Ion Xpress Library Preparation and Sequencing

Barcoded libraries were prepared using Ion Xpress plus Fragment Library Kit (Thermo Fisher Scientific) and Ion Xpress barcode Adaptors 1-16 Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Each barcoded library was mixed in equal amounts and diluted to 100 pM. Using 2.5 μl of the 100 pM mixed barcoded library, we prepared sequencing templates with Ion One Touch™ 2 (Thermo Fisher Scientific) and Ion One Touch™ ES (Thermo Fisher Scientific). Sequencing was performed using the Ion PGM™ Sequencing 400 Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions for the 318v2 chip. Torrent Suite software (Thermo Fisher Scientific) was used to extract reads for individuals (BAM files). For each locus, one of the HLA sequences in RefSeq (https://www.ncbi.nlm.nih.gov/refseq/) was used as a reference sequence. BAM files were converted to SAM files using SAMTools [40 (link)].