Extended Data Figure 2: Schematic of strategy for identifying the segregating structural forms of the C4 locus.

a, Molecular assays for measuring copy number of the key, variable C4 structural features—the length polymorphism (HERV insertion) that distinguishes the long (L) from the short (S) genomic form of C4, and the C4A/C4B isotypic difference. Each primer–probe–primer assay is represented with the combination of arrows (primers) and asterisk (probe) in its approximate genomic location (though not to scale). b, Measurement of copy number of C4 gene types in the genomes of 162 individuals (from HapMap CEU sample). The absolute, integer copy number of each C4 gene type in each genome is precisely inferred from the resulting data. To ensure high accuracy, the data are further evaluated for a checksum relationship (A + B = L + S) and for concordance with earlier data from Southern blotting of 89 of the same HapMap individuals⁵¹. c, To measure the copy number of compound structural forms of C4 (involving combinations of L/S and A/B), we perform long-range PCR followed by quantitative measurement of the A/B isotype-distinguishing sequences in droplets. d, Analysis of transmissions in father–mother–offspring trios enables inference of the C4 gene contents of individual copies (alleles) of chromosome 6. Three example trios are shown in this schematic. e, Examples of the inferred structural forms of the C4 locus (more shown in Fig. 1c). For the common C4 structures (AL–BL, AL–BS, AL–AL, and BS), genomic order of the C4 gene copies is known from earlier assemblies of sequence contigs in individuals homozygous for MHC haplotypes due to consanguinity¹⁷ and other molecular analyses of the C4 locus¹⁸. For the rarer C4 structures, the genomic order of C4 gene copies is hypothesized or provisional.