Exomes were sequenced in a total of two siblings with Miller syndrome (kindred 1 in Table 1) and two additional unrelated affected individuals (kindreds 2 and 3 in Table 1), i.e. a total of 4 affected individuals in three independent kindreds. An average of 5.1 gigabases (Gb) of sequence was generated per affected individual as single-end, 76 bp reads. After discarding reads that had duplicated start sites, we achieved ~40-fold coverage of the 26.6 Mb mappable, targeted exome defined by Ng et al. (2009)2 (Table 2). About 97% of targeted bases were sufficiently covered to pass our thresholds for variant calling. To distinguish potentially pathogenic mutations from other variants, we focused only on nonsynonymous (NS) variants, splice acceptor and donor site mutations (SS) and coding indels (I), anticipating that synonymous variants were far less likely to be pathogenic. We also predicted that the variants responsible for Miller syndrome would be rare, and therefore likely to be novel. A novel variant was defined as one that did not exist in the datasets used for comparison, including dbSNP129, exome data from 8 HapMap individuals previously sequenced by us2, and both (Table 1).
