A Comparison of Molecular Detection Methods for …...A Comparison of Molecular Detection Methods...

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?