Exome Sequencing of Genomic DNA

Patient genomic DNA was extracted from peripheral blood using Nucleon® kit (Gen-Probe Life Sciences Ltd). Exome library was captured from 3 µg of genomic DNA using Agilent SureSelect XT Human All Exon v.4 kit. The libraries were sequenced by 100 nt paired-end reads on Illumina HiSeq platform. The obtained sequences were mapped to human genome build hg19 by using Novoalign software (Novocraft Technologies). Variants were called using Samtools program (Li et al., 2009 (link)). Variants were filtered out if their population frequency was ≥0.01 according to 1000 Genomes Project (European subset) (Genomes Project et al., 2010 (link)). We used ANNOVAR software to annotate and separate non-synonymous substitutions, indel variants and splicing mutations (Wang et al., 2010 (link)). As CMS are usually inherited in an autosomal recessive manner, we focused on all genes that have either one or more homozygous variant, or two or more heterozygous variants in the same gene. We further filtered the obtained variants against an in-house database of 14 exomes from cases with unrelated disorders, and manually removed misaligned or low quality reads.