1. Droplet Digital PCR Competition Overview
Version 1.0 – November 16

2. Bio-Rad QX100™ Droplet Digital™ PCR System
System designed specifically for digital PCR
Sample partitioned into 20,000 droplets
Unparalleled precision at +/- 10%
105 linear dynamic range (1 to 100,000 copies)
Flexible assay format for running experiments
Simple, yet elegant workflow
Easy to use software
Droplet Reader
Droplet Generator

3. Digital PCR Competition
Life Technologies
QuantStudio 12K Flex
Fluidigm
EP1 System
Biomark HD System
Raindance Technologies
System TBD

4. Life Technologies QuantStudio 12K Flex Real Time PCR system
Combination of Viia7 and OpenArray technologies
Real-time PCR: TaqMan array card (TLDA), 384-well block, Standard or Fast 96-well blocks
Digital PCR: OpenArray block
Promote several applications, including viral load, mutant sequence detection against wt background, GMO quality control, gene expression and genotyping
Stand-alone operation is possible with LCD touch screen, but data is not shown on the system, need software on computer

5. OpenArray Plates Each plate is essentially a microarray chip
Through-hole accepts 33 ± 1 nL of sample and supermix
Reagents retained in through-holes via surface tension

6. OpenArray Plates
Plates come preloaded from Life Technologies, can be predesigned or custom

7. OpenArray Plate Loading
AccuFill System loads samples into plate, transfers samples from 384-well plates into OpenArray Plates

8. OpenArray Plate Preparation
Users have ~90 seconds to load samples before they begin to evaporate
Contributes to ~50% dead volume compared to 30% with QX100
After loading the plate, apply adhesive lid, fill the case with immersion fluid, then plug and load 4 plates into holder

9. Running OpenArray Plates
Run up to 4 plates
Software fits real-time PCR data to a Poisson statistical model and displays digital results as copies-per-through-hole

10. OptiFlex System QuantStudio uses the same optical system as the Viia7
White light LED, filter wheels and camera
Claim unlimited flexibility for dye selection, but fluorescence excitation and emission of real-time PCR dyes are limiting
Only 2-targets for dPCR – Fam and Vic (ROX normalization)

11. Life Technology Claims on Throughput
Maximum resolution of 12,000 data points (4 plates)
The system allows you to process up to 1,728 genotyping samples and 2,304 samples for gene expression in an 8 hr day
PCR offline using the Dual Flat Block 9700, process a total of over 5,184 genotyping samples in one day
Can generating up to 110,000 data points or more per 8 hour day, completing your project in days rather than weeks

12. Higher Throughput Option Only For Genotyping
Requires the purchase of Twister 2 robot and several thermal cyclers

13. Digital PCR Sample Dilutions
Claim multiple sample dilutions possible for increased input dynamic range
We do not need sample dilutions because we use 20,000 droplets

14. Digital PCR Claims
Life Tech states you can detect as little as 1.5-fold changes in target quantities in singleplex reactions
We can do 1.2-fold changes in multiplex reactions
They acknowledge precision is determined by the number of replicates
Better confidence intervals with more replicates
This approach forces the user to choose between throughput (number of samples analyzed) versus experiment precision
At most, run 12,888 partitions for a single sample, but it will cost $600 and requires 400 μL of sample

15. Bio-Rad Versus Life Tech
Bio-Rad
Life Tech
Cost per sample
$3.00
$150.00
Number of partitions
20,000
3072
Cost per data point (cents)
0.0001 5
Partition Volume
1 nL
33 nL
Sample volume
20 μL
100 μL
# of chips to match 20,000 partitions 1 7
Sample Volume Required to match 20,000 partitions
700 uL
Cost to match 20,000 partitions
$1,050
Dead Volume
30%
50%
Price
$89,000
$190,000

16. ddPCR Versus Life Technologies
Sample partitioning into 20,000 droplets enables independent, PCR micro-reactions, resulting in unmatched precision, accuracy and sensitivity for quantification
ddPCR +/- 10% precision allows the ability to distinguish very small fold differences and detect low copy number targets
Low sample volume translates into reduced reagent and sample costs
Significantly lower cost per sample $3 vs $150
Flexible format is not determined by a microfluidic chip layout, multiple independent experiments can be run with ddPCR
Serial dilutions are not required, eliminates pipetting error between dilutions and saving reagent and consumable costs

17. ddPCR Versus Life Technologies
Yes, you can perform real-time PCR and dPCR with one system, but it costs $190,000 for the dPCR upgrade and the need to have data from both approaches is redundant
The OpenArray technology was developed for high throughput, low volume real-time PCR and genotyping, not digital PCR
The OpenArray format forces a compromise between precision and sample throughput
You must order chips pre-loaded with primers and probes, so assay flexibility is limited and the experiment is locked

18. Fluidigm BioMark HD System
Integrated Fluidic Circuitry (IFC) technology
Microfluidic plates with varying throughput (~$360/plate)
Real-time PCR: 48 samples x 48 assays (2034 rxns) 96 samples x 96 assays (9216 rxns), 192 samples x 24 SNPs
Digital PCR: 12 samples X 765 partitions (9180 rxns), 48 samples x 770 partitions (39,960 rxns)
Promote gene expression, single cell analysis, genotyping, NGS sample preparation, CNV

19. Fluidigm Sells 2 Systems
Biomark HD system – includes integrated cycler and real-time detection camera. It costs $240K
EP1 System – separate thermal cycler and camera, can’t be used for single cell gene expression. It costs $100K
BioMark HD System
EP1 System

20. Integrated Fluidic Circuitry (IFC) technology
Microfluidic chip that uses pressure to move liquids and nano-scale valves to trap samples into small wells
Sample Inputs
Chip
IHS Underneath Chip
Pressure Accumulator
Inside Chip
RC – Reaction Chamber
IV – Interface valve
CV – Containment Valve

21. IFC Chip Loading Sample CDNA or DNA concentration: 1/5,000 nl
Target concentration: 1,000 greater, or 1/5nl

22. Workflow Part 1
Prime. Prime chip in IFC controller to close interface valves, preventing premature mixing of samples and assays (10 min)
Transfer. Pipette samples premixed with master mix and primer, probes into separate sample inlets on frame of the chip
Chip – minimum load 4 ul per well
Chip – minimum load 8 ul per well
IFC Controller

23. Workflow Part 2
Load samples. Place the chip back on IFC controller, and pressure load assay into reaction chambers (40 minutes)
Run. Place chip on BioMark HD System for thermal cycling and fluorescence detection, OR place chip in FC1 cycler for PCR and then into EP1 reader
Analyze. Software automatically counts and reports the number of positives for each reporter, provides color coded heat maps

24. Results

25. Fluidigm
Focused much more on single cell gene expression and high throughput/low volume screening

26. Fluidigm
Trying to adapt, instead of develop technology for digital PCR
Fewer partitions per sample negatively impacts precision
For more precision, need to run panel replicates
Panels of 765 wells

27. Bio-Rad Versus Fluidigm
Bio-Rad
Fluidigm
Cost per sample
$3.00
$30.00
Number of Partitions
20,000
765
Cost per data point (cents)
0.0001 4
Partition volume
1 nL
6 nL
Sample volume
20 μL
10 μL per well
# of chips req for 20,000 partitions 1
2 (for chip)
Sample Volume req
240 uL
Cost
$2.38
$720
Dead Volume
30%
50%
Price
$89,000
$240,000

28. ddPCR Versus Fluidigm
Sample partitioning into 20,000 droplets enables independent, PCR micro-reactions, resulting in unmatched precision, accuracy and sensitivity for quantification
ddPCR +/- 10% precision allows the ability to distinguish very small fold differences and detect low copy number targets
Low sample volume translates into reduced reagent and sample costs
Significantly lower cost per sample $3 vs $30
Flexible format is not determined by a microfluidic chip layout, multiple independent experiments can be run with ddPCR

29. ddPCR Versus Fluidigm
Yes, you can perform real-time PCR and dPCR with one system, but it costs $240,000 for the BioMark HD1 and the need to have data from both approaches is redundant
IFC format was developed for high throughput, low volume real-time PCR and genotyping, not digital PCR
IFC format forces a compromise between precision and sample throughput
Changing throughput on Fluidigm system requires chip and hardware modifications
We have the ability to optimize reactions using a gradient

30. Raindance
Small, venture-backed company

31. Raindance Also Uses Droplets

32. Raindance System
System combines pre-made primer droplets with sample droplets, creating many million drops of partitioned sample, each drop has one of 20,000 different primers
PCR is carried out in standard TC
20,000 independent amplifications from a single sample

33. Raindance
Researchers are not currently using Raindance technology to quantify target DNA based on a digital readout

34. Raindance
RDT 1000 is a high-priced (~$225K) droplet generator for next generation sequencing sample preparation. The RDT 1000 does not analyze gene targets in droplets like the Bio-Rad system
New system (Thunderstorm) enables 8 primer libraries by 96 samples
Lowered chip price from ~$600 down to ~$150
This is a per sample price
New pre-made libraries

35. Pre-Made Primer Libraries

36. Targeting Next Gen Sequencing Sample Prep

37. Raindance Is Working on Digital PCR

38. Bio-Rad Versus Raindance
The ability to produce 100K to 1M droplets does not give Raindance an advantage for ddPCR
Generating and reading more droplets can improve statistical analysis for rare mutant detection, but for CNV, allelic discrimination and gene expression there is limited benefit to analyzing that many droplets
We expect the Raindance system to be very expensive if they “fit” digital PCR into their current format and system