Schematic for bisulfite sequencing (BSS) analysis of FSHD-associated 4qA chromosomes and 4q D4Z4 repeat units. (A) Cartoon depicting the location of bisulfite (BS) PCR products for the 4qA BSS assay (blue), the 4qA-L BSS assay (orange), and the DUX4 5’ BSS assay (green). For the DUX4 5’ reaction, the nested primer has a preference for a 4q D4Z4 polymorphism (red “x”); however, a fraction of D4Z4 units are amplified from chromosome 10q arrays (denoted by thin green lines), (*) including chromosome 4q-type D4Z4 units present on chromosome 10q due to trans chromosomal rearrangements found in ~6% of subjects . The proximal Bsa AI and Fse I methylation-sensitive restriction enzyme sites analyzed by Southern blotting are indicated (B and F, respectively) and highlighted in yellow. (B and C) Diagrams of the distal-most D4Z4 repeat that produces the polyadenylated DUX4-fl mRNA and is analyzed in the (B) 4qA BSS assay and (C) 4qA-L BSS assay. Arrows indicate BS PCR primers and red “X” indicates sequence differences with 4qA; rare 10A or 4A166 products amplified in the absence of 4A alleles and due to primer degradation are detected and eliminated from analysis by specific sequence polymorphisms (Additional file 1: Figure S1). Neither 4qA nor 4qA-L BSS assay amplifies the 4qB allelic variant.