1. FECH gene marker site (Single-plex assay design)
Same forward primer with the
degenerate base denoted by Y
Assay for
methylated sequences Y CG CG CG CG
VIC
MGB
Probe targeting 2 of the 4 CpG sites within the DMR
Different reverse primer targeting 2 of the 4 CpG sites near the 3’ end of the DMR
Same forward primer with the
degenerate base denoted by Y
Assay for
unmethylated sequences Y TG TG TG TG
FAM
MGB
Probe targeting 2 of the 4 CpG sites within the DMR
Different reverse primer targeting 2 of the 4 CpG sites near the 3’ end of the DMR B
Ery-1 & Ery-2 marker sites (Duplex assay design)
Same forward primer with the
degenerate base denoted by Y Y CG CG CG
VIC
MGB
Probe targeting the 3 CpG sites within the DMR
Same reverse primer
Assay for both methylated and
unmethylated sequences
Same forward primer with the
degenerate base denoted by Y Y TG TG TG
FAM
MGB
Same reverse primer
Probe targeting the 3 CpG sites within the DMR
Supplemental Fig. 6. Schematic diagram of the design of digital PCR assays targeting the three erythroblast-specific DMRs: (A) FECH-gene marker site (B) Ery-1 and Ery-2 marker sites.