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