Hi Siti,

I took a quick look at the data you messaged me and it looks like you've got way too much DNA. You can see there are three levels of droplet amplitude in Ch1. Ideally you will see two levels, the negative droplets (no template molecules in the droplet) at a lower amplitude and positive droplets (one template molecule in the droplet) at a higher amplitude. When you see a third level, this often means that in addition to having negative (no template) and positive (one template) droplets, you also have droplets with two or more template molecules/droplet. Additionally, your Ch2 negative droplets have higher amplitude than I would expect for typical negative droplets, suggesting you have only positive droplets (1 molecule/droplet) and multi-positive droplets (>=2 molecules/droplet). It is difficult, though, to assess these results without a no-template control included where you omit the DNA so we can see the normal amplitude of negative droplets. The ‘no call’ is because the program cannot decide which droplets are positive and negative.

I would recommend you perform an experiment where you include no-template controls, as well as a series of dilutions of one of your samples. Performing a serial 10-fold dilution on one sample and running those dilutions on ddPCR will allow you to determine whether you are seeing positive and multi-positive droplets. This will also help you determine an optimal DNA concentration for your samples. 20ng/uL seems very high, although I have never used ddPCR for CNV analysis specifically. BioRad’s documents (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf page 44) suggests 10-66ng/well at the high end, while you have 400ng/well with 20ng/uL x 20uL/well. I have previously come across situations where I had to dilute my sample significantly to get meaningful ddPCR results.

Let me know once you’ve performed additional controls or if you have any questions about setting up those essential controls.