Aim: A procedure was validated for combined next-generation sequencing (NGS) of HLA typing and chimerism tests to minimize test cost and time.
Method: Two NGS library pools, HLA typing (AllType FASTplex, ThermoFisher, Waltham, MA, USA, 1.5 ng/µL of 800bp, 2.84nM) and a chimerism (AlloSeq Tx, CareDx, Brisbane, CA, USA, 0.22 ng/µL of 250 bp, 1.33nM) were sequenced in combination using V2 MiSeq reagent kits (combined2 × 150 bp, 300 cycle) or separately using V2 micro kits (2 × 150 bp, 300 cycle) and V3 kits (2 × 75 bp, 150 cycle), respectively. Combined NGS pools were prepared at the volume ratio of X:2Y and a maximal sample number of X+2Y ≤96 (X: typing sample number; Y: chimerism sample number). Before sequencing, a chimerism sample sheet was uploaded to the BaseSpace Sequence Hub. After sequencing, chimerism FASTQ download, automatic read-trimming (75bp), and 202 single nucleotide polymorphisms analysis was performed using the AlloSeq Assign software, version 2.2 (CareDx). HLA typing sample sheet was uploaded and re-queued to index HLA typing reads. Typing FASTQ download and HLA 11 loci analysis was performed using the TypeStream Visual (TSV) software, version 2.1 (ThermoFisher).
Results: Combined sequencing was validated from the batches of 16 or 24 HLA typing samples in combination with 31-34 chimerism samples. Median reads (range) per sample were 788K (433K-1192K) for typing in combination; 1048K (575K-1452K) for typing alone; 899K (326K-1931K) for chimerism in combination; 1697K (670K-3834K) for chimerism alone. All met the validated read number (250K for typing; 300K for chimerism). List prices of MiSeq reagent kits were $1749 ($1178 +$571) when sequenced separately and $1359 (saving of $390) in combination. Time on MiSeq was 40 hours (21 hours + 19 hours) when sequenced separately and 24 hours (saving of 16 hours) in combination.
Conclusion: Combined NGS assay of HLA typing and chimerism are validated to save cost and time.
