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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
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Environmental DNA (eDNA)
DNA that is collected from non-
biological samples (i.e. soil, air, water)
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Early detection of invasive species is
essential for control
eDNA As A Conservation Tool
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Bighead Carp Distribution in North America
Map Created by USGS 9-3-16
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Silver Carp Distribution in North America
Map Created by USGS 9-3-16
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Study Sites
Expected Carp Density
POOL 13
POOL 17
POOL 19
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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
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‘Transect’ Sampling (2014)
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‘Targeted’ Sampling (2015)
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eDNA Extraction
River water samples were extracted
and tested for bigheaded carp DNA by
qPCR and dPCR
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TESTING WORKFLOW
qPCR Analysis
PCR inhibition?
(R script)
Remove inhibitors
dPCR analysis
INHIBITED
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Estimate target DNA copy number with a standard curve
qPCR Basics
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PCR reaction is partitioned into a large
number of separate wells prior to PCR
dPCR Basics
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Endpoint fluorescence is measured after PCR
and positive wells (copies) are counted
dPCR Basics
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We used a chip based digital PCR system (QuantStudio™ 3D)
dPCR Assay
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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
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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
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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%
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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
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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%
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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
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dPCR Threshold Gating
SVC +
NEGATIVE BHC +
SVC +
BHC +
These boundaries define positive and negative samples
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dPCR Gating: Negative Controls
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dPCR Gating: Standards
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dPCR Gating: Standards
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Conclusions
• qPCR detections were consistent
with expected carp densities
• dPCR data was variable; results
vary based on threshold gating
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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
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QUESTIONS?