Abstract
Our lab has established the NXType™ kit on the Ion-Chef and Ion-S5 workflow for high resolution HLA typing. This work was aimed to evaluate the analytical performance of the method for accuracy, precision, sensitivity, specificity, reportable range, repeatability, and reproducibility. We also determined optimal run size and workflow within the lab to strike a balance between suitable number of reads per locus and number of samples to avoid low coverage and dropouts. Using NGS as primary typing method for hematopoietic stem cell (HSC) and the solid organ (SO) programs was assessed.
Class I and HLA Class II were amplified with NXType™ or ALLType™ primer sets. Automated template preparation and chip loading were performed in the Ion Chef™ system and run on Ion S5™. Data was analyzed with TypeStream™ v1.1.0.11 and TypeStream™ Visual NGS Analysis Software.
We found that the optimal run size was a minimum of 24 samples, a 3-fold increase of previous capacity. To maximize the use of chip space the number of samples/run was increased with confirmatory solid organ typings. We determined that running 24 samples with SSOP or NGS required similar manpower and instrument time. Although lab work was less with SSOP, analysis time and effort was greater than with NGS. After two years of experience with NGS HLA typing (∼1500 samples), we considered using NGS as the primary typing method for the HSC and the solid organ programs. Our historical data showed only 2 cases where NGS gave non-concordant results with SSOP or SSP. However, these samples were flagged by the software and both had associations that were red-flagged during analysis. The repeat rate for SSOP was 2.8% while for NGS was 0.3%. All NGS repeats were technical failures and not ambiguity resolution. We evaluated the ALLtype kit, which includes DQA1 necessary for solid organ initial typing, and a liquid handler for automation of library preparation. In addition, we implemented the TypeStream Visual (TSV) analysis software that includes serological equivalents.
We confirmed that NGS is a robust methodology for HLA typing. It provides results first time is run, reducing reflex testing. Combining automation with ALLType™ kit and TSV software, the analysis will be used as the primary method and SSOP as a reflex method for HSC and SO.
