Exome Sequencing with Ion S5 XL

Next-generation sequencing (NGS) was performed as previously described [4 (link), 7 (link)] by Fujita and Damiati in 2017 and 2016, respectively. In brief, 100 ng of DNA was amplified for genomic library preparation using the exome enrichment kit (Ion AmpliSeq™, Life Technologies, USA) in order to sequence the key exonic regions (> 97% of CCDSs) of the genome. Ion Chef™ System (Life Technologies, USA) was used for template preparation and enrichment using Ion 540™ Kit – Chef (Life Technologies, USA). The same automated platform was used for loading Ion 540™ Chips with template-positive Ion Sphere™ Particles. Exome sequencing was performed on Ion S5™ XL Sequencer (Life Technologies, USA) with the loaded chips. Data analysis was done by Torrent Suite™ Software (v 5.2.2; Life Technologies, USA). Coverage analysis was performed using the Coverage Analysis plug-in (v5.2.0.9). Variant Caller plug-in (v5.2.0.34) was used for mutation/variant detection against the reference genome (hg19).