Schematic workflow of the phase-defined HLA gene sequencing. (A) The individual tagging method for HLA homozygous samples. The 2 × 250 bp paired-end reads of the pooled amplicons were aligned to six HLA gene sequences from the hg19 and the consensus sequences were determined. Most of the analytical tools shown here are standard use for genome sequence alignment and variant detection. For generating consensus sequence, we used original perl scripts to list variants and to construct HLA gene sequences. (B) The gene-tagging method for HLA heterozygous samples. The 2 × 250 bp paired-end reads of the each amplicon were aligned to the corresponding gene and six genes were separately analyzed to avoid mismapping. In the alignment step, 2 × 250 bp paired-end sequence reads were aligned to reference sequence using BWA and SAMtools. SNVs were detected by UnifiedGenotyper in GATK. Paired-end reads harboring SNVs in both forward and reverse reads were extracted to construct two phased HLA gene haplotype sequences using our original perl script. Finally, two HLA gene haplotype sequences from an individual were generated with phase-defined SNVs and Indels as HLA gene haplotypes.