EvaGreen-based droplet digital PCR:

a rapid and inexpensive method to

confirm and enhance breakpoint

resolution of CNVs detected by CMA

August 8, 2017

Hou-Sung Jung, PhD

Clinical Postdoctoral Fellow

Cameron Griffin, Courtney Studwell, Stephanie E. Vallee, Scott A.

Turner, Mary Beth Dinulos, Gregory J. Tsongalis, Joel A. Lefferts

Dartmouth-Hitchcock Medical Center

Copy Number Variation Measurement

Methods

Genomics (2013) 101:86-93

PFGE: Pulse Field Gel Electrophoresis;

MAPH: Multiplex Amplifiable Probe Hybridization;

PRT: Paralogue Ratio Test

High Resolution Microarray

for Copy Number Determination

Confirmatory Copy Number Assays

Are Required Because of relatively sparse marker distribution

in an area of interest

http://people.ee.duke.edu/~mz1/NB_CRT_Pois_Log.png

Ideal

marker

distribution

Real

marker

distribution

NGS

Candidates for Confirmatory Copy Number

Assay

Genomics (2013) 101:86-93

Inexpensive (easy reimbursement)

Less DNA High Resolution Easily

Customizable PRT qPCR

Microarray NGS Microarray

Fiber Fish Southern

Blot PFGE

MLPA MAPH MAPH

Fiber Fish Southern

Blot PFGE

MLPA

PRT

Rapid

qPCR: Indirect Quantification Using Ct with Known Amounts

https://www.neb.com/~/media/Catalog/All-Products/ECCE5D577424448D983A70BA9BDA8613/Long%20Description/Luna_RNA_SYBR_Figure1.jpg

NGS

Confirmatory Copy Number Assay

Genomics (2013) 101:86-93

Fiber Fish

Rapid Inexpensive

Less DNA High Resolution Easily

Customizable

Southern

Blot PFGE

MAPH MLPA

PRT qPCR

Microarray NGS Microarray

Fiber Fish Southern

Blot PFGE

MAPH MLPA

PRT

Accurate

qPCR

Digital PCR

http://www.bio-rad.com/en-us/applications-technologies/absolute-quantification-pcr-targets-with-droplet-digital-pcr-system

Digital PCR Principles and Procedures

https://www.abmgood.com/marketing/knowledge_base/polymerase_chain_variation_system.php

Dilution

(20 uL 1 nL)

1 molecule per partition

PCR amplification

in individual partitions

Sensitivity ↑

1 0 1 0

Digital Detection Accurate

qPCR vs Digital PCR (dPCR) measuring lengths using different rulers

qPCR

dPCR

Droplet Digital PCR (ddPCR)

for Copy Number Assessment

http://ars.els-cdn.com/content/image/1-s2.0-S002751070500031X-gr1.jpg www.bio-rad.com/en-us/product/qx200-autodg-droplet-digital-pcr-system

Accurate and Precise

Absolute Quantification

Droplet Digital PCR TaqMan

Probe Production

Cost and Time

Droplet Digital PCR (ddPCR)

for Copy Number Assessment

https://biotium.com/wp-content/uploads/2013/07/FEM-8.gif www.bio-rad.com/en-us/product/qx200-autodg-droplet-digital-pcr-system

Accurate and Precise

Absolute Quantification

Droplet Digital PCR

Rapid and Inexpensive

EvaGreen

Outline

1. Proof-of-Principle EvaGreen-based ddPCR

for Copy Number Assessment

using the NIPA1 gene CNV

2. Cases

Validation of EvaGreen-based ddPCR for

Copy Number Determination Sample:

CNVs of x3 and x1

in the NON-IMPRINTED IN PRADER-

WILLI/ANGELMAN SYNDROME 1 (NIPA1) gene

Sample:

CNVs of x3 or x1 in NIPA1 gene

Reference genes:

RIBONUCLEASE P RNA COMPONENT H1

(RPPH1) gene (14q11)

or

TELOMERASE REVERSE TRANSCRIPTASE

(TERT) gene (5p15)

Validation of EvaGreen-based ddPCR for

Copy Number Determination

EvaGreen Signals from ddPCR CN Assay

NIPA1-Exon 1 NIPA1-Exon 5

RPPH1 TERT

Reference

Genes

Target

Genes

The Number of EvaGreen-Positive Droplets

10 ng → ~ 3000 copies

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

x2 NTC x3 x1 x1 x3 x1 x1 x3 x1 x1 x1 x3 x3 x3

Po

sitiv

e D

rop

lets

NIPA1-exon 1 NIPA1-exon 5 RPPH1 TERT

Positive

Droplets of

Target

Gene in

Patient

Copy Number Calculation

Positive

Droplets of

Reference

Gene in

Patient

Positive

Droplets of

Target

Gene in

Calibrator

Positive

Droplets of

Reference

Gene in

Calibrator

÷ X 2 = Copy

Number

Proof of Concept: EvaGreen-ddPCR

Case 1: Single Copy Deletion (48 kb) in

AUTS2 arr[hg19] 7q11.22(70,188,374-70,236,724)x1

Exon 7 Exon 19 Exon 6

48 kb

Log2 Ratio

Copy Number State

AUTS2 (AUTISM SUSCEPTIBILITY CANDIDATE 2): OMIM 607270

Neurodevelopmental defects, haploinsufficiency

Exon 7 Exon 11 Exon 19

12 kb

EvaGreen ddPCR Copy Number Assay for

AUTS2 Exon 7~ Exon 11 Targets: AUTS2 Exon 7 and Exon 11

Controls: AUTS2 Exon 2 and Exon 19 and NIPA1 1x

Normal Copy Numbers in AUTS2 Normalized to RPPH1

0.0

0.5

1.0

1.5

2.0

2.5

Co

py N

um

be

r

Patient NIPA1 x1 control

Case 1:

Refined Breakpoints by EvaGreen ddPCR arr(1-22,X)x2

Exon 7 Exon 19 Exon 6

Case 2: Deletion in MBD5 arr[hg19]2q23.1(149,034,377-149,094,573)x1

60 kb

Exon 5

65 kb

or

MBD5 (Methyl-CpG Binding Domain Protein 5): OMIM 611472

Mental retardation, autosomal dominant 1

CN Assay MBD5 Intron 4 and Exon 5 Normalized to TERT

Exon 5 2327 bp

3981 bp 7389 bp

MBD5-

IN4-4

MBD5-

IN4-3

MBD5-

IN4-2

MBD5-

IN4-1

MBD5-

EX5-2 ?

5000 bp

0.0

0.5

1.0

1.5

2.0

2.5

IN4-4 IN4-3 IN4-2 IN4-1 EX5-2

Copy N

um

ber

Patient Control

Case 2: Results of Refining Breakpoints

60 kb

Exon 5

~ 62 kb

No Deletion in MBD5 Exon 5

EvaGreen-based ddPCR CNV Assay Accurate, Rapid, and Inexpensive

Day 1 Day 2 Day 3

Primer Design

Droplet

Generation

PCR

Result Analysis

Report

≤ 48 hours

AM

PM

AM

Further Validation/Development

Greater than x3 Gain

FFPE DNA

Automatic Copy Number Analysis

Acknowledgement Clinical Genomics and Advanced Technology (CGAT)

CGAT- Admin

Heather Steinmetz

Amber Erskine

Wendy Wells

CGAT- Postdocs

Aaron Atkinson

Precision Summer Interns

Jennifer Kilbourn

Rachel Alibozek

CGAT-Core

Samantha Allen

Leanne Cook

Sophie Deharvengt

Betty Dokus

Torrey Gallagher

Donald Green

Arnold Hawk

Rachel Henne

Michael Johnston

Jason Peterson

Terri Wilson

CGAT- Histology

David Beck

Rebecca O’Meara

Scott Palisoul

CGAT- Clinical

Mark Cervinski

Francine de Abreu

Deana Denault

Eric Loo

Laura Tafe

Questions?

Thank You~

Current Affiliation:

Hou-Sung Jung

Clinical Genomic Scientist I

Children’s Hospital of Philadelphia

1. Primer Design

Primer3 (version 4.0.0)

UCSC in-silico PCR

Primer-BLAST

Procedures of EvaGreen-based ddPCR

for Copy Number Determination

2. Droplet Generation

10 ng genomic DNA

without restriction enzyme digestion

AUTS2

Exon 2 AUTS2

Exon 7 AUTS2

Exon 11

AUTS2

Exon 19 NIPA1

Exon 1

NIPA1

Exon 5 RPPH1

TERT

EvaGreen Signals of AUTS2 CN Assay