1. Setting Up Copy Number Variation Assays
Droplet Digital PCR
Setting Up Copy Number Variation Assays
Ben Bunce

2. Outline Droplet Digital PCR (ddPCR) overview Applications
Implementation of a new workflow
Requirements
Challenges
Solutions
Case studies
X-LAG
tNGS confirmations
Summary

3. ddPCR Principle Standard PCR qPCR ddPCR
Quantitation of an end product in a bulk reaction
qPCR
Quantitation of product in real time in a bulk reaction
ddPCR
Bulk reaction separated into smaller partitions.
Each partition intended to contain zero or one copy of DNA.
The fraction of positive partitions is fitted to a Poisson distribution to determine the absolute starting copy number of DNA.

4. ddPCR Principle: Process
Bio-Rad QX200 System

5. Bio-Rad Quantasoft
1D Amplification
Channel One
Channel Two

6. Bio-Rad Quantasoft Concentration Channel 1 concentration

7. Bio-Rad Quantasoft Events Total droplets Channel 1 droplets

8. ddPCR Applications Detection of Single Nucleotide Variants (SNV)
Utilise TaqMan-type hydrolysis probes
Greater statistical power for low level detection (ctDNA, cfDNA, transcript abundance)
Absolute quantitation possible without standard curves
Detection of gene dosage/CNV
Utilise either dye-binding (external reference) or hydrolysis probes (internal reference)
comparison of positive partitions for target and reference allow gene dosage calculations
absolute quantitation possible without standard curves
Phasing of variants

9. Integration: Requirements
Exporting Quantasoft Setup File
Bio-Rad QX200 System
Quantasoft Analysis Package
Import patient results

10. Challenges Quantasoft Setup File Patient demographics Primers/Probes
Reagents
Experiment type
Results Import File
Patient demographics
Quality control data
Assay results
Software derived
Externally calculated
Interpretation of numerical results
Manual entry
Modify an existing LIMS export function
Commission LIMs provider to create new feature
Quantasoft output – “example” Contains redundant information
Hydrolysis probe assay results are calculate within the software (internal reference) i.e. Duplex probes. EvaGreen assays use an external and can’t be calculated within the software. Requires outside calculating.
All data is numerical – requires interpretation
Quality data, pass and fail samples - OK for a few samples, but for the possibility of larger batches in the future it is more prudent to automate this process too. Consistency between users.
Manual entry
Modify existing LIMS export function
Commission LIMS to create new feature

11. Challenge: Importing results
63 columns of data

12. Solution: Importing results
15 columns of data

13. Minor Challenges Technical challenges DNA quantification
Increased automation
Transitioning from each step of implementation
Informatics challenges
Networking

14. High throughput ddPCR copy number assay services
Solution: Importing results
Modified Quantasoft output
Quantasoft Analysis Package
Quantasoft Setup File
High throughput ddPCR copy number assay services
Bio-Rad QX200 System

15. Case Study 1: X-Linked Acrogiganitsm

16. Case Study 1: X-Linked Acrogiganitsm
Positive female
Positive male
Normal female
Normal males

17. Case Study 2: tNGS confimations
CDC73 exon 8 duplication
CDC73 exon 8 primer
Reference primer

18. Summary
QX200 system - a replacement for current qPCR and dosage techniques?
Increased sensitivity and statistical robustness
Workflow implementation
Underestimating IT project time frames
Utilising staff with additional skills (e.g. advanced knowledge of Excel)
Communicating concepts clearly between clinical, technical and bioinformatic staff
Any questions?