1. Droplet digital PCR Overview and applications
Genetic Technologist Training Day
Thursday 20th November 2014
Natalie Brace

2. Contents Introduction into Digital PCR Workflow Advantages of ddPCR
JAK2 testing
Future developments

3. 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.

4. ddPCR work flow QX100 Droplet generator 2) Bio-Rad Thermal cycler
Droplet reader

5. ddPCR work flow 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
droplets
Sample
Make droplets
Place loaded cartridge onto QX100 droplet generator
Partitions reagents and sample to create up to 20,000 droplets per well
oil
Oil
Sample
Oil

6. ddPCR work flow 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
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

7. 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

8. Droplet Digital PCR platforms
We have two digital PCR systems currently in use in the lab. The biorad QX100 is the machine we are currently using for clinical work. This is capable of producing 20 thousand droplets and costs around 3 pounds per sample to run. The Raindance is capable of producing between 5-10 Million droplets so is more sensitive but is much more expensive to run. Because of the greater sensitivity the raindance is currently being explored for BCR-ABL1 testing and non-invasive prenatal diagnosis.
20K droplets/20ul PCR reaction
0.9nL droplets
Biorad ddPCR Supermix
(~£3 per reaction)
5-10M droplets/25ul PCR reaction
0.5pL droplets
Open system
(~£19 per reaction)

9. 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%).
JAK2 is short for Janus Kinase 2 which is the gene that provides instructions for making a protein that promotes the growth and division of cells. This protein is part of a signalling pathway called the JAK/STAT pathway, which transmits chemical signals from outside of the cell to the cell's nucleus.
The JAK2 protein is especially important for controlling the production of blood cells from stem cells located in bone marrow.
When there is a mutation in the JAK2 gene it results in the production of a JAK2 protein that is constantly activated and so leads to the overproduction of abnormal blood cells.
Because there are so many extra cells in the blood stream, blood clots are more likely to form and so the blood is thicker and flows more slowly and can result in the organs not receiving enough oxygen.
Nearly all people with Polycythaemia Vera and about half those with Essential thrombocythemia and Chromic idiopathic myelofibrosis have the JAK2 mutation and many of the signs and symptoms are related to a lack of oxygen in body tissues.

10. 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.
Previously we had two systems for JAK2 testing in house. All new diagnostic samples were tested using an ASO PCR which looked at the ratio between the WT and mutant. This test was only sensitive to 5% and all results between 1.5-5% were classed as equivocal and had to be repeated or ran on the taqman which is sensitive down to 0.09%. The RQ-PCR was not only used to resolve equivocal results but also as a monitoring tool for positive patients. Because we have few patients who are monitored using JAK2 levels the samples had to be batched up meaning turn around times could up to two months.

11. 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.
ddPCR was suggested as an appropriate platform to replace the old two tier strategy with the aim of improving turn around time as well as sensitivity.
The QX100 was selected as the most appropriate platform for JAK2 testing because it allows us to detect 1% mutation load using just one well. 1% is deemed the clinically relevant mutation load according to best practice guidelines.
We receive roughly 200 samples into our laboratory each month for JAK2 testing and so to keep up with this we do 3 runs of 20 patients on each run 3 times a week and so we have no backlogs and by doing numerous runs a week we can get fast confirmation of positive patients that have to be repeated.

12. 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)
To test for JAK2 using ddPCR we use an assay designed by Quantalife. This is a duplex assay meaning we can look at two signals in one reaction. The assay contains two probes, one FAM labeled specific probe for the JAK2 mutation V617F and the other VIC labelled probe for Wild type JAK2.

13. 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.
This slide shows an example of a typical positive JAK2 sample. The droplets emitting FAM (Mutant) positive signals are plotted on the pictogram along with any VIC (WT) droplets and any negative droplets that haven’t produced a signal.
The pictogram also plots any dual occupancy droplets. These duel occupancy droplets represent any droplet that may contain more than one target of interest. This could mean two positive targets, two negative targets or many different combinations depending on how many ‘chunks’ of target DNA there is in the droplet.
The QX100 assigns these duel occupancy targets by counting the positive an negative droplets and then fits these concentrations to a Poisson distribution to account for the possibility of having more than one target in each droplet.

14. 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

15. Thank you for listening.
Any Questions?