTTAAA GGCCCGCCGG CCG------- A--------- --CGCGGTGT GTGGCGTGCG TGCGCGGCGC Meonis sp. AF398722 Harpalinae ----GGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TGCTAACTAG GCGTA----A CAAAATACCG GTATCTTGAA GGCTCCGACA CTT------- G--------- --CTTTTTGA TTTCATTCAC TGCATTTCTT Moriosomus seticollis AF398721 ----GGTGGT GGTGCATGGC CGTTCTTAGT TGGTGGAGCG ATTTGTCTGG TTAATTCCGA TAACGAACGA GACTCTAGCC TACTAAATAG ACGTT----A CACT-TACCA GTATCTCGAA GGCTC--ACA TCT------- C--------- --TCGTTTAC TTT---TCAC TATATAA---
E137_AY922318_GenBank (0.0068)A71_6 (0.0000)A22_11 (0.0034)A99_4 (0.0060)
A108_3 (-0.0034)A29_9 (0.0034)
A116_3 (0.0068)A30_9 (0.0000)
A137_2 (0.0000)A74_5 (0.0000)
A15_13 (0.0034)A129_2 (0.0000)
A111_3 (0.0034)A35_9 (-0.0034)
A113_3 (0.0000)A82_5 (0.0000)A85_5 (0.0000)A86_4 (0.0000)A122_3 (0.0000)
A123_3 (-0.0034)A63_6 (0.0034)A12_14 (0.0034)
A40_8 (-0.0034)A139_2 (-0.0034)
A83_5 (0.0034)A146_2 (0.0000)
A64_6 (0.0034)A8_18 (-0.0034)
A100_4 (0.0000)A135_2 (0.0068)
A96_4 (0.0051)A1_3756 (0.0000)
A101_4 (0.0069)A97_4 (0.0000)
A102_4 (0.0069)A103_4 (0.0034)A38_9 (0.0034)A104_4 (0.0034)A37_9 (0.0034)A105_4 (0.0034)A117_3 (0.0034)A106_3 (0.0000)A98_4 (0.0000)
A140_2 (0.0000)A6_22 (0.0000)
A107_3 (0.0069)A109_3 (0.0069)A10_16 (0.0069)A110_3 (0.0069)A112_3 (0.0069)A114_3 (0.0069)A115_3 (0.0069)A118_3 (0.0069)A119_3 (0.0069)A11_15 (0.0069)A120_3 (0.0034)A138_2 (0.0034)A121_3 (0.0069)A124_3 (0.0069)A125_3 (0.0034)A27_10 (0.0034)A126_3 (0.0034)A18_12 (0.0034)A127_3 (0.0069)A128_3 (0.0034)A84_5 (0.0034)A130_2 (0.0069)A131_2 (0.0069)A132_2 (0.0034)
A133_2 (0.0068)A2_67 (0.0000)A47_7 (0.0043)A134_2 (0.0034)A80_5 (0.0034)A136_2 (0.0051)
A41_8 (0.0205)A95_4 (0.0000)A13_14 (0.0069)A141_2 (0.0069)A142_2 (0.0069)A143_2 (0.0069)A144_2 (0.0069)A145_2 (0.0069)A14_14 (0.0069)A16_12 (0.0069)A17_12 (0.0069)A19_11 (0.0069)A20_11 (0.0034)A28_10 (0.0034)A21_11 (0.0069)A23_11 (0.0069)A24_10 (0.0069)A25_10 (0.0069)A26_10 (0.0069)A31_9 (0.0069)A32_9 (0.0069)A33_9 (0.0069)A34_9 (0.0069)A36_9 (0.0034)A45_7 (0.0034)A39_9 (0.0069)A3_34 (0.0069)A42_8 (0.0069)A43_8 (0.0069)A44_8 (0.0069)A46_7 (0.0069)A48_7 (0.0069)A49_7 (0.0069)A4_23 (0.0069)A50_7 (0.0069)A51_7 (0.0069)A52_7 (0.0069)A53_6 (0.0069)A54_6 (0.0069)A55_6 (0.0069)A56_6 (0.0069)A57_6 (0.0069)A58_6 (0.0069)A59_6 (0.0069)A5_23 (0.0069)A60_6 (0.0069)A61_6 (0.0069)A62_6 (0.0069)A65_6 (0.0069)A66_6 (0.0069)A67_6 (0.0069)A68_6 (0.0069)A69_6 (0.0069)A70_6 (0.0069)A72_6 (0.0069)A73_5 (0.0069)A75_5 (0.0069)A76_5 (0.0069)A77_5 (0.0069)A78_5 (0.0069)A79_5 (0.0069)A7_22 (0.0069)A81_5 (0.0069)A87_4 (0.0069)A88_4 (0.0069)A89_4 (0.0069)A90_4 (0.0069)A91_4 (0.0069)A92_4 (0.0069)A93_4 (0.0069)A94_4 (0.0069)A9_17 (0.0069)
CSIRO APDP
nMds, PERMANOVA,dbRDA, INDVAL (indicator analysis).
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De-noise & chimera clean up
Blast NCBI
Morgan et al., in review
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson
Results
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Phylum
• >1 million sequences produced, 731,992 retained producing 2,966 OTUs• All control sequences recovered with no artificial inflation of OTUs • Richness was significantly greater in Logan than the other locations • Only 1 OTU, Naviculale (diatom) observed in all 125 samples• Diatoms are the most widely distributed taxa (>80% of the samples)
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson
Results
Chlorophyta and nutrients?
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Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson
2D Stress: 0.13
full dataResemblance: S7 Jaccard
LocationNoosaMaroochydoorePineCurrumbinLogan
2D Stress: 0.13
Compositional comparisons among estuaries
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Estuary Score (2010)Noosa B+Maroochydore CPine C-Currumbin CLogan F
nMDS ordination plot of 18S rDNA derived benthic assemblages
PERMANOVA identified that all estuaries contained unique compositions
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson
-60 -40 -20 0 20 40
dbRDA1 (29.5% of fitted, 21.8% of total variation)
-40
-20
0
20
40
dbR
DA
2 (2
1%
of f
itted
, 15 .
6% o
f tot
al v
aria
tion) Noosa
Maroochydoore
Pine
Currumbin
Logan
Depth
Chlorophyll-a
Conductivity
Secchi depth
Ammonia
N (organic)
N (oxidised)
N (total)Dissolved oxygen
pHP (reactive)
P (total)
TemperatureTurbidity
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Relationships between biotic composition and the physico-chemical properties of the overlying waters (distance-based linear modelling)
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson
Bacillario
phyta
Alveolata
Amoebozoa
Chlorophyta
stramenopile
s
Unknown
Chytridiomyco
ta (Fungi)
Foraminife
ra
Porifera
Basidiomyco
ta(fungi)
Cercozoa
Gastopoda
Gastrotri
cha
Glomeromycota(Fu
ngi)
Arthropoda
Coleoptera
Blastocla
diomycota (F
ungi)
Centrohelio
zoa
Cnidaria (p
arasitic)
dinoflagellate
Flabellin
ea
Mucoromyco
tina (Fugi)
Oligoch
aeta
Rotifera
Sphagnum
Telonemida
Bacillario
phyta
Foraminife
ra
Ascomyco
ta (Fungi)
Cnidaria (p
arasitic)
Harpactico
ida
Platyhelm
inthes0
2
4
6
8
10
12
OTUs present in >80% of Logan samples and absent from the NoosaOTUs present in >80% of Noosa samples and absent from the Logan
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Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson
Where to from here?South East Queensland• Develop and test predictive models which clarify the
relationships between OTUs and environmental variables.
• 2012 Estuaries were re-sampled for biota and greater number of environmental parameters and sites were examined.
• Biosentinals: Identify key taxa which characterise and discriminate environments e.g. Kinorhyncha, Diatoms.
• Develop species specific PCR.• Integrate ecotoxicology.
• Integrate the structural and functional attributes of the systems.
-
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Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson
Metagenetics: Are we meeting the needs ecologists?
Challenges• Unknown eukaryotes.• Presence / absence only.• 18S copy number.• OTU confidence: Only as good as
database e.g. SILVA, NCBI.• Extracellular DNA• How many replicates? Rare taxa.
Solutions• Metagenomic and deeper sequencing.• Target ITS for Fungi.• Complimentary target CO1 for
eukaryotes.• Biological/ Technical replicates.• ‘Mock community’ positive control.• Microcosm and lab manipulation studies.
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CSIRO LAND AND WATER
CSIRO LAND AND WATER
Holly Bik, SMBE and UC Davis
Anthony Chariton
Andy Steven
CLW Lucas Heights and CSIRO North Ryde
CLW field crew Dutton Park
Mathew Morgan
Chris Hardy and Leon Court
Australian Genome Research Facility
The traditional owners of Jagera, Turrbal and Kallangur, SEQ
Acknowledgements
CSIRO Land and WaterSarah StephensonResearch Project Officert +61 2 9E [email protected]
www.csiro.au/loremw www.csiro.au
Thank you
CSIRO LAND AND WATER LUCAS HEIGHTS
Metagenetic examinations of benthic eukaryl estuarine communities| Sarah Stephenson
Results
• >1 million sequences produced, 731,992 retained producing 2,966 OTUs
• All control sequences recovered with no artificial inflation of OTUs
• Richness was significantly greater in Logan than the other locations
• Only one OTU, Naviculale (diatom) was observed in all 125 samples
• Diatoms were the most widely distributed taxa (>80% of the samples)
Streptophyta?
Chlorophyta and nutrients?
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Ascomycota and nutrients?