Combined next generation sequencing for HLA typing and chimerism testing

Two NGS-based assays, HLA typing (HLA-A, B, C, DRB1/3/4/5, DQA1/B1, DPA1/B1) and chimerism testing (202 single nucleotide polymorphisms) were combined and sequenced together, using the MiSeq V2 reagent kit. The HLA typing library pool (sample number: X, 1.42 nM) and the chimerism library pool (sample number: Y, 1.33 nM) were combined at the volume ratio X:2Y (X + 2Y ≤ 100). Chimerism MiSeq sample sheets (dual index) were edited to support 150 bp read length and uploaded to the BaseSpace Sequence Hub. After NGS, chimerism FASTQ files were downloaded and analyzed. An HLA typing MiSeq sample sheet was then uploaded to requeue indices for FASTQ file generation. Both the combined and the separate NGS achieved sufficient read numbers, sequence quality, chimerism assay sensitivity of 0.1 %, and comparable test results on clinical samples (HLA typing, N = 44; chimerism genotyping, N = 24, % recipient chimerism, N = 95). Potential benefits and considerations in combining NGS-based tests are discussed.