A Comparison of Molecular
Detection Methods for Bigheaded
Carp DNA Copy Number Estimation
In the Mississippi River
CRAIG JACKSONCHRIS MERKES
JON J. AMBERG
U.S. GEOLOGICAL SURVEY
UPPER MIDWEST ENVIRONMENTAL
SCIENCES CENTER
Environmental DNA (eDNA)
DNA that is collected from non-
biological samples (i.e. soil, air, water)
Early detection of invasive species is
essential for control
eDNA As A Conservation Tool
Bighead Carp Distribution in North America
Map Created by USGS 9-3-16
Silver Carp Distribution in North America
Map Created by USGS 9-3-16
Study Sites
Expected Carp Density
POOL 13
POOL 17
POOL 19
Study Design and Sampling
3 sampling trips
2014 Trip 1
(June 2014)
2014 Trip 2
(August 2014)
2015
(June 2015)
3 Study Sites
Pool 13: few carp
Pool 17: some carp
Pool 19: many carp
‘Transect’ Sampling (2014)
‘Targeted’ Sampling (2015)
eDNA Extraction
River water samples were extracted
and tested for bigheaded carp DNA by
qPCR and dPCR
TESTING WORKFLOW
qPCR Analysis
PCR inhibition?
(R script)
Remove inhibitors
dPCR analysis
INHIBITED
Estimate target DNA copy number with a standard curve
qPCR Basics
PCR reaction is partitioned into a large
number of separate wells prior to PCR
dPCR Basics
Endpoint fluorescence is measured after PCR
and positive wells (copies) are counted
dPCR Basics
We used a chip based digital PCR system (QuantStudio™ 3D)
dPCR Assay
qPCR
• The ‘gold’ standard
• Quick runs
• Price is relatively cheap
• Set-up is easily
automated
dPCR
• No standard curve
required
• Counting copies, not
calculating
• Thought to be
resistant to inhibition
• Does not rely on
amplification
efficiency
Positives
qPCR
• Requires a standard
curve for quantification
• Difficult to quantify low
copy numbers
• Sensitive to inhibition
• Dependent on
amplification efficiency
dPCR
• Not easily automated
• Expensive
• Longer runs
• Requires gating for
determination
Negatives
Silver Carp Detections
POOL 13 qPCR dPCR2014 TRIP1 0% 17%2014 TRIP2 0% 11%
2015 0% 6%
POOL 17 qPCR dPCR2014 TRIP1 6% 19%2014 TRIP2 6% incomplete
2015 10% 8%
POOL 19 qPCR dPCR2014 TRIP1 42% incomplete2014 TRIP2 15% incomplete
2015 16% 2%
Silver Carp Copy Numbers
POOL 13 qPCR dPCR2014 TRIP1 - 10.72014 TRIP2 - 6.6
2015 - 14.2
POOL 17 qPCR dPCR2014 TRIP1 1.8 11.12014 TRIP2 <1 incomplete
2015 <1 24.4
POOL 19 qPCR dPCR2014 TRIP1 2.7 incomplete2014 TRIP2 <1 Incomplete
2015 <1 41.4
Bighead Carp Detections
POOL 13 qPCR dPCR2014 TRIP1 0% 4%2014 TRIP2 0% 13%
2015 0% 8%
POOL 17 qPCR dPCR2014 TRIP1 29% 0%2014 TRIP2 8% incomplete
2015 10% 6%
POOL 19 qPCR dPCR2014 TRIP1 48% incomplete2014 TRIP2 20% incomplete
2015 30% 2%
Bighead Carp Copy Numbers
POOL 13 qPCR dPCR2014 TRIP1 - 15.12014 TRIP2 - 8.3
2015 - 7.1
POOL 17 qPCR dPCR2014 TRIP1 5.1 -2014 TRIP2 <1 incomplete
2015 <1 39.7
POOL 19 qPCR dPCR2014 TRIP1 4.8 incomplete2014 TRIP2 <1 incomplete
2015 <1 45.3
dPCR Threshold Gating
SVC +
NEGATIVE BHC +
SVC +
BHC +
These boundaries define positive and negative samples
dPCR Gating: Negative Controls
dPCR Gating: Standards
dPCR Gating: Standards
Conclusions
• qPCR detections were consistent
with expected carp densities
• dPCR data was variable; results
vary based on threshold gating
THANK YOU!
In The Lab
• Jon Amberg
• Bridget Ladell
• Jenna Malinauskas
• Grace McCalla
• Chris Merkes
• Theresa Schreier
• Justin Smerud
• John Steiner
• Emily Ziegler
In The Field
• Pete Boma
• Brent Knights
• Mark Roth
Acknowledgements
QUESTIONS?