A duplex ddPCR assay was adapted using separate indicators for PfMGET and Pfs25 markers in a single reaction, and Pfs16 was quantified in a separate reaction (Pomari et al., 2020 (link)). For both assays, each reaction included 11 μL Supermix for Probes (no dUTPs) (Bio-Rad Laboratories Inc (2018) ., Hercules, CA, US), 2 μL cDNA template, nuclease free water as needed, 455 nM of each primer and 250 nM of each probe in a final reaction volume of 22 μL. Droplets were generated using the QX200 Droplet Generator (Bio-Rad Laboratories Inc (2018) , Hercules, CA, US) within a range of 10,000 to 20,000 droplets. Reactions were performed on the C1000 Touch Thermal Cycler (Bio-Rad Laboratories Inc (2018) , Hercules, CA, US) under the following programs (ramp rate 2°C/second): Duplex PfMGET/Pfs25 - 95°C for 10 minutes, 40 cycles of 94°C for 30 seconds and 50°C for 1 minute, 98°C for 10 minutes; Pfs16 - 95°C for 10 minutes, 40 cycles of 94°C for 30 seconds and 55°C for 1 minute, 98°C for 10 minutes. Droplets were analyzed and counted on a QX200 Droplet Reader (Bio-Rad Laboratories Inc (2018) , Hercules, CA, US).
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