Whole Corneal Exome Sequencing

This pilot ES study was conducted with 50 ng of genomic DNA of whole corneal tissues (KC15, KC16, KC17, KC18, and KC19) using the SureSelectQXT Reagent Kit combined with the SureSelectXT Human All Exon V5 (Agilent Technologies, Cedar Creek, TX, USA) according to manufacturer’s instruction. Prepared libraries were paired-end sequenced (2 × 100 bp) on an Illumina HiSeq1500 (Illumina, San Diego, CA, USA). For each cornea sample > 80 million read pairs were generated resulted in >100× of mean coverage. Sequence readouts were initially analyzed with bcl2fastq software to generate reads in fastq format. These reads were mapped against a human genome reference sequence (GRCh37) using the Burrows–Wheeler Alignment (BWA), which was followed by BAM post-processing and variant calling using HaplotypeCaller and the GATK suite (McKenna et al., 2010 (link)). Finally, ANNOVAR (Wang, Li & Hakonarson, 2010 (link)) was used to annotate relevant information about gene names, predicted variant pathogenicity, reference allele frequencies and metadata from external resources and then to add these to the variant call format (VCF) file.