Whole genome SNP array and sequencing analysis

Genome-wide SNP array was performed using Illumina HumanOmni2.5 BeadChip (Illumina, San Diego, CA) containing ∼2.5 million markers, for all nine members of 1617 family. Copy number variation (CNV) was called out by CNV partition plug-in in the Beadstudio software. SNP genotyping data were exported to PLINK (35) and non-parametric linkage analysis was done by MERLIN software package (36). Shared genomic segments among all affected individuals were collected for whole-genome sequencing analysis (Supplementary Material, Fig, S7A and B).

Germline DNA from individuals I-3, II-1, II-3 and II-4 from family 1617 were subjected to whole-genome sequencing, as previously described (37). Briefly, paired-end libraries with 100-bp sequencing were prepared and genome sequencing was performed on an Illumina HiSeq2000 instrument. Next generation sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under accession number SRX1942054.

An in-house pipeline was used to align reads against the hg19 genome build (GRCh37) and to call single-nucleotide variants and small insertion/deletions (indels). The alignment and quality score recalibration were performed using Burrows-Wheeler Aligner (BWA) and the Genome Analysis Toolkit (GATK, see Web Resources) according to GATK best practice (38,39). Targeted regions were sequenced to an average depth of 34x, with 98% of the regions covered by 1x and 96% covered by 10x (Supplementary Material, Fig. S7C). Common polymorphisms (with reported frequency of >1%) were removed by comparison with dbSNP135, the 1000 Genomes Project database using ANNOVAR (40) (see Web Resources) and a proprietary database of exomes from in house non-CS individuals. All variants were manually inspected using the Integrative Genomics Viewer (IGV, see Web Resources) (41). The accession numbers for naming mutations in USF3 sequences reported in this paper is {"type":"entrez-nucleotide","attrs":{"text":"NM_001009899.3","term_id":"613410204","term_text":"NM_001009899.3"}}NM_001009899.3.