FMR1 promoter sequences, with inferred methylation states of CpG sites, recovered from male fragile X patients using hairpin-bisulfite PCR with linker barcoding and batch-stamping. Methods are as described in the text. Unconverted (methylated) CpG dyads are black, and converted (unmethylated) CpG dyads are boxed. Within the 26 nt linker (boxed region at left), the randomized 7 nt variable barcodes are shaded at far left; the designated variable batch-stamps (A:T or T:A) are shaded at right. All sequences show 100% conversion of non-CpG cytosines. (a) A distinctive hypermethylated sequence. (b and c) Redundant hypermethylated sequences recovered from independent bacterial colonies, with identical barcodes and methylation patterns. (d) A hypomethylated sequence with a distinctive barcode. (e and f) Redundant hypomethylated sequences with identical barcodes recovered from independent bacterial colonies. These are distinguishable as redundant and as different from the hypomethylated sequence ‘d’ only because of barcoding. (g) A contaminant sequence bearing our original hairpin linker that predates the addition of the barcode and batch-stamp. This sequence was recovered during analysis of the same sample that generated sequences ‘a–c’. Sequences ‘a–c’ carry a different batch-stamp than sequences ‘d–f’, with the inversion of the A–T base pair, confirming that these sequence sets came from different DNA samples. Redundant hypermethylated sequences are denoted with asterisks (^*), and redundant hypomethylated sequences with plus signs (+).

Schematic of barcoded and batch-stamped hairpin linker, designed for ligation to DraIII-cut genomic DNA of FMR1. The letter D represents a nucleotide randomly selected from A, G and T.