Real time analysis pipeline

Whole-exome capture libraries were prepared according to standard protocols using SureSelect Human All Exon 50Mb and 38Mb kit (Agilent technologies). We performed paired-end (2 × 100 bp) sequencing using the Illumina HiSeq2000 instruments, imaging analysis and base calling using the Illumina Real Time Analysis (RTA) Pipeline version 1.9. After removing reads with sequence matching the sequencing adaptors and low-quality reads (reads with more than 50% of bases with Phred quality score of < 5), high-quality reads were aligned to the reference human genome (hg19, http://genome.ucsc.edu/) using Burrows-Wheeler analysis (BWA) with default parameters. The reference human genome assembly hg19 was used for mapping all samples. SNVs were called with GATK and filtered with recommended threshold (SNV quality ≥ 20, > 3 reads covered and depth ≥ 10).

Genome DNA re-sequencing was performed by IntegraGen (Evry, France). Forty-eight genomic DNA libraries were prepared using TruSeq DNA sample preparation kit (v3) followed by paired-end 100 bases massively parallel sequencing on Illumina HiSeq 2000. Briefly, 3 μg of each sample of genomic DNA were fragmented by sonication and purified to yield fragments of 400–500 bp. Paired-end adaptor oligonucleotides from Illumina were ligated on repaired A-tailed DNA fragments, then purified and enriched by PCR cycles. Each library was quantified by qPCR before equimolar pooling of the 48 libraries. The 48-plex pool was sequenced on one flowcell lane of Illumina HiSeq 2000 platform as paired-end 100 bp reads. Image analysis and base calling were performed using Illumina Real Time Analysis (RTA) Pipeline with default parameters.

Genomic DNA was extracted from peripheral blood samples using the standard salting-out method [12 (link)]. First, the entire coding region of GJB2 and flanking splice acceptor site was analyzed by Sanger sequencing in one patient of each family, i.e. DF11-VI.1, DF25-V.3, DF99-IV.1, and DF103-III.2, aged 6, 20 (with severe intellectual deficiency), 4 and 1 years, respectively [13 (link)]. These patients then underwent WES on Illumina HiSeq2000 plattform (IntegraGen: Evry, France) using Agilent Sure Select Human All Exon v2 enrichment kit. Image analysis and base calling were performed using the Illumina Real-Time Analysis Pipeline version 1.14 with default parameters. Bioinformatic analysis was based on the Illumina pipeline (CASAVA 1.8) which aligns reads to the human reference genome (hg19) with the alignment algorithm ELANDv2 (it performs multispeed and gapped alignments). Genetic variation was annotated with the IntegraGen in-house pipeline [10 (link),14 (link)]. We achieved an average percentage of 95% of covered CCDS (Consensus Coding DNA Sequence) at 10X and 90% at 25X.The mutations identified in MYO7A and USH1G were confirmed by Sanger sequencing of the corresponding exons, using primers designed with Primer3 (http://primer3.ut.ee/) (sequences are available on request).