Dg8 cartridges and gaskets

Digital droplet PCR
was performed using the QX200 Droplet Digital PCR System (Bio-Rad)
according to the manufacturer’s instructions. The ddPCR Supermix
for Probes (no dUTP, Bio-Rad) was mixed with 0.9 μM primers,
0.25 μM probe, and water. 20 μL of PCR reagent mix was
supplemented with 5 μL of solution containing target DNA. Droplets
were generated using the QX200 Droplet Generator with DG8 cartridges
and gaskets (Bio-Rad), transferred to a 96-well plate as recommended
by the manufacturer, and sealed using the PX1 PCR plate sealer (Bio-Rad).
Thermocycling was performed in a Mastercycler Nexus Gradient (Eppendorf)
with an initial denaturation of 10 min at 95 °C, followed by
40 cycles of 30 s at 94 °C and 60 s at 60 °C. After thermal
cycling, the plate was held at 98 °C for 10 min followed by cooling
to 4 °C. The plate was transferred to the QX200 Droplet Reader
for data collection. Results were analyzed using the QX Manager 1.2
Standard Edition software. The QX200 workflow took about 30 min for
wet lab sample processing with droplet generation, 120 min PCR run
time, and about 60 min detection and analysis with the QX200 device.

Among various ddPCR applications, Rare Mutation and Sequence Detection were chosen. To specifically target sequences differing by only one nucleotide, two probes labelled with different fluorophores were designed together with a pair of primers. Because of the high dilution of the sample, probes bind to templates carrying specific SNP variants with a high specificity. After the readout based on the fluorescence, data were analyzed using Poisson statistics to determine the concentration of template carrying each SNP variant (and its flanking sequence) in the original sample. ddPCRs were prepared using DG8 cartridges and gaskets, Droplet Generation Oil for Probes, and ddPCR Supermix for Probes (no dUTP) (Bio-Rad), and they were performed on the QX200 Droplet Digital PCR System (Bio-Rad), according to the manufacturer’s protocols and established MIQE guidelines for ddPCR [67 (link)]. ddPCR assays targeting identified SNP variants were designed by a Bio-Rad’s technician and their sequences are unknown; however, their assay IDs are listed in Additional File 10: Table S2. As a reference, ddPCR assay targeting β-actin was used, while for determination of HTT transgene copy number, an assay targeting mouse Rpp30 was used.