….no, not the French word for fish.
Poisson distribution is a mathematical principle that becomes incredibly important at low levels. So why am I blogging about it? Well these ‘low levels’ can be found in cell free DNA (cfDNA) and copy number variants (CNV) due to the ‘dilution principle’. Although most pathologists would be more than happy to spend a day sectioning, mathematics ultimately is an important concept that we all need to get our heads around.
Definition: Poisson is defined as ”a discrete probability distribution that expresses the probability of a given number of events occurring in a fixed interval of time and/or space if these events occur with a known average rate and independently of the time since the last event. The Poisson distribution can also be used for the number of events in other specified intervals such as distance, area or volume.”
So what does this actually mean?
Let’s have a look at digital PCR as an example of poisson distribution in action. Below, you can see digital PCR in action on a 100 well plate. In a ‘dream’ world, the 100 molecules would slot into the 100 wells, however we know that this is far from being the case. Actually, one possibility would be for 37 wells to have 0 molecule, 39 wells with 1, 13 wells with 2, 9 wells with 3 and 2 wells with 4 molecules (example below).
Taking this one step further, you can see that, using poisson distribution that 99 molecules have been calculated with a confidence limit of 77-129 (below)
Comparison to Analog
Let’s compare the ‘traditional’ (analog) qPCR sequencing to digital PCR.
The best way to describe the differences is in the figure above and the table below draws out the points. Ultimately, digital PCR is read almost like a binary code in comparison to the amplified fragments in qPCR.
|Rare allele may be difficult to detect in presence of abundant wild-type||Rare allele easily detected in presence of abundant wild-type|
|Standard curve required for quantitation||No need for reference standards or controls|
Compared to real-time PCR, digital PCR is more:
- Sensitive – can go down to lower allelic frequencies
- Precise – repeatable results every time
- Specific – can detect the right mutation
- Less affected by PCR inhibitors
- No need to worry about PCR efficiency
- Multiplexing possible
- Absolute quantitation possible
- Without a standard sample/curve
- Assay optimisation not necessary
So, will you be performing cfDNA sequencing using poisson distribution?