Because of the moderate sample size in the sequencing stage and our focus on rare variants, the power to detect a significant association with a single RV and SD is very low. The discovery stage was employed only for variant discovery, not for association discovery. Based on the sequencing results, we selected a subset of the RVs and genotyped them individually in an independent sample of 6751 similarly assessed subjects for follow up, and in the 1520 subjects included in the sequencing stage, to validate the genotype calls in that sample. The criteria for selecting these RVs were: first, MAFs ≤ 1% in both EAs and AAs; second, location in coding regions or introns within 5 bp of a splice junction; third, variants were found in at least three more case pools than control pools, or vice versa, so they could be either deleterious or protective. A total of 17 RVs met these criteria. Because three TaqMan assays failed manufacturing quality control, and another three assays performed poorly, in the end, 11 RVs were successfully genotyped.
