1. FURTHER FACTS FROM FAECES:
Dietary DNA Barcoding Using High Throughput Sequencing
Bruce Deagle and Simon Jarman

2. Why study animal diet?
“Statistics show that of those who contract
the habit of eating, very few survive.”
George Bernard Shaw
Decline of Steller sea lions
From Trites and Donnelly (2003) Mammal Review 33:3-28

3. Baleen Flighted
whales birds
Penguins
Seals Toothed whales
Demersal fish Pelagic fish Squid
Amphipods Krill Salps Copepods
Unicellular Organisms

4. 1. Observe feeding
3. Collect gut contents
4. Collect faeces
2. Tissue sample identify hard-parts
‘the one-eyed man is king
in the land of the blind’
Different approaches to studying diet

5. Captive Feeding Trial
Steller sea lions
(Eumetopias jubatus)
• Reliability of prey DNA recovery from
faeces?
• Persistence of genetic signal?
• Quantitative estimates of diet?
• Quality of prey DNA?

6. Feeding Trial - Methods
Surf Smelt Squid
(Hypomesus pretiosus) (Loligo sp.)
Pulse prey items
Sockeye Salmon Pacific Herring
(Oncorhynchus nerka ) (Clupea pallasii ) Group Specific PCR
- DNA was extracted from soft
material (n=108)
- Sea lion DNA was dominant
component Jarman et al. (2004) Molecular Ecology 13:1313-1322

7. Feeding Trial - Results
Frequency of detection • Reliable prey DNA detection
for basic dietary items
• Each species has a equal chance
% prey positives
(n=108) of being detected, squid (6% of
diet) is consistently detected
Squid 94%
• DNA from pulse prey items turned
Herring 94% up in faeces produced between 12
and 48 hours after ingestion
Smelt 92%
Salmon 87%
Mean 92%
Deagle et al. (2005) Molecular Ecology 14:1831-1842

8. Quantitative Estimates
Do proportions of DNA in faeces reflect intake?
Proportion of DNA in Faeces (qPCR)
• Prey DNA in faeces
from 10 diets fed to
Steller sea lions.
4 Diet Items
Herring
Eulachon
Squid
Rockfish
Proportion in diet by mass
Bowles et al. (2011) Molecular Ecology Resources 11: 530-540
Deagle and Tollit (2007) Conservation Genetics 8: 743-747

9. Prey DNA Quality
Herring DNA- Scat 1
25000
- Mini-barcodes best
20000
(< 200 bp)
Copy number
Copy Number
15000
10000
5000
0
0 50 100 150 200 250 300
PCR product size
PCR product size
Deagle et al. (2006) Frontiers in Zoology e3:11

10. Prey DNA Quality
Herring DNA- Scat 1
25000
F(x)= αe-λ x - Allows quantification of
20000
DNA damage
Copy number
λ = probability of a break
Copy Number
15000
10000
5000
0
0 50 100 150 200 250 300
PCR product size
PCR product size
Deagle et al. (2006) Frontiers in Zoology e3:11

11. Passmore et al. (2006) Marine Biotechnology
Casper et al. (2007) Marine Biology
Dunshea (2009) PLoS ONE
Deagle et al. (2007) PLoS ONE

12. Next generation sequencing
Marguilies et al. 15 September 2005: Volume 437: 376-380

13. Australian fur seal
(Arctocephalus pusillus doriferus)

14. Fur Seal Diet Questions
• Benthic foragers, pelagic prey?
• Are more large commercially important
fish consumed than has been estimated?
• Are sharks and/or rays consumed?

15. - 3 sites, 90 faecal samples each

16. DNA extraction from
individual faecal
samples
PCR 1* PCR 2* PCR 3 PCR 4
Chordata Chordata/Cephalopoda Cephalopod Bilateria
mtDNA 16S mtDNA 16S nuclear 28S nuclear 18S
*With blocking primer *With blocking primer
Pool PCR amplicons
Pyrosequencing
Roche GS FLX
Bioinformatic sorting of
sequences
Prey species
ID

17. Choice of Genetic Markers
MtDNA COI
(protein coding gene)
Sequence position (bp)
MtDNA 16S 1. mtDNA 2. mtDNA
(ribosomal DNA gene) short long
Based on alignment of DNA sequences from 100 teleost fish
(data from Miya et al. 2003, Molecular Phylogenetics and Evolution 26: 121-138)

18. Methods- PCR blocking
mtDNA Bony fish 1
short Bony fish 2
mtDNA
long Bony fish 3
Fur seal Blocking Oligo
Shark
Squid
0.05

19. Methods- Melting curve analysis of PCR
PCR without blocker PCR with blocker
Fur seal
PCR
products
∆ fluorescence dF/d(T)
∆ fluorescence dF/d(T)
Fish PCR
products
Temperature Temperature
Figure: Melting profiles of PCR products from six fur seal faecal DNA extracts.
Vestheim et al. (2011) Methods in Molecular Biology 687:265-274

20. > 1000 PCRs
Prey Reference
database:
Sequence 16S mtDNA from
voucher specimens

21. 70 %
10 ? 1? 2?
Images from: www.fish.gov.au and www.marine.csiro.au

22. Results- Comparison of mtDNA primer sets
Marker
Short Primer Set
~150 bp mtDNA 16S
n= 10585
Seal Rocks
Jack Mackerel
(Trachurus sp.)
n= 2959
Redbait
Location
Lady Julia Percy
(Emmelichthys nitidus)
Blue Mackerel
(Scomber australasicus)
Barracouta
n= 2990 (Thyrsites atun)
Other Fish
The Skerries
n= 4636
Deagle et al. (2009) Molecular Ecology 18:2022-2038

23. Results- Comparison of mtDNA primer sets
Marker
Short Primer Set Long Primer Set
~150 bp mtDNA 16S ~300 bp mtDNA 16S
n= 10585 n= 2102
Seal Rocks
Jack Mackerel
(Trachurus sp.)
n= 2959 n= 524
Redbait
Location
Lady Julia Percy
(Emmelichthys nitidus)
Blue Mackerel
(Scomber australasicus)
Barracouta
n= 2990 n= 908 (Thyrsites atun)
Other Fish
The Skerries
n= 4636 n= 670
Deagle et al. (2009) Molecular Ecology 18:2022-2038

24. One-eyed king?
Who is eating what: diet assessment using Next Generation Sequencing
Pompanon et al. (In Press) Molecular Ecology

25. Nick Gales
Paige Eveson
Acknowledgements
Roger
Kirkwood
Mark Hindell
Ella Bowles
Andrew Trites
Nuka, Hazy et al.
Dom
Tollit
Bob Ward