Droplet digital PCROverview and applications

Genetic Technologist Training DayThursday 20th November 2014

Natalie Brace

Contents

• Introduction into Digital PCR

• Workflow

• Advantages of ddPCR

• JAK2 testing

• Future developments

What is droplet digital PCR?

• A real-time PCR reaction that is partitioned into many droplets – a PCR reaction happens in each droplet.

• A portion of these reactions contain the target molecule while others do not.

• Positive reactions are counted.

• Fraction of negative reactions is used to deduce the absolute number of target molecules in the sample.

• Currently in use to test for JAK2 V617F mutation.

ddPCR work flow

1) QX100 Droplet generator

2) Bio-Rad Thermal cycler

3) QX100 Droplet reader

ddPCR work flow1. Prepare sample and reagent mixture

• Primers and fluorescent probes are combined with the Biorad ddPCR supermix

• DNA is then added to create PCR ready samples• Loaded into individual wells of the 8 channel disposable droplet generator cartridge

Sample

droplets

oil2. Make droplets• Place loaded cartridge onto QX100 droplet generator• Partitions reagents and sample to create up to 20,000 droplets per

well

Sample

Oil

Oil

ddPCR work flow3. PCR droplets on Thermal Cycler

• Transfer the droplets from the cartridge to a 96 well PCR plate• Seal the plate using Biorad PX1 plate sealer• Perform PCR to endpoint (40 cycles) using a thermal cycler

4. Read droplets• Post PCR, load 96 well plate onto the QX100 droplet reader• Droplets are streamed single file past a two colour optical detection

system to determine if they contain a target (positive) or not (negative)• Analyse concentrations using QuantaSoft software

Key advantages of ddPCR

• Absolute quantification means no standard curve is needed.

• Cost effective when compared with quantitative PCR.

• Highly flexibility technique-

- High throughput with single well measurements for 96 samples

- High sensitivity, dependent on the number of droplets

Droplet Digital PCR platforms

20K droplets/20ul PCR reaction0.9nL droplets

Biorad ddPCR Supermix(~£3 per reaction)

5-10M droplets/25ul PCR reaction0.5pL dropletsOpen system

(~£19 per reaction)

JAK2- Janus kinase 2 • Protein involved in JAK/STAT signalling pathway.

• The JAK2 protein is especially important for controlling the production of blood cells from hematopoietic stem cells.

• The V617F JAK2 gene mutation results in the production of a JAK2 protein that is constantly activated, this leads to the overproduction of abnormal blood cells.

• Excess platelets can cause abnormal blood clotting.

• With so many extra cells in the bloodstream, abnormal blood clots are more likely to form. In addition, the thicker blood flows more slowly throughout the body, which prevents organs from receiving enough oxygen.

• Somatic JAK2 V617F mutation is found several chronic myeloproliferative neoplasms• – Polycythemia vera (97%), essential thrombocythemia (57%), and chronic

idiopathic myelofibrosis (50%).

Previous JAK2 testing strategy Two tier strategy;Diagnostic Allele Specific Oligonucleotide (ASO) PCR (~£3)• TAT- 14 days.• Lots of re-runs due to issues with ABI- size standard drop outs etc.• ‘Equivocal’ results sometimes given by ASO (Ratios between 1.5% and

5.0%).

Follow up RQ-PCR(~£30)• Resolve ‘equivocal’ results.• Monitor Post transplant patients .• Monitoring for positive patients.• Sensitivity to detect JAK2 V617F down to 0.09%.• Relatively few patients so batching required; turn around times may be

up to 2 months.

ddPCR JAK2 testing strategy and service

• Use a single test to replace the two tier strategy with the aim to Improve turnaround times Improve sensitivity

• To reliably detect 1% mutation load using 1 well of QX100 1% is the clinically relevant mutation load according to Best

Practice Guidelines (Bench et al.2013)

• Approx. 200 samples a month received into the laboratory• 3 runs of 20 patients each week usually sufficient to clear all

samples and repeats for positives• Regular runs means fast confirmation of positives.

JAK2 V617F ddPCR assay using QX100

We use an assay designed by Quantalife-

This is a Duplex assay using hydrolysis probes specific for JAK2 wild type (VIC) and JAK2 V617F mutation (FAM)

JAK2 Results

• QX100 generates a maximum of 20,000 droplets

• Its possible that when droplets are generated some droplets contain more than one target DNA.

• Positive and negative reactions are counted by the QX100 reader and are fit to a Poisson distribution to account for the possibility of having more than one target in each droplet.

Future ddPCR uses and developments

• KIT mutations in Myeloproliferative Disorder (MPD) patients – SOP being developed

• BCR-ABL testing for Chronic Myeloid Leukemia (CML) using the Raindance platform – higher sensitivity – ongoing research

• Fetal sexing using the QX100- new project

Thank you for listening.

Any Questions?