Whole exome sequencing was initially performed on DNA extracted from peripheral blood leukocytes of 111 individuals from the 18 most extensive families from our cohort (76 with DP and 35 controls). The exome sequences were aligned to the UCSC hg19 reference genome. Picard tools and the genome analysis toolkit were used to mark PCR duplicates, realign around indels, recalibrate quality scores and call variants. Variants were analyzed further and filtered for potential causal variants using filters for quality control, predicted functional annotation, minor allele frequency (MAF), segregation with trait, variants in multiple families and biological relevance (See methods and Table EV1 for further information on filtering criteria). Targeted exome sequencing using a Fluidigm array of 28 candidate genes identified post-filtering was then performed in a further 42 families from the same cohort (288 individuals, 178 with DP and 110 controls). Variants post targeted re-sequencing were filtered using the same criteria as the whole exome sequencing data. Rare variant burden testing was performed for all genes selected for targeted re-sequencing, in order to rank candidate genes post targeted re-sequencing. A multiple comparison adjustment was applied to the set of 28 p values post hoc (Benjamini et al., 2001). Screening of 100 further cohort controls was via conventional Sanger sequencing. Functional annotation of the variants as described elsewhere in methods. DP - delayed puberty. *data unpublished. 