Ingenuity variant analysis platform

Considering that the twins were monochorionic identical twins and twin A presented with situs inversus, WES was first performed on twin A and his parents. Twin A’s DNA sample, obtained through amniocentesis and parents’ peripheral blood DNA were used for whole-exome sequencing to identify causal genetic variants. Briefly, 3 μg DNA was sheared to fragments of 150–200 bps in size. An adaptor-ligated library was prepared using the paired-end sequencing library prep kit (Agilent Technologies, Santa Clara, CA, United States). Both coding exons and flanking intronic regions were enriched using the Agilent SureSelect XT Human All Exon V6 reagent kit (Agilent Technologies, Santa Clara, CA, United States). Clusters were then generated by isothermal bridge amplification using the Illumina cBot station, and sequencing was performed using the Illumina HiSeq 2500 System (Illumina, San Diego, CA, United States). Burrows-Wheeler Alignment tool (BWA) v0.2.10 was used for sequence alignment to the Human Reference Genome (NCBI build 37, hg 19). Data quality was assessed using FastQC (version 0.11.2). The read data were uploaded to the Ingenuity Variant Analysis platform (Qiagen, United States) for mutation screening and interpretation. Copy number detection and visualization of WES were performed using the bioinformatics tool CNVkit.2.3.