Table 2.

Conditions of testing and optimization of ddPCR assays.

Gene, SNP rs Number	Allele	Primer F (5′–3′)	Primer R (5′–3′)	Probe (5′–3′)			PCR Product bp	Specificity to cffDNA	Optimal Annealing Temperature
				Fluorophore	Sequence of the Probe	Quencher			55 °C **	60 °C
RHD	RHD	GGGTGTTGTAACCGAGTGCTG	CTCCAAGCAGACCCAGCAAG	56-FAM	CCCACAGCTCCATCATGGGCTACAA	3IAkFQ	72	* YES	YES	YES
		GGGTGTTGTAACCGAGTGCTG	CCGGCTCCGACGGTATC				134	YES	YES	YES
β-globin	---	GTGCATCTGACTCCTGAGGAGA	CCTTGATACCAACCTGCCCAG	5HEX	AAGGTGAACGTGGATGAAGTTGGTGG	3IAkFQ	74	* YES	YES	YES
RHCE, rs609320	RHE	ATTCTTCCTTTGGATTGGAC	CTTGTGGATGTTCTGGC	5HEX	TCAGCAGAGGAGAGTTG	3IAkFQ	64	* YES	YES	YES
		TGGCATTCTTCCTTTGGATTGG	CTCAGACCTTTGGAGCAGGAGT				134	YES	YES	YES
	RHe	ATTCTTCCTTTGGATTGGAC	CTTGTGGATGTTCTGGC	56-FAM	TCAGCAGAGCAGAGTTG	3IAkFQ	64	* YES	YES	YES
		TGGCATTCTTCCTTTGGATTGG	CTCAGACCTTTGGAGCAGGAGT				134	YES	YES	YES
RHCE, rs676785	RHC	AATACCTGAACAGTGTGATG	CTGCTGGACGGCTTC	5HEX	CCTTCCCAGAAGGGAAC	3IAkFQ	74	* YES	YES	NO
		CCAGCCACCATCCCAATACC	TGTGCAGTGGGCAATCCTG				94	YES	YES	NO
	RHc	AATACCTGAACAGTGTGATG	CTGCTGGACGGCTTC	56-FAM	GATGACCACCTTCCCAGGAGGGAA	3IAkFQ	74	* YES	YES	NO
		CCAGCCACCATCCCAATACC	TGTGCAGTGGGCAATCCTG				94	YES	YES	NO
KEL, rs8176058	KEL1	ddPCR Mutation Assay: KEL p.T193M, Human		56-FAM	ddPCR Mutation Assay: KEL p.T193M, Human	3IAkFQ	65	* YES	YES	NO
		GGTAAATGGACTTCCTTAAAC	CTGAAGAAAGGGAAATGG	56-FAM	TAACCGAATGCTGAGACTTCTGATGAGTCAG	3IAkFQ	77	NO	NO	NO
	KEL2	ddPCR Mutation Assay: KEL p.T193M, Human		5HEX	ddPCR Mutation Assay: KEL p.T193M, Human	3IAkFQ	65	* YES	YES	NO
		GGTAAATGGACTTCCTTAAAC	CTGAAGAAAGGGAAATGG	5HEX	TAACCGAACGCTGAGACTTCTGATGAGTCAG	3IAkFQ	77	NO	NO	NO

* Assays selected for ddPCR as proper for plasma samples analysis. ** Annealing temperature 55 °C was used for plasma samples analysis, as optimal.