ddPCR

Introduction to Digital PCR
Yann Jouvenot, PhD
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What is ddPCR?
ddPCR
(droplet digital PCR) is a breakthrough technology,
a potential game changer in the fields of life science and
clinical diagnostics
It is a method that uses droplet technology to divide
complex samples into small, manageable sub-units
This technology is (so far) used for DNA and RNA, but
should soon expand to other targets
It provides absolute and extremely high resolution for
target quantification
ddPCR Publications
Benefits of ddPCR
•
Absolute Quantification: No need for a standard curve because number
of targets in a given volume (i.e. concentration) is counted directly
(and adjusted for multiple targets/droplet by Poisson statistics)
•
Precision: The ability to consistently make the same measurement
on replicates of a sample (minimal CV across msmts)
•
Reproducibility: The ability of a researcher to obtain the same measurement
on the same sample from experiment to experiment in the same lab OR in
another lab across the globe.
•
Sensitivity: The ability to measure very few copies of a target with precision
mostly limited by the technology independent statistics of sampling
•
Accuracy: The ability to make the correct measurement – this requires
validation before this can be claimed, but is then an intrinsic property of the
ddPCR assay used.
Digital Biology Center
Bio-Rad QX200™ Droplet Digital PCR System
Droplet Reader
Droplet Generator
Reagents
Consumables
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Droplet Digital PCR Workflow
Partition reagents and sample into thousands of droplets
Perform PCR on thermal cycler
Count droplets with a positive PCR product (fluorescent) and a negative PCR
product
Digital readout provides concentration of target DNA
“X” target
copies
Make Droplets
PCR Droplets
Read Droplets
Results
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Droplet Fluorescence Converted to a Digital Signal
Positive droplets contain at least one copy of target DNA (cDNA)
Positive droplets have increased fluorescence vs. negatives
Quantasoft software measures the number of positive and negative droplets per
fluorophore per sample
Each positives counted as 1
Each negatives counted as 0
Simultaneous Detection of Two Targets
Data Output by QuantaSoft
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Rare Event Detection
Rare Event Detection (RED)
Rare Sequence
Detection (RSD)
→ analysis of sequences
with no relation to their
background
•
•
•
Microbial genomes (virus,
bacteria, yeast)
Environmental studies
GMO
Rare Mutation
Detection (RMD)
→ analysis of sequences
closely related to the
background DNA (wildtype DNA)
•
•
•
Cancer mutations
Prenatal diagnosis
Transplanted organs
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Rare Event Detection
Method
Sensitivity
Nb of
Targets
Throughput
Cost
Abs.
Quant
qPCR
++
+
++++
Low
+
CAST-PCR
+++
+
+++
High
NA
Sanger/CE
+
+
++++
Low
NA
NGS
+
++++
+
Very High
NA
ddPCR
++++
++
+++
Moderate
++++
ddPCR is very well suited for studies with limited number of
targets, where sensitivity is of the essence. The robustness of
this method to PCR inhibitors is also an asset for detection of
rare targets in complex matrices (blood, plasma, environmental
samples).
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Tolerance of Droplet-Digital PCR vs Real-Time Quantitative PCR
to Inhibitory Substances (Dingle et al., Clinical Chemistry 59:11)
Analysis
of PCR inhibition by heparin, SDS and EDTA
ddPCR shows higher tolerance to SDS and heparin in comparison
to qPCR
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New assays for monitoring residual HIV burden in effectively
treated individuals (Strain et al., Curr Opin HIV AIDS 2013, 8)
CV improved 4-fold over qPCR results
“Digital PCR improves the accuracy and precision of HIV DNA measurement
over real-time PCR, and this general approach should be applicable to any
quantification of any nucleic acid.”
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New assays for monitoring residual HIV burden in effectively
treated individuals (Strain et al., Curr Opin HIV AIDS 2013, 8)
• Assay LOD: Between 0.7 and 3
copies per million cells
• Shaded area: below the
theoretical LOD for one single
cell
15
One-step RT-droplet digital PCR: a breakthrough in the
quantification of waterborne RNA viruses (Racki et al., Anal. Bioanal.
Chem. 2014)
• Serial dilutions of Rotavirus
in water
• 5 log dynamic range for
ddPCR
• Improved CV over qPCR
• Lower LOD
RT-ddPCR
RT-qPCR
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One-step RT-droplet digital PCR: a breakthrough in the
quantification of waterborne RNA viruses (Racki et al., Anal. Bioanal.
Chem. 2014)
• Spikings of Rotavirus RNA in different water samples
• Improved quantification and CV over qPCR
• Higher tolerance to PCR inhibition
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Quantification of Zoonotic Bacterial Pathogens within
Commercial Poultry Processing Water Samples Using Droplet
Digital PCR (Rothrock et al., Adv. Microbiol. 2013)
Improved detection sensitivity by
ddPCR for C. jejuni and L.
monocytogenes
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Quantitative Analysis of Food and Feed Samples with
Droplet Digital PCR (Morisset D et al, PLoS One 2013 May 2;8(5))
ddPCR ensures precise
measurement of GMO
content through a large
range corresponding to
routine laboratory use
(<0.1% - 30%)
Reduced cost in time and reagents
Increased tolerance to PCR inhibition
observed in different matrices
Cancer Mutation Detection: JAK2 V617F Detection from
Whole Blood – Pyrosequencing vs ddPCR (Hindson et al.,
2012)
Results:
Excellent concordance between 2 methods for
samples with JAK2 V617F mutant abundances
above several percent (R2 ~0.91)
However, at least 3 samples were judged
normal by pyrosequencing, but determined by
ddPCR to have the JAK2 mutation present at
~0.1 and 0.02% abundance
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Gene Expression Analysis
Analysis
Usually
of RNA (transcripts) levels in biological samples
performed in duplex (in combination with reference gene)
Includes
detection of miRNA
Growing
research on single cell transcriptome
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Gene Expression Analysis
Method
Precision
Nb of
Targets
Throughput
Cost
Abs.
Quant
RT-PCR
++
+
++++
Low
+
Array
+
+++
+
High
NA
RNA-Seq
+
++++
+
Very High
NA
NanoString
++
+++
+
High
++
ddPCR
++++
+
+++
Moderate
++++
ddPCR is very well suited for studies with limited number of
targets, where changes in levels of expression are <2-fold. It
is also useful for studies of alternate species (RNA editing,
allelic expression), in single cell studies or for low abundance
targets.
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ADAR Regulates RNA Editing,Transcript Stability, and Gene
Expression (Wang et al., Cell Reports 5, 1–12)
• Use of ddPCR to validate RNA editing events
• Confirms role of ddPCR as complementary technology to NGS
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Reduced C9orf72 gene expression in c9FTD/ALS is caused by
histone trimethylation, an epigenetic event detectable in blood
(Belzil et al., Acta Neuropathol, 013-1199-1)
“As
transcript variant 1 was difficult to detect through standard qRT-PCR, we
assessed its expression using a highly precise and absolute nucleic acid
quantification technique, termed droplet digital PCR (ddPCR)”
ddPCR
allows scientists to quantify very low levels of transcripts
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MicroRNA-137 represses Klf4 and Tbx3 during differentiation of
mouse embryonic stem cells (Jiang et al., Stem Cell Research (2013) 11)
qRT-PCR
Use
of ddPCR to quantify miRNA during stem
cells differentiation
While
ddPCR
qRT-PCR works well to characterize
large changes (Fig A), but ddPCR is needed
when the differences are more subtle (Fig B)
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Copy Number Variation
• Analysis of the change in ploidy of certain genes, genomic
regions or chromosomes
• Can be associated with normal developmental processes or
pathological evolution
• Important field of study for cancer, human genetics, crop
studies…
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Copy Number Variation
Method
Sensitivity
Nb of
Targets
Throughput
Cost
Linkage
qPCR
+
+
++++
Low
+
FISH
+
+
+
High
++
CGH Array
+++
++
+
High
+
NGS
+/++++
++++
+
Very High
+
ddPCR
++++
++
+++
Moderate
+++
ddPCR is very well suited for studies with limited number of
targets, where sensitivity is of the essence (5 vs. 6 copies). It
also allows the researcher to obtain information on the physical
linkage of the studied targets (is it a tandem amplification or an
additional chromosome?)
Quantitative and Sensitive Detection of Cancer Genome
Amplifications from Formalin Fixed Paraffin Embedded
Tumors with Droplet Digital PCR
Nadauld et al., Transl. Med. 2012, 2:2
ddPCR
FGFR2 genome amplification
(copy number variation, or
CNV, assay)
21 FFPE gastric and colorectal
tumors
5 matched normal FFPE
samples
Cell line with known FGFR2
amplification titrated in normal
DNA
is a robust method for quantify genome amplification in FFPE samples
across a wide range of concentrations
Structural haplotypes and recent evolution of the human
17q21.31 region
Boettger et al., Nat. Genetics 2012 Jul 1;44(8)
• Copy number analysis of 3
regions of 17q21.31 by wholegenome sequencing (b, c, d),
and by ddPCR (e, f, g).
• Copy number determination in
234 samples by NGS and
ddPCR >99% concordant (h, i, j)
• ddPCR provides easy,
inexpensive, accurate way to
validate and further study CNVs
discovered by NGS.
Best Reasons to use ddPCR
Sensitivity
–Detect rare mutations in complex backgrounds
–Detect rare mutations earlier
–10-1000x fold improvement over qPCR
–Works with FFPE samples
–Works with blood, tissues, environmental samples…
Absolute Quantification
–Answers in absolute numbers of molecules (not Cq)
–Quantify lower levels of targets
–No standard curve
Precision
–Measure more subtle differences in expression or mutation
–Detect structural variants in cancers
–Higher tolerance to PCR inhibitors
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