We performed a CNV detection method with Ion AmpliSeq sequencing and multiplex PCR-based targeted genome enrichment. The detailed protocol has been described elsewhere [40 (link)]. The read depth data was used for copy number analysis. From the results of the CNVs analysis of the 2262 probands, we picked up 14 patients with OTOA gene CNVs.
We designed a custom aCGH for 68 genes previously reported as genetic causes of non-syndromic hearing loss using the Agilent web software (Agilent SureDesign, Agilent Technologies, Santa Clara, CA, USA), with the probes covering specific chromosomal regions of those genes at 150–200 bp intervals as a design-setting on the Agilent 8 × 60 K platform (Agilent Technologies, Santa Clara, CA, USA) [41 (link)]. There were 235 probes laid across the OTOA region (chr16:21,740,000–21,772,500). We used the same DNA samples as used for the amplicon resequencing, with quality assessment also performed. Five micrograms of genomic DNA were fragmented, and labeled with cyanine-3 for reference DNA samples and cyanine-5 for subjects, and then hybridized. We performed scanning of the array with a G2600D SureScan Microarray Scanner (Agilent Technologies) according to the manufacturer’s recommended protocols, and analyzed scanned aCGH data using CytoGenomics software version 3.0.6.6 (Agilent Technologies).
We designed a custom aCGH for 68 genes previously reported as genetic causes of non-syndromic hearing loss using the Agilent web software (Agilent SureDesign, Agilent Technologies, Santa Clara, CA, USA), with the probes covering specific chromosomal regions of those genes at 150–200 bp intervals as a design-setting on the Agilent 8 × 60 K platform (Agilent Technologies, Santa Clara, CA, USA) [41 (link)]. There were 235 probes laid across the OTOA region (chr16:21,740,000–21,772,500). We used the same DNA samples as used for the amplicon resequencing, with quality assessment also performed. Five micrograms of genomic DNA were fragmented, and labeled with cyanine-3 for reference DNA samples and cyanine-5 for subjects, and then hybridized. We performed scanning of the array with a G2600D SureScan Microarray Scanner (Agilent Technologies) according to the manufacturer’s recommended protocols, and analyzed scanned aCGH data using CytoGenomics software version 3.0.6.6 (Agilent Technologies).
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