droplet digital pcr competition overview

Droplet Digital PCR Competition OverviewVersion 1.0 – November 16

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 GeneratorDroplet Reader

Digital PCR Competition

Life Technologies QuantStudio 12K Flex

Fluidigm EP1 System Biomark HD System

Raindance Technologies System TBD

Life TechnologiesQuantStudio 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

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

OpenArray Plates

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

OpenArray Plate Loading

AccuFill System loads samples into plate, transfers samples from 384-well plates into OpenArray Plates

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

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

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)

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

Higher Throughput Option Only For Genotyping

Requires the purchase of Twister 2 robot and several thermal cyclers

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

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

Bio-Rad Versus Life Tech

  Bio-Rad Life TechCost 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 20 μL 700 uL

Cost to match 20,000 partitions $3.00 $1,050

Dead Volume 30% 50%

Price $89,000 $190,000

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

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

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

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

Integrated Fluidic Circuitry (IFC) technology

Microfluidic chip that uses pressure to move liquids and nano-scale valves to trap samples into small wells

Sample Sample InputsInputs

ChipChipIHS Underneath ChipIHS Underneath Chip

Pressure AccumulatorPressure Accumulator

RC – Reaction ChamberIV – Interface valveCV – Containment Valve

Inside Chip

IFC Chip Loading

Sample CDNA or DNA concentration: 1/5,000 nl Target concentration: 1,000

greater, or 1/5nl

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 12.765 Chip – minimum load 4 ul per well 48.770 Chip – minimum load 8 ul per well

IFC Controller

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

Results

Fluidigm

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

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

Bio-Rad Versus Fluidigm

  Bio-Rad FluidigmCost 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 12.765 chip)

Sample Volume req 20 μL 240 uL

Cost $2.38 $720

Dead Volume 30% 50%

Price $89,000 $240,000

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

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

Raindance

Small, venture-backed company

Raindance Also Uses Droplets

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

Raindance

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

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

Pre-Made Primer Libraries

Targeting Next Gen Sequencing Sample Prep

Raindance Is Working on Digital PCR

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