Bruce Deagle and Simon Jarman
FURTHER FACTS FROM FAECES: Dietary DNA Barcoding Using High Throughput Sequencing
“Statistics show that of those who contract the habit of eating, very few survive.”
George Bernard Shaw
From Trites and Donnelly (2003) Mammal Review 33:3-28
Decline of Steller sea lions
Why study animal diet?
Unicellular Organisms
Copepods Krill Salps Amphipods
Demersal fish Pelagic fish Squid
Seals
Baleen
whales
Flighted
birds
Toothed whales
Penguins
Different approaches to studying diet
1. Observe feeding
2. Tissue sample
3. Collect gut contents
4. Collect faeces identify hard-parts
‘the one-eyed man is king in the land of the blind’
Captive Feeding Trial
• Reliability of prey DNA recovery from
faeces?
• Persistence of genetic signal?
• Quantitative estimates of diet?
• Quality of prey DNA?
Steller sea lions
(Eumetopias jubatus)
Pacific Herring (Clupea pallasii )
Squid (Loligo sp.)
Surf Smelt (Hypomesus pretiosus)
Sockeye Salmon (Oncorhynchus nerka )
Feeding Trial - Methods
- DNA was extracted from soft
material (n=108)
- Sea lion DNA was dominant
component
Pulse prey items
Group Specific PCR
Jarman et al. (2004) Molecular Ecology 13:1313-1322
• Reliable prey DNA detection
• Each species has a equal chance
of being detected, squid (6% of
diet) is consistently detected
Frequency of detection
for basic dietary items
% prey positives
(n=108)
Squid 94%
Herring 94%
Smelt 92%
Salmon 87%
Mean 92%
• DNA from pulse prey items turned
up in faeces produced between 12
and 48 hours after ingestion
Deagle et al. (2005) Molecular Ecology 14:1831-1842
Feeding Trial - Results
Quantitative Estimates
Do proportions of DNA in faeces reflect intake?
Deagle and Tollit (2007) Conservation Genetics 8: 743-747
• Prey DNA in faeces
from 10 diets fed to
Steller sea lions.
Herring
Eulachon
Squid
Rockfish
4 Diet Items
Proportion in diet by mass
Pro
port
ion o
f D
NA
in F
aeces (
qP
CR
)
Bowles et al. (2011) Molecular Ecology Resources 11: 530-540
Herring DNA- Scat 1
0 50 100 150 200 250 300
05
00
01
00
00
15
00
02
00
00
25
00
0
PCR product size
Co
py N
um
be
rPrey DNA Quality
Deagle et al. (2006) Frontiers in Zoology e3:11
Co
py
nu
mb
er
PCR product size
- Mini-barcodes best
(< 200 bp)
0 50 100 150 200 250 300
05
00
01
00
00
15
00
02
00
00
25
00
0
PCR product size
Co
py N
um
be
r
F(x)= αe-λ x
Herring DNA- Scat 1
Deagle et al. (2006) Frontiers in Zoology e3:11
Prey DNA Quality
λ = probability of a break
PCR product size
Co
py
nu
mb
er
- Allows quantification of
DNA damage
Passmore et al. (2006) Marine Biotechnology
Deagle et al. (2007) PLoS ONE
Casper et al. (2007) Marine Biology
Dunshea (2009) PLoS ONE
Marguilies et al. 15 September 2005: Volume 437: 376-380
Next generation sequencing
Australian fur seal (Arctocephalus pusillus doriferus)
Fur Seal Diet Questions
• Are more large commercially important
fish consumed than has been estimated?
• Are sharks and/or rays consumed?
• Benthic foragers, pelagic prey?
- 3 sites, 90 faecal samples each
PCR 1*
Chordata
mtDNA 16S
*With blocking primer
Bioinformatic sorting of
sequences
PCR 2*
Chordata/Cephalopoda
mtDNA 16S
*With blocking primer
PCR 3
Cephalopod
nuclear 28S
PCR 4
Bilateria
nuclear 18S
Pool PCR amplicons
Pyrosequencing Roche GS FLX
DNA extraction from individual faecal
samples
Prey species ID
MtDNA COI
(protein coding gene)
Sequence position (bp)
Choice of Genetic Markers
Based on alignment of DNA sequences from 100 teleost fish (data from Miya et al. 2003, Molecular Phylogenetics and Evolution 26: 121-138)
MtDNA 16S (ribosomal DNA gene)
1. mtDNA
short
2. mtDNA
long
mtDNA
short
mtDNA
long
Methods- PCR blocking
Bony fish 1
Bony fish 2
Bony fish 3
Fur seal
Shark
Squid
0.05
Blocking Oligo
PCR without blocker
Fur seal
PCR
products
Fish PCR
products
Methods- Melting curve analysis of PCR
Figure: Melting profiles of PCR products from six fur seal faecal DNA extracts.
Vestheim et al. (2011) Methods in Molecular Biology 687:265-274
∆ flu
ore
sce
nce
d
F/d
(T)
Temperature
PCR with blocker
∆ flu
ore
sce
nce
d
F/d
(T)
Temperature
> 1000 PCRs
Prey Reference
database: Sequence 16S mtDNA from
voucher specimens
Images from: www.fish.gov.au and www.marine.csiro.au
10 ? 1 ? 2 ?
70 %
Seal R
ocks
L
ad
y J
ulia P
erc
y
Th
e S
kerr
ies
Lo
cati
on
Jack Mackerel
(Trachurus sp.)
Redbait
(Emmelichthys nitidus)
Blue Mackerel
(Scomber australasicus)
Barracouta
(Thyrsites atun)
Other Fish
Marker
Results- Comparison of mtDNA primer sets
Deagle et al. (2009) Molecular Ecology 18:2022-2038
Short Primer Set ~150 bp mtDNA 16S
n= 10585
n= 2959
n= 4636
n= 2990
Seal R
ocks
L
ad
y J
ulia P
erc
y
Th
e S
kerr
ies
Lo
cati
on
Jack Mackerel
(Trachurus sp.)
Redbait
(Emmelichthys nitidus)
Blue Mackerel
(Scomber australasicus)
Barracouta
(Thyrsites atun)
Other Fish
Marker
Results- Comparison of mtDNA primer sets
Deagle et al. (2009) Molecular Ecology 18:2022-2038
Long Primer Set ~300 bp mtDNA 16S
n= 2102
n= 524
n= 670
n= 908
Short Primer Set ~150 bp mtDNA 16S
n= 10585
n= 2959
n= 4636
n= 2990
One-eyed king?
Who is eating what: diet assessment using Next Generation Sequencing Pompanon et al. (In Press) Molecular Ecology
Mark Hindell
Nick Gales
Roger
Kirkwood
Dom
Tollit
Paige Eveson
Andrew Trites
Acknowledgements
Nuka, Hazy et al.
Bob Ward
Ella Bowles
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